WALNUT

Editör

Muhammet Ali GUNDESLİ

Authors

Aysen Melda COLAK

Cafer Hakan YILMAZ
Hakan KELES
Halil İbrahim OGUZ
Hayat TOPCU
İlbilge OGUZ
Kamil SARPKAYA
Mozhgan ZARIFIKHOSROSHAHI
Muhammet Ali GUNDESLİ
Murat GUNEY
Naim OZTURK
Nesibe Ebru KAFKAS
Remzi UGUR
Selim ERDOĞAN
Serhan CANDEMİR
Sule POLAT
Yılmaz UGUR
Zeynep ERGUN
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ISBN: 978-625-8061-35-2
Cover and Photo Design: Muhammet Ali GUNDESLİ and Hakan KELES
December / 2021
Ankara / Turkey
Size = 16x24 cm
 Table of Contents

--------------------------------

Page number
Part authors
Contributors 1
Preface 3
Acknowledgements 6
Part I: Walnut Production, Trade and Forecast 7
Serhan Candemir
Part II: Flowers and Fertilization Biology in 23
Walnuts
Hakan Keles
Part III: The use of Rootstock in Walnut Cultivation 41
Remzi Ugur
Part IV: Soil Management, Fertilization in Walnut 65
Cafer Hakan Yılmaz
Part V: Micropropagation of Walnut 87
Sule Polat, Muhammet Ali Gundesli and Hayat Topcu
Part VI: Sustainable Farming Systems and Organic 107
Walnut
Aysen Melda Colak
Part VII: Industrial Use of Walnut 133
Zeynep Ergun and Mozhgan Zarifikhosroshahi
Part VIII: Diseases in Walnut Orchard 153
Kamil Sarpkaya
Part IX: Important Pests of Walnut and Their 177
Control
Naim Ozturk and Remzi Ugur
Part X: Walnut (Juglans regia L.) Breeding and 203
Walnut Breeding Criteria
Halil İbrahim Oguz and İlbilge Oguz
Part XI: Advances in Walnut Breeding 231
Murat Guney
Part XII: Conventional and Molecular Breeding in 249
Walnut
Hayat Topcu
Part XIII: Secondary Metabolites in Walnut 311
Mozhgan Zarifikhosroshahi, Murat Guney and Nesibe
Ebru Kafkas
Part XIV: Fatty Acids Composition of Walnut 339
Yılmaz Ugur and Selim Erdoğan
Part XV: Nature's Miracle Walnut and Human 365
Health
Muhammet Ali Gundesli
 Wal nut |1

Contributors

--------------------------------
Dr. Serhan CANDEMİR; General Directorate of Agricultural Research and
Policies, East Mediterranean Transitional Zone Agricultural Research of Insttute,
Department of Agriculture Economy, E-mail: serhan_candemir@hotmail.com

Assoc. Prof. Hakan KELES; Yozgat Bozok University, Agriculture Faculty,

Department of Horticulture, Yozgat, Turkey, E-mail: hakan.keles@yobu.edu.tr

Dr. Remzi UĞUR; General Directorate of Agricultural Research and Policies, East
Mediterranean Transitional Zone Agricultural Research of Insttute, Department of
Horticulture, E-mail: remzibey@hotmail.com

Dr. Cafer Hakan YILMAZ; General Directorate of Agricultural Research and

Policies, East Mediterranean Transitional Zone Agricultural Research of Insttute,
Department of Soil Science and Plant Nutrition,
E-mail: c_hakanyilmaz@hotmail.com

Şule POLAT; Department of Horticulture, Agriculture Faculty, Cukurova

University, , Adana, Turkey, E-mail: polatsule@outlook.com

Assoc. Prof. Aysen Melda COLAK; Department of Horticulture, Faculty of

Agriculture, Usak University, Usak, Turkey, E-mail: aysenmelda.colak@usak.edu.tr

Dr. Zeynep ERGUN; Department of Bioengineering, Faculty of Engineering,

Adana Alparslan Turkes University, E-mail: zergun@atu.edu.tr

Dr. Kamil SARPKAYA; Department of Forest Engineering, Faculty of Forestry,

Karabuk University, E-mail: kamilsarpkaya@karabuk.edu.tr

Dr. Naim OZTURK; General Directorate of Agricultural Research and Policies,

Biological Control Research Institute, Yuregir, Adana, Turkey, E-mail:
naim.ozturk@tarimorman.gov.tr,

Prof. Dr. Halil İbrahim OGUZ; Nevsehir Haci Bektasi Veli University, Faculty of
Architecture, Department of Biosystem Engineering, E-mail:
hioguz@nevsehir.edu.tr

Ilbilge OGUZ; Cukurova University, Agriculture Faculty, Department of

Horticulture, Adana, Turkey, ilbilge94@gmail.com
2|Walnut

Assoc. Prof. Murat GUNEY; Department of Horticulture, Faculty of Agriculture,

Yozgat Bozok University, Yozgat, Turkey, E-mail: murat.guney@yobu.edu.tr

Dr. Hayat TOPCU; Department of Agricultural Biotechnology, Tekirdag Namık

Kemal University, ,

E-mail: hayattopcu@nku.edu.tr

Dr. Mozhgan ZARIFIKHOSROSHAHI; Cukurova University, Agriculture

Faculty, Department of Horticulture, Adana, Turkey, E-mail: mn_zarifi@yahoo.com

Prof. Dr. Nesibe Ebru KAFKAS; Department of Horticulture, Faculty of

Agriculture, Cukurova University, Adana, Turkey, E-mail:
ebruyasakafkas@gmail.com

Dr. Yılmaz UGUR; Vocational School of Health Services, Pharmacy, Inonu

University, E-mail: yilmaz.ugur@inonu.edu.tr

Prof. Dr. Selim ERDOGAN; Faculty of Pharmacy, Inonu University, Malatya,

Turkey, E-mail: selim.erdogan@inonu.edu.tr

Assoc. Prof. Muhammet Ali GUNDESLI; Department of Plant and Animal

Production, Nurdağı Vocational School, Gaziantep University, E-mail:
maligun46@gantep.edu.tr
 Wal nut |3

Preface

--------------------------------
Walnut is one of the most important fruits which has been defined as a
"Super food" in recent years that appreciated by many for its taste and
aroma and evaluated in the Nuts category. This food is consumed with
love, is a special fruit with a nutritional and consumer-friendly
dimension that provides physiological benefits for human health.
Consumers are willing to pay good money for these features that help
ensure strong growth in worldwide production and contribute to the
country's economy. Walnut production in both nuts and other products
has increased significantly in the last two decades in traditionally
leading walnut producing countries such as China, USA, Chile,
Turkey and Ukraine. Also during this time, China and the USA are the
largest producers in the world, and production in China is also
increasing dramatically with the rise of the middle class in the world's
most populous country. The relatively long juvenile period and many
labor-intensive processes take place in walnut cultivation, from pollen
collection, hand pollination and fruit protection from
birds/insects/diseases to manual harvesting. However, as a result of
rapid developments in the field of molecular genetics in recent years,
it shows great promise for wider improved diversity characteristics
and more efficient studies in walnut breeding.

I think it is of greater importance among future walnut growers, who

will have to adopt more complex, site-specific production strategies in
terms of plant materials and cultural practices. Considering global
warming in recent years, taking into account current and future
4|Walnut

climatic conditions, potential genetic advances (such as proprietary

varieties resistant to biotic and abiotic stress factors), may cause
limitations in the application of certain modern orchard technologies
for walnut producers. Therefore, these issues need to be addressed.
For this purpose, characterization and conservation of the diversity of
walnut genetic resources and broad and multidisciplinary action plans
are needed for this purpose.

For this reason, in recent years, there are brochures, books, etc., about
the book on walnuts, which he has included in his new studies, and the
human health effects of walnuts. There were not many documents and
it was stated that both students and those who were interested in
walnut needed a new book about walnuts.

We need vitamins and minerals for our health. These nutrients occur
naturally in plants. However, our busy lives make it difficult to
maintain a balanced diet on a regular basis. Our daily activities often
prevent us from consuming enough of grains, fruits, vegetables and
other products. Daily multivitamin and mineral support may not
provide us with all the nutrients we need. In addition, we cannot get
enough antioxidant substances that the body needs on a daily basis.
For this, some of the necessary nutrients can be supplemented with
many plants that are functional food sources and nutritional
supplements obtained from these plants.

I thought this was mainly due to the fact that new books were not
written. For this reason, I wanted to bring to the agenda the bioactive
compounds it contains and walnuts, which have become increasingly
popular as a support product for human health, which has been very
 Wal nut |5

important to both the country's economy and the producer economy

and the last years. While the book was being prepared, it was prepared
by scanning current sources together with our expert academician
friends.

In the book; will help students, research scientists and individuals who
are curious about walnuts to better know the walnut, which is
undoubtedly the most interesting area of human health in recent years,
to understand more, to research more deeply and finally to benefit
more from them. It has been prepared in the light of scientific findings
and academic rules in order to help you. Almost every subject related
to walnut is also explained in detail with charts, graphics, pictures and
figures. The book provides readers with some innovations in terms of
topics and approach. With this compiled book, it is aimed to inform
the producers about walnut cultivation, which is currently grown in
our country and which is in demand every day, and I hope it will be
useful to all our readers.

I would like to thank all my friends and IKSAD International

Publishing House publication staff who contributed to the preparation
of the walnut book. I hope that this printed book study called "Super
Food" in Walnut will be useful to all academicians, producers and
students, as well as those who are interested in walnuts.

December 2021
Editor
Assoc. Prof. Muhammet Ali GUNDESLİ
6|Walnut

Acknowledgements

--------------------------------
This publication helps connect scientific research initiatives and
enables scientists to share their ideas with colleagues and enhance
their research, careers and innovation.
 Wal nut |7

Part I
--------------------------------

Walnut Production, Trade and

Forecast
Dr. Serhan Candemir1

Walnuts are grown all over the world, from the Carpathians to Turkey,
Iran, Iraq, Afghanistan, Southern Russia, India, and Korea (Sen et al,
2006). Although it is documented in many scientific publications that
people in Anatolia have known this plant for at least 3000 years and
have benefited from various products of this plant, walnut cultivation
is carried out at an altitude of 1000-2000 m in the Swiss Alps and an
altitude of 1730 m in Turkey's Munzur Mountains (Ferhatolu, 2001;
Kilci, 2015).

The fossils obtained from cultivation areas are the most important
evidence that this plant has been grown in wide geography for
centuries due to the walnut tree's high adaptability to environmental
conditions. According to some sources, the walnut dates back to BC.
They claim that it took 750-500 years to travel from Iran to Greece.
According to the sources cited above, the history of walnut cultivation

1
General Directorate of Agricultural Research and Policies East Mediterranean
Transitional Zone Agricultural Research of Insttute, Department of Agriculture
Economy Kahramanmaras, Turkey, ORCIDs: Dr. Serhan CANDEMIR,
https://orcid.org/0000-0003-4248-7024, serhan_candemir@hotmail.com
8|Walnut

dates back to ancient times (Akarçay, 2007). Turkey, which has a long
and deep-rooted fruit-growing culture, is the home of walnuts and
many other fruit species (Koyuncu and Aşkın, 1999).

Preferring fruit varieties that can be adapted to various climate and

soil conditions around the world is important because it creates
alternatives that can serve a variety of functions (Gülcan et al 2000).
The fruits produced not only supply raw materials to many sectors, but
also meet the food needs of people. The production and consumption
of hard-shelled fruits, which contain valuable components such as
protein, fat, and carbohydrates for a healthy diet, is increasing daily.
Walnut is a popular species for hard-shell fruit cultivation.

With the impact of epidemics all over the world, healthy nutrition has
become an important issue. Hard-shelled fruits, a fast-consuming food
product, are among the producers' investment preferences in terms of
aquaculture, economy, and nutrition. Walnut is regarded as an
important agricultural product throughout the world due to its wide
range of applications, with its fruit, leaves, timber, and green shell all
being economically valuable. The fact that it is a raw material in many
industrial sectors such as food, furniture, and medicine has allowed for
an increase in demand for walnuts both globally and Turkey.

Walnut cultivation in Turkey is mostly done for domestic

consumption and domestic consumption. The walnuts produced
cannot meet the consumption, especially in recent years, walnuts are
imported at an increasing rate, but although it changes according to
the years, very little of it is exported to different countries as shelled
 Wal nut |9

and kernel walnuts. While Turkey's walnut proficiency index was

approximately 94% in 2000, it was 73% in 2019 (Turkstat, 2021).

The majority of walnut cultivation in Turkey is done for domestic and

household consumption. Walnuts are being imported at an increasing
rate, but very little of it is being exported to various countries as
shelled and kernel walnuts, even though production cannot keep up
with demand, particularly in recent years. While Turkey's walnut
proficiency index was around 94% in 2000, it is now only around 76%
in 2019 (Turkstat, 2021).

Walnuts in the World

In many countries around the world, 4.5 million tons of production is

made on a total area of 1.3 million hectares. While there were no
major changes in the walnut planted areas between 1965 and 1985, it
was determined that the walnut planted areas increased rapidly every
year as of 1986. Between 1965 and 1985, walnut planted areas, which
changed between 160,000 ha and 175,000 ha, reached 600,000 ha in
the early 2000s, 1 million ha in 2010 and 1.3 million today (FAO,
2021).
10 | W a l n u t

5000 1400
1200
4000
1000
3000 800
2000 600
400
1000
200
0 0
1965 1975 1985 1995 2001 2003 2005 2007 2009 2011 2013 2015 2017 2019

Planty Area (000 ha) Production Quantity (000 ton)

Figure 1. World Walnut Planting Area and Change in Production Quantity

Walnut production is the subject of statistics in 59 countries around

the world, and the world walnut production is around 4.5 million tons.
China produces about half of this production with a production of 2.5
million tons. China is followed by the USA with 0.6 million tons of
production, Iran with 0.3 million tons and Turkey with 0.2 million
tons of production. With these production values, 81% of the world
walnut production is realized by these four countries (FAOSTAT,
2021).

When the walnut production amount is analyzed by countries,

according to 2019 data, the largest planting area in the world is
630,000 ha, 48% of which belongs to China. China is followed by
USA with 11%, Turkey with 9% and Iran with 3%. Turkey, which is
3rd in the world in terms of planting area, ranks 4th in terms of
production amount due to differences arising from productivity. 56%
of the world walnut production is carried out by China, 13% by USA,
7% by Iran and 5% by Turkey. Yield is one of the most important
 W a l n u t | 11

factors affecting the amount of production. When the yield data by

countries were examined, it was determined that the lowest yield was
found in Mexico with 1.68 tons/ha, while the highest yield was found
in Uzbekistan with 10.43 tons/ha. Factors such as the preferred
variety, production system and climate can change the yield
(FAOSTAT, 2021).

Table 1. Production, cultivation area and yield based on countries

Production Quantity Cultivation Area Yield

(ton) (ha) (ton/ha)
China 2521504 631330 3,99
USA 592390 147710 4,01
Iran 321074 44780 7,17
Turkey 225000 124553 1,81
Mexico 171368 102068 1,68
Ukraine 125850 13900 9,05
Chile 122950 40801 3,01
Uzbekistan 50660 4857 10,43
Other
367646 195350
Countries
World 4498442 1305349 3,45
Source: Faostat, 2021

World Foreign Trade

In the world's foreign trade, walnuts are traded in two different ways
as shelled walnuts and unshelled walnuts. While the world's (shelled +
unshelled) walnut import was around 154 thousand tons in 2000, it
increased to 711 thousand tons in 2019. World walnut (shelled +
unshelled) exports, on the other hand, were 180 thousand tons in 2000
and increased to 850 thousand tons in 2019. It has been determined
12 | W a l n u t

that the share of the walnut market in the world foreign trade shows an
increasing trend.

Table 2. World walnut foreign trade by years

Import Quantity (ton) Export Quantity (ton)
Without Shell Shelled Total Shelled Without Shell Total
2000 88029 65880 153909 72567 106565 179132
2005 91234 115378 206612 137678 129018 266696
2010 198973 150329 349302 191379 214325 405704
2015 189968 205836 395804 267123 282831 549954
2016 238162 218002 456164 271162 364204 635366
2017 251410 246882 498292 295886 320008 615894
2018 281442 243860 525302 294194 335226 629420
2019 434217 277403 711620 344049 503730 847779
Source: FAOSTAT, 2021

According to the data of 2019, Germany is the largest walnut importer

country in the world with approximately 42.000 tons, followed by
Japan, Turkey and Spain. When the shelled walnut export data is
analyzed, USA comes to the forefront as the most important walnut
exporting country with 122 thousand tons, followed by Mexico,
Ukraine and Chile.

Table 3. World walnut in shell foreign trade by countries

Shelled Import (ton) Shelled Export (ton)

Import Quantity Share (%) Export Quantity Share (%)
Germany 42425 15,3 USA 121980 35,5
Japan 17412 6,3 Mexico 54899 16,0
Turkey 16978 6,1 Ukraine 36829 10,7
Spain 16702 6,0 Chile 31646 9,2
Korea 12539 4,5 China 20524 6,0
France 11972 4,3 Moldova 15836 4,6
Netherlands 11712 4,2 Germany 13632 4,0
Canada 11637 4,2 Turkey 6200 1,8
Other Countries 135990 49,0 Other Countries 42503 12,4
World 277367 100,0 World 344049 100,0
Source: FAOSTAT, 2021
 W a l n u t | 13

Turkey ranks first in the import of unshelled walnuts with 88

thousand, while United Arab Emirates ranks second with 55 thousand
(12.6%). Together with Italy, Iran and Kyrgyzstan, 5 countries realize
approximately 60% of the world's unshelled walnut imports. When the
export of unshelled walnuts is analyzed, USA stands out as the largest
exporting country with 157 thousand tons. USA is followed by China,
Chile and United Arab Emirates. 4 countries realize 60% of the
world's shellless walnut exports.

Shelled walnut producer prices by country are examined in table 5.

According to 2019 data, while the average world walnut price is
$3.12, it is $3.55 in Turkey. The highest walnut price is in Palestine
with $10.2 and the lowest price is in Peru with $0.32. Prices in the
USA, which is the largest walnut exporter in shell, were at the level of
$2.2, below the average world walnut prices. This situation provides
the USA with competitive power in world trade due to its price
advantage.

Table 4. World without shell walnut foreign trade by countries

Without Shell Import (ton) Without Shell Export (ton)

Import Share Export Share
Quantity (%) Quantity (%)
Turkey 88053 20,3 USA 157554 31,3
United Arab
54797 12,6 China 74193 14,7
Emirates
Italy 45893 10,6 Chile 73792 14,6
United Arab
Iran 41436 9,5 49487 9,8
Emirates
Kyrgyzstan 36956 8,5 Mexico 47800 9,5
Mexico 35904 8,3 France 25396 5,0
14 | W a l n u t

Spain 15331 3,5 Ukraine 20027 4,0

India 14973 3,4 Turkey 16587 3,3
Iraq 12606 2,9 Argentina 6445 1,3
Morocco 10840 2,5 Kyrgyzstan 4434 0,9
Germany 10504 2,4 Australia 3266 0,6
Other Countries 66923 15,4 Other Countries 24749 4,9
World 434216 100,0 World 503730 100,0

Table 5. Shelled walnut producer prices by country

Countries Price (USD/kg)

Palestine 10,2
China 7,5
Iran 4,8
Germany 4,5
Mexico 3,8
Turkey 3,6
USA 2,2
Azerbaijan 1,5
Peru 0,4
World 3,12

Walnut Production in Turkey

Turkey is an important country that ranks 4th in terms of world walnut

production. The amount of walnut production by years is examined in
chart 2. It is observed that the amount of production has been on an
increasing trend since the beginning of the 2000s, and there has been
an increase in the amount of consumption with the increase in the
amount of production. Both as a requirement of a healthy life and with
the effect of population growth, the rate of increase in the amount of
consumption after 2014 was above the rate of increase in the amount
 W a l n u t | 15

of production. This situation caused a decrease in the proficiency level

of walnuts in Turkey. Walnut proficiency level, which was around
90% at the beginning of the 2000s, declined sharply to 70% after
2014.

While trying to fill the supply gap with imports, support policy tools
are used by the Ministry of Agriculture and Forestry for the
establishment of new walnut orchards. For the establishment of walnut
orchards, the Ministry of Agriculture and Forestry provides input
support, organic agriculture support, good agriculture support and
certified sapling use supports. In addition to these supports, 25% of
the project investment value is given as a grant in walnut orchard
facilities within the scope of private afforestation. In addition, low
interest loans applied in agricultural production reduce interest rates
up to 95% in walnut production (TOB, 2021).

400000 100

300000 80
60
200000
40
100000 20
0 0
2008

2012

2016
2000
2001
2002
2003
2004
2005
2006
2007

2009
2010
2011

2013
2014
2015

2017
2018
2019

Consumption Quantity (ton) Production Quantity (ton)

Qualification Degree (%)

Figure 2. Turkey Walnut Production, Consumption and Sufficiency Degree

16 | W a l n u t

According to 2019 data, Turkey produced around 225 thousand tons

of shelled walnuts. Hakkari with 11.5 thousand tons and
Kahramanmaraş with 11.4 tons are among the most important
provinces in walnut production in Turkey. The provinces of Hakkari
and Kahramanmaraş, Mersin, Bursa and Denizli follow it. While
Denizli stands out as the walnut planting area in Turkey, the provinces
of Manisa and Bursa follow Denizli. It is expected that the leading
provinces will change for production, as the production amount of
Turkey increases as the trees that do not bear fruit reach the yield age.

Table 6. Walnut production by provinces in Turkey

Production Number of fruit Number of non-

Quantity bearing trees fruiting trees Planting
(ton) (Number) (pieces) area (ha)
Hakkâri 11682 501500 212000 2725
Kahramanmaraş 11436 494290 417831 6587,5
Mersin 10838 306389 183076 3161,8
Bursa 10837 372023 573938 7270,2
Denizli 8941 563218 708511 8073
Çorum 8581 605444 301920 3602,1
Sakarya 5830 240765 172725 2691,3
Antalya 5825 263605 103766 1928,7
Manisa 5394 351106 924314 7712,2
İzmir 5305 208297 95613 1468,9
Other provinces 140331 7343889 6310623 79332
Türkiye 225000 11250526 10004317 124552,7
Source: Turkstat, 2021
 W a l n u t | 17

Forecast in Turkish Walnut Production

In this part of the study, the time series of production, consumption,

export and import amounts of walnut production between 2000-2019
were examined. 4-year estimations were made with time series and
double exponential correction method.

Table 7. Self-sufficiency indexes and calculations in Turkish walnut production

Parameters Formulation 2019 values

Production Quantity (ton) 225000
Consumption Quantity 294698
(ton)
Import Quantity (ton) 90525
Export Quantity (ton) 8180
Self-Sufficiency Ratio SFL = Production / %76,3
(SFL) Consumption
Import Dependency Index IDI = %30,7
(IDI) Import/Consumption
Exportability Index (EI) EI = Export %2,8
/Consumption

According to the estimation results, Turkish walnut proficiency

indices were calculated. The formulations of the proficiency indices
and their 2019 actual values are given in table 7.

Walnut Production, Consumption and Foreign Trade Forecast

The normality test for the time series of 2000-2019 was applied
(Kolmogorow-Smirnow) and it was determined that the data set had a
normal distribution. 3-year (2020-2022) forecasts for walnut
18 | W a l n u t

production, consumption, exports and imports were made using the

double exponential recovery method.

a) Forecast Export Quantity b) Forecast Import Quantity

Figure 3. Forecast of walnut foreign trade amount between 2020-2022

a) Forecast Production b) Forecast Consumption

Quantity Quantity

Figure 4. Forecast of walnut production and consumption trade amount between

2020-2022
 W a l n u t | 19

Turkey walnut production, consumption, export and import

quantity have been estimated separately and given in table 8. The data
for 2019 shows the realized values. While the walnut production
amount in Turkey was 225 thousand tons in 2019, it is expected to
increase to approximately 240 thousand tons in 2022 with an
increasing trend in the forecast years. Similarly, it is estimated that the
amount of consumption will increase and reach the level of
approximately 320 thousand tons. It is predicted that the export
amount, which is 8 thousand tons, will reach the level of 10 tons in
2022, and the import quantity will reach the level of 120 tons.

Table 8. Forecast of aquaculture production and foreign trade between 2020-2022

Production Consumption Export Import

Quantity Quantity (ton) Quantity Quantity
(ton) (ton) (ton)
2019* 225000 294698 8180 90525
2020 227517 297593 8819 99405
2021 233143 308032 9474 109441
2022 238770 318471 10177 120490
* realized value

Forecast of Qualification Indices

Self-Sufficiency Ratio (SCR) and Import Dependency Indices (IDI)
are used to measure the extent of countries' self-sufficiency in any
product. Both indices measure how much of a country's total supply is
met by domestic production or by imports. In addition to the two
20 | W a l n u t

ratios, the Exportability Index is used to show how much of the

production is exported (Candemir and Dagtekin, 2020). Examining the
adequacy indices related to walnut production will enable to comment
on the future of the sector.

Table 9. Forecast of proficiency indices for 2020-2022

Self- Import
Exportability
Sufficiency Dependency
Index (%)
Ratio (%) Index (%)
2019* 76,3 30,7 2,8
2020 76,5 33,4 3,0
2021 75,7 35,5 3,1
2022 75,0 37,8 3,2
* realized value

In Table 9, proficiency indices related to Turkish walnut data were

calculated. In 2019, the self-sufficiency rate of walnut production was
76.3%. It is estimated that walnut production self-sufficiency rate will
decrease to 75% in 2022. In addition, it is estimated that the import
dependency ratio, which is 30%, will increase to 37.8% in 2022 and
the exportability ratio will be 3.2% with a small increase.

GENERAL EVALUATION

Turkey is a country with a high walnut production potential. It is

estimated that the demand for walnuts all over the world has increased
over the years and will increase in the coming years. Walnut, which is
 W a l n u t | 21

a healthy fast consumption product, seems likely to increase for

consumption due to the increase in the world population and the effect
of epidemics.

The fact that Turkey walnut self-sufficiency rate will decrease to 75%
in 2022 will increase Turkey's import dependency ratio. For this
reason, it is very important for Turkey to increase its walnut
production areas in order to gain a place in the world walnut market
and become a self-sufficient country.

Walnut production in private afforestation areas should be an

obligatory production activity, and it is necessary to turn the products
produced into processed products with high benefit. The regions
where walnut cultivation is carried out are generally disadvantaged
regions where there are sloping lands, where the altitude is high, and
one-year agricultural production cannot be done. An important way to
ensure regional development is to carry out studies that increase the
welfare level of producers in their own regions. In this respect, it is
thought that by increasing the organization in walnut production
regions, the realization of processing facilities through organized
structures will contribute to regional development.
22 | W a l n u t

References

Akarçay, H., (2007). Türkiye Ceviz Gen Kaynaklarının Tanıtımı, Yüzüncü Yıl
Üniversitesi, Fen Bilimleri Enstitüsü
Candemir, S., Dağtekin, M., (2020). Türkiye su ürünleri üretimi ve yeterlilik
endekslerinin tahmini. Acta Aquatica Turcica, 16(3), 409-415.
FAOSTAT, (2021). Production, Consumption and Trade Statistics
Ferhatoğlu, Y., (2001). Ceviz yetiştiriciliği. Tarım ve Köyişleri Bakanlığı yayınları.
Gülcan, R.,, Tekintaş, E., Mısırlı, A., Sağlam, H., Günver, G., Adanacıoğlu, H.,
(2000). Meyvecilikte Üretim Hedefleri. V. Türkiye Ziraat Mühendisliği
Teknik Kongresi, 17-21
Kilci, M., (2015). Tokat ili Niksar ilçesi Ceviz Üretim ve Pazarlama Yapısı,
Gaziosmanpaşa Üniversitesi, Tarım Ekonomisi Anabilim Dalı.
Koyuncu, M. A., Aşkın, M.A., (1999). Van Gölü çevresinde yetiştiriciliği yapılan
bazı ceviz tiplerinin depolanması üzerine çalışmalar. Journal of Agriculture
and forestry 785-796
Sen, S.M., Kazankaya, A., Yarılgaç, T., Doğan, A., (2006). Bahçeden Mutfağa
Ceviz, Maji Yayınları (1) 233.
TURKSTAT, (2021). Crop production statistics
 W a l n u t | 23

Part II
--------------------------------

Flowers and Fertilization Biology in Walnuts

Assoc. Prof. Hakan KELES1

In fruit species, the presence and shape of flowers on the tree are
defined in 3 different ways. Hermaphrodite is the condition in which
stamens and pistils are present on the same flower and the same plant.
The presence of anthers and pistils on the same plant but in different
places is called monoic. The presence of stamens and pistils both on
different plants and in different places is called dioecious (Figure 1)
(Bilir and Ulusan, 2010).

Figure 1. 1: Hermaphrodite flower structure: Male and female flowers together and
on the same plant, 2: Monoic: Male and female flowers in separate places and on the

1
Yozgat Bozok University, Agriculture Faculty, Department of Horticulture,
Yozgat, Turkey, ORCİDs: Assoc. Prof. Hakan KELES, https://orcid.org/0000-0002-
8225-931X hakan.keles@yobu.edu.tr
24 | W a l n u t

same plant, 3: Male and female flowers in separate places and on different plants
(Anonymous, 2021a)

Walnut (Juglans regia L.) trees are among monoecious plants in terms
of flower biology (Sharma et. al., 2016; Eser et. al., 2019; Kafkas et
al., 2020; Okatan et al., 2021). This word, called monoic, is actually of
Greek origin and is formed from the combination of the words
"Monos" meaning "single" and "Oikos" meaning "house" expressing
that the male and female flower lives in the same house (Schmidt and
Joker, 2001; Mueller et. al., 2015). This means that male and female
flowers are found separately on the same tree, but in different places
(Polito, 1998).

The presence of male and female flowers in different places on the

same tree does not require that these flowers bloom at the same time.
In this regard, in J. regia, male and female flowers become active at
different times according to a certain order and sequence. This
situation is called dichogamy (Akça and Şen, 1995; Shu-Ganga et. al.,
2011, Guney et. al., 2021).

In monoic structure, male and female flowers are not found in the
same structure and flowers can occur on shoots of different ages. The
flowers that occur at the ends of the annual shoots of the same year are
called female flowers, and the catkins formed on the shoots of the
previous year are called male flowers (Figure 2).
 W a l n u t | 25

A
B
Figure 2. (A) Female flowers (Photo by: Carlos Coutinho, Anonymous, 2021b ), (B)
Male flowers (Photo by: H. Zell, Anonymous, 2021c)

1. Female Flowers

Female flowers are usually formed at the ends of the shoots and 1-3
are found together. However, cases, where 10-18 female flowers
coexist at the ends of the shoots in walnut trees, have also been
encountered. These trees can be used as parents in studies with the
aim of productive variety breeding. As in all fruit varieties, the
formation and time of female flowers in walnut trees cultivars
according to the genetic structures of the cultivars. Although this
timing sometimes seems to change due to ecological differences, it
usually shows a stable attitude. In a study on the inheritance of
different characters in walnuts, it was reported that the heritability of
characters such as leafing date, female flower activation date, first
pollen dispersal date was high (Janick and Moore, 1979; Akça, 2009).
This shows that ecology has little effect on these characters. (Table 1).
26 | W a l n u t

Table 1. Heritability of some important characters in walnuts (Janick et al.,

1979)
Characters Heritability

Leafing date 0,96

Receptive time of female flowers 0,93

Pollen distribution 0,91

Harvest date 0,85

Fruit height 0,82

Number of quality walnut kernel 0,08

Yield 0,07

Figure 3. Female Flower (Anonymous, 2021d)

 W a l n u t | 27

There are no petals in female flowers in walnuts. Although there are

3-6 sepals, the four-part cover leaves (perianth) and bracticle leaves of
the flowers are fused with the ovary. (Şen, 2011). The ovary of female
flowers is inferior and has two carpels. The stigma, where the pollen is
attached, is large lobed and has a recessed structure for the pollen to
hold more easily (Polito, 1998; Akça, 2009; Jankovic et al., 2021).
Female flowers begin to develop in April. Towards the period when
the stigma accepts pollen (receptive period), the stigmas turn from
yellow to a darker orange color and the stigmas are opened
(approximately 45°). In the receptive period, the stigmas are covered
with a sticky liquid to better capture the pollen. This period, when the
embryos of female flowers are fertile, can be detected by the presence
of sticky fluid on the stigmas (Şekil 4). Under optimum ecological
conditions, stigmas of the female flowers are receptive for up to seven
days. At the end of the flowering period, which lasts for an average of
20 days, the stigmas have completed their tasks and then dry up
(Krueger, 2000; Akça, 2009).

A B
Figure 4. (A) Non-receptive Female Flower (Photo by: Anil Thakur, Anonymous,
2021e), (B) Receptive Female Flower (Anonymous, 2021f)
28 | W a l n u t

2. Male Flowers

On the contrary female flowers formed on annual shoots, male flowers

are located on structures called catkins formed on the shoots of the
previous period (Latorse, 1985). Catkins, which contain male flowers
that distribute pollen, are first miniature cone-shaped, conical and gray
on the branches, and they begin to develop with the spring period
(Krueger, 2000). The catkins, which are 2-3 mm in length when they
first form, reach 10-15 cm in length when they complete their
development, and the maturing catkins hang down. An axis passes
through the center of the catkins and the male flowers are attached to
this axis by a stem. Brackets and perianth leaves of male flowers
together formed a six-part cover structure (Şen, 2011) (Figure 5).

Figure 5. A: Catkin, B: Male flowers attached to the catkin axis, C: Bottom view of
male flower, D: Anthers (Modified from, Köhler, 1887)
 W a l n u t | 29

Each male flower has an average of 12-18 stamen. When all flowers
are considered in a catkin, there are 150 male flowers, and about 2
million pollen is produced from a catkin that is ready for distribution.
Catkin buds that are formed in the previous development period on the
shoots are differentiated with the development period of the new year.
It is known that a ripe walnut tree can produce 5-20 billion pollen
from an average of 5000 catkins. 10-25% of the produced pollen can
survive and pollinate female flowers (Wood, 1934). A catkin that has
reached the end of pollen distribution loses its vitality, turns black,
and finally falls off.

A B C D

Figure 6. Male flower development stages

3. Fertilization Biology

Walnut (J. regia) flowers do not have attractive petals and tempting
nectar extracts like insect-pollinated (anemophilous) plants. For this
reason, pollination between walnut trees is provided by wind and
walnut trees produce a lot of pollen to avoid pollination problems.
30 | W a l n u t

There is no evidence of infertility or incompatibility problems that can

be encountered in different fruit types in walnut trees that do not have
problems in fertilizing each other. Although all walnut trees can
fertilize each other, the most important issue in fertilization is the
periods of receptive when male and female flowers are active.
Because of this situation, walnut plants are classified in the group of
dichogamy plants.

3.1. What is dichogamy?

In walnuts, the pollen distribution time of male flowers and the period
in which female flowers accept pollen are at different times (Forde
and Griggs, 1975). This situation is called dichogamy which term was
first used by Sprengel in 1793 (Stout, 1928).

3.2. Factors affecting dichogamy

Dichogamy tendencies of cultivars are fixed by genetic traits, but

some factors can push or reverse flowering times. These changes are
very limited. The most important factors affecting the flowering times
are the age of the plants and environmental factors (Batu et al., 2009).
It is very important to know which cultivars to choose in order to
ensure pollination in the walnut orchard and to know which factors
will affect the flowering behavior and how. Factors affecting
flowering times are mentioned below:

- The age of plants is one of the important features that affect the
dichogamy. Young walnut trees have a greater tendency to
 W a l n u t | 31

dichogamy and, on these trees, male flowers mature earlier than

female flowers (protandrous) (Wood, 1934; Akça, 2009).
- Warm weather is another important factor affecting the
dichogamy. In particular, staminate flowers react more quickly
to warm weather than pistillate flowers (Wood, 1934;
Cosmulescu et. al., 2010).
❖ In a warm season following a cold winter, all parts of the
plant tendency to grow rapidly. However, this is more valid
for male flowers compared to female flowers. In such cases,
male flowers develop much more rapidly, and in protandry
cultivars, the flowering of male flowers ends long before the
female flowers become receptive, and dichogamy is certainly
occur.
❖ In warm spring times accelerate catkins development, but
female flowers are not affected this condition much. Under
these conditions, protogenetic cultivars may behave like
homogamy, especially in cultivars where the flowering
interval between the two flower types is less than 3-4 days.
❖ Cool weather after a warm winter can cause the opposite
situation in flower behavior. In such cases, the protogenic
feature is emphasized. The effects of weather and climate can
be so great that, in some cases, protandry cultivars may act as
protogeny.
32 | W a l n u t

3.3. Types of dichogamy

Walnut trees show two types of dichogamy. Cultivars or genotypes

whose male flowers mature first are called protandry. Protandry is the
most common type of flowering in walnut trees. A large part of
Turkish walnuts is protandry. At the same time, protandry
characteristic is predominant in California and French walnut
cultivars. Cultivars or genotypes whose female flowers mature before
the male flowers are called protogynous. Walnuts with this feature are
more common in Iran and the central regions of Asia (Sutyemez,
2006; Akça, 2009). Unlike protogynous and protandrous types, there
are some cultivars or types whose male and female flowers mature at
the same time on the same tree. This situation is known as homogamy.
In a study conducted for the determination of promising walnut
genotypes and 20 types were determined, it was identified that 11
genotypes were protandry, 5 types were protogynous and 4 types were
homogamy in terms of flowering times (Keles, 2014). In addition, in
some walnut selection studies carried out in the world, it was stated
that the majority of the types showed protandry characteristics in
terms of flowering in walnut types examined. (Rouskas et. al., 1997;
Koyuncu and Görgün, 2003; Kaymaz, 2005; Oğuz and Aşkın, 2007).

3.4. The importance of dichogamy for walnut orchard

establishment

Flowering time of walnut cultivars affects yield that should be

considered before orchard establishment. Dichogamy is one of the
 W a l n u t | 33

most important issues to be given priority for homogeneous and

regular yield in the walnut orchards (Shu-Ganga et. al., 2011). In the
planning of the walnut orchard, it is necessary to coincide with the
period when the female organs of the cultivar to be considered as the
main cultivar are receptive and the period when the male organs of the
cultivar to be considered as pollinators distribute pollen. For this
reason, using more than one cultivar in the planning of a walnut
orchard will eliminate the pollination problem and provide regular
yield. Otherwise, it should be expected that pollination will be done
by pollen from different orchards or by artificial pollination. This
situation will either lead to a decrease in productivity values or an
excess of labor. Keeping an average of 5% pollinator cultivars in a
walnut garden is important for a good fruit set. As shown in Figure 4,
the opening of the stigmas at an angle of 45° in the female organ of
the main variety and the red color of the tips give clues that they
accept pollen. During this period, the male organs of the pollinator
cultivar should also change color and be ready for pollen distribution
as shown in Figure 6/D. In this case, pollination and fertilization will
occur as a result, an optimum fruit set will be achieved.

Depending on the ecological characteristics of the regions where they

are located, the opening times of male and female flowers of walnut
trees may be different. Different researchers working on walnuts have
determined the flowering times of some cultivars according to their
regions (Cosmulescu et. al., 2010; Cosmulescu and Botu, 2012; Keles,
2014). Farmers who will grow walnuts can also choose pollinator or
34 | W a l n u t

main cultivars according to these tables. In Figure 7, male and female

flowering times of some walnut varieties found in California
ecological conditions are shown.

Figure 7. Maturation times of male and female flowers in California (USA)

/modified from original (Anonymus, 2021g)

In the table prepared by İbrahim BALKAL, who is known for his studies on
walnuts in Turkey (Figure 8), the maturation dates of male and female
flowers of some American, French and Turkish walnut varieties grown in
Turkey are shown.

Figure 8. Maturation times of male and female flowers in Inegol-Bursa/ Turkey

(Anonymus, 2021h)
 W a l n u t | 35

Considering the tables before mentioned and similar tables, when

establishing a walnut orchard, cultivars in which the maturation times
of male and female flowers coincide can be selected. In this way, one
of the most important steps for optimum and regular efficiency is
taken.
36 | W a l n u t

REFERENCES

Akça, Y. (2009). Ceviz Yetiştiriciliği. Ankara, 371 p, Turkey.

Akça, Y., Şen, S. M. (1995). The relationship between dichogamy and yield-nut
characteristics in Juglans regia L. In III International Walnut Congress 442,
pp 215-216.
Anonymous, (2021a). Bitkilerde Eşey Durumu. https://sorhadi.net/d/136023-
monoik-bitki-nedir-dioik-bitki-nedir/2. (Date accessed: 01.10.2021).
Anonymous, (2021b). Juglans regia. https://gd.eppo.int/taxon/IUGRE/photos. (Date
accessed: 05.10.2021).

Anonymous, (2021c). Walnut (Juglans regia), inflorescence, Germany.

https://www.feedipedia.org/content/walnut-juglans-regia-inflorescence-
germany. (Date accessed: 05.10.2021).
Anonymous, (2021d). Walnut tree. https://www.daviddarling.info/encyclopedia
/W/walnut_tree.html. (Date accessed: 10.10.2021).
Anonymous, (2021e). Flowers of India. Retrieved October 23, 2021, from
https://www.flowersofindia.net/catalog/slides/Walnut.html. (Date accessed:
10.10.2021).
Anonymous, (2021f). The Many Faces of the Walnut – Up close and personal.
https://frogenddweller.wordpress.com/2015/05/16/the-many-faces-of-the-
walnut-up-close-and-personal/ (Date accessed: 10.10.2021).
Anonymous, (2021g). Walnuts. Male and Female Blooming Dates at UC Davis.
Average since 1990 (In harvest date order). https://burchellnursery.
com/walnuts/2/ (Date accessed: 4.10.2021).
Anonymous, (2021h). Cevizin Tozlanma Durumu. http://www.ozyurtardafidan.com/
sayfa.asp?sayfano=44 (Date accessed: 4.10.2021).
Bilir, N., Ulusan, M. D. (2010). Bitkilerde Döllenme Varyasyonunun Tahmini.
Süleyman Demirel Üniversitesi Yaşam Dergisi, 2(2), pp 14-16.
Botu, M., Cosmulescu, S., Baciu, A., Achim, G. H. (2009). Environmental Factors
Influence on Walnut Flowering. In VI International Walnut Symposium 861.
 W a l n u t | 37

Cosmulescu, S., Botu, M. I. H. A. I. (2012). Walnut biodiversity in south-western

Romania resource for perspective cultivars. Pak J Bot, 44(1), pp 307-311.
Cosmulescu, S., Baciu, A., Botu, M., Achim, G. H. (2010). Environmental factors’
influence on walnut flowering. Acta horticulturae, 861: pp 83-88.
Eser, E., Topcu, H., Kefayati, S., Sütyemez, M., Islam, M. R., Kafkas, S. (2019).
Highly polymorphic novel simple sequence repeat markers from Class I
repeats in walnut (Juglans regia L.). Turkish Journal of Agriculture and
Forestry, 43(2), 174-183.
Forde, H. I., Griggs, W. H. ( 1975). Pollination and blooming habits of walnuts.
Univ. Calif. Div. Agr. Sci. Lflt. 2753.
Guney, M., Kafkas, S., Keles, H., Zarifikhosroshahi, M., Gundesli, M. A., Ercisli,
S., Necas, T., Bujdoso, G. (2021). Genetic Diversity among Some Walnut
(Juglans regia L.) Genotypes by SSR Markers. Sustainability, 13(12), 6830.
Janic, J., Moore, J. N. (1979). Advences in Fruit Breeding, ISSN, ISBN, 0-911198-
36-9.
Janković, S., Stanković, J., Janković, D., Milatović, D. (2021). Morphology and
morphogenesis of female reproductive organs in some walnut (Juglans regia
L.) genotypes. Scientia Horticulturae, 289, 110471.
Kafkas, N.E., AttaR, Ş.H., Gundesli, M.A., Ozcan, A, Ergun, M., (2020). Phenolic
and Fatty Acid Profile, and Protein Content of Different Walnut Cultivars
and Genotypes (Juglans regia L.) Grown in the USA. International Journal of
Fruit Science. 20 (3), 1711-1720.
Kaymaz, Ö. (2005). Hizan (Bitlis) Merkez İlçe Ceviz (Juglans Regia L.)
Popülasyonlarında Ümitvar Genotiplerin Seleksiyonu Üzerine Bir Araştırma.
Yüksek Lisans Tezi. Yüzüncü Yıl Üniversitesi Fen Bilimleri Enstitüsü, Van.
Keles, H., Akca, Y., Ercisli, S. (2014). Selection of promising walnut genotypes
(Juglans regia L.) from inner Anatolia. Acta Scientiarum Polonorum
Hortorum Cultus, 13(3), pp 167-173.
Koyuncu, M.A., Görgün, O. (2003). Ağlasun (Burdur) yöresi cevizlerinin ön
seleksiyonu. Türkiye IV. Ulusal Bahçe Bitkileri Kongresi 8-12 Eylül, s. 298-
300, Antalya.
38 | W a l n u t

Köhler, F. E. (1887). Köhlers Medizinal-Pflanzen in naturgetreuen Abbildungen und

kurz erläuterndem Texte. Köhler, Gera, Germany.
Krueger, W. H. (2000). Pollination of English walnuts: practices and
problems. HortTechnology, 10(1), 127-130.
Latorse, M.P. (1985). Etude de divers aspects de la reproduction sexuée chez le
noyer (Juglans regia L.). Ph.D. Thesis, Sciences de la vie, Université de
Bordeaux II, Bordeaux, France, 155 p.
Mueller, W. A., Hassel, M., Grealy, M. (2015). In Preparation for New Life I: Sex
Determination and Sexual Development. In Development and Reproduction
in Humans and Animal Model Species (pp. 215-240). Springer, Berlin,
Heidelberg.
OkataN, V., Bulduk, I., Kaki, B., Gündeşli, M.A. Usanmaz, S., ALAS, T., HelvacI,
M., Kahramanoğlu, I., Hajizadeh, H.S., (2021). Identification and
Quantification of Biochemical Composition and Antioxidant Activity of
Walnut Pollens. Pak. J. Bot, 53(6): DOI:
http://dx.doi.org/10.30848/PJB2021-6(44).
Oğuz, H.İ., Aşkın, A. (2007). Ermenek Yöresi Cevizlerinin (Juglans regia L.)
Seleksiyon Yoluyla Islahı Üzerine Bir Araştırma. Yüzüncü Yıl Üniversitesi,
Ziraat Fakültesi, Tarım Bilimleri Dergisi (J. Agric. Sci.), 17(1): pp 21-28
Polito, V.S. (1998). Floral biology: flower structure, development, and pollination.
In: Ramos, D.E. (ed.), Walnut Production Manual. University of California,
Oakland, pp. 127–132.
Rouskas, D., Katranis, N., Zakynthinos, G. and Isaakidis, R. (1997). Walnut
(Juglans regia L.) seedlings selection in Greece. Acta Hort., 442:109-116.
Schmidt, L., Joker, D. (2001). Glossary of seed biology and technology. Danida
Forest Seed Centre.
Sharma, O. C., Singh, D. B., Zahoor, S., Padder, B. A., Haji, S. A. (2016).
Gynodioecious behaviour in some walnut genotypes-a new report. Journal of
Hill Agriculture, 7(2), pp 283-285.
Shu-Ganga, Z. H. A. O., Hong-Xiab, W. A. N. G., Zhang, Z. H. (2011). Research
Advances on Dichogamy of Walnut [J]. Hubei Agricultural Sciences, 17.
 W a l n u t | 39

Stout, A. B. (1928). Dichogamy in flowering plants. Bulletin of the Torrey Botanical

Club, pp 141-153.
Sutyemez, M. (2006). Comparison of AFLP polymorphism in progeny derived from
dichogamous and homogamous walnut genotypes. Pak J Biol Sci, 9, pp 2303-
2307.
Şen, S. M. (2011). Ceviz Yetiştiriciliği, Besin Değeri, Folklorü. ÜÇM yayınları, 220
p,
Wood, M. N. (1934). Pollınatıon and Bloomıng Habıts Of The Persıan Walnut In
Calıfornıa. Technical Bulletin, No. 387
40 | W a l n u t
 W a l n u t | 41

Part III
--------------------------------

The Use of Rootstock in Walnut Cultivation

Dr. Remzi UGUR1

1.Introduction

Fruit trees can be described as the combination of two different plant

section known as rootstock and scion through grafting. Although
many years have been passed, the textural difference between these
two different plant parts is immediately understandable with careful
examination. It is very important for the rootstock as the soil part of
the plant to have a very vigorous and healthy root system (Gundesli et
al., 2018; Sharma et al., 2020). Walnut rootstock is usually obtained
from seeds called the seedling rootstock. In recent years, thanks to
breeding and biotechnological methods developed, rootstocks that can
be propagated clonally in walnuts have also been obtained. To
establish more homogeneous gardens, we see that clonal rootstocks
are more preferred in recent years. Another reason why clonal
rootstocks are preferred is that they provıde resistance to biotic and

1
General Directorate of Agricultural Research and Policies, East Mediterranean
Transitional Zone Agricultural Research of Insttute, Department of Horticulture,
ORCIDs: Dr. Remzi UGUR, https://orcid.org/0000-0001-6717-1689,
remzibey@hotmail.com
42 | W a l n u t

abiotic soil conditions in terms of breeding aim. However, when a

garden will be established, it is necessary to know the characteristics
of rootstock and the variety (Ozcan and Sutyemez, 2017). Thanks to
the advances in agricultural biotechnology in recent years even
although vegetative propagation of the later developed varieties is
carried out in vitro, it is very important to know that rootstock
breeding has a different process and aim than breeding variety.
Sometimes, it is possible to obtain trees that are clonally propagated
from standard walnut varieties and formed on their roots. However,
this situation has not been an obstacle to the breeding of rootstocks
dependent on extreme soil conditions.

1.2 Walnut Rootstock Breeding Studies in the World

Although the use of rootstock in walnut cultivation varies according to

the region of cultivation, it has mostly been in the form of seedling
rootstocks. Since the beginning of the 1900s, selection breeding
studies have been started on the use of walnut rootstocks in the United
States and Europe (especially France). With these studies, the use of
suitable rootstocks for walnuts began to spread.

In the 1960s, clonal rootstock production started due to reasons such

as establishing more homogeneous walnut orchards, increasing yield
and quality values, and resistance to soil-borne diseases and pests.
Today, although seedling rootstocks are used in walnut orchards, there
is a rapid increase in the use of clonal rootstocks (Kluepfel et al.,
2015; Granahan et al., 1987).
 W a l n u t | 43

Towards the end of the 1800s, in the United States of America,

California, the use of Juglans regia rootstocks brought from France as
rootstocks in the walnut orchards started, and by 1912, grafting, that
is, the use of rootstock in walnut cultivation, started to be
recommended. However, in the following years, rootstocks originating
from J.regia started to be left gradually due to the developmental
differences in the grafted walnuts and the problems in the adaptation
ability of these rootstocks (Granahan ve Forde,1985). In studies on
species and hybrid tests, the use of Juglans hindsii rootstocks as an
alternative to J.regia has been an increased. After the 1950s, J. hindsii
rootstocks have been used in almost all gardens in California.
However, until the end of the 1800s, hybridization studies were started
between J.regia and J. hindsii species, and garden trials were started
on Paradox rootstocks, the first generation hybrid rootstock obtained
from these hybridization studies (Potter et al., 2002). After these
crossing studies, Paradox rootstock, a hybrid of J.regia and J. hindsii,
was bred. During the passing time, these rootstocks have been used in
almost 80% of the walnut orchard in America. The hybrid rootstock
Royal (J.regia x J. hindsii) which has the same parents as Paradox,
has a certain cultivation area, but it is not as widespread as Paradox.
Since Paradox rootstocks have vigorous and disease resistance than
their parents, they have been easier to spread. Although Paradox
provides resistance to some diseases such as Armillaria and
Phytophtora, the idea of breeding resistance to other root diseases and
blacklines has become widespread. For this reason, the breeding of
44 | W a l n u t

rootstock resistant to blackline disease has been started in California

(Browne et al., 2015). At the end of these studies, rootstocks that can
also be produced vegetatively (Vlach, RX1 and VX211) were bred.
Some walnut growers that some rootstocks from English walnuts are
resistant to blackline disease and have started to use them. From these
three rootstocks bred, although Vlach showed vigorous growth,
disease resistance was not determined, RX1 is a P.microcarpa x J.
Regia hybrid which is resistant to nematodes, and VX211 is a J.
hindsii x J.regia hybrid that is resistant to nematodes (Kluepfel et al.,
2011).

Walnut rootstock breeding studies in France were started using J.nigra

seeds brought from America between 1920 and 1930. At the end of
studies performed, promising rootstocks types were obtained in
J.nigra and J.regia origin walnuts, and it was determined that
rootstocks from J.nigra origin provide better results than J.regia
walnuts in poor soils. In addition, in other performed studies related to
the rootstock characteristics of J. Hindsii walnut, it was not preferred
since it could not survive cold soil conditions (Ramos and
Doyle,1984; Bernard et al., 2018). In the following years, some
J.nigra ve J.regia walnuts selected in France were left to open
pollination, and in the studies carried out the hybrids obtained them,
hybrid of J.nigra x J.regia which are more resistant to Armillaria than
J.regia and has the same growth strength as Paradox rootstocks were
obtained. İt is estimated that Paradox rootstock will be difficult to
replace J.nigra x J.regia hybrid rootstocks in France as it is more
 W a l n u t | 45

sensitive to anthracnose. INRA continues to work as the leading

research institution in the breeding of walnut rootstock by
crossbreeding. Lozeronne, RA 8 611 and Culplat rootstocks are some
of them (Ramos,1997; Germain,1999).

Rootstocks breeding studies have been going on for a long time in

China where grafting in walnut cultivation based is performed during
ancient times. In 2011, The Jin-RS-1 rootstock series was bred and
released by Shanxi Academy of Forestry Science. Within this
rootstock series, Jin-RS-2 and Jin-RS-3 rootstocks were found to be
resistant to cold and soil pathogens in the northern regions of China
(Gunn et al., 2010; Han et al., 2016).

Four different walnut cultivars grafted on J.regia and J.nigra seedling

were studied in Hungary. It has been determined that the success of
grafting in both rootstock types is around 30-60% and at similar rates.
The walnut cultivars Alsoszentivana 117 and Tiszacsecsi 83
performed better on J. regia than on J. nigra. The success of grafting
was 49.00 % in Alsoszentivana 117 variety and 38.2% in Milotai
variety grafted on another species J. rupestris. It has been reported that
the growth of plants grafted on J. regia species in the garden plant is
better than other species (Szentivanyi and Lantos, 1997).

Achim et al., 2007 reported that walnut cultivation is an important

product for Romania and the importance of rootstock selection for
modern cultivation. For this purpose, he reported that a new seedling
rootstock named Portval which was obtained by selection made in the
46 | W a l n u t

existing natural walnut population was detected. It has been

determined that this rootstock shows vigorous growth, flowering type
is protandry and resistant to environmental conditions and diseases. It
was determined that the growth in the nursery experiment germination
rate 80% , the seedling diameter 23.8 mm and the seedling length 160
cm.

In general, seedling rootstocks have been used in Turkey, which is the

homeland for walnut genetic resource (Akca and Sutyemez,2016).
However, recently, the importance of rootstock breeding has begun to
be better understood due to developmental disorders caused by
seedling rootstocks and susceptibility problems to some diseases such
as phytophthora (Avanzato et al., 2014). In rootstock breeding
studies, drought, salinity, and lime resistance should also be
considered. Although clonal rootstock breeding studies have just
begun in Turkey, it is noteworthy that Paradox and Vlach rootstocks
have been used recently.

An improvement work with drought resistance and stunted growth

was started in Iran after 2003 year. Educational studies continue by
the Tehran University Walnut Research Center (Atefi,1993; Arzani et
al.,2008)
 W a l n u t | 47

2. Rootstock Types

2.1 Rootstocks

The use of rootstock suitable for walnut cultivation can be created

between species as well as in-species hybrids. The lack of desired
characteristics in a single species has led researchers to use
interspecies hybridization (Rom and Carlos,1987). İt is thought that
suitability for clonal production, which is the most important feature
in rootstocks breeding, can be obtained more easily with interspecies
hybridization. Until today, the use of seedling rootstocks was more
common in walnuts. These rootstocks include other species within the
genus Juglans, including J.hindsii, J.regia and J.nigra (McGranahan
et al., 1986).

2.1.1 J.hindsii

It has been the most widely used rootstock for walnuts among the
black walnut seedlings of Northern California (Figure 1). On its
narrow leaves, approximately 15-19 leaflets per leaf, 2.5-3.5 cm wide,
firm and rough fruits are known. This walnut species, which was first
found by Hinds in Sacramento in the USA, was later identified
botanically as Juglans californica var hindsii by researcher Jepson
(Rom and Carlos,1987). It has been reported that the main reason for
this definition is that J.hindsii and J.californica are essentially the
same species, and the differences are due to climate and soil
conditions (Brinkman,1974). J.hindsii is in danger of extinction
48 | W a l n u t

because it can easily pollinate with other walnut species. J.hindsii is

recommended as a rootstock in deep and fertile soils. There are
problems in places where there is a danger of nematode and blackline
disease (Sen,1986; McGranahan and Leslie,2012). It is reported that
these rootstocks, which are resistant to armillaria and root cancer, do
not perform well in high pH and calcareous soils (Woeste et al., 1996;
Sen,1986; Gregory et al., 2015; Ramesh et al., 2021).

A- Juglans hindsii tree in its natural environment; B-Juglans hindsii fruit

Figure 1. Tree and fruit of the genus Juglans hindsii are grown in their natural
environment.

2.1.2 J.regia

J.regia is the most commonly used rootstock in Turkey and in many

countries of the world where walnuts are grown. J.regia has a smooth
gray color with a soft shell structure, the number of leaflets varies
between 5 and 9 and has moderately rough fruit. The stratification
time of J.regia seeds is about eight weeks on average (Rom and
 W a l n u t | 49

Carlos,1987). However, as the uniformity in the seed deteriorates, the

yield from seed stratification will gradually decrease, and there will be
differences in the germination of seeds in a certain period time
(Brinkman,1974). It would be beneficial to perform homogeneous
germination tests for a suitable J.regia seed rootstock. There is a
possibility that Cherry Leaf Roll Virus (CLRV), which causes
blackline disease, can be found in J.regia seeds. Although J.regia is
generally not affected by blackline disease, it is very sensitive to
Armillaria, Phytophthora, root meadow nematode, water-saturated
soils and salinity (Sen,1986; McGranahan and Leslie,2012). The
resistance of J.regia to blackline disease began to be widely used in
California, especially in Oregon, in the 1950s. For this purpose, they
have started breeding studies by selection from genetic sources
created by planting J.regia seeds brought from outside the USA,
especially from China. When comparing some selected rootstock
candidates with J.hindsii, it was observed that J.regia rootstocks
formed more vigor crowns. In another study conducted in California,
it was determined that some walnut varieties grafted on J.regia
rootstock were lower in terms of yield efficiency (kg/cm2) than those
grafted on J.hindsii, Juglans microcarpa and J.major rootstocks.

Although J.regia is used as a common rootstock in many parts of the

world, there is almost no rootstock that can be defined as a true
seedling rootstock through selective breeding from J.regia. In Turkey,
in this sense, some saplings in the Marmara region still use some
J.regia seedlings, which they believe to develop homogeneously and
50 | W a l n u t

form hairy roots, as rootstocks. In addition, the use of rootstocks from

some standard varieties such as Maraş-18 is common in our country.
However, since the J.regia species is very rich in genetic variation, it
is quite possible to develop more effective rootstocks with appropriate
rootstock breeding studies. However, since the J.regia species is very
rich in genetic variation, it is quite possible to develop more effective
rootstocks with appropriate rootstock breeding studies (Woeste et al.,
1996; Sen,1986; Gregory et al., 2015; Ramesh et al., 2021).

A-Juglans regia tree in its natural environment; B-Juglans regia fruit

Figure 2. Tree and fruit of the genus Juglans regia are grown in their natural
environment

2.1.3 J.nigra

The homeland of Juglans nigra, known as the eastern black walnut, is

naturally distributed in the region between New Hampshire-Georgia-
Minnesota and Texas in the region from the Middle East-Western
 W a l n u t | 51

parts of the United States to the south. J.nigra has been brought to
Europe especially for timber since the early 1600s. Since it is resistant
to cold in Europe, it has spread to Scandinavian countries. J.nigra
forms tall trees with 15-23 leaflets. Its fruits are 3-4 cm in diameter
with irregular stripes (Rom and Carlos,1987). Compared to J.regia,
the seed stratification period takes longer (12-16 weeks). J.nigra has
been used as a rootstock in the United States because it is easy to find
(Brinkman,1974; Ozcan and Sutyemez,2017). While walnuts grafted
on J.nigra formed smaller crowns, they were found to be more
resistant to cold, nematodes, Armillaria and Phytophtora than walnut
varieties grafted on J.regia (Kluepfel et al., 2011). However, it was
determined that the yield efficiency of rootstocks grafted on J.nigra
rootstocks was lower. Although J.nigra rootstocks are more sensitive
to blackline disease than J.regia rootstocks, they are still used in
Europe and America as a suitable parent in crossing studies due to
their resistance to some other diseases mentioned above and their
unique superior characteristics (Gregory et al., 2015; Ramesh et al.,
2021).
52 | W a l n u t

A- Juglans nigra tree; B-Juglans nigra fruit

Figure 3. Juglans nigra tree and fruit

2.1.4. J.californica

This type, known as the California black walnut, spreads naturally

from the Santa Mountains to the south in the state of California, USA.
It spreads in the form of a bush, its leaflets are less in number than
J.hindsii (11-15 pieces) (Rom and Carlos,1987). Compared to other
Juglans species, J.californica has a shorter vegetation period. Seeds
can begin to germinate after 12 weeks of stratification (Özcan and
Sutyemez,2017). Due to its weak growth, this species cannıt hold
onto the soil very well if it is used as a rootstock. In addition, root rot
was frequently encountered in walnut varieties grafted on
J.californica rootstocks, and its use as rootstock decreased in the
following years (Gregory et al., 2015; Ramesh et al., 2021).
 W a l n u t | 53

2.1.5 J.microcarpa

This species, known as Texas black walnut, has chosen Texas and
New Mexico as its natural distribution area. This species, which has
the most leaflets and the smallest fruit among the Juglans species,
shows dwarf development in terms of its general feature (Rom and
Carlos,1987; Gregory et al., 2015). This species, which forms dwarf
trees even in comfortable soil conditions, has successfully performed
as a rootstock in soils with high pH, high boron and chlorine toxicity.
Although it showed dwarf growth, yield efficiency in walnut varieties
grafted on it was the same as other rootstocks.

2.1.6 J.major

It is known as the Arizona black walnut and the distribution area of

this species is around New Mexico, Arizona, Colorado and Northern
Mexico. This species has an average of 9-13 leaves and 2-3 cm
diameter fruit with a deeply corrugated rind. This species, which has
been tested on rootstock characteristics in different soil conditions, has
not been found to have many superior characteristics to J.hindsii
(Brinkman,1974). However, it will be beneficial to study the
rootstock characteristics of this species, which is not selective in terms
of soil in later years (Gregory et al., 2015; Rom and Carlos,1987).

2.1.7 Other Juglans Species

Juglans show a lot of diversity as a species. In many of these species,

studies on rootstock traits are still ongoing. Juglans mandshurica is a
54 | W a l n u t

native plant of North China and intensive studies are carried out on its
rootstock characteristics. Particular emphasis is placed on crossing
J.mandshurica with J.regia. Similar hybridization studies are carried
out in Argentina between Juglans australis x J.regia, Juglans cinerea
x J.regia species (Rom and Carlos,1987; Vahdati et al., 2021).
However, the intensive use of black walnuts, which grow naturally in
the central and southern parts of the United States, in rootstock
breeding studies by hybridization would be the right decision.

2.1.8 Hibrids

In modern fruit growing, it is often not possible to find a rootstock that

meets the desired criteria in rootstock breeding within a single species.
For this reason, interspecies hybridization studies have been initiated.
Suitable rootstocks in many fruit species have been obtained by
interspecies hybridization. These rootstocks have achieved a very high
commercial value and have been in high demand by growers (Rom
and Carlos,1987; Gregory et al., 2015; Zhu et al., 2019). There are
also a large number of species in the genus Juglans. It varies quite
widely from the difference of tree vigor to different soil requirements.
However, there are also variations in the resistance of these species to
different diseases and pests (Gregory et al., 2015; Ramesh et al.,
2021). Most of the species in the genus Juglans can form hybrid
plants by pollinating among themselves. This situation focused on
some species, and the previously known superior rootstock
characteristics of these species were tried to be increased by
interspecies hybridization. In many studies, crosses of J.hindsii x
 W a l n u t | 55

J.regia, J.hindsii x J.nigra and J.nigra x J.regia have been performed.

At the end of these studies, rootstocks called Paradox ,Royal and
Persian hybrids, which are used extensively, were obtained. More
detailed analyzes of these rootstocks are still being made since the
1950s when these studies were carried out (Rom and Carlos,1987;
Kluepfel et al., 2011; Zhu et al., 2019; Vahdati et al., 2021). Paradox
rootstocks were found to be more resistant to Phytophthora and
nematodes than both parents. Walnut varieties grafted on Paradox
rootstocks show more vigorous growth than the same varieties grafted
on J.regia rootstocks. Due to the susceptibility of Paradox rootstock
mostly to blackline disease, it will be important to select this hybrid
and search for more suitable clonal rootstocks that can be produced by
tissue culture (Ramesh et al., 2021; Vahdati et al., 2021).

2.1.9 Other Juglans Varieties.

Research on the rootstock characteristics of Juglans species of

different genera under the Juglandaceae branch has been limited, only
studies on the rootstock characteristics of Pterocarya stenoptera to
J.regia have been conducted in China. Studies have shown that
Pterocarya stenoptera gives excessive bottom shoots, this increases
even more in anhydrous conditions and shows incompatibility with
some J.regia walnuts (Vahdati et al., 2021). This genus, which is
highly resistant to nematodes, would be beneficial to cross with the
Juglans genus and it would be more appropriate to investigate the
rootstock characteristics of the hybrids obtained.
56 | W a l n u t

2.2 Clonal Rootstocks

2.2.1 Vlach

It is a useful rootstock obtained by selection from Paradox hybrids

(J.hindsii x J.regia) and can be produced clonally by
micropropagation (Zhu et al., 2019). Walnuts grafted on this
rootstock, which is tolerant to root-knot nematodes, show vigorous
growth. Although Vlach clonal rootstock varies according to the
variety, it forms more vigorous trees than RX1 and VX211 rootstocks
(Brown et al. 2010; Baumgartner et al.2013). It has been determined
that it has the same water consumption as VX211 in terms of water
stress.

2.2.2 RX1

This rootstock is a hybrid (J.microcarpa x J.regia) rootstock obtained

by crossbreeding. It is moderately resistant to Phytophthora bred by
the University of California and is also tolerant to Agrobacterium
(Brown et al. 2010; Baumgartner et al.2013). Walnuts grafted on RX1
have a medium-strength crown, while yield efficiency is quite high.
Classic J.hindsii x J.regia hybrids formless vigorous trees than
rootstocks. It can be easily reproduced by tissue culture. It is
moderately tolerant of drought and waterlogging.

2.2.3 VX211

It is another clonal rootstock obtained by selection from Paradox

hybrids (J.hindsii x J.regia).
 W a l n u t | 57

A group of researchers from the University of California and USDA-

ARS selection breeding studies from open pollinated Paradox
seedlings, taking into account their genetic relationship carried out in
1996. Growth vigor, nematode resistance and vegetative propagation
possibilities were taken into consideration in the study (Potter et al.,
2002). Only Vlach clonal rootstock was used until the years of this
study. Between 1997 and 2002, the production possibilities of a total
of 3000 seedlings consisting of 2000 paradox and 1000 J.hindsii
seedlings by tissue culture and their resistance to nematodes were
investigated. Field trials of promising clones obtained from the
selections made in these studies were carried out (Buzo et al.2009). It
was observed that this rootstock was resistant to nematodes and
Phytophthora in field trials and produced vigorous trees (Gregory et
al., 2015; Zhu et al., 2019). Compared to the Vlach rootstock, it forms
relatively semi vigorous trees but forms more vigorous trees than the
RX1 rootstock.VX211 rootstocks can be easily produced by
micropropagation.

2.2.4 Grizzly

It is a random seedling that has drawn attention with its healthy

development in a closed walnut garden in the United States and has
been bred as a clonal rootstock with studies (Gregory et al., 2015;
Vahdati et al.,2021). As a result of more than 20 years of studies on
this rootstock, it has been determined that it forms very healthy and
vigorous trees in different soil conditions.
58 | W a l n u t

A- Walnut trees grafted on paradox rootstock; B- Royal rootstok grafted Juglans

regia (Preece and Granaham,2015)

Figure 4. Juglans nigra tree and fruit

 W a l n u t | 59

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(2010). Etiology and management of crown and root rots of walnut, Walnut
Research Reports. California Walnut Board. p. 225–236

Buzo T., McKenna J., Kaku S., Anwar S.A., McKenry M.V.(2009). VX211,A
Vigorous new walnut hybrit clone with nematode tolerance and useful
resistance. The Journal of Nematology 41 (3):211-216.
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(Mahrabaşı) Üzüm Çeşitlerinde Aşı Başarısı ve Fidan Kalitesi Üzerine Etkisi.
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of wild walnut Juglans spp. for resistance to crown gall disease.
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crown gall disease. Phytopathology 101:92-103
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McGranahan GH, TuleckeW, Arulsekar S, Hansen J.J. (1986) Intergeneric

hybridization in the Juglandaceae: Pterocarya sp. x Juglans regia. Journal of
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Carlson, R.F. (Eds.), Rootstocks for Fruit Crops. John Wiley and Sons. New
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selection trait expression in mature walnuts. Journal of American Society for
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Ramos D.E. (1997). Walnut production manual, V: 3373. UCANR Publications,

Oakland. USA. Germain E. 1999. Le Noyer. Centre Technique Interpr. des
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Davis, California. p:415-450.
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64 | W a l n u t
 W a l n u t | 65

Part IV
--------------------------------

Soil Management and Fertilization in Walnut

Dr. Cafer Hakan YILMAZ1

1. Introduction

Walnut, which has been produced since the Neolithic age, is one of
the important hard-shelled fruit species with a wide spread area in the
world. Among many species included in the genus Juglans in the
family Juglandaceae, Juglans regia L. is the most important species
traded due to its superior fruit characteristics (McGranahan and Leslie,
1991; Kafkas et al., 2020; Guney et al., 2021). According to the data
of 2019, walnut is among the first three with a share of 9.3% among
the hard-shelled fruits in the world, and in terms of production
amount, it ranks first with a share of 25.8%. China covered 56.1% of
the total world walnut production in 2019. Turkey, on the other hand,
ranked fourth in walnut production with a share of 5.0%. China
accounts for 48.4% of walnut production areas, the USA for 11.3%
and Turkey for 9.5% (Anonymous, 2021a). It is grown in many
provinces of Turkey (Şen, 1986); (Şimsek, 2010); (Akça, 2012). In

1
General Directorate of Agricultural Research and Policies, East Mediterranean
Transitional Zone Agricultural Research of Insttute ORCIDs: Dr. Cafer Hakan
YILMAZ, https://orcid.org/0000-0003-3680-453X c_hakanyilmaz@hotmail.com
66 | W a l n u t

this respect, our country is among the most important countries in the
world in terms of walnut production.

Walnut is a plant that adapts well to different climatic conditions, the

need for vernalisation varies between 400-1800 hours, it also grows in
locations up to 1700 m high from the sea (Akça 2012).

Walnut, which is an indispensable food in terms of human health due

to its nutritional value, has not achieved a significant increase in both
the yield per tree and the amount of production despite the high
demand today, and even serious problems have been encountered
(Oğuz et al., 2016; Okatan et al., 2021). Although many selection
studies have been carried out in our country from the past to the
present, the existence of a limited number of studies on applications to
increase quality and yield in standard varieties is noteworthy. In this
respect, conscious fertilization applications can be effective in
perennial species such as walnuts. It has been reported that plant
nutrient deficiency causes problems such as low yield and internal
shrinkage and darkening, which are important for walnuts (Şen, 1986).
In commercial cultivation, it is stated that macro and microelements
are significantly effective in applied plant feeding programs (Yıldız et
al., 2007). In a study conducted in the Niksar district of Tokat, it is
stated that the leaf samples taken from 72 walnut orchards have
deficiencies in N, P, K, Mg, Ca, Fe, Zn, Mn and Cu (Adıman, 2013).
In a study conducted in 46 walnut orchards in Tekirdağ, where
nutritional status was determined, it was determined that most of the
soils were insufficient in terms of N, P, K, Ca, Mg, S, Fe, Cu, Mn and
 W a l n u t | 67

Zn nutrients (Solmaz, 2014). Yıldız and Uygur (2016) determined that

61% of walnut orchards in Uşak province had lime problems, 69%
had phosphorus, 78% were poor in magnesium, 96% were zinc and
98% were potassium poor. In addition, according to the results of the
leaf analysis, they reported that 29% of the walnut orchards had
phosphorus, 76% potassium, 80% magnesium, 78% iron, 94% zinc
and copper deficiencies. In 157 soil samples collected from the
orchards where walnut cultivation is carried out in Mersin province
and its districts, it was determined that the orchards were poor in
terms of P and K (Arslan and Aydın, 2017). Direction and Sönmez
(2021) found the N, P, Cu and Zn contents insufficient, according to
the results of leaf analysis in their study in walnut gardens in Burdur
region. In addition, they stated that they determined excessive and
high lime in soil samples collected from 0-30 and 30-60 cm depths
and that this could cause some problems in terms of cultivation and
nutrition. Again in the same study, they reported that the organic
matter contents of the soil samples were with low hummus and poor,
partially insufficient in terms of available P, and available Fe and Zn
contents were insufficient.

Mills and Jones (1996), of some macro and micro plant nutrient levels
that should be in walnut leaves are N: 2.47-2.98%, P: 0.16-0.24%, K:
1.32-1.47%, Ca: 1.90-2.01%, Mg: %. 0.51-0.63, S: 1500-1600 mg kg-
1
, Fe: 69-129 mg kg-1, Mn: 207-274 mg kg-1, Cu: 10-12 mg kg-1, Zn:
33-55 mg kg-1 and B: 66-81 mg kg-1 they reported that it should be in
the ranges of. Soil fertility levels of walnut orchard should be in range
68 | W a l n u t

of pH 6.5 to 7.2, 2.0 to 3.5% OM, 0.25 to 0.3% N, 27.2 to 36.3 kg da-1
P, 102.2 to124.9 kg da-1 K, 908 to 1360 kg da-1 Ca, and 113.5 to 136.2
kg da-1 Mg. However, it was also stated that in most cases, the
correlation between nutrient supply in the soil and tree growth and nut
production is poor (Ponder, 1981). Walnut trees are not a very
selective fruit type in terms of soil. It grows better in deep, low ground
water, permeable, not holding much water, well-drained, well-
ventilated, keeping its moisture, organic matter-rich, alluvial soils
(Kramer, 1983); (Anonymous, 2008a); (Anonymous, 2008b);
(Anonymous, 2021b).

2. The Importance of Soil Fertility in Walnut Cultivation

Although walnut is a more tolerant plant in terms of soil fertility, it

shows significant increases in the amount and quality of the product
taken per tree in highly fertile soils. In walnut cultivation, it is
necessary to consider the fertility parameters of the soil when
establishing a new garden. These parameters are:

▪ Soil depth,
▪ Soil texture (water holding and aeration capacities of the soil),
▪ Soil reaction (pH),
▪ Soil electrical conductivity (EC),
▪ Soil lime (CaCO3),
▪ Soil organic matter (SOM) and
▪ Soil is plant nutrients (macro and micronutrients and their
amounts).
 W a l n u t | 69

2.1. Soil Depth

For the walnut plant, generally deep and well-drained soils are defined
as suitable soils. In studies in the USA, a soil depth of at least 90-100
cm is recommended for restrictive and unsuitable soils (Losche,
1973); (Funk, 1979). According to Ponder (2004), effective soil
rooting depth should be 90 to 150 cm or more and should not be
limited to a layer of sand or gravel, compacted massive clay layer or
bedrock. However, it has been detected that walnut roots can also
benefit from deeper soils and absorb water at a depth of 300-370 cm
(Ponder, 1981). Walnut trees grow best in soils where their roots can
easily grow up to 2.5-3 m. For this reason, 3 m below the soil should
be checked before the garden is established (Anonymous, 2021c).
Considering that walnut roots can go down to 3-3.5 m, it requires deep
soil for a good cultivation. Soil depth should not be less than 1.5-2 m.
For this reason, when considering the establishment of an economic
garden, it is useful to know the soil depth and the underlying soil
structure (Vural, 2009).

2.2. Soil Texture

Texture is the size limitation of the sand, silt and clay that make up the
soil. It affects root performance by inhibiting the soil's water holding
capacity, aeration, plant growth and in some cases, plant nutrition
(Ponder, 1981); (Begg, 1985). Although walnut trees are not selective
in terms of soil, they grow well in deep soils with a ground water level
of 2.5-3.0 meters, which do not hold water, maintain their moisture. It
70 | W a l n u t

can not grow in water-retaining clay soils and stagnant watery places.
Walnut roots are taprooted and since they grow into the deep, soils
with moist undersides, deep and loam, silty loam, silty clay loam,
silty, clay loam, sandy loam and fine sandy loam are more suitable for
cultivation. Excess moisture and stagnant water prevent the roots from
getting the necessary oxygen, so root growth slows down and the tree
stops growing (Ponder, 2004); (Anonymous, 2021d).

2.3. Soil Reaction (pH)

Soil reaction (pH) is one of the most important properties of the soil
solution. The pH of the soil causes the movement of plant nutrients,
their accumulation in the soil or making them unavailable, preventing
them from being uptaken by the roots or hindering growth and
development by making toxic effects. Fruit trees develop best in soils
between pH 5.5–6.5. However, soils with a pH of up to 8.5 are also
used in fruit cultivation (Arslan and Aydın, 2017). For walnuts, soil
pH is required to be 6.5-7.2 (Ponder, 2004); (Anonymous, 2021b). In
a study they conducted in the Burdur region, Yön and Sönmez (2021)
reported that the pH of walnut-grown soils varied between 6.58-7.59
at 0-30 cm and 6.84-7.64 at 30-60 cm depth. In another study, it was
stated that the pH value of the soil should be slightly acid and neutral
(6.0-7.5), and that iron and zinc deficiency are generally observed in
the young leaves of the trees in walnut orchards established in soils
with high pH value (more than 8) (Anonymous, 2021e).
 W a l n u t | 71

2.4. Electrical Conductivity (EC)

Electrical conductivity is directly related to salinity. Walnuts are also

in the moderate fruit group in terms of resistance to soil salinity
(Anonymous, 2021c). Since walnuts are sensitive to salinity, the
amount of salt in the soil and irrigation water must comply with the
specified standards. As salinity increases, yield loss also increases
(Sesli, 2014); (Anonymous, 2016). Values of 1.5 dS m-1 and below are
suitable for walnut cultivation. If the EC is higher than 1.7 dS m-1,
yield loss begins. When EC is 2.3 dS m-1, 10%, when EC is 4.8 dS m-
1
, there is a 50% efficiency decrease (Akça, 2000); (Anonymous,
2021f).
Root, trunk and shoot length of walnut trees exposed to saline stress,
in leaf area and number, significant decreases were observed in the
amount and yield of chlorophyll. When the plant was exposed to
salinity stress for a long time, it was observed that signs of ion toxicity
and water deficiencies on old leaves, and carbohydrate deficiencies
appeared in young leaves (Meddamaz and Ellialtıoglu, 1994);
(Sivritepe, 1995).

2.5. Soil Lime (CaCO3)

Especially in the form of CaCO₃, the presence of calcium more than

desired causes many plant nutrient elements to be bonded. For this
reason, it is desirable to have the appropriate amount of calcium in the
soil. The effect of lime on the walnut plant, which is more tolerant to
soil lime than other fruit species, is indirect due to relationship of
72 | W a l n u t

walnut with pH (Arslan and Aydın, 2017). In walnut orchards

established on very calcareous soils, iron and zinc deficiency are
usually seen in the young leaves of the trees (Anonymous, 2021e). At
the same time, it is known that phosphorus precipitates in the form of
tricalcium phosphates in very calcareous soils and turns into a form
that the plant cannot uptake. Direction and Sönmez (2021), in a study
they conducted in walnut orchards in Burdur region, reported that
more than 80% of the soils were high and extremely high in lime, and
that the Fe and Zn contents, and partly phosphorus contents, were
insufficient in all of the soils. Arslan and Aydın (2017), in their study
in districts of Mersin, where walnut cultivation was carried out,
reported that approximately 50% of the soils were too calcareous and
also 60% were low in phosphorus. Fe deficiency is often called lime
chlorosis (Carlson, 1985).

2.6. Soil Organic Matter (SOM)

Organic matter is very important for both the nutrition of the tree, root
health and drainage. Walnut grows well in soils rich in organic matter
content. The amount of organic matter desired in the soil in walnut
cultivation is 2.0-3.5% (Ponder, 2004). Therefore, it is necessary to
increase the organic matter content of the soil by applying suitable
farmyard manures. However, increasing the amount of organic matter
of the soil with farm fertilizers is both expensive and if poorly burned
manure is used, weeds and some soil diseases and pests, if any, are
infected to the soil. The most economical and optimal way to increase
the amount of organic matter is to make a green manuring program.
 W a l n u t | 73

Green manure plant to be determined in accordance with the region,

preferably leguminous plants such as vetch, broad bean and sainfoin,
are grown between rows from the beginning of autumn to the spring
period, and green manuring is made by mixing the plants with the soil
in the middle of the flowering period. Since there will be no weed
growth between rows by growing green manure plants, weed control
may not be necessary (Anonymous, 2021g).

2.7. Plant Nutrient Elements

Plant nutrients consist of some chemical elements. Therefore, the

nutrients needed by green plants are inorganic. Plants uptake inorganic
nutrients through their roots or leaves. Plants need at least 17 plant
nutrients to ensure their normal growing. Three of these elements are
carbon, hydrogen and oxygen. Since these elements are mostly
uptaken from air and water, they are considered as non-mineral plant
nutrients. In addition, they are almost ignored in plant nutrition due to
their sufficient resources (Jones and Jacobsen, 2001); (Fageria, 2009);
(Kacar and Katkat, 2010). Plants uptake 14 other essential elements
(nitrogen (N), phosphorus (P), potassium (K), calcium (Ca),
magnesium (Mg), sulfur (S), iron Fe), manganese (Mn), copper (Cu) ,
zinc (Zn), boron (B), molybdenum (Mo), chlorine (Cl) and nickel
(Ni)) directly from the soil. Elements can be taken by plants in the
form of anions and cations, as well as in the form of molecules. The
proportional amounts of these soluble elements are also different from
each other (Bolat and Kara, 2017). In perennial plants such as walnuts,
it is necessary not only soil analysis, but also leaf analysis to find out
74 | W a l n u t

the nutritional status of the plants. It is of particular importance to

interpret the soil and plant analyzes together for walnuts. Balanced
fertilization is essential to increase walnut yield and quality (Ponder
and Schlesinger, 1986); (Garrett et al., 1991); (Jones et al., 1995);
(Jacobs et al., 2005).

2.7.1. Uptaking Soil and Leaf Samples

Upaking soil samples is closely related to climatic conditions. Soil

samples can be collected at any time during the year if the humidity
and temperature conditions are suitable. However, in order for the
analysis results to be in the hands of the farmers on time, soil samples
should be collected 1.5-2 months before planting or fertilizing. Before
the soil samples are collected, a sketch of the land is drawn if the land
is very large and showing differences. In this sketch drawn, the
differences are indicated. Different samples are taken from each
different region. Soil samples are collected from 10 different points by
walking in a zig-zag (S or Z shape) on the land where the walnut
garden will be established. Soil samples are collected from 20-40-60
cm depths of these points separately. If necessary, samples are also
taken from 90 and 120 cm depths. For leaf samples, middle leaflet
pairs are collected from mature shoots with 5, 9 or 13 leaflets 6-8
weeks after the full flowering period (in July or August) from at least
25 trees (Kacar and İnal, 2010).
 W a l n u t | 75

2.7.2. Fertilizer Application

For a fertilization program to be carried out in walnut orchards, soil

and leaf analyzes must be done. The results from leaf samples are
important in determining the amounts of fertilizer to be given.
Fertilization in walnuts, 1. Fertilizing when establishing a garden, 2.
Sapling fertilization, 3. Fertilization in the juvenil period and 4. Four
separate periods are taken into account as fertilizing during the full
yield period. In addition, when necessary, foliar fertilization can be
carried out as well as from the soil. In general, fertilization is carried
out in two different periods in walnuts of both in sapling age and in
full yield age. The first fertilization is carried out 2-3 weeks before the
bud swell on the shoots at the beginning of spring. This fertilization is
called base dressing. The second fertilization is applied before
irrigation (in the period between fruit set and fruit growth) and
watered. This fertilization is called top dressing.

2.7.2.1. Fertilizing when establishing a garden

Fertilizing when establishing a garden, which is the fertilization

applied to the whole area or to planting pits without planting saplings.
According to the results of soil analysis; farmyard manure,
phosphorus and potassium fertilizers should be used. In addition,
liming or sulfur applications should be carried out according to the
status of pH and organic fertilization also according to the state of
organic matter into the sapling planting pits. In cases where organic
fertilizers of animal origin are insufficient, green manure crops can be
76 | W a l n u t

sown to interrows after planting saplings. Since walnut trees grow

well in light-textured alluvial soils, the soils are generally poor in
phosphorus, potassium and magnesium. Saplings are planted in pits
containing a mixture of 100-150 g of Normal Super Phosphate and
Potassium Sulphate fertilizers, 50-60 g of Magnesium Sulphate and 2-
3 g of Zinc Sulphate. Then, the top soil is mixed with barnyard
manure and the pits are filled. According to irrigation methods after
planting saplings, a fertilization program is applied until the saplings
reach the age of yield (Anonymous, 2021b and h).

2.7.2.2. Sapling fertilization

Walnut saplings become yielding when they reach 4-7 years of age,
depending on the variety. During this period, it is necessary to fertilize
the saplings in a way to ensure that they have a strong root structure
and healthy development of the above-ground part. Fertilizers given to
the crown projection of the saplings or to the part where the drip
irrigation pipes pass (60-70 cm) are mixed to a depth that will not cut
the roots. In base dressing, all of the phosphorus and potassium and
half of the nitrogen are given according to the soil analysis result. The
other half of the nitrogen is applied 1-2 times before irrigation (fruit
set-fruit growth) and irrigated. In fertilization with drip irrigation,
30% of nitrogen fertilizer, 60-70% of phosphorus fertilizer and 50%
of potassium fertilizer are applied during the bottom fertilizer
application period. The remaining part of the fertilizers is applied in
each irrigation in accordance with the irrigation program with
fertilizers suitable for the drip irrigation system. The amounts of
 W a l n u t | 77

fertilizers that should be given according to the age of the sapling are
given in Table 1.

Table 1. Fertilizer Amounts to Be Given According to The Age of the Saplings

(Anonymous, 2021g).

Sapling Age (g fertilizer/sapling)

Growth Period Fertilizer Type
1 2 3
Bottom Fertilizer
(before bud swelling 15-15-15 400 800 1000
on shoots)
Ammonium
Tassel Fall Period 100 200 200
Sulphate
Calcium
Fruit Set-
Ammonium - - 100
Fruit Growth
Nitrate (CAN)

2.7.2.3. Fertilization sapling in the juvenil period

Nitrogen is the most needed nutrient element of walnut saplings,

which are in the juvenil period, in the spring flowering and shoot
formation period. The amounts of fertilizers that should be given to
saplings of the age of yield (in the juvenil period) are given in Table 2.

Table 2. The Amounts of Fertilizers to Be Given to The Saplings During The

Yielding Period (Anonymous, 2021g).

Sapling Age (g fertilizer/sapling)

Growth Period Fertilizer Type
4 5 6
Bottom Fertilizer
(before bud swelling 15-15-15 1000 1200 1200
on shoots)
Ammonium
Tassel Fall Period 200 300 300
Sulphate
Calcium
Fruit Set-
Ammonium 200 200 200
Fruit Growth
Nitrate (CAN)
78 | W a l n u t

2.7.2.4. Fertilizing in fully yielding walnut trees

Depending on the light, medium and heavy texture of the soil, bottom
fertilizers are mixed into the soil at a depth that will not cut the roots
and at the distance of the tree's crown projection. Top dressing should
be pre-precipitation. Fertilizer should be used according to the soil
analysis and the amount of product that can be taken per tree. In Table
3, amounts of fertilizer that should be given according to the amount
of product that can be taken per tree is given. Composite or potassium
fertilizers containing chlorine should not be used on walnut trees that
do not like salinity. Base dressing with potassium sulfate should be
preferred for the increase in %fat, %protein and aroma in the edible
part of walnuts.

Table 3. Amounts of Fertilizer to Be Given According to Amounts of Product That

Can Be Taken Per Tree (Anonymous, 2021g).

Yield (kg/tree, g fertilizer/tree)

Growth Period Fertilizer Type
10-20 20-40 40-60 > 60
Bottom
Fertilizer
1500- 2500- 3500- 4500-
(before bud 15-15-15
2000 3000 4000 5000
swelling on
shoots)
Tassel Fall Ammonium
600 800 1000 1200
Period Sulphate
Calcium
Fruit Set-
Ammonium 400 500 600 800
Fruit Growth
Nitrate (CAN)
 W a l n u t | 79

2.7.2.5. Fertilization according to the drip irrigation system

Base Dressing: 10 kg of 15-15-15 fertilizer per decare is divided by

the number of trees per decare and given to the part where the drip
irrigation pipes pass and mixed with the soil. The amounts of fertilizer
given for one-year-old saplings in Table 4 should be applied by
increasing by 15% each year until the saplings reach the full yield
period.

Table 4. Amounts and Application Intervals of Fertilizers to Be Given by Drip

Irrigation for One-Year-Old Saplings (Anonymous, 2021g).

g fertilizer/acre/month
Growth Period
15-15-15 MAP KNO3 ZnSO4 MKP
April 500 500 500 50 -
May 832 1500 1000 100 -
June 664 1000 1500 100 -
July 332 500 1500 50 -
August 1000 -
September 3
MAP: Monoammonium phosphate, KNO3: Potassium nitrate, ZnSO4: Zinc sulfate,
MKP: Monopotassium phosphate

All fertilizers and doses given in this study are for exemplary
purposes. The main thing is the recommended fertilization program
according to the results of soil and plant (leaf) analysis.

2.7.2.6. Foliar Fertilization

Foliar fertilizers can be in solid (powder and crystal) and liquid form.
Leaf fertilizers in liquid form are generally obtained by dissolving
solid form mineral fertilizers in water, acid and special solutions and
preparing them as concentrated solutions. The total amount of active
80 | W a l n u t

substances is 2-3 times less than those in solid form. The application
dose in these fertilizers should be 1-2% in macroelements and 0.1-
0.2% in microelements on the basis of effective substances. It should
not be used in windy weather and before precipitation. Since very
little of it is taken by the plant, it should be applied 2-3 times at certain
times (Aslan, 2013). Application doses and times depend on the
growth period of the plant and the thickness of the cuticula layer in the
plant leaf. It should be applied in the proportions written on the
package. In case of excessive use, burns may occur on the leaves of
the plant. The symptoms and symptoms locations of the deficiency of
plant nutrients in walnuts are given in Table 5.

Tablo 5. Plant Nutrient Deficiency Symptoms and Symptom Locations in Walnuts

(Anonymous, 2021h).

Deficient Element Symptom Symptom Location in the Plant

N, (S), Fe Chlorosis Old and Ripe Leaves
Mg, (Mn) Chlorosis
Fe, (S) Chlorosis Young Leaves
Zn, Mn, (B) Chlorosis
K, Mg Necrosis Old and Ripe Leaves
Mg, (Mn) Necrosis
B, Cu, Cl Necrosis Young Leaves
B, Mo, Ca, Zn Deformation Young Leaves
P Dark Gray Color Very Old Leaves

3. Conclusion

Plant nutrient deficiencies in our soils reveal the importance of plant

nutrition practices in the production process. The internal fruit quality
 W a l n u t | 81

of walnut, which is one of the hard-shelled fruit types, is important

and is a clear reason of preference for consumers. In order to increase
the yield and quality in walnuts (fruit internal weight, width, height
and height, yield, shell thickness, internal shrinkage, color values,
shoot length, lateral branch efficiency, etc.), a balanced fertilization is
required according to the analysis results of soil and leaf samples
(Ponder and Schlesinger, 1986); (Garrett et al., 1991); (Jones et al.,
1995); (Jacobs et al., 2005); (Adiloğlu and Adiloğlu, 2005).
82 | W a l n u t

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 W a l n u t | 87

Part V
--------------------------------

Micropropagation of Walnut
Sule POLAT1
Assoc. Prof. Muhammet Ali GUNDESLI 2
Hayat Topcu3

Walnut has a taxonomy that includes a total of 21 species belonging to

the genus Juglans from the Juglandaceae family. The most important
of these species are Juglans regia L. Juglans nigra L. and Juglans
cinerea L.. Wild walnuts as natural habitats have spread as far as
Alaska in Europe, Asia and North America. They are walnuts
belonging to the Juglans regia species, which are naturally grown for
their nut and investigation subject to the breeding studies. In addition,
Juglans nigra is grown for its timber. Juglans hindsii, which is called
California black walnut, is mostly used in rootstock breeding studies
(McGranahan and Leslie, 1990). Walnut is a temperate climate fruit.
It’s wood has been used for a long time due to its high quality, its
leaves and fruit skin due to its pharmacological properties, and its fruit
1
Department of Horticulture, Agriculture Faculty, Cukurova University, Adana,
Turkey, E-mail: polatsule@outlook.com
2
Department of Plant and Animal Production, Nurdagı Vocational School,
Gaziantep University, E-mail: maligun46@gantep.edu.tr
3
Tekirdag Namık Kemal University, Agricultural Biotechnology Tekirdag, Turkey,
ORCIDs: https://orcid.org/0000-0003-3108-4393, hayattopcu@nku.edu.tr
88 | W a l n u t

due to its nutritional properties. The fruit of the walnut is very rich in
protein, fat components and energy. Walnut, which spreads naturally
on rich and high quality soils and streams, can also form healthy
plants in arid and rocky areas (Pijut, 2004).

Walnut species are usually propagated by seeds. Walnut (Juglans

spp). the dormant embryo reproduction and hybridization method are
very effective in the development of high-yielding varieties. Walnut
varieties are also propagated vegetatively by grafting onto seedling
rootstocks. Different rates of success have been achieved with
different intra- and inter-species grafting techniques (Ki and Ding,
1990). Vegetative propagation in walnut cultivation is labor, time
wastage and grafting, which is expensive. at the same time, vegetative
propagation is a very difficult situation as their rooting ability is low
(Land and Cunninham., 1994). They stated that in many studies
conducted in previous years, the desired success in walnut production
and reproduction is still not achieved, and the reason for this is
irregular and low rooting rates (Claudet et al., 1992; Heloir et al.,
1996). There are different studies with walnuts for research, clonal
reproduction, and genetic development. Progress in progressive
techniques for tissue culture production and hybrids of walnuts has
been very successful in recent years. Plantlets were obtained through
shoot tip propagation, cultured node segments, and somatic
embryogenesis (Payghamzadeh, 2008)

The use of grafting and rootstock in walnut cultivation dates back to

ancient times. In the beginning years, rootstocks were generally used
 W a l n u t | 89

as seedling rootstocks. Orchards were prepared from walnut cultivars

grafted on walnut seedling rootstocks of different species. However,
in the following years, the use of clone rootstocks began in order to
establish more homogeneous orchards and to provide resistance
against some soil pathogens. Hardwood production in walnuts is very
difficult and has not been preferred because it provides low success.
With the emergence of Paradox rootstocks, micro production
possibilities began to be sought in rootstock candidates obtained from
these hybrids (Kaur et al. 2006). In fact, micropropagation has become
a vejetatif propagation method that has been economically used in
many fruit and rootstock varieties by the private sector in recent years.
Successful results are obtained especially in the reproduction of stone
and pome fruit rootstocks. Many private companies produce rootstock
with this method and sell until export. This success achieved in stone
and pome fruits could not be achieved in walnuts in the early days.
One of the most important reasons for this is that explants taken to in
vitro secrete phenolic compounds that cause darkening from the
cutting site (Rout et al.1999). This situation was seen as the most
important obstacle to the micropropagation of walnut. Some
researchers have tried to prevent this situation by adding substances
such as citric acid, ascorbic acid, activated charcoal, polyvlinyl
pyyrolidone (PVP), thirourea, L-cysteine and glutamine to the
medium. In other studies, polyphenol activity was tried to be reduced
with different dark applications (Payghamzadeh, 2008). In another
study, it was concluded that activated charcoal added to the first
culture medium was effective. In fact, the biggest problem in walnut
90 | W a l n u t

micropropagation is the release of Juglon compounds, which are

allelochemicals, into the medium after they are taken into the medium.
In addition to the above-mentioned applications, it is reported that the
problem is solved to a great extent by taking explants to fresh nutrient
media at intervals of 1-3-5 days (Driver and Kuniyuki, 1984;
McGranahan et al., 1984).

IN VITRO RESEARCH PROTOCOL

In the spring season, the material should be taken from walnut trees
that show 2-4 weeks of development, 0.5-0.8 cm in diameter, 2-5 cm
between the knuckles and leaf buds. Antioxidant liquor and fungicide
liquor should be used for pre-sterilization before the materials are
taken to the laboratory, and then they should be processed in the
laboratory. In August and September, semi-woody cuttings are taken
from walnut trees aged 2-4 years, 20 cm tall. The surface is
disinfected by mixing with the fungicide mixture for 20 minutes in the
laboratory and then washed three times with sterile distilled water.
The cuttings are placed on filter paper until dry and left with 50 ml of
water in 200 cc bottles under sterile conditions, then closed with
transparent plastic bags. It is kept in the climatic chamber for a 16-
hour photoperiod at 25 ± 1 C until vegetative leaf bud formation.
Walnut the widely accepted operation includes surface sterilization of
beginning explants with 50 - 70% (v/v) ethanol for 20 – 30 s traced by
0.1 - 15% (v/v) sodium hypochlorite implicating 0.01% Tween 20 for
10 - 20 min wait. Later three 5 min washes were in sterile distilled
water (Payghamzadeh and Kazemitabar, 2011). Kour et al. (2006),
 W a l n u t | 91

Netar Akhort, Gobind, Solding walnut kinds vis., of the choice kinds
made embryo culture in ripe fruit. Ripe fruits were bathed in flowing
water faucet water, the epicarp was taken, and stayed fruit piece was
sterilized by treating with 0.1% sodium hypochlorite solution and
washed twice for 5 minutes with sterile distilled water. San and
Dumanoglu (2006), Yalova-1, Şebin, Bilecik, KR-1, KR-2, Sen-2, 07-
KOR-1, Tokat-1, Kaman-1, walnut kinds somatic embryogenesis
made works. Kaman-5 cultivars were sterilized with 3.75% bleach in
the raw fruit. Fruits were sterilized by dip in 3.75% (v/v) sodium
hypochlorite for 25minute dip three 5-minute washes in sterile
distilled water. Roschke and Pijut (2006); Micropropagation studies
were carried out on shoots in walnuts and shoots in leaves.
Sterilization was done separately for leaves and shoots. For shoots:
shoots are washed under the flowing water for 30 minutes. Later
shoots were dissected and dipped in 70% ethanol for 30 seconds and
disinfected for 20 minutes in 15% sodium carbonate 0.01% Tween
liquor on an orbital shaker. Rinsed in sterile water four times for 30
seconds. For leaves: the surface of the explants was washed in flowing
tap water for 5 minutes, and sterilized in 10% sodium hypochlorite
liquor for 10 minutes. Later washed four times with sterile water.

A successful micropropagation is studied in four stages, which

includes different methods:

1-Sterilization and initial culture,

2-Shoot formation and multiplication,

92 | W a l n u t

3- Formation and rooting of microshoots,

4-Hardening and acclimatization of plants from in vitro

(Payghamzadeh and Kazemitabar, 2011)

1-Sterilization and initial culture

Embryo, cotyledon or axillary buds are used in walnut

micropropagation. In recent years, the most commercially used shoot
meristem. At this stage, it is very important that the explant is not
adversely affected by the sterilization material. A careful sterilization
process will also prevent future contamination. The shoots taken from
the explant source plant are cleaned with the help of liquid soap and a
brush and kept under running water for 2-3 hours. Then it is kept in
70% ethanol for 2-3 minutes and rinsed with pure water. After this
stage, the explants are sterilized in a sterile cabinet with 70% Actigen
((Natural Protection System, NPS Biocidal) 100% natural water-based
and 0.015% active chlorine containing liquid, floor and surface
disinfectant) for 5 minutes and then 5' 3 times. rinsed with sterile
distilled water for each minute.

Figure 1. Obtaining meristematic nodal tissue from the explant source

 W a l n u t | 93

Table 1. Different sterilization methot of explants.

Species/ Explants Study Disinfectant and Reference
Cultivars dosage
J. regia L.cv. Mature Embryo culture 0.1% sodium Kour et al.
fruit hypochlorite (2006)
J. regia L.cv. Immature Somatic 3.75% sodium San and
fruit embryogenesis hypochlorite Hatic
(2006)
J. regia L. Shoot and Adventitious For shoots: 70% Roschke
leaf shoot ethanol, 15% and Pijut
regeneration and bleach solution (2006)
micro propagation
J. cinerea L. Nodal Axillary bud 0.8% (v/v) Pijut
segment culture sodium (1997)
hypochlorite
(15% clorox
bleach)
J. regia L. Fruit Nut Micropropagation 0.5% NaClO, Revilla et
rootstock Embryo culture 75% EtOH al. (1989)

2-Shoot formation and multiplication

In many different studies, it has been reported that walnut explants

micropropagated very well to appropriate in vitro conditions. Axillary
buds that have grown in the starting medium are taken into Driver and
Kuniyuki Walnut (DKW) medium supplemented with approximately
8-9 μM BAP (Benzylaminopurin). In other studies, it has been
revealed that modified MS nutrient media supplemented with BAP at
rates ranging from 4.4-8.9 μM can also give successful results for
J.regia. Sister shoot formation varies highly according to genotypes,
even in similar media (Payghamzadeh and Kazemitabar, 2011).
Scaltsoyiannes et al. 1997 reported that they obtained different results
in micropropagation of 11 different walnut genotypes. Payghamzadeh
and Kazemitamar 2010, who made micropropagation experiments in
94 | W a l n u t

different walnut varieties, reported that the media to be used may vary
according to the genotypes.

Micropropagation of walnuts different media by Driver and Kuniyuki

(1984) (DKW), Murashige and Skoog (1962) (MS), Cheng (1975),
Gamborg et al (1968) and Lloyd and McCown (1981), (WPM),
Rodríguez (1982), medium K(h) are used. Among these media, Driver
and Kuniyuki (1984) (DKW), Murashige and Skoog (1962) (MS) are
the most used in walnut micropropagation. The DKW medium is
especially optimized for the production of Paradox rootstocks.
Micropropagation of many Juglans species is successfully carried out
with this medium. However, some researchers reported that they
obtained successful results from the MS medium. Saadata and
Hennerty 2002 reported that J.regia needs more salt minerals in
micropropagation, and the most suitable medium for this should be
DKW. Although DKW salts are similar to MS in terms of minerals
and nitrogen, it has a higher concentration in terms of other ions.
Saadata and Hennerty 2002 reported that DKW and MS gave higher
results than WPM in terms of callus weight and average shoot length.
Driver and Kuniyuki 1984 reported that DKW gave more positive
results in the results obtained from micropropagation of Paradox
rootstock in DKW and WPM media. In a similar study by the same
researchers in black walnut, they found better results in shoot length,
shoot thickness, leaf width and color of DKW medium.

MS medium has higher nitrate concentration compared to WPM

medium. This situation is thought to cause metabolic stress in plants.
 W a l n u t | 95

It is reported that this nutrient medium does not cause stress in the
plant cell, since many components in the MS nutrient medium are not
in WPM. It has been determined that leaf deformations and plant
stresses occur in potato plants taken into MS medium due to the cobalt
content. WPM does not contain cobalt or nickel. While potassium
iodide is present in MS, there is no iodine source in WPM. Some
studies have shown that iodine sources have a toxic effect on the
micro-propagation of golden weed (Haplopappus gracilis).

Payghamzadeh and Kazemitamar 2008(a) used modified DKW with

MT mediums for walnut micropropagation. At the end of the study,
they determined that sister shoot formation was higher than DKW
medium. Positive results were obtained in embryo germination from
the medium developed for walnut micropropagation (NGE) by
Murciano de Investigacióny Desarrollo Agrarioy alimentario
(IMIDA). Later, researchers compared DKW, WPM and NGE media.
The highest germination percentage was obtained in WPM with the
highest 81%, then 54% and 62% values were obtained from DKW and
NGE, respectively. In the micropropagation of J.cinerea, it was
revealed that the explants taken in DKW and NGE mediums showed
similar growth rates. However, contrary to these results, it has been
reported in many studies that DKW medium is most suitable for
micropropagation of J.regia (Kaur et al.2006).
96 | W a l n u t

Figure 2. Shoot multiplication of walnut explants

3- Formation and rooting of microshoots

Phenolic compounds and some internal bacterial contaminations are

the main barriers to regeneration and rooting in successful in vitro
studies of walnut protocol. Many studies on in vitro propagation of
some walnut genotypes have reported that these plants bear fruit early
compared to genotypes propagated by conventional methods. They
also stated that they have a good rooting system and that there is no
incompatibility (Lopez, 2004; Nomiya et al., 2004; Vahdati et al.,
2009; Zarghami and Salari, 2015). Rooting of walnut micro shoots
depends on internal and external factors. In the rooting studies of
hybrid walnut rootstocks (J.nigra X J.regia), 24.6 μM IBA was
applied and it was determined that the rooting rate was very high after
a 5-day dark period. It has been observed that some internal hormones
and peroxidase enzyme activity increase rooting in micro shoots.
Leslie and McGranahan (1992) reported that they obtained the highest
rooting rate from 4.9 or 24.6 μM IBA application. It has been reported
that rooting of microshoots taken to medium with proportionally low
salt concentration is higher. Scaltsoyiannes et al. (1997) found that
 W a l n u t | 97

rooting was higher in DKW medium where the macro element was
reduced by ¼ and the NO3/NH4 ratio was increased from 0.1 to 3.0.
Hyndman et al. (1982) determined that DKW medium containing
reduced KNO3 and NH4NO3 concentration was more effective in
rooting. Long et al. (1995) observed that rooting was higher in DKW
medium containing high sugar concentration and reduced nitrogen
(456.2 mg/L NH4NO3 and 634.0 mg/L Ca (NO3)2). It has been
revealed that rooting rate, average root length and root number are
high in all media where activated charcoal is used. In addition, it was
revealed that micro shoots taken to the medium with activated
charcoal needed less time for rooting.

Revilla et al. (1989) observed that micro shoots that were kept in 2
mg.l-1 liquid nutrient medium for 24 hours and then taken to solid
nutrient medium with the same hormone ratio rooted more easily.
Researchers stated this situation as some polyphenols that prevent
rooting on the cut surface of the microshoots were transferred to the
liquid medium and then rooting increased in the solid medium taken.
Payghamzadeh and Kazemitabar (2008) investigated the effects of
different phase, activated charcoal and BAP concentrations on rooting
in DKW medium, and reported that double phase nutrient culture
provided more effective rooting. Many studies so far have shown that
auxin hormones are quite effective in rooting. The best result in
rooting can be obtained by:

When the microshoots grow to 4-5 cm, they are taken into DKW
medium containing ¼ reduced macro element supplemented with 24.6
98 | W a l n u t

μM IBA and 40 g.l-1. Then it is kept at 24 °C for 6 days and at 21 °C

for 6 hours. After this application is completed, the micro shoots are
transferred to DKW medium containing sterile vermiculite and gelled
¼ reduced macro element. Scaltsoyiannes et al (1997) reported that
the use of medium type vermiculite in rooting increases rooting and
root quality as it allows the roots to breathe more easily.

Figure 3. In vitro rooting of walnut

 W a l n u t | 99

Figure 5. In vitro propagation of walnut (a, b) preparation, (c)

Establishment, (d, e) Proliferation and (f) Rooting (Zarghami and Salari, 2015)

Table 2. Salts composition (mg.l-1) of culture medium

Component MS DKW WPM NGE B5 LP

NH4NO3 1650 1416 400 908 - 908

KNO3 1900 - - 723 2500 -

Ca(NO3)2.4H2O - 1968 556 2249 - 1262

CaCl2.2H2O 440 149 96 699 150 122.2

K2SO4 - 1559 990 - 250 1274.5

MgSO4.7H2O 370 740 370 2053 134 555

(NH4)2SO4 - - - - 10 -

MnS04.H2O - - - - 150 27.9

100 | W a l n u t

NaH2PO4.H2O - - - - - -

KH2PO4 170 265 170 155 - 217.5

MnSO4.4H2O 22.30 33.5 22.3 22.3 0.25 -

Na2MoO4.2H2O 0.25 0.39 0.25 0.25 2 0.32

ZnSO4.7H2O 8.6 - 8.6 8.6 - 4.3

Zn(NO3)2.6H2O - 17 - - 0.75 8.5

Kl 0.83 - - 0.83 3 -

H3BO3 6.2 4.8 6.2 6.2 0.025 5.5

CuSO4.5H2O 0.025 0.25 0.25 0.025 0.025 0.25

CoCl.6H2O 0.025 - - 0.025 - -

NiSO4.6H2O - 0.005 - - - -

FeSO4.7H2O 27.8 33.8 27.8 27.8 37.3 30.8

Na2EDTA.2H2O 37.3 45.4 37.3 37.3 100 41.35

Myo-inositol 100 100 100 100 10 100

Thiamin-HCL 0.1 2 1 0.1 1 1.5

Nicotinic acid 0.5 1 0.5 0.5 1 0.75

Pyridoxine-HCL 0.5 - 0.5 0.5 - 0.25

Glycine 2 2 2 2 - 2

Glutamine - - 2 - - -

Sucrose 30.000 30.000 30.000 30.000 20.000 30.000

 W a l n u t | 101

Table 3. Commonly used hormones, mediums and explants for in

vitro propagation of walnut (Payghamzadeh and Kazemitabar,2011)
Ex
Species Medi References
Plant growth regulator Study pla
Cultiva um
nt
rs
TD 2,4 IA NA G
BAP Kn IBA
Z -D A A A3
J. - - - - 2.5 - - - Rooting 1/2 Ms Pijut (1997)
cinerea μM MS
L
J. 8.9 - - - - - - - Axillary bud MS Ns Pijut 1997)
cinerea μM culture
L
J. regia - - - - 3 - - - Rooting of DKW S Heloir et al.
L mg/l shoot (1996)
J. regia 1 - - - - - - - Axillary MS S Heloir et al.
L mg/l shoot (1996)
proliferation
J. nigra 1-5 - - - - - - - Bud MS - Sommers et
L. mg/l proliferation or al.(1982)
DKW
J. regia 1 - - - 0.01 - - - Shoot DKW Saadat and
L. mg/l mg/l multiplicatio , MS St Hennerty
n and (2002)
WPM
J. regia - - 6.8 - - - 1 - Adventitious DW L Roschke and
L μM μM shoot (1/2 Pijut
regeneration DKW (2006)
+1/2
WPM
)

J. regia 4.44 - - - 0.005 - - - Shoot DKW Ge Jay-

L. μM μM multiplicatio Allemand et
n al.(1992)

J. regia 1 - - - - - - - Shoot MS Eje Penula et al.

L mg/l multiplicatio (1988)
n and
rhizogenesis
induction

J. regia 1 2 - - 0.01 - - - Macro Modif Sb Rodriguez et

L. mg/l mg mg/l morphologic ied al.(1993)
/l al and MS
histological
analyses.

J. regia 1 - - 0.1 0.0 - 0.1 Micro MS Jns Revilla et al.

L mg/l mg/l 5 mg propagation ,Ea (1989
mg /l
/l
J. regia 4.4, - - - - - - - Culturing Modif Ns Gruselle et
L 8.9 nodal ied al.
μM segement MS (1987)

J. regia 0.4 - - - - - - 0.8 Axillary bud MS Ns Chalupa

L. μM μM elongation (1981)
J. nigra - - - 5 0.1 - - - Adventitious WPM - Long et al.
L μM μM regeneratio (1995)
n
102 | W a l n u t

4-Hardening and acclimatization of plants from in vitro

Acclimatization of successfully micropropagated plants is very
important for commercial use. Plant losses experienced at this stage
can cause all efforts to be wasted. Acclimatization of plants after
micropropagation is actually a difficult step (Sanchez et al.2005).
Ambient humidity and temperature are important at this stage. During
acclimatization, plants are likely to be damaged by fungal diseases. In
studies on this subject, it is important to use ventilated transparent
containers and to perform the acclimatization process in fully
controlled greenhouses. The survival probability of plants transferred
to suitable humidity and temperature environments is quite high. As a
matter of fact, in walnut research reports, it has been reported that
more positive results are obtained from plants that are acclimatized in
heat and humidity controlled greenhouses after in vitro (Stevens and
Pijut, 2018).

Figure 3. Acclimatized walnut seedling

Result

The most important problem encountered in in vitro reproduction of

walnuts is juglone, endogenous internal bacterial contamination and
phenolic compounds that affect the materials used. The use of shoot
 W a l n u t | 103

tips, meristems and epicormic shoot tips at different physiological

periods appear to be the best alternatives for in vitro propagation.
There are many difficulties faced by the tissue culture industry in
production. These; cost-effectiveness, automation, control and
optimization of the microenvironment. A major challenge in walnut
studies should be focused on genetic improvement of elite trees with
desired treatments through transformation systems and identification
with molecular markers, and these issues need to be studied in more
detail in the future. Micropropagation of walnut with nodal segment
culture has now become the most suitable method for clonal
propagation of precious plant materials. Contrary to popular belief,
walnuts can be easily reproduced vegetatively. Today, a large number
of protocols have been established for the in vitro propagation of
walnuts (Table 3). Thanks to micropropagation techniques, rootstock
breeding studies for walnuts have gained momentum. In this way,
mass rootstock production has begun. Currently, micropropagation
companies have to deal with problems such as the effective cost of
reproduction media, air conditioning and optimization. Recently, the
use of agar-free liquid media has become widespread due to the cost
of agar media.
104 | W a l n u t

References

Claudet A.C, Drauet A, Jay-Allemand, C. (1992). Tissue distribution of phenolic

compounds in annual shoots from adult and rejuvenated hybrid walnut trees.
Plant Physiol. Biochem. 30(5): 565-572
Driver, J. A., & Kuniyuki, A. H. (1984). In vitro propagation of Paradox walnut
rootstock. HortScience, 19(4), 507-509.
Heloir, M.C., Kevers C., Hausman, J.F, Gaspar, T. (1996). Changes in the
concentrations of auxins and Polyamines during rooting of in vitro
propagated walnut shoots. Tree Physiol. 16(5): 515-519
Hyndman, S. F., Hasegawa, P. M., & Bressan, R. A. (1982). of Mineral
Salts. HortScience, 17(1), 82-83.
Kaur, R., Sharma, N., Kumar, K., Sharma, D. R., & Sharma, S. D. (2006). In vitro
germination of walnut (Juglans regia L.) embryos. Scientia
Horticulturae, 109(4), 385-388.
Payghamzadeh, K., and Kazemitabar, S.K. (2011). In vitro propagation of walnut -
A review African Journal of Biotechnology Vol. 10(3), pp. 290-311
Land SB, Cunningham M (1994). Rooted cutting macropropagation of hardwoods.
In:-Applications of vegetative propagation in forestry‘‘.Proc. of the Southern
regional information exchange group biennial symposium on forest genetics.
Foster GS and Diner AM. (eds.). Published by Southern Forest Experiment
Station New Orleans, Louisiana. pp. 75-96.
Leslie, C. & McGranahan, G. (1992) Micropropagation of Persian Walnut (Juglans
regia L.). In Bajaj, Y.P.S. (Ed), Biotechnology in Agriculture and Forestry
18. High Technology and Micropropagation II. SpringerVerlag. Berlin
Heidelberg pp. 137–150.
Long LM, Preece JE, Van Sambeek JW (1995). Adventitious regeneration of
Juglans nigra L. (eastern black walnut). Plant Cell Rep. 14: 799-803.
Lopez, J.M., 2004. Walnut tissue culture: research and fields applications.
Proceedings of the 6th walnut council research symposium, July 25-28, 2004,
Lafayette, pp: 146-152
 W a l n u t | 105

McGranahan G, Leslie C (1990). Walnuts (Juglans). In: Moore JN, Ballington JR

(eds). Genetic resources of temperate fruit and nut crops, Int. Soc. Scientia
Horticulturae Wageningen, 2: 907-951.
McGranahan G, Leslie CA, Driver JA (1988). In vitro propagation of mature Persian
walnut cultivars. HortScience 23(1): 220
Nomiya, K.,A. Yoshizawa, K. Tsukagoshi, N.C. Kasug, S. Hirakawa and J.
Watanabe, 2004. Synthesis and structural characterization of silver(I),
aluminium(III) and cobalt(II) complexes with 4-isopropyltropolone
(hinokitiol) showing noteworthy biological activities. Action of silver(I)-
oxygen bonding complexes on the antimicrobial activities. J. Inorg.
Biochem., 98: 46-60.
Payghamzadeh K (2008). Somatic embryogenesis from immature cotyledons and
meristemic culture of walnut (Juglans regia L.). The MSc thesis. College of
agriculture, Dep of Plant breeding and Biotechnology, University of
Agricultural and Natural Resources of Sari, Iran. pp. 48-77.
Payghamzadeh K, Kazemitabar SK (2008a). Comparison effects of MT novel
medium with modified DKW basal medium on walnut micropropagation.
Proceeding book of the 1st national conference of student biology and modern
world, p. 204.
Payghamzadeh, K., Kazemitabar, S.,K. (2011) In vitro propagation of walnut - A
review. Accepted 19 November, 2010 African Journal of Biotechnology Vol.
10(3), pp. 290-311, 17 January, 2011 Available online at
http://www.academicjournals.org/AJB ISSN 1684–5315 © 2011 Academic
Journals.
Pijut PM (2004). Vegetative propagation of butternut (Juglans cinerea) Walnut
Council Research Symposium; Gen. Tech. Rep. NC-243. St. Paul, MN: U.S.
Department of Agriculture, Forest Service, North Central Research Station.
pp. 37- 44.
Revilla, M. A., Majada, J., & Rodriguez, R. (1989). Walnut (Juglans regia L.)
micropropagation. In Annales des Sciences Forestières (Vol. 46, No.
Supplement, pp. 149s-151s). EDP Sciences.
106 | W a l n u t

Roschke, C., and Pijut, P.M. (2006). Micropropagation of Juglans nigra L. in liquid
culture. http://ncrs.fs.fed.us/4157/localresources/downloads/posters/2006/
Roschke.pdf.
Şan, B., & Dumanoğlu, H. (2006). Somatic embryogenesis from immature
cotyledons of apomictic and non-apomictic seeds in walnut (Juglans regia
L.). Turkish journal of agriculture and forestry, 30(2), 111-117.
Sanchez-Olate, M., Rios, D., Revilla, M. & Rodriguez, R. (2005) Factores
involucrados en el enraizamiento in vitro de leñosas de interés agroforestal.
In: Sánchez-Olate, M. & Ríos, D. (Eds)
Scaltsoyiannes, A., Tsoulpha, P., Panetsos, K. P., & Moulalis, D. (1997), (1998).
Effect of genotype on micropropagation of walnut trees (Juglans
regia). Silvae Genetica, 46, 326-331.
Stevens, M. E., Pijut, P. M. (2018). Rapid in vitro shoot multiplication of the
recalcitrant species Juglans nigra L. In Vitro Cellular & Developmental
Biology-Plant, 54(3), 309-317.
Vahdati, K., J.R. McKenna, A.M. Dandekar, C.A. Leslie and S.L. Uratsu et al.,
2002. Rooting and other characteristics of a transgenic walnut hybrid
(Juglans hindsii x J. regia) rootstock expressing rolABC. J. Am. Soc. Hortic.
Sci., 127: 724-728.
Zarghami R., and Salari, A. 2015. Effect of Different Hormonal Treatments on
Proliferation and Rooting of Three Persian Walnut (Juglans regia L.)
Genotypes. Pakistan Journal of Biological Sciences, 18: 260-266
Rout, G. R., Samantaray, S., Mottley, J., & Das, P. (1999). Biotechnology of the
rose: a review of recent progress. Scientia Horticulturae, 81(3), 201-228.
 W a l n u t | 107

Part VI
--------------------------------

Sustainable Farming Systems and Organic

Walnut Growing
Assoc. Prof. Aysen Melda COLAK1

Department of Horticulture, Usak, Turkey People and groups who saw

that this increase in agricultural production, which was called the
"Green Revolution" in the 1950s-60s, when chemical fertilizers,
drugs, and chemical additives began to be used, did not bring a
solution to the problem of hunger in the world in general, but on the
contrary, deteriorated the natural balance, soil quality, and human
health. They started various studies on the subject. Although the
amount of production increased with these chemicals used over time,
it killed beneficial organisms in the soil in the long term, causing a
decrease in nutrient quality and soil fertility (Sinha and Herat, 2009).
This situation necessitated the development of human and
environment-friendly input and production systems in order to
establish the natural balance lost as a result of these faulty practices in
the ecological system.

1
Department of Horticulture, Faculty of Agriculture, Usak University, Usak, Turkey
ORCIDs: Assoc. Prof. Ayşen Melda ÇOLAK: https://orcid.org/0000-0003-0113-
2104 aysenmelda.colak@usak.edu.tr
108 | W a l n u t

The first goal in organic farming is to preserve soil vitality and

productivity. For this purpose, it is the addition of organic substances
to the soil that ensures the continuity of vital activities without a
natural-synthetic additive in the cycle of nature (Karacalar, 2008).

The amount and property of organic matter is one of important

characteristics of soils. Organic matter plays an important role in
many physical, chemical, and biological properties of soil. Organic
matter improves the physical properties of heavy clay and light sandy
soils, increases the water holding capacity and aeration of the soil. It
also positively affects the chemical properties of the soil. It is a source
of nutrients and N. It increases the cation exchange capacity, balances
the soil pH, reduces the effects of toxic substances such as lime and
excess fertilizer in the soil. It enables the plant to take insoluble plant
nutrients into a soluble form and improves the living conditions of
organisms in the soil (Karaman et al., 2007).

As a result of the intensive use of synthetic chemical inputs in

traditional agriculture, chemical residues in products have increased.
Organic farming includes the complete avoidance of the use of
synthetic chemicals used in traditional agriculture that harm the
environment (Altındişli, 1998).

Although most of the walnuts produced in our country are not named,
they are organic. Walnut is one of the rare plants among the fruit
species that did not use commercial fertilizers and pesticides in
 W a l n u t | 109

cultivation so far. This will provide great convenience in the transition

to organic cultivation of walnuts.

The points to be considered in setting up a garden for organic walnut

cultivation are similar to traditional (conventional) principles. The
most important factor in establishing a garden for organic products is
that the variety to be grown is suitable for the ecological conditions of
the region (Özkan, 2005). It is a very important factor that fruit
species and varieties are resistant to diseases and pests. While the size
is an indicator of quality in conventional fruit growing, small fruit
obtained without using any synthetic chemicals can be called high
quality in organic fruit growing (Yalçınkaya, 2001). The most
important issue to be considered in the walnut garden plant is the
appropriate selection of the variety and pollinator variety. In addition,
care should be taken to ensure that the grafted part of the seedlings to
be used in the garden facility is an old and organic certified part. In
the production of seedlings, non-organic or growth regulators that are
not allowed to be used should not be used. Planting distances vary
depending on the variety, rootstock, terrain, pruning, and harvesting.
In addition, a planting distance that will minimize the risk of diseases
and pests should be applied.

When weeds compete with trees in the intake of water and plant
nutrients in organic fruit cultivation, weeds in the tree crown
projection should be understood. Herbicides should never be used in
the fight against weeds. Instead, mechanical methods, crop rotation,
110 | W a l n u t

and mulch can be used. As a mechanical method, machine or manual

cutting should be preferred (Anonymous, 2002).

Making green manure 1-2 years before planting will be an important

gain, especially in terms of nitrogen source and soil improvement.
Green fertilization after planting can be done by planting a legume
plant such as vetch on the tree crown projection. The main food
source is farm manure. According to the nutritional needs of the plant
(between 1-5 tons per decare), well-burned farm manure should be
given or organic farm manures prepared specially for their nutrient
content should be used in recommended doses. It is appropriate to
give fertilizers containing essential nutrients to the seedlings during
the establishment of the garden. Then, other plant nutrients should be
given according to soil and leaf analysis. One of the main reasons for
the lack of yield and quality in walnut cultivation in our country is the
plant nutrition problem. In order to determine this, soil analysis must
be done and the appropriate fertilization program must be determined.
In order to understand that the fertilization program of the plant is
done correctly, leaf analysis should be done and according to this
result, the missing plant nutrients should be supplemented. In addition
to these, humic acid, seaweed, mycorrhiza, bacteria, etc. organic
materials such as The most important issue that we will pay attention
to in fertilization is that the products to be used are approved as
organic fertilizers by the control and certification bodies (Altındişli,
1998).
 W a l n u t | 111

Fertilization is the main factor affecting the yield and quality of trees.
Walnut trees take many plant nutrients from the soil due to the
development of roots, shoots, branches, leaves, buds, and fruits with
the help of their roots. It is necessary to give these plant nutrients back
to the soil.

The use of chemical fertilizers, against organic or natural fertilizers, is

extremely common in the world and in our country. One of the main
reasons for this is that it is easily available. Since chemical fertilizers
reduce the vitality population in the soil, they make the soil
increasingly self-dependent. In recent years, due to increasing
fertilizer prices, producers have begun to be unable to obtain fertilizer,
and they have turned the excrement of their animals into
vermicompost, either directly or with the help of worms, and applied
it to their gardens.

Organic farming is a sustainable farming system and a practice that

includes the use of organic fertilizers (plant residues, manures of
animal origin), based on specific production techniques such as
disease and weed control. The main organic fertilizers used are;

Vermicompost

Vermicompost is a kind of natural organic fertilizer. Worms feed by

carrying the plant residues that are thrown into the soil and start to rot,
to the channels they open in the soil at night. As a result of their
feeding, humus is formed from their feces and this humus plays an
112 | W a l n u t

important role in increasing soil fertility. The common name of

vermicompost in the world is vermicompost, and the cultivation of
vermicompost is called vermicomposting (Şimşek, 2007).

Vermiculture is the general name for worm growing and

vermicompost production. The main purpose of worm growing in
vermiculture is to obtain vermicompost. But it is also part of the
process to grow worms as a source of live and/or dried feed.
Vermicompost is a product obtained as a result of composting various
organic wastes (animal manure, straw, straw, household fruit, and
vegetable waste, garden leaf waste, sawdust, waste paper, etc.) by
some earthworms during their digestion and used as organic fertilizer
and soil conditioner in the agricultural industry. Vermicompost is also
called black gold. vermicompost; Due to its physical, chemical, and
biological interactions in the soil and its slow release, it has become
one of the most popular fertilizers of recent times (Türkmen, 2016).

Vermicompost is a very rich mixture in terms of plant nutrients and

minerals, it gives root development to the plant with the fertilizer
added to the soil. It increases productivity and improves the water
holding capacity of the soil. Since it is an organic fertilizer, it
regulates soil pH and soil structure. In addition, worm castings do not
contain weed seeds (Anonymous, 2016).
 W a l n u t | 113

sheep wool manure

Wool consists of keratin protein, which contains nitrogen, carbon, and

sulfur, which is important in plant nutrition. It has been determined in
the experiments that it has beneficial effects on the productivity of
many plant species. Washed wool and its hydrolysates can be used as
a valuable and environmentally friendly fertilizer (Tüfekçi and Olfaz,
2014).

seaweed fertilizer

Although seaweed is a soil improver, it is only suitable for local use

due to its high transportation costs. The use of seaweed extracts as a
foliar spray is quite common. In many crops, foliar sprays have
increased yield. This is due to the micronutrients it contains along
with the growth hormones it contains such as auxins. Seaweed
extracts can be applied as liquid fertilizers as well as foliar sprays.

Seaweeds:

By providing strong root development, plants take more nutrients and

water from the soil,

increase the green parts by accelerating the formation of chlorophyll

in plants, therefore more carbohydrates, proteins, etc. They enable
plants to be more resistant to diseases and pests, to make plants
resistant to environmental stresses such as frost, drought, insufficient
sun, excessive water, extreme heat, and extreme cold. It is a source of
114 | W a l n u t

macro and micronutrients for plants. By putting the microelements,

which cannot be taken by the plant in the soil, into chelate form, it
ensures that the plant receives them at the highest rate and makes them
balanced in the plant. It increases side branching and fruit set in fruit
trees. It also reduces flower and fruit drop. It provides up to 30%
increase in yield in plants. It increases the storage durability of the
products. It inhibits the proliferation of viruses and reduces the
damage of nematodes. By providing a balanced and long-term intake
of macro and micronutrients from the soil, it increases the yield,
improves the quality, and increases the market and export value
(Blunden et al., 1992).

farm manure

Farm manure (barn manure) consists of the excrement of large and

small cattle and the bedding laid under the animals in the barns. On
the one hand, barn manure positively affects the structure of the soil,
on the other hand, it has a positive effect on the amount of product by
providing the necessary nutrients for the plants. These effects can be
listed as follows: It increases the water holding capacity of the soil. It
prevents water from flowing independently on the soil surface,
evaporating and transporting arable land. It allows the soil to come to
the pan easily. It makes the soil temperature suitable for plant growth.
It has an effect on the pH of the soil. Barn manure has a positive effect
on soil aeration due to its organic structure. On the other hand, carbon
dioxide and organic acids formed as a result of the breakdown of barn
manure in the soil make plant nutrients useful for plants. A large
 W a l n u t | 115

amount of microorganisms is given to the soil with barn manure.

Thus, the rate of biological changes in the soil increases (Soyergin,
2003).

Humic acid

Humic acid is used as a fertilizer in plant nutrition. Humic acid, which

is produced from lignite coal or leonardite by the extraction method, is
impregnated with clay or compost and offered to the market as pellets
or in the concentrated form under the name of organic fertilizer.
Humine substances are important in plant nutrition, especially by
increasing the rate of use of nitrogen and phosphorus fertilizers by
breaking them down (Karaçal and Tüfenkçi, 2010).

It provides the ideal pH balance (5.5–7), the amount of organic matter

(4–6%) in the root zone, regulates microbiological activity, balances
the soil reaction of the environment by lowering the high pH value of
salty and calcareous soils. It removes salt and lime from the root area
by buffering. It has the feature of making the soil healthier, stronger,
and a suitable environment for microorganism activities (Tunç, 2006).

Humic acids convert iron into an absorbable form and protect plants
from chlorosis. It also helps the iron complex in the soil to be taken up
by plants and the formation of chlorophyll in the leaves (Özbay,
2012).

Some studies on walnuts in the world and in Turkey are listed below
with their results.
116 | W a l n u t

The study named “Investigation of the Effects of the Use of

Composed Microbial Fertilizer on Yield and Quality Parameters in
Walnut (cv. Chandler)” was carried out in the Demirci district of
Manisa province between 2018-2019. (before the male flowers open,
when the male flowers open and the fruits reach the size of hazelnuts)
it was applied by spraying with the help of a leaf sprayer at a dose of 2
cc/L per tree, the highest results were obtained from the EM.A
application in terms of fruit width, weight, and height in terms of the
average of two years. In two applications, it was observed that the
color of the fruits became darker but duller compared to the control
group, in general, the amount of linoleic acid in the fruits was higher
in the EM.5 application, followed by linoleic acid in the control group
and oleic acid in the EM.A application, respectively, the highest yield
was EM. In 5 applications (2.52 kg/tree), the lowest yield was
determined in the control group (0.78 kg/tree). (Bilgin et al., 2020).

In a study conducted in the Kashmir region of India, four selections of

walnut (SKAU/002 (S1), SKAU/008 (S2), SKAU/024 (S3), and
SKAU/040 (S4)) were used in 6 different combinations of organic and
inorganic fertilizers (T1). : all inorganic NPK fertilizer; T2: 50%
manure, 25% vermicompost, 25% poultry manure; T3: 75% NPK
fertilizer, 25% farm manure; T4: 75% NPK fertilizer, 25%
vermicompost,; T5: % 75 NPK fertilizer, 25% poultry manure; T6:
75% NPK fertilizer, 25% evenly distributed worm, farm and poultry
manure) the study was carried out, and looking at the results obtained,
 W a l n u t | 117

the best result was T4 application (tree height (16.12%), tree

circumference (1.40%), tree canopy volume (37.85 m3), shoot
elongation (0.81 m), fruit set (40.52%), fruit holding (58.21%), yield
maximum increase. In addition, maximum fruit set (38.96%) and fruit
holding (57.53%) were measured as higher results in S1 variety
compared to other select varieties, S2 flood The highest yield (5.86
kg/da) was obtained from the cultivation compared to other cultivars
(Wani et al., 2016).

In a study called "Organic Walnut Cultivation and Its Importance for

Turkey", it was stated that cultural practices are very important for our
country to be in the top places in terms of organic walnut production
in the world, and this can be possible by combining traditional
methods with new cultivation methods (Özkan, 2005).

In a study on walnut yield and soil quality in Kashmir, India, four

selections [(SKAU/002 (S1), SKAU/008 (S2), SKAU/024 (S3), and
SKAU/040 (S4)] and six fertilizer application in the first week of
December [T1 (100% NPK fertilizer), T2 (50% farm manure, 25%
vermicompost, 25% poultry manure), T3 (75% NPK fertilizer, 25%
farm manure), T4 ( 75% NPK fertilizer, 25% vermicompost), T5
(75% NPK fertilizer 25% poultry manure) and T6 (75% NPK
fertilizer, 25% fertilizer (1/3 farm manure + 1/3 vermicompost + 1/3
poultry manure) fertilizer)], the highest yield was obtained as a result
of T4 application from S3 selection with 6.82 kg/tree, the highest
nitrogen (338.59 kg/ha), phosphorus (20.80 kg/ha), calcium (1312.25
ppm), zinc (1.19 ppm), manganese (66.66 ppm), iron (55.95 ppm) and
118 | W a l n u t

copper (2.74 ppm) contents were found as a result of T5 application,

the highest potassium (259.27 kg/ha) and magnesium (268.86 ppm)
T2 application was obtained (Wani et al., 2017).

Dr. In a study conducted in a 10-year-old walnut garden at YS Parmar

University of Horticulture and Forestry, 13 different fertilizer
applications were made, and according to the results obtained, 750 g
nitrogen, 375 g phosphorus, 750 g potassium + 50 kg vermicompost,
and 562.50 g nitrogen, It was observed that 281.25 g phosphorus,
562.50 g potassium + 68.50 kg vermicompost applications were
effective on the leaf nutrient status of the trees (Bhattarai and Tomar,
2009).

In a study conducted in the Otovalo canton of Imbabura province of

Ecuador, 13 different applications (T1= 25% Humus + 75% Rice
husk, T2= 25% Humus + 75% Pomina, T3= 25% Humus + 75% Sand,
T4=% 25 Humus + 75% Soil, T5= 50% Humus + 50% Rice husk,
T6= 50% Humus+ 50% Pomina, T7= 50% Humus + 50% Sand, T8=
50% Humus + 50% Soil, T9= % A study on the germination time of
walnut seeds with 75 Humus + 25% Rice hull, T10= 75% Humus +
25% Pomina, T11= 75% Humus + 25% Sand, T12= 75% Humus +
25% Soil, T13= Control) After the study, as a result of 50% Humus +
50% Pomina application, the highest germination and seedling
number was reached with 99.38% (Cabascango, 2011).

In a three-year study with pecans, the combination of vermicompost

and 3 different fertilizers (urea, potassium nitrate, Hydro complex (N
 W a l n u t | 119

12.40%, P2O5 11.40%, K2O 17.70%, S 8%, Fe 0.20%) , Mn, 0.02%,

Zn 0.02%, B 0.015)) with 4 replications, 4 different applications were
made, in the applied applications (F1= 30 g/wood synthetic fertilizer,
F2=60 g/wood synthetic fertilizer, 5 kg) /tree vermicompost and
control group) soil properties and plant growth were examined, it was
observed that there was no significant change in soil pH value, an
increase in salinity was observed in the last two years of the study,
provided with synthetic fertilization with a high stem diameter or
organic improvement, the highest P in soil and C levels were obtained
as a result of vermicompost application (Giuffré et al., 2017).

In a study conducted in the Kashmir region of India, the bio-efficacy

of rhizobacteria isolated from walnuts grown in the Himalayas against
five fungal pathogens in the future was investigated. As a result of the
in vitro evaluation of rhizobacteria in terms of biocontrol effectiveness
against 5 fungal pathogens, namely Dematophora necatrix, Alternaria
solani, Pythium aphanidermatum, Fusarium oxysporum and
Phytophthora capsici, it was determined that most of the bacteria
belonged to the Bacillus genus. D. necatrix, A. solani of WI 90 (66%),
WI 63 (55.6%), WI 62 (43.8%), WI 63 (45.5%) and WI 65 (49%)
respectively. It was found to be antagonistic to F. oxysporum, P.
phanidermatum and P. capsici and had maximum inhibition. The 12
most effective isolates were characterized morphochemically and
molecularly and based on the 16S rDNA sequence, Bacillus
licheniformis (Isolates WI90), Bacillus subtilis (Isolates WI63 and
WI65), Bacillus tequilensis (Isolate WI62), Bacillus cereus (Isolate
120 | W a l n u t

WI36), Micrococcus lute, WI41, and WI80 isolates), Micrococcus

yunnanensis (WI30 and WI60 isolates) and Micrococcus sp. (WI11
and WI91 isolates) (Sofi and Dar, 2018).

In a study titled "The Effect of Microbial Liquid Fertilizer on Rooting

of Fig (Bursa Black), Reverse Mulberry, Red Mulberry, Walnut
(Kaplan-86) Wood Cuttings", wood cuttings were taken from Fig,
Reverse Mulberry, Red Mulberry, Walnut trees in two different
periods during the winter resting season. , the cuttings were planted in
plastic black pots containing perlite after being kept in 0, 25, 50, and
100 ml/L solutions of 10×104 (w/w) microbial organic liquid fertilizer
overnight. While in mulberry, no rooting was observed in walnut,
while increasing doses of organic microbial liquid fertilizer used in the
experiment had a negative effect on rooting, it was observed that the
optimal dose of 50 ml/L had a positive effect on root length, root
diameter, shoot length and shoot diameter in cuttings planted in
autumn (Dalkılıç et al., 2019).

The mineral nutrition status of walnut orchards in Uşak was

investigated by soil and leaf analyzes. and copper; In the leaf samples,
additional nitrogen and boron mineral analyzes were made to these
elements, according to the analysis results, 39% of the soils of the
gardens are loamy, 57% are clay loam, salinity is between normal
values, approximately 61% of the soils have lime problems, pH value
It was determined that it is neutral or slightly alkaline, mostly in the
middle class in terms of organic matter content, 69% of the orchards
are poor in phosphorus, 78% in magnesium, 96% in zinc and 98% in
 W a l n u t | 121

potassium; According to the results of leaf analysis, it was observed

that 29% of walnut orchards were deficient in phosphorus, 76% in
potassium, 80% in magnesium, 78% in iron, and 94% in zinc and
copper (Yıldız and Uygur, 2016).

In a study on the “Western” variety of pecan nut, anaerobically

digested biosolids were applied to pecan trees for three years, and it
was observed that the fruit shoots increased by 16% and the yield per
tree increased by 11.3% as an average of three years, and heavy metal
concentrations ( As, Cd, Cr, Hg, Ni and Pb) were found to be
decreased, also Salmonella spp. And Escherichia coli was not found
(Tarango-Rivero et al., 2011).

In a study on pecans, it was observed that one of the most important

factors in the development of walnuts is nutrition, and some of the
direct-acting elements for the best development of the fruit are
nitrogen, phosphorus, potassium, zinc, and boron (Acevedo-Barrera et
al., 2013).

In order to make the rock phosphate in the soil useful, two different
nitrogen-fixing bacteria (Pseudomonas chlororaphis and Bacillus
megaterium) and two different phosphate-solving bacteria
(Arthrobacter pascens and Burkholderia cepacia) were inoculated
into the soil, the highest phosphorus solubility was obtained from the
mixture of Pseudomonas chlororaphis and Arthrobacter pascens
bacteria. , a strong correlation was found between total organic acid
production and phosphorus solubility as well as pH and soluble
122 | W a l n u t

phosphorus correlation. In addition, the concomitant use of Bacillus

megaterium and Arthrobacter pascens was unsuccessful, and it was
concluded that co-vaccination of Pseudomonas chlororaphis and
Arthrobacter pascens bacteria would be a good alternative to
fertilization (Yu et al., 2012).

In a study conducted in California, “Paradox”, which is widely used as

a walnut rootstock, is sensitive to Agrobacterium tumefaciens, and the
use of methyl bromide and Telone-C35 (1,3-dichloropropene and
chloropicrin) is common in that region against this important problem.
It was stated that these applications significantly changed the
microbial activity in the soil over time. It was assumed that the
increase in microbial activity and diversity in fumigated soil would
cause competition against A. tumefaciens and decrease its density, the
study was initiated by commercially procuring three soil types
(vermicompost and two different fermented microbial fertilizers), the
study continued for 4 weeks, two Although different microbial
fertilizers did not have any effect, it was determined that
vermicompost reduced the A. tumefaciens population, and the reason
for this was biotic (Strauss et al., 2015).

In a study, a trial was set up in walnut plant using 3 synthetic and 3

natural and microbial inputs, N=0 per cm of trunk diameter; 16; 80;
160 and 320 g; P2O5=0; 7; 35; 70 and 140 g; K2O 0; 7.5; 37.5; 75 and
150 g; compost 0; 250; 1250; 2500 and 5000 g; vermicompost 0; one
hundred; 500; 1000 and 2000 g; mycorrhiza 0; 3.81; 19.05; It was
applied as 38.00 and 76.20 g, and as a result of the study, 226 kg/ha
 W a l n u t | 123

nitrogen, 121 kg/ha P2O5, 94 kg/ha K2O, 3111 for 1.94 t/ha, 149
fruit/kg and 59% edible walnut production kg/ha compost, 1905 kg/ha
vermicompost and 33.02 g/cm mycorrhiza were used. It was also
observed that the required doses for production were 30% nitrogen
and organic improvers, 50% phosphorus and potassium, and
mycorrhiza contributed 95% to fruit quality; it was also determined
that fertilizer costs constitute 40.8% of the total expenses (Flores-
Cordova et al., 2018).

In order to control A. tumefaciens and other soil and seed-borne

phytopathogens in walnut nurseries, anaerobic soil disinfection was
applied considering it could be an alternative option compared to
fumigation, and after this application, its potential was examined
together with vermicompost. In this study, 20 metric tons/ha of soil
was covered with rice bran, covered with TIF for 6 weeks after
watering for 24 hours. To examine the effects of anaerobic soil
disinfection efficiency and vermicompost application after anaerobic
soil disinfection on disease incidence, Paradox walnut rootstock seeds
were dipped into A. tumefaciens inoculum before planting and planted
in such a way that anaerobic soil disinfection was applied to the place
where anaerobic soil disinfection was applied. The amount of A.
tumefaciens was found to be significantly less than the areas without
vermicompost compared to the control plots planted with walnut seeds
and vermicompost (Sarah et al., 2014).

In a study, walnut plants were grown in 10-liter pots in soil mixed

with three different organic materials (vineyard compost, liquid
124 | W a l n u t

vermicompost, fulvic acid), solid fertilizers were added to the soil at

the first planting and liquid ones were added to the soil every week for
16 weeks, plant growth parameters were determined during the
season. During the incubation and pot experiments, Zn extractability
was increased by C ratio (p<0.05), and a significant positive
correlation was found between water-soluble C and DTPA-Zn, Zn
extractability was highly affected by the Zn and C source in the pot
experiment ( p<0.05), it was observed that humic substances were
more effective in terms of Zn extractability compared to the control,
vermicompost and fulvic acid provided the highest Zn extraction, the
highest number of shoots and leaves with 24 kg Zn/ha-C equivalent
application and the highest obtained with 12 kg Zn/ha+FA at low C
ratio (Molina et al., 2015).

In this study to examine the effect of liquid vermicompost on the

survival and growth of walnut seedlings, it was found that the use of
bio-preparation had a positive effect for this culture, plant survival
rate after diversion was 28% and growth parameters (average number
of branches per plant, number of leaves). , average branch growth rate
per plant) by 11.5% – 54.1% (Faleyev et al., 2019).

In a study on plant growth in 1+0 aged Humic acid and biohorm

walnut seedlings of vermicompost, a product of Biohumus®, grown in
Bartın-Turkey, seedlings were placed in black seedling bags in loem
soil, a sandy soil, and humic acid (0, 2, 4 ) ml/L and biohorm (0, 3, 6)
ml/L solutions containing different concentrations were applied by
spraying on the leaves at 15-day intervals on three dates, respectively,
 W a l n u t | 125

20 April, 5 May and 20 May, data were collected in September and

There was a significant increase in shoot number, leaf number, shoot
diameter, shoot dry weight (gm), root number and leaf surface area
(cm2) in seedlings treated with 4 ml/L humic acid solution and 6 ml/L
biohorm solution compared to the control group. It was observed that
the highest chlorophyll pigment was observed in the seedlings applied
3 ml/L biohorm (Taha, 2017).

In the study carried out at an average altitude of 900 m in the

Yeşilyayla neighborhood of Kula district of Manisa province in 2019,
the domestic walnut varieties Bilecik, Kaman-1, and Şebin were found
to be treated with Solid Vermicompost, Liquid Vermicompost, Solid
Sheep Wool Pellet, Liquid Sheep Wool Pellet, Solid Seaweed and
Sebin. Some fruit quality characteristics of liquid seaweed fertilizers
applied from soil were investigated. 20 fruits were selected from each
application. As a result of the study, the fruits with the highest fruit
weight were obtained as a result of the application of Solid Worm
Fertilizer in Sebin variety with an average of 11.85 g, the fruits with
the highest internal weight were found in the Kaman-1 variety with
7.10 g. As a result of the application of Liquid Sheep Wool Pellet, the
fruit internal yield The highest amount of fruits was observed in the
Solid Seaweed application of Kaman-1 variety with 62.04%, the
highest protein ratio was found in Kaman-1 variety with Liquid Worm
Fertilizer application with 30.54%, and the highest Phenolic
Compound amount was 1145 ppm in Bilecik. It was observed that it
was obtained as a result of the application of liquid Sheep Wool
126 | W a l n u t

Pellet. In addition, it was observed in this study that the organic

fertilizers that most affect fruit quality characteristics are Solid
Vermicompost, Liquid Vermicompost, and Liquid Seaweed (Çolak
and Karaca 2021).

Conclusion

Turkey is a country suitable for organic production with many factors

such as its geographical conditions, climate, product variety, and the
high number of people working in agriculture, and the demand for
organic agriculture is increasing day by day. Organic (ecological)
agriculture includes the complete avoidance of the use of synthetic
chemicals used in traditional agriculture that harm the environment.
The goal in organic fruit cultivation is to create the best conditions for
human and environmental health in all stages from the starting
material of production to the delivery of the fruit to the consumer,
without the producer incurring economic loss compared to other
agricultural systems. The organic farming system has an important
place in agricultural practices. It is necessary to carry out the
necessary studies for the spread of this agricultural practice. With the
increase in support and marketing opportunities, the demand for this
system will increase even more. The majority of walnuts produced in
our country are organic, although they are not named. It is one of the
rare fruit species that does not use synthetic fertilizers or pesticides in
cultivation so far. This situation provides great convenience in the
transition to organic cultivation of walnuts. The most important factor
in creating a walnut garden is that the variety to be grown is suitable
 W a l n u t | 127

for the ecological conditions of the region and is not damaged,

especially from late spring frosts. The grafted part of the seedlings to
be used in the garden facility must be an old and organic certified part.
In organic farming, issues such as fertilization and fight against pests,
garden location, variety selection, seedling quality, training system
and pruning technique, irrigation, tillage, and harvest are important
cultural processes.
128 | W a l n u t

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132 | W a l n u t
 W a l n u t | 133

Part VII
--------------------------------
Industrial Use of Walnut

Dr. Zeynep ERGUN1*,

Dr. Mozhgan ZARIFIKHSOSROSHAHI2

Introduction

Walnut (Juglans regia L.) is one of the members of Juglandaceae

family. Juglans genus has 20 species of many with economic
significance. These species are distributed in the temperate and
subtropical climate zone of the world. Among these species, the most
economically important species is Juglans regia L. Walnut (Juglans
regia L.) is one of the hard-shelled fruit taxa that is deciduous in
winter (Akyüz and Serdar, 2017; (Doğu et al., 2001); (Dönmez and
Güler, 2015).

Today, walnut is a type of fruit that is grown in many countries of the

world due to valuable fruits (Akyüz and Serdar, 2017); (Dönmez and
Güler, 2015); (Mitrović et al., 2007). Walnut is a native plant of a

1*
Department of Bioengineering, Faculty of Engineering, Adana Alpaslan Turkes
Science and Technology University, Adana, Turkey, ORCID: Dr. Zeynep ERGUN:
https://orcid.org/0000-0002-9868-9488, zergun@ata.edu.tr
2
Department of Horticulture, Faculty of Agriculture, Cukurova University, Adana,
Turkey, ORCID: Dr.Mozhgan ZARIFIKHSOSROSHAHI: https://orcid.org/0000-
0001-5491-1430, mn_zarifi@yahoo.com
134 | W a l n u t

wide region from the Carpathian mountains to Turkey, Iraq, Iran,

Afghanistan, Southern Russia, India, Manchuria, and Korea (Sandal et
al., 2008).

In 2019, 3.7 million tons of shelled walnuts are produced in the world.
China ranks first in the cultivation area and production amount of
walnut in shell followed by America and Iran (Table 1). Turkey as the
fourth proceeding country in world walnut production is both an
importer and an exporter of walnut. The countries with the highest
income from walnuts worldwide are the USA and France. However,
walnut production in Turkey is increasing every year (FAOSTAT,
2021).

In Turkey, walnut cultivation is carried out in almost every province

except for a few ones. Providing technical support from production to
processing, organization, and marketing of fruits are factors affecting
the economical value in walnut production both in domestic and
foreign markets (Aksoy and Kaymak, 2021); (Akyüz and Serdar,
2017); (Dokuzlu, 2020); (FAOSTAT, 2021).

Table 1. Walnut (with Shell) Production in 2019 for Countries with a Descending
Order (Top 20 Countries with Tonnes) (FAOSTAT, 2021)
Area Value
1 China 2521504
2 United States of America 592390
3 Iran (Islamic Republic of) 321074
4 Turkey 225000
5 Mexico 171368
6 Ukraine 125850
 W a l n u t | 135

7 Chile 122950
8 Uzbekistan 50660
9 Romania 49580
10 France 34950
11 Greece 31040
12 Egypt 24013
13 Argentina 18488
14 Belarus 18431
15 Republic of Moldova 17706
16 Spain 17540
17 Pakistan 14862
18 Afghanistan 14690
19 Azerbaijan 11581
20 Italy 10800
FAOSTAT (Access date: 2nd Descember, 2021)

Walnut cultivation is common in Anatolia due to its place in the diet

of people living in Mediterranean countries and the long storage
period of walnut (Amaral et al., 2003).

The cultural history of the walnut is old and it is a species with high
genetic diversity. Moreover, the ability of walnut to adapt to different
environmental conditions makes its cultivation worldwide. It is a
species that has been researched a lot due to its nutritional content,
industrial use, and economic return. The global popularity of walnut
fruit is due to its high nutritional, health, and sensory properties
(Bayazit et al., 2016); (Martínez, 2010).
136 | W a l n u t

There are numerous studies on the chemicals, phytochemical and

biochemical properties of walnut fruits. Studies on the contribution of
walnut fruits to human health and nutrition cannot be ignored. Walnut
kernels have a high amount of vitamins, minerals (K, P, Mg, Ca, S,
Cu, P, etc.), fat, protein, and polyphenols (Guney et al., 2021);
(Okatan et al., 2021) (Kafkas et al., 2020); (Rusu et al., 2018);
(Simsek, 2016); (Panth et al., 2016). Protein, vitamins, and minerals
increase the biological and nutritional value (Ebrahimi, 2018); (Yiğit,
2005).

Walnuts are a rich source of vitamin E. Walnut kernel also contains

vitamins A, C, B6, thiamine (B1), riboflavin (B2), niacin (B3),
pantothenic acid (B5), and folic acid (B9). Moreover, it has been
reported that walnuts are a good source of magnesium, calcium,
manganese, potassium, sodium, and copper (Şen and Karadeniz,
2015); (Yiğit et al., 2005), flavonoids and phenolic acids Arcan et al.,
2021);(Ebrahimi et al., 2018); (Martínez, 2010); (Trandafir et al.,
2016).

Walnut has a wide range of uses in preventing diseases and supporting

plant-based drugs used in diseases treatment. Due to this feature,
walnuts are considered as a 'functional food'. One of the most
important components that make the walnut functional is the oil it
contains (Bakkalbaşı et al., 2010); (Yiğit et al., 2005). Walnut kernels
usually contain about 60% oil, but this can range from 52% to 70%
depending on the variety, geographic location, and irrigation rate
(Oliveira, 2002); (Moser, 2012); (Yiğit et al., 2005).
 W a l n u t | 137

In terms of nutrition, the composition of the oil is more important than

the amount of oil. While some of the nuts (E.g. almonds) are rich in
monounsaturated fatty acids (MUFA), walnuts contain
polyunsaturated fatty acids (PUFA) at a higher rate. One of the most
important features of walnut oil is its high amount of linoleic acid
(Sabaté and Hook, 2020); (Yiğit et al., 2005). There is a lot of
literature reporting the total SFA (Saturated Fatty Acid) (4-7.86%;
8.8%), total USFA (Unsaturated Fatty Acid), total PUFA (84%;
72.84%; 71.43 - 62.73%) and total MUFA (1%; 28.53 - 22.17%; )
values of walnuts (Copolovici et al., 2017); (Simsek, 2016);
(Tsamouris, 2002). About 80% of unsaturated fatty acids are linoleic
acid (omega-6), and 20% are α-linolenic (omega-3) acids. The ratio of
omega-6:omega-3 has been determined as 5.06 which could help in a
human healthy diet (Copolovici et al., 2017); (Şahin, 2005); (Willett et
al., 1995).

Walnut application in the pharmaceutical industry:

Walnut has a long history in folk medicine, especially in ancient

China. Although the use of chemical and synthetic drugs became more
common in the last half-century, their harmful side effects quickly led
to a re-emergence of natural base drugs. Plant-derived drugs have
always been one of the most effective methods throughout history.

The studies proved that walnut has a protective effect against

cardiovascular diseases and raises good cholesterol (HDL) in the
blood (Şahin et al., 2005); (Savage et al., 1999); (Wu et al., 2014);
138 | W a l n u t

(Zibaeenezhad et al., 2005); (Zibaeenezhad et al., 2003). Moreover, it

has been reported that walnut has the ability to delay or reduce the
development of very strong diseases such as Parkinson's and
Alzheimer's. Walnut consumption can increase the level of melatonin
in the blood. Walnut has a positive effect on the intelligence
development of children. Due to its fiber content, it plays a beneficial
role in the human digestive system. It can contribute to the elimination
of disorders such as sleep disorders (Şimşek and Gülsoy, 2016);
(Serrano, 2005); (Reiter et al., 2005).

Modern pharmacy studies reported that walnut regulate blood

circulation, shows antiallergic properties, regulate liver functions, and
promotes protein synthesis. For the past years, it has been reported
that people have been using walnuts as a diuretic, stone remover, anti-
vomiting during pregnancy, weight gain, and calming (Kim and Choi,
2021); (Yiğit et al., 2005); (Milind and Deepa, 2011).

In addition to the inner part of the walnut fruit, it is known that the
walnut leaf and shell have been used in alternative medicine for many
years. Walnut leaf pharmacologically has vasoconstrictor,
hypoglycemia, antifungal, wart removal, diarrhea suppressant, and
skin cleansing effects. It has also been reported to be vascular
protective and tumor inhibitory (Girzu, 1998). Walnut leaves are used
in the treatment of skin inflammations and ulcers, as well as in
alternative and modern medicine treatments thanks to their antiseptic
properties. It has also been reported that walnut leaves are used
externally in skin inflammation, hand and foot sweating, acne and
 W a l n u t | 139

wounds, eczema, herpes, bee stings (Almeida, 2008); (Yiğit et al.,

2005). In addition, it has been reported that the porridge obtained from
walnut leaves contributes to the healing of acne, wounds, and swollen
glands (Şimşek and Gülsoy, 2016). Walnut fruit shell also has
therapeutic properties for various diseases (Yiğit et al., 2005).

Walnut application in dying and food industry:

In addition to walnut application in the pharmaceutical industry,

different parts of walnut also are used in other various industries.
Walnut leaves, husk, or tree bark are among the important natural
dyestuffs. Thus, before the development of the dye industry, it has
been used for centuries to obtain different brown tones, especially for
dyeing wool yarn (Şahin, 2005); (Arifeen et al., 2021).

It has been stated that the outer shells of walnuts are used as a
flavoring agent in the production of some traditional liqueurs
(Stampar, 2006). Moreover, walnut shells have the potential to be
used in different areas of the food industry (Doğan et al., 2014).

In recent years, wastes generated as a result of rapid industrialization

constitute a major problem for living life and the environment. The
application of appropriate adsorbents in the removal of colored and
colorless organic pollutants in industrial wastewater draws attention as
a significant usage of the adsorption process. In a study, it was aimed
to obtain a cheaper, more abundant new natural adsorbent substance
that can be an alternative to activated carbon, which is widely used in
heavy metal purification. For this purpose, walnut wood chips were
140 | W a l n u t

used as adsorbents. As a result of this study, it has been reported that

walnut wood sawdust can be an alternative adsorbent to activated
carbon (Çakır et al., 2013); (Kaya et al., 2011). In addition, walnut
wood sawdust can be a suitable adsorbent in chrome removal (Çakır et
al., 2013).

Walnut tree has very valuable wood. For this reason, it is used in
furniture production, parquet production, sports, and musical
instruments, carving, dowry chest production. In addition, it is used in
the arms industry for the manufacture of some parts of rifles and
pistols (Dönmez and Güler, 2015).

Walnut application Energy industry

Walnut pruning and shell materials can be used in obtaining electricity

and biofuel (Dönmez and Güler, 2015); (Halil, 2005); (Moser, 2012);
(Folaranmi et al., 2016); (Ardebili et al., 2011).

The high amount of oil content makes the walnut to be considered a

good source for the biodiesel industry. In parallel with the increase in
the world population and developing technology, the energy demand
is constantly increasing. The fact that fossil energy resources are
limited and will be depleted in the near future has revealed the
necessity of evaluating alternative energy resources today. Factors
such as the depletion of industrial oils, the increase in their prices, the
damage they cause to the environment, and the increase in
environmental awareness have increased the interest in renewable
energy sources such as biodiesels. Biodiesel is an alternative fuel for
 W a l n u t | 141

diesel engines which can be produced from renewable resources such

as vegetable and animal oils (Knothe, 2010); (Borugadda and Goud,
2012); (Mekhilef et al., 2011); (Hosseini and Wahid, 2012); (Halil,
2005); (Van Gerpen, 2005); (Demirbas and Karslioglu, 2007);
(Krawczyk, 1996). Biodiesels are non-toxic, renewable, and
biodegradable with low CO2 and NOx emission profiles and,
therefore, are environmentally useful (Krawczyk, 1996); (Hossain et
al., 2018); (Folaranmi et al., 2016). Although biodiesel and diesel fuel
have similar physical and chemical properties, biodiesel fuel
properties are better than petrodiesel fuel (Balaji and Cheralathan,
2013); (Demirbas, 2007); (Canakci, 2007); (Sharma, 2005). Cetane
number which reduces the ignition delay is a significant criterion in
evaluating the quality of biodiesel fuel. Biodiesel has a higher cetane
number (Sivaramakrishnan and Ravikumar, 2012); (Shahid and Jamal,
2011). Biodiesel emits fewer emissions such as CO2 (carbon dioxide),
CO (carbon monoxide), SO2 (Sulfur dioxide), PM (particulate matter),
and HC (hydrocarbon) compared to diesel (Gupta and Agarwal,
2015); (Mofijur et al., 2016); (Mofijur et al., 2015); (Agarwal et al.,
2018); (Macor and Pavanello, 2009); (Agarwal et al., 2016). Biodiesel
is safe to process and transport and does not need to be refined
(Demirbas, 2007).

Appropriate raw material selection is very important for biodiesel

production. More than 350 oil-bearing crops have been identified as
potential sources for biodiesel production (Atabani et al., 2012);
(Balat and Balat, 2008); (Mofijur et al., 2012); (Silitonga et al., 2011).
142 | W a l n u t

The most common edible oil sources are soybean, palm, sunflower,
cottonseed, rapeseed, peanut, castor, jojoba, corn, olive, coconut
(Meneghetti, 2007); (Obadiah et al., 2012); (Pimentel and Patzek,
2005); (Sandouqa and Al-Hamamre, 2019).); (Veljković, et al., 2018);
(Yee et al., 2009); (Venkatesan and Sivamani, 2019).

Walnut seed oil is among the edible oils that can be processed as
biodiesel (Folaranmi et al., 2016). Properties of walnut oil such as
density at 25oC, acid value, saponification value, iodine value,
peroxide value, ester value, and kinematic viscosity are as in Table 4
(Folaranmi et al., 2016) and walnut biodiesel properties are given in
Table 5 (Aydoğan et al., 2020).

Table 4: Physicochemical Properties of Walnut (juglans regia L) Oil (Folaranmi et

al., 2016).
Parameters Walnut (Juglans regia L) oil

Relative Density (Kg/l) 0.873

Flash Point (oC) 156
Iodine Value (Wij) 75.05
Acid Value (Mg KOH/g) 0.846
Saponification Value (Mg KOH/g) 308
Peroxide Value 56
Free Fatty Acid (Mg KOH/g) 0.423
Ester Value 307.577
Kinematic viscosity (mm2/s) 34.5
 W a l n u t | 143

Table 5 . Physical Properties of Walnut Biodiesel and Eurodiesel (Aydoğan et al.,

2020).
Specifications Walnut Biodiesel Eurodiesel
Density (gr/m3) 0,893 0,827
Viscosity (mm2/s) 5,23 2,82
CFPN (˚C) -3 -20
Calorie Value (MJ/kg) 40,6 47,5
pH 5.5 6

Biodiesel produced from walnut oil and its mixtures with euro diesel
fuel can be considered as an alternative fuel for diesel engines
(Aydoğan et al., 2020).

To summarize, walnuts can be used primarily in the food sector, but

also in the chemical, pharmaceutical, cosmetic, furniture, paint, and
fuel sectors (Bakkalbasi et al., 2015); (Beiki et al., 2018); (Grosu et
al., 2012); (Qin et al., 2005); (Saxenaa, 2009).
144 | W a l n u t

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 W a l n u t | 153

Part VIII
--------------------------------
Diseases in Walnut Orchard
Dr. Kamil Sarpkaya1

Walnut Blight:

The causal agent of the disease is the bacteria, Xanthomonas

campestris pv. juglandis (Pierce (Dye), which is previously named
Bacillus juglandis, Bacterium juglandis, Phytomonas juglandis,
Xanthomonas juglandis.

Xanthomonas campestris pv. juglandis is aerobic, gram-negative, non-

spore-forming, rod-shaped bacteria.

Walnut blight is regarded as an important disease from the economic

point of view, and it can be easily detected where walnut is grown in
the World.

Causal agent bacteria, mainly overwinter in buds and catkins of

walnut and dormant buds, can lead to an infestation of internal bud
parts and developing fruits (Miller and Bollen, 1946; Mulrean and
Schroth, 1982). Rain splash also causes suspend of bacteria in the air,
and it might be the main factor for carrying inoculum to developing

1
Karabuk University, Faculty of Forestry, Department of Forest Engineering,
ORCIDs: Dr. Kamil SARPKAYA, https://orcid.org/0000-0001-5794-668X,
kamilsarpkaya@karabuk.edu.tr
154 | W a l n u t

fruits by winds (Stall et al., 1993). It is also claimed that infected

pollens may also transport the inoculum, but it has not been proved
yet. Walnut blight makes infection on fruits (mainly), catkins, shoots,
and leaves (Figure 1).

Figure 1. Initial Symptoms of Walnut Blight on Catkins (a) Developing Fruits (b)
and Shoots (c)

Initial symptoms of the disease are seen on catkins with several black
spots. Infection occurs at the apical or blossom end initially and
lesions of bacteria increase in the side or apical end of developing
fruits with black and water-soaked bands surrounding lesions (Figure
2). According to the severity of the disease, lesions size enlarges in
infected areas. The band may disappear thoroughly when the pathogen
stops colonization in unaffected tissues. Infection may develop in
whole or part of the kernel, as well. In the case of favorable conditions
for the occurrence of this disease in spring, fruits may drop
prematurely, and yield significantly declines.
 W a l n u t | 155

Figure 2. Apical Lesions and Developing Water-Soaked Bands on Fruit (Photo

Credit: Florent Trouillas)
As the plant aged, the formed lesions are suppressed and bacteria form
ooze in the lesions together with the decomposed cell materials in
higher humidity levels. Lesions on fruit may range from a few mm to
over half the size of the outer part of the fruit. Early infections develop
with blackening or rotting in the hull before the hardening of the shell.
Shrivel of kernel causes premature fruit dropping. Lesions develop on
outer parts of the fruits in later infections and such fruits remain on the
tree until harvesting time. The quality of the nut is also affected
because of the possible adherence of the infected hull to the hard shell,
resulting in discoloration and stain after dehulling (Lang and Evans,
2010). In favorable conditions for disease, lesions may develop in the
kernel and cause the rotting of kernels (Figure 3).
156 | W a l n u t

Figure 3. Progression of Walnut Blight Severity in Kernel (Left to Right) (Photos:

https://shop.agriad.ir)

Control of Disease: Using healthy plants for launching an orchard is

essential. Distance between the trees is also important for reducing the
risk of disease incidence. Water level should be kept balanced in
irrigation, and sprinkler irrigation should be avoided especially in
flowering time. Cultural practices, such as balanced fertilization, weed
control, ought to be implemented properly.

Due to the uncertainty of epidemiological aspects, management

strategies of disease are not well defined (Moragrega and Ozaktan,
2010). Although a spray prediction model has been improved in recent
years, the control of disease is not guaranteed because of the effects of
several factors for disease development. Walnut blight is not well
controlled in the areas where the climatic condition is favorable.
However, protective treatments can be used according to rainfall
duration in spring at ten days interval starting bud break to fruit set of
walnut orchards, having a disease history of walnut blight (Ninot et
al., 2002). It is highly recommended for full coverage spraying.
Mummified fruits should be removed for reducing inoculum.
 W a l n u t | 157

Walnut Anthracnose

Walnut anthracnose, caused by Ophiognomonia (=Gnomonia)

leptostyla (Fr.) Sogonov, has been reported as the most important and
common fungal disease of black walnut (Juglans nigra L.) or English
walnut (J. regia L.) throughout all walnut production regions in the
World (Belisario, 2002; Belisario et al., 2008; Hassan and Ahmad,
2017).

The causal agent is well described by many researchers. Fungus

produces acervuli in early spring with black specks on abaxial of
leaves. Conidiophores are short, elliptical, hyaline, and one-celled
packed together in a small layer bearing conidia at the tips. The shape
of conidia can be straight, ovoid, falcate, or be rounded at one end,
while the other end is pointed. The characteristic of fungi is
possessing one septum and two globular unequal cells with the size of
15- 26 × 2-5 μm. Immersed perithecia are composed of fallen leaves
in brown color at the end of the season, while the beak protrudes
considerably onto the leaf surface (Hassan and Ahmad, 2017).

O. leptostyla makes infections by ascospores during May, and it

causes initial lesions on leaves (Figure 4). Seconder infections are
made by conidia form of fungus, and the number of lesions increases
in early summer. Furthermore, the development of disease reaches a
maximum level in late July and early August. Defoliation might be
seen on previously infected leaflets (Kessler, 1984). According to
Rosnev and Naidenov (1986), the fungus is favored of the temperature
158 | W a l n u t

of 15-30°C, frequent precipitation, and humidity over 65 percent. The

severity of infection is even more on older leaves and 10 times more
frequent with adaxial inoculation (Matteoni and Neely, 1977).

Figure 4. Initial lesions of Anthrachnose on Walnut

Decreasing in nut yield due to anthracnose varies from variety to

variety. Based on the report, there is a relationship between the size of
the spots on the leaf and fruit pericarp and size, nut development, and
yield. Because of spots forming after infections, metabolic processes
disturbance and changes in biochemical indices in leaves are expected
(Shirnina and Kotljarova, 2000). As a result, the fruits may be smaller
than their normal size, poorly filled, of low quality with darkened
kernels (Black and Neely, 1978; Zamani et al. 2011), as well as low
yielding (Pinter et al. 2001) (Figure 5)
 W a l n u t | 159

Figure 5: Effect of Various Severity Levels of Anthrachnose on Walnut Fruits

(http://www.baghbantak.com/index.php/nuts-trees/walnut/walnut-anthracnose)

The initial symptoms of the disease can be seen on current year

leaves, fruits, and twigs, while shoots might be rarely infected. At the
first stages of the disease, infected areas on leaves are seen as brown
to black colors with regular or irregular circular spots. Later on, large
necrotic areas are composed by enlarging and coalescing of these
spots. Eventually, these infected leaves change to yellow and early
defoliation is seen on the trees according to the severity of the disease
(Kalkism, 2012) (Figure 6)
160 | W a l n u t

Figure 6. Characteristics of Lesions and Early Defoliation on Trees

Control of Disease: Management of disease, based on both cultural

practices and chemical control, was broadly studied by various
researchers in different countries. Fallen leaves need to be buried to a
depth of 10-15 cm, on the other hand, infected twigs should be pruned
well, and eradicated, and adequate nitrogen fertilizer will be helpful to
control the disease in orchards (Pscheidt and Ocamb, 2014).
Moreover, some resistant or less susceptible varieties are
recommended to set an orchard in the area where the climatic
conditions are favorable for disease.

To ensure the efficiency of chemical control of the disease, diagnosis

the proper and most effective time of chemical application in the
initial stages of the disease, aerial spore dispersing, is of high
importance. Application of Bordeaux mixture in late winter and
copper compounds in early spring, just before sprouting, is far
effective to control walnut anthracnose (Zamani et al. 2011).
Furthermore, new generations of systemic fungicides have been
widely used in different countries in recent years.
 W a l n u t | 161

Thousand Cankers Disease

Thousand cankers disease is actually caused by a combination effect

of canker-producing fungus (Geosmithia morbida), which is inserted
into trees by an insect vector named as the walnut twig beetle
(Pityophthorus juglandis). Those organisms cause a decline in
branches of trees that is resulted from the cumulative injury.
More likely, two organisms (fungus, twig beetle) are steadily found
together to indicate the occurrence of thousand cankers disease. A
second fungus (Fusarium solani) is also associated with trunk cankers
in advanced stages of Thousand Canker Disease (Cranshaw and
Tisserat, 2012). (Figure 7).

Figure 7. Damage of Walnut Twig Beetle and Developing Canker Tissues on

Walnut Twigs (Photo: http://www.invadingspecies.com/invaders/forest/thousand-
cankers-disease/#bwg23/88)

Defoliation on the crown part of the tree is a common characteristic of

disease which is followed by yellowing or sudden leaf wilting.
162 | W a l n u t

Generally, such symptoms on trees cause dieback or death of all trees

in subsequent years because of faster progress of the disease after
infestation (Figure 8). Development of the disease varies according to
plant vigor, host cultivars or species, climatic conditions, and natural
controls of the walnut twig beetle.

Figure 8. Dieback of Walnut Trees by Thousand Cankers Disease

(Photos:https://www.udel.edu/academics/colleges/canr/cooperative-extension/fact-
sheets/thousand-cankers-walnut/)

Based on symptoms, the disease is well identified in mid-summer.

Furthermore, research has shown resides in the vascular tissue of
trees, and until a change in climatic conditions (such as water stress)
activates it, stays hidden. Therefore, in stress conditions, such as
drought or high temperature, the disease progress can be faster. The
 W a l n u t | 163

severity of the disease in both cases depends on the sensitivity of the

host, the length of the incubation period, and the presence of the
wounds.

Control of disease: It is recommended neither apply insecticide to

control walnut twig beetle nor fungicide. Instead of chemical control,
some cultural practices, such as sanitation of infested materials,
adequate fertilization, and proper irrigation in orchards, can be
implemented. In the condition of less than 50% of the alive crown,
main branches and scaffolds should be removed. To avoid the onset
activity of walnut twig beetle, plant materials after any practices like
pruning should be removed from orchards.

Shallow bark canker

Shallow bark canker is caused by a bacterium belonging to genus

Brenneria (called Erwinia previously), and subsequently named as B.
nigrifluens (Hauben et al., 1998). The disease is characterized by the
symptoms demonstrated blacken spots or areas on the trunk or main
branches of trees. Areas with a marginal water-immersed lesion on
bark with vertical oozing represent recently infected areas, which is
followed by dried spots with a thick resin (Figure 9). When the
surface of lesions is scratched, forming of dying tissues can be
detected under the bark. General symptoms of the disease appear as
weakness, deterioration, and dieback of the scaffolds. Initial dieback
starts unilaterally and finally the whole tree dies. Bacteria overwinter
inside the canker or its secretions and in spring, it goes out with the
164 | W a l n u t

exudates. Then, spread out to other trees through wind and rain, and
penetrates inside especially through wounds on trees. Shallow bark
canker remains active for many years and are very active, especially in
summer, but become inactive during autumn and winter.

Figure 9. Necrotic Lesions of Shallow Canker (left) and Vertical Oozing from
Infected Areas (Photos: K. Keshavarzi and H. Karimipourfard, 2021)

The disease occurs due to the weakening of the tree resulting from
environmental stresses such as soil compaction, nutritional
deficiencies, over-irrigation, crown, and root rot. Even recovering
trees will be still prone to show the symptoms if they are exposed to
these stresses again.

Control of Disease: The disease develops rapidly in poor and dry

soils. Proper nutrition and regular irrigation as well as the use of
resistant cultivars are the most important strategies for managing this
disease. Tissues involved with the disease can be removed, then
treatment with copper compounds is helpful.
 W a l n u t | 165

Crown Gall

Crown gall is caused by Agrobacterium tumefaciens which is a rod-

shaped and gram-negative bacterium, and it often appears as tumors or
galls of various sizes on roots, crown, at or beneath the soil surface.
Galls rarely occur on the upper parts of trees or the trunk. Wounds on
plants and also the inoculum attached to the injured areas are general
infection points, so some operations resulting in an injury either in
nurseries or orchards drastically increase the risk of disease. After
penetration of bacteria, the infected parenchymatous and vascular
tissue grow rapidly with a bright color, and swelled galls darken later.
Active galls are mostly soft and flabby (rather than being hard); they
get decayed in centers when aged. An important characteristic of the
disease is to give no exudate on tumors, unlikely the other bacteria do
in walnut (Figure 10). In young trees, the growth process can be
affected remarkably according to the severity of galling, while in older
trees wood-rotting is a prevalent occurrence. Care should be taken not
to confuse the symptoms of this disease with galls caused by insects or
nematodes.
166 | W a l n u t

Figure 10. Tumors on Older Walnut Trees (Photo: https://www.sjvtandv.com/)

Nurseries are the first risky sites that provide desired conditions in
favor of agrobacteria activity (Figure 11). The pathogen may enter the
soil (through organic residues for instance) and reside there for 1-2
years and can be detected on roots regardless of being host or not.
Since the entrance of pathogen is only possible through wounds,
subjecting to mechanical injury (during seedling transferring or any
horticultural practices) is regarded as a potential risk for disease.
Uncontrolled infected plant materials most possibly result in the
dissemination of inoculum to the orchards (Teviotdale et al. 2002).
 W a l n u t | 167

Figure 11. Characteristics Galls of A. tumefaciens on Young Plants

(Photo: https://www.growingproduce.com)

Control of disease: A. tumefaciens is on the quarantine list in most

countries, and strong precautions are taken by nurseries to produce
clean plant materials. Moist control in orchards is essential for
reducing the contamination risk and development of crown gall. It is
necessary to use healthy seedlings for setting up an orchard. On the
other hand, it is needed to abstain from activities resulting in physical
wounds for planting. Although preventive biological control agents
(not eradicating) are available to root dip application before
transferring plant materials, their efficiency can be different on walnut
trees. In the possible use of diseased plant materials, a combination of
some treatments such as doing surgery, flaming, and applying
bactericide are suggested in older trees only. It should be taken into
consideration that the success of operations is extremely limited and
168 | W a l n u t

expensive. Plant stunting should be removed completely from the

orchards. Crop rotation (especially with grass) may help degrade
remaining host materials and reduce the pathogen dispersion.

Phytophthora Root and Crown Rot

Several Phytophthora species cause root and crown rot of walnuts.

General symptoms of the disease are necrosis of the collar and main
roots; dark, flame-shaped necrosis, spreading up into the trunk
(Belisario et al. 2006).

Phytophthora infection generally progresses in roots or crown, and the

trees decline after the moderate spring season. Symptoms on the trees
depend on infection rates on roots or crown (Figure 12). Generally,
infection of fungi makes progress a couple of years. At the beginning
of the disease, the growth of trees is reduced, followed by more severe
symptoms in subsequent years gradually. Defoliation and dieback can
occur in the final years in the condition of no precautions for disease.

Figure 12. General Symptoms of Phytophthora Disease on Walnut Trees

(Photo: https://www.sacvalleyorchards.com)
 W a l n u t | 169

Control of Disease: The first principle of controlling the disease is to

prevent the crown from getting wet and also to avoid waterlogging of
soil around the crown with the duration of 18-24 hours, which favors
for Phytophthora infections. On the contrary, water drainage in
orchards reduces the risk of the causal agent. Water management in
orchards, limitation to 18 hours for irrigation is highly recommended
for preventing of spore production of fungi. The susceptibility of
walnut cultivars or rootstocks against the fungi is varied. However,
none of them are resistant to all Phytophthora species causing crown
or root rot. Although the existence of some fungicides against the
disease, the resistance of Phytophthora species in different cultivars
are taken into consideration in walnut.

Botryosphaeria and Phomopsis Cankers

Cankers on walnut are described by two groups of fungi, which are

Botryosphaeria dothidea,
Diplodia mutila, D. seriata, Dothiorella iberica, Lasiodiplodia
citricola, Neofusicoccum mediterraneum, N. nonquaesitum, N.
parvum, N. vitifusiforme, Neoscytalidium dimidiatum, belonging to the
family Botryosphaeriaceae and Diaporthe neotheicola, and D.
rhusicola, belonging to the family Diaporthaceae (Chen et al. 2013).
Botryosphaeria is accepted as an endophytic fungus in plants
(Slippers and Wingfield, 2007) may cause disease in several woody
plants (Chen et al. 2013). Generally, this fungus makes infection
because of stress conditions (Blodgett and Stanosz, 1997). Disease
170 | W a l n u t

symptoms develop rapidly on the condition that stress factor is

prolonged.
Shoot wilting and leaf flagging on branches are regarded as the initial
symptoms (Figure 13). Brown or black color on cambium tissues is
obvious under the bark. In the progress of the infection, pycnidia
(containing fungal spores) or perithecia of both species can be
detected under the bark. Shoots of young trees commonly get black
after infection, while branches of older trees might die.
Botryosphaeria fungi may colonize after infection of branch wilting
fungus, Neoscytalidium dimidiatum (=Scytalidium dimidiatum,
Hendersonula toruloidea), which has similar symptoms to cankers.

Figure 13. Initial Symptoms of Botryosphaeria Canker in Walnut Orchard

(Photo: https://ccfruitandnuts.ucanr.edu)

Symptoms of disease are also remarkable on nuts. When the fruits

become mature (during August or September), decay on the outer part
of the nut is seen (Figure 14). The initial symptoms come in black
 W a l n u t | 171

color, then turn into brown to beige, and subsequently drop down
prematurely. Contamination occurs from one fruit to another and thus,
the disease spreads all through the tree.

Figure 14. Special Characteristic of Botryosphaeria is to Embroil Peduncle and

Result in Nut Decay (Photo: https://ccfruitandnuts.ucanr.edu)

Botryophaeria and Phomopsis fungi usually make infection via

wounds or prune areas by spreading spores on previously infected
tissues in the tree, while direct infection occurs in favorable
conditions.

Control of Disease: Cultural practices such as the pruning of infected

shoots, twigs, etc. are essential to control the disease, and it also
172 | W a l n u t

affects the efficiency of the chemical application. To prevent

infections, protective fungicides should be applied for penetrating the
inoculum through wounds or pruned areas. Registered fungicides in
different countries can be applied in May or early summer to control
the disease. Frequent precipitation in spring and temperatures over 10
0
C are favorable conditions for disease. Because the success of
chemical management is highly dependent on climatic circumstances,
the weather conditions during spring such as heavy rainfall and
moderate temperatures should be taken into account, therefore
repeating of chemical application may be needed.

Fusarium Wilting

Fusarium solani and F. incarnatum are causal agents of the disease.

Apart from thousand cankers disease on walnut, F. solani is
determined as the primary pathogen in the trees. Fusarium-related
wilting has been demonstrated on newly established walnut orchards.
It is reported that symptoms of Fusaria lack sprouting on buds after
transplantation, several injuries are seen in roots, color changes can be
detected on the woody part of the plants, root necrosis is obvious in
young trees (Seta et al. 2004; Singh et al. 2011; Mulero-Aparicio et al.
2019), and dieback allies with the discoloration of vascular bundles of
shoots and twigs in mature trees (Chen and Swart, 2007).
 W a l n u t | 173

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Rev. v.21, p. 90-106.
Stall R.E., Gottwald T.R., Koizumi M., Schaad N.C., 1993. Ecology of plant
pathogenic xanthomonads. In: Swings J.G., Civerolo E.L. (eds).
Xanthomonas, pp. 265-290.
Teviotdale, B.L, Michailides T.J. and J.W. Pscheidth, 2002. Compendium of nut
Crop Diseases in Temperate Zones. The. Am. Phyt. Soc., p.10.
Zamani, A. R., Imami, A., Mirza, M. A and Mohammadi, R. 2011. A study and
comparison of control methods of anthracnose disease in walnut trees of
Roodbar region. Int. J. Nuts and Related Sci. 2: 75-81.
176 | W a l n u t
 W a l n u t | 177

Part IX
--------------------------------
Important Pests of Walnut and Their Control
Dr. Naim OZTURK1 and Dr. Remzi UGUR2

Introduction

Anatolia is the homeland of walnut (Juglans regia L.) as well as many

fruit species. Walnut; It is a very valuable fruit and wood species with
wild forms in Anatolia, deciduous in winter, up to 30 m in height,
cultivated in almost every part of our country. While the world walnut
production is approximately 4.5 million tons (Anonymous, 2021),
Turkey has 9,875,000 fruit-bearing walnut trees and ranks 3rd in the
world after China and the USA in terms of walnut production with
225,000 tons (TUIK, 2020. As with other fruit varieties grown in
Turkey, in parallel with the increase in production in recent years,
many harmful insect and mite species in walnut orchards that cause
plant health problems and cause significant product loss (Uygun et al.,
2010; Canıhoş et al., 2014; Anonymous, 2017; Çakır, 2018). To
obtain higher quality and abundant products, many methods are used

1 1
General Directorate of Agricultural Research and Policies, Biological Control
Research Institute, Yuregir/Adana/Turkey, naim.ozturk@tarimorman.gov.tr,
2
General Directorate of Agricultural Research and Policies, East Mediterranean
Transitional Zone Agricultural Research of Insttute, Department of Horticulture,
ORCIDs: https://orcid.org/0000-0001-6717-1689, remzibey@hotmail.com

*
178 | W a l n u t

in the fight against walnut pests. One of these methods is chemical

control. However, unconscious, indiscriminate and intensive use of
drugs has brought along many problems such as deterioration of
natural balance, human and environmental health, durability and
residue. To minimize these problems caused by pests seen in walnut
orchards and to obtain products of higher quality and market value;
Attention should be paid to "Integrated Struggle Programs" in which
all warfare methods are used together with quarantine measures
applied meticulously as well as producer training. As it is known,
chemical control is the last method to be applied in integrated control
programs. Therefore, this method should not be preferred unless it is a
necessity. If chemical control is obligatory, environmentally friendly
and specific drugs should be preferred and spraying should be done at
the right time and dose.

Table 1. Common properties of walnut pests

Common name Species name Order, Family
Lepidoptera:
Codling moth Cydia pomonella L.
Tortricidae
Chromaphis juglandicola
Hemiptera:
Walnut aphids (Kaltenbach)
Aphididae
Panaphis juglandis (Goeze)
Walnut eriophyid Aceria erinea Nalepa Acarina:
mites Aceria avanensis (Bagdasarian) Eriophyidae
Leopard moth Lepidoptera:
Zeuzera pyrina L.
borer Cossidae
Hemiptera:
Pear lace bug Stephanitis pyri (Fabr.)
Tingidae
Caloptilia roscipennella Lepidoptera:
Walnut leaf miner
(Hübner) Gracillariidae
 W a l n u t | 179

This study; It is aimed to contribute to the solution of phytosanitary

problems in walnut orchards by giving brief information about the
definition of the pests. The form of damage, and the control of their
symptoms, as well as the practical information obtained from the
studies on "Walnut Pests" in the walnut orchards of Turkey.

Important Walnut Pests and Control

Codling moth (Cydia pomonella L.)

The adults of the Codling moth (Cydia pomonella L.) are small and
gray, with dark, transverse wavy lines on their front wings (Figure 1).

Figure 1. The adults of Codling moth (Cydia pomonella L.)

The mature larva is 15-20 mm long and has a whitish-pink appearance

(Figure 2a). Butterflies that emerge in the spring usually lay their eggs
on fruits and rarely on leaves and twigs close to the fruits (Figure 2b).
Walnut fruits are phenologically the size of hazelnuts when the first
moths lay eggs and the first damage occurs (Figure 3).

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Figure 2. Codling moth's larval (a) and egg on fruit (b).

The pest usually spends the winter between the cracked bark of the
tree trunk and the fallen plant remains, inside the fruits, in the storage
houses in the mature larval stage. The first butterflies start in April-
May and continue until July. The second-generation adults are seen in
July-August. For the adults to mate and lay eggs, the twilight
temperature must be above 15 °C two evenings in a row (Anonymous,
2017).

Figure 3. First offspring damage and larval entry sites of codling moth
 W a l n u t | 181

Larvae that emerge from the eggs usually enter from the parts where
the fruits come into contact with each other or the leaves, the stem
base, the flower pot, and the fruit sides (Figure 4). Young larvae may
die at a high rate before they enter the fruit (because they are very
sensitive to adverse conditions such as wind, rain, cold, etc., or due to
natural enemies). Since it is essential to kill the larvae before they
enter the fruit, it is significant in the fight against the pest during this
period. The larva, which matures by feeding in the fruit for 30-40
days, leaves the fruit from the hole it entered and becomes a pupa in a
suitable place.

The Codling moth (Cydia pomonella L. ) directly harms the walnut

fruit. The larva feeds on the inner and outer shell of the walnut,
causing the fruit to fall, reducing the quality and market value of the
product. The first generation is fed in the fruit, and the second
generation is usually fed on the peel. The first progeny larvae, which
enter through the green shell of the walnut, open a gallery and pierce
the inner shell, which is not yet hardened, and feed inside the fruit,
while the second progeny feed mostly on the green shell and open a
gallery under the shell (Figure 5). However, since they cannot pierce
the hardened shell during this period, they cannot enter and there is no
damage to the fruit. In varieties with an opening in the fruit shell, the
second generation larvae can enter through the hole in the fruit stem
pit or the opening at the junction of the peel, feed in the fruit and
cause damage.

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182 | W a l n u t

Figure 4. Fruit entry points and damage of Codling moth (Cydia pomonella L. )

The Codling moth (Cydia pomonella L. ) is a polyphagous pest.

Walnut, apple, pear and quince are among the important hosts. In
addition, it has been determined that it rarely harms plum, apricot,
peach and wild berry.

Figure 5. The damage of Codling moth (Cydia pomonella L. ) in walnut fruit.

Control

Cultural measures: It should be preferred to establish a closed

walnut orchard and care should be taken not to plant fruit trees such as
apple, pear and quince, which are the other hosts of the Codling moth
(Cydia pomonella L. ), in and around the garden. Contaminated fruits
 W a l n u t | 183

spilled on the ground throughout the year should be collected and

destroyed weekly (Canıhoş et al., 2014; Anonymous, 2017).

Biological control: The apple wolf has many natural enemies in

nature. First of all, care should be taken to increase the efficiency of
these species by protecting them.

For this, random drug applications should be avoided, and when

necessary, specific drugs with low impact on the environment and
natural enemies should be used. Perennial shelter plants (wild rose,
blackberry, etc.) and flowering plants such as mint, wild carrot and
fennel, which can be a food source (prey, pollen, nectar, etc.) for
natural enemies, should be found or grown in the garden edges.

Biotechnical control: The technique of inhibition of mating is

successfully applied in the control of the codling moth (Cydia
pomonella L. ) (Figure 6).

Figure 6. Codling moth (Cydia pomonella L. ) trap and pheromone dispenser.

*
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Chemical control: The aim of the fight against the codling moth
(Cydia pomonella L. ) is to kill the larvae before they enter the fruit.
In walnuts, the first generation causes economic damage and the
second generation does not cause any harm in general. However, in
some cultivars, spraying may be required for the second generation, as
the bark is not completely closed and the opening remains and the
progeny are mixed. For this purpose, in general, when the fruits are
phenologically hazelnut-sized, and 15 days after that, 2 sprayings can
be done for the first generation, and if necessary, 1 spraying can be
done for the second-generation 15 days after the second spraying.

Walnut aphids [Chromaphis juglandicola (Kaltenbach) and

Panaphis juglandis (Goeze)]

Walnut aphids are generally lemon yellow or yellowish greenish. C.

juglandicola scattering colonies under walnut leaves and feeding
(Figure 7a). P. juglandis species feeds by localizing along the midrib
on the upper surface of the leaves (Figure 7b). Both species spend the
winter in the egg stage. All of the individuals that hatch in the spring
consisting of female individuals, reproduce without fertilization
(fertile) throughout the summer and give a large number of offspring
per year (Anonymous, 2017).
 W a l n u t | 185

a b

Figure 7. The under and upper leaf appearance of Chromaphis juglandicola (a)
and Panaphis juglandis (b)

Walnut aphids; It feeds on the leaves and fruits of the walnut by

absorbing the plant sap. As a result of absorption, photosynthesis is
adversely affected by the breakdown of chlorophyll, yellowing of
leaves, and shedding in dense populations. In addition, fumagin is
formed on leaves and fruits due to the honey-like substance they
secrete (Figure 8). This situation causes weakening of the tree,
deformation of the leaves, decrease in product quality and yield.
C. juglandicola and P. juglandis species often coexist in the plant
organs they feed on. If the presence and damage of aphids will
continue in the near-harvest period at the end of summer, shriveling
will occur in the inner part of the fruit and the quality will decrease
due to this situation (Canıhoş et al., 2014). Of the walnut aphids, P.
juglandis is a monophagous species. On the other hand, C.
juglandicola species, Ricinus communis, Crataegus sp., and Prunus
amygdalus (Canıhoş et al., 2014; Anonymous, 2017).

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186 | W a l n u t

Figure 8. Fumagin damage of walnut aphids on leaves and fruits

Control

Cultural measures: Aphids; It is more of a problem in gardens that

are planted frequently, are not regularly pruned, have high humidity,
and use excessive and broad-spectrum insecticides. For this reason,
frequent planting, excessive irrigation, and nitrogen fertilizer
application should be avoided in new plant gardens. Care should be
taken to clean weeds in and around the garden, and trees should be
pruned regularly every year to ensure airflow in the garden.

Biological control: Walnut aphids have many natural enemies in

nature. First of all, care should be taken to increase the efficiency of
these species by protecting them. For this, random drug applications
should be avoided, and when necessary, specific drugs with low
impact on the environment and natural enemies should be used.
Perennial shelter plants (wild rose, blackberry, etc.) and flowering
plants such as mint, wild carrot, and fennel, which can be a food
source (prey, pollen, nectar, etc.) for natural enemies, should be found
 W a l n u t | 187

or grown on the garden edges. In addition, ant exits to trees should be

prevented to feed on the honey-like substance secreted by aphids.

Chemical control: In general, a single application in the spring is

sufficient against walnut aphids. For this purpose, 20 trees are
determined to represent the garden in the early period in the gardens
known to be infected from the previous year, and 5 leaves are taken
from each tree, from different directions and heights. When the
density of 15 individuals/leaf per 100 leaves taken is determined,
spraying is done. If necessary, 2-3 more sprayings can be done
according to the effective time of the drug used in the summer months
(Canıhoş et al., 2014; Anonymous, 2017).

Walnut eriophyid mites [Aceria erinea Nalepa and Aceria

avanensis (Bagdasarian)]

Adults of Walnut Eriophyid mite are yellowish-cream colored,

transparent and carrot-shaped. These species are very small, about 0.1-
0.3 mm in size, and move slowly (Figure 9). However, they are known
for the damage they cause to walnut leaves and fruits.

Winter, They live in buds, under the bark, in slits and cracks in the
stem. Adult individuals start to feed on young leaves and fruits by
leaving the winter when the buds start to appear in the spring. Then
they lay eggs in the gall and ridges they have formed on the leaves and
fruits.

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188 | W a l n u t

Adult individuals leave the ridges and galls and migrate to young
leaves and fruits. They do the main damage towards the end of the
summer season when they are at their peak. They are generally
mechanically transported by wind, birds, and insects. In addition,
sapling transportation plays an important role in the transportation of
these species to clean areas. They give many offspring per year
(Canıhoş et al., 2014; Anonymous, 2017).

Figure 9. The appearance of nymphs and adults belonging to Eriophyid mite species

Walnut Eriophyid mites feed by sucking the sap on the leaves and
fruits of the walnut. The toxic substance they secrete during sucking
causes deformation (gal, blistering and spalling) in the plant tissue. A.
erinea species feed only on leaves and causes large galls on the upper
surface of the leaf and perineum (hairs) on the lower surface (Figure
10). A. avanensis, on the other hand, feeds on the leaves and fruit of
the walnut, causing wart-shaped small galls on the lower and upper
surfaces of the leaves and fruit, and deformation in the form of
incrustation on the bark (Figure 11). The gall and ridges, which are
first light greenish, gradually turn red, brown, and black. In heavy
 W a l n u t | 189

contamination, the leaves fall prematurely, the shape of the fruit

deteriorates and its quality decreases.

Walnut Eriophyid mites are monophagous species and no hosts other

than walnuts have been encountered so far (Canıhoş et al., 2014;
Anonymous, 2017).

Figure 10. Damage of Aceria erinea (blister mite) species on walnut leaf upper and
lower surfaces

Figure 11. Damage of Aceria avanensis (gall mite) species on walnut fruit and
leaf

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190 | W a l n u t

Control

Cultural measures: Saplings and cuttings to be used while establishing

a garden should not be contaminated with pests. Annual maintenance
of the garden should be done regularly, trees should be kept healthy
and excessive nitrogen fertilizer use should be avoided. Infected
shoots should be pruned at the end of spring, and leaves that fall to the
ground in autumn together with pruning residues should be collected
and destroyed. In addition, weed control in and around the garden
should be done regularly.

Biological control: Eriophyid mites have many effective predators in

nature. First of all, care should be taken to increase the efficiency of
these species by protecting them. For this, random drug applications
should be avoided, and when necessary, specific drugs with low
impact on the environment and natural enemies should be used.
Perennial shelter plants (wild rose, blackberry, etc.) and flowering
plants such as mint, wild carrot and fennel, which can be a food
source (prey, pollen, nectar, etc.) for natural enemies, should be found
or grown in the garden edges.

Chemical control: In the gardens known to be contaminated with

pests in previous years, leaves and fruits are checked in spring. Galls,
which are signs of nutrition, are searched, and when signs of damage
are seen on leaves and fruits, pesticides are applied. However, in the
gardens where the contamination is intense every year, as the
appropriate spraying time; It is the April-May period in which the
 W a l n u t | 191

adults migrate to the leaves and fruits of the plants where they spend
the winter in the spring and the October-November period when they
migrate to their hosts to spend the winter again in the autumn.

In nursery gardens and young gardens (under 5 years old), at least one
spraying should be done when damage is observed. If the damage
continues on the newly emerging leaves, another spraying can be
recommended considering the effect time and damage status of the
drug (Canıhoş et al., 2014; Anonymous, 2017).

Leopard moth borer [Zeuzera pyrina L.)

Adults are large and have a wingspan of approximately 41-43 mm. Its
wings are white with dark spots on the wings (Figure 12a). The larva
is large and dark yellowish with dark dotted spots (Figure 12b).

Figure 12. Adult (a) and larva (b) of Leopard moth borer [Zeuzera pyrina L.).

The overwintering larvae of the woodworm start to feed in March-

April and juice flows from the gallery openings (Figure 13a). The first
butterfly flights are seen in May-June. Butterflies are active at night
and lay eggs (Figure 13b). Eggs; It is usually left in groups near the

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adult exit holes, in the cracks on the stem, and between the bark
(Canıhoş et al., 2014; Anonymous, 2017).

Figure 13. Feeding sign of Leopard moth borer [Zeuzera pyrina L.) larva - sap
discharge (a) and eggs (b).

The larvae open a gallery on the annual shoots, branches and trunks of
the walnut. Feeding scum (Figure 14a) and plant sap flow are present
at the mouth of newly entered galleries (Figure 14b). Due to the
opened galleries, the transmission bundles in the tree branches and
trunk are damaged and the transmission of water and nutrients to the
upper parts of the plant is prevented. Therefore, there is a sign of
growth retardation and a general stagnation in the plant. Contaminated
trees and branches can dry completely in about 2-3 years. Opened
galleries cause breakage in twigs or saplings due to windy weather or
fruit load (Canıhoş et al., 2014; Anonymous, 2017).
 W a l n u t | 193

Figure 14. Gallery mouth feeding waste (a) and plant sap discharge (b) of Leopard
moth borer larva.
This type of damage causes yield loss and distortion of the crown
shape. The sap flow from the larval entrance holes is typical of this
pest (Figure 13a, 14b). Z. pyrina is mostly a problem in old, neglected,
barren and high-ground water gardens.

The pest usually gives offspring once a year and sometimes once
every two years.

The leopard borer moth is a polyphagous pest. It is harmful to forest

trees and shrubs, usually fruit trees. Walnut, pomegranate, olive,
apple, pear, quince, cherry, plum are among the important hosts.

Control

Cultural measures: Steel and saplings to be used while establishing a

garden should not be contaminated with pests. Garden maintenance
should be done well, trees should be kept healthy and attention should
be paid to weed cleaning, especially under the tree crown. Trees
should be pruned regularly every year, and the infected branches

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194 | W a l n u t

should be cut and destroyed both during pruning and during the
controls during the year. In orchards with low density, the larvae
should be killed by inserting steel wire into the galleries or injecting a
suitable insecticide.

Chemical control: Since the larvae of the pest feed in the gallery in
the wood tissue of the tree, it is very difficult to control. In May-June,
the first spraying should be done 7-10 days after the first butterfly is
seen, probably in June. If the butterfly flights continue, one more
spraying can be done 20-30 days after the first spraying, possibly in
August and October (Canıhoş et al., 2014; Anonymous, 2017).

Pear lace bug (Stephanitis pyri (Fabr.))

In adults, the body is dark-colored, flat and wide, and it is an

ornamental insect. When viewed from above, its wings are transparent
and have a bee-comb pattern (Figure 15a).

a b

Figure 15. Adults and nymphs of the pear lace bug (a) and feeding waste (b).
 W a l n u t | 195

Winter; They spend their adult life under tree bark, in soil crevices
and roofs between stones, and under dried leaves. As of April, they
leave their winter quarters and move to fruit trees. It lays its eggs one
by one in the tissue under the leaf. The eggs are covered with a sticky
liquid such as pitch for protection (Figure 15b).

Adults are withdrawn to winter from October and the pest gives 2-3
offspring per year.

The nymphs and adults of the pear tiger suck the sap of the leaves,
causing the chlorophyll to break down and yellowish-white spots to
form on the leaf (Figure 16). As a result of feeding on the underside of
the leaf, dark-colored scum accumulated in small spots and the honey-
like substance they secrete prevents the leaf from respiration and
causes drying and shedding starting from the leaf tips. In high
populations, growth retardation occurs in trees, shoots cannot fully
mature, fruits remain small, so the quality and yield of the product
decrease.

Figure 16. Nutritional damage and symptoms of the Pear Lage Bug on the leaves

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196 | W a l n u t

The pear lage bug is a polyphagous pest. Walnut, pear, apple, quince,
cherry, cherry, peach, apricot, plum, chestnut, medlar, hazelnut,
blackcurrant, poplar, willow, elm, plane tree and ornamental plants are
among its important hosts (Canıhoş et al., 2014; Anonymous). , 2017).

Control

Cultural measures: frequent planting should not be done in new plant

gardens. Excessive irrigation and nitrogen fertilizer application should
be avoided. Care should be taken to clean weeds in and around the
garden. Trees should be pruned regularly every year and airflow
should be provided in the garden.

Biological control: The pear tiger has many natural enemies in

nature. First of all, care should be taken to increase the efficiency of
these species by protecting them. For this, random drug applications
should be avoided, and when necessary, specific drugs with low
impact on the environment and natural enemies should be used.
Perennial shelter plants (wild rose, blackberry, etc.) and flowering
plants such as mint, wild carrot and fennel, which can be a food
source (prey, pollen, nectar, etc.) for natural enemies, should be found
or grown in the garden edges.

Chemical control: As of April, 3 leaves are checked from one shoot

in 4 different directions of 10 trees determined to represent the garden.
If the average number of adults is 1 individual/leaf or more in the
counts, spraying is done. If the number of nymphs+adults is 4
 W a l n u t | 197

individuals/leaf in the controls made in June, second spraying may be

recommended (Canıhoş et al., 2014; Anonymous, 2017).

Walnut leaf miner (Caloptilia roscipennella (Hübner))

C. roscipennella is a micro butterfly species. Adults range in color

from grayish-white to light brownish gray or dark yellow to reddish-
brown (Figure 17a). There are silvery blackish spots on the wings and
fringe-shaped hairs that vary from yellow to black on the edges
(Canıhoş et al., 2014).

Larva; It’s 9 mm long, the yellowish-green body is transparent and its

intestines are visible (Figure 17b).

Figure 17. Adult (a) and larva (b) of the walnut leaf miner

It spends the winter in the sheltered parts of the tree in the adult
period. C. roscipennella is a monophagous species and feeds on the
leaves and shoots of walnuts. It lays its eggs on the leaves of trees.
The larva opens a gallery in the leaf and causes curling in the form of
a roll. In dense populations, leaves and shoots dry out (Figure 18).
This damage is more important in nurseries and young gardens, it

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198 | W a l n u t

prevents seedling growth and leads to deterioration of the crown

structure. In older trees, this damage can be tolerated with copious
shoot yields. The feeding damage of the larva on the leaves is first
seen in April. The density and damage of larvae in the shoots occur
mostly in the months of June-July (Canıhoş et al., 2014).

Figure 18. The damage of walnut leaf miner on walnut leaves.

Control

Cultural measures: Excessive irrigation and nitrogen fertilizer

application should be avoided. Trees should be pruned regularly every
year and airflow should be provided in the garden. The gluttonous
shoots and pest-infested shoots on the tree should be cut and destroyed
during the year.

Biological control: C. roscipennella has many natural enemies in

nature. First of all, care should be taken to increase the efficiency of
these species by protecting them. For this, random drug applications
should be avoided, and when necessary, specific drugs with low
impact on the environment and natural enemies should be used.
Perennial shelter plants (wild rose, blackberry, etc.) and flowering
plants such as mint, wild carrot and fennel, which can be a food
 W a l n u t | 199

source (prey, pollen, nectar, etc.) for natural enemies, should be found
or grown in the garden edges.

Chemical control: In the control of C. roscipennella, there is no need

for additional spraying because the damage is tolerated on large trees
and the pesticides applied against other walnut pests also control this
pest. However, since the damage is more important in the nurseries, 1
spraying is done when the leaves and shoots show signs of damage
during the controls to be made in April-May. If necessary, 1-2 more
sprayings can be recommended after 15-20 days, taking into account
the effect time of the drug.

Result

In Turkey, many closed walnut orchards have been established due to

the increasing domestic and foreign market demand, especially after
the 2000s. This situation has brought along many plant protection
problems as well as aquaculture problems. Along with these problems,
human and environmental health should be prioritized against possible
plant health problems in the future, and more importance should be
given to "Integrated struggle and "Organic agriculture", which are
agricultural techniques that protect and support the natural balance.
While planning these studies, the agro-ecosystem should always be
considered as a whole and sufficient information should be obtained
about the important pests seen in walnut orchards and their control. In
addition, to leave a healthier environment for future generations with
sustainable agricultural activities, efforts should be made to preserve

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200 | W a l n u t

the natural balance in the ecosystem by using environmentally

friendly-specific drugs with the right techniques at the right time,
instead of intensive pesticide applications in herbal products.
 W a l n u t | 201

References

Anonymous, 2017. Ceviz Entegre Mücadele Teknik Talimatı. Tarımsal Araştırmalar

ve Politikalar Genel Müdürlüğü, Bitki Sağlığı Araştırmaları Daire Başkanlığı,
Ankara, 94 p. Web page: www.tarim.gov.tr/TAGEM/Menu/28/Yayinlar
_veriler (Date of access: December 2021).

Anonymous, 2021. Tarım Ürünleri Piyasaları (Ceviz). Tarımsal Ekonomi ve Politika

Geliştirme Enstitüsü, 4 p. Web page: https://arastirma.tarimorman.gov.tr/tepge
/Belgeler/ (Date of access: December 2021).
Canıhoş, E., N. Öztürk, M. Sütyemez, S. Toker Demiray & A. Hazır, 2014. Ceviz.
Türkiye Bilimsel ve Teknik Araştırma Kurumu (Tübitak) Tarım, Ormancılık ve
Veterinerlik Araştırma Grubu Yayını, 69 s.

Çakır, Ş., 2018. Türkiye’de Ceviz Zararlıları İle İlgili Yapılan Çalışmalar. Ondokuz
Mayıs Üniversitesi, Fen Bilimleri Enstitüsü, Bitki Koruma Anabilim Dalı
(Seminer), Samsun, 51 p.
TÜİK, 2020. Türkiye İstatistik Kurumu, Türkiye’de Meyve Üretimi Verileri. Web
page: https://biruni.tuik.gov.tr/bitkiselapp/bitkisel.zul. (Date of access:
December 2021)
Uygun. N., M.R. Ulusoy, İ. Karaca & S. Satar, 2010 Meyve ve Bağ Zararlıları.
Çukurova Üniversitesi, Ziraat. Fakültesi, Bitki Koruma Bölümü, Özyurt
Matbaacılık, Adana, 347 p.

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 W a l n u t | 203

Part X
--------------------------------

Walnut (Juglans regia L.) Breeding and Walnut

Breeding Criteria
Prof. Dr. Halil İbrahim OĞUZ1, İlbilge OĞUZ2

1. Introduction

Considering the distribution and habitat of walnuts throughout the

world; it is a type of fruit that spreads over a wide area from the
Carpathian Mountains, from the South to Eastern Europe, and from
Turkey, Iraq, east of Iran to the Himalayan Mountains. Although there
are many species of walnuts, the most commercially produced and
consumed species in the world is Juglans regia L., which is generally
diploid with a chromosome number of 2n=2x=32. In addition, there
are wild forests of Juglans regia L. especially in the western border
mountains of Kyrgyzstan, Tajikistan, Uzbekistan, and China. (Akça,
2001; Şen 2005; Guney et al., 2021).

1 *
University of Nevsehir Hacı Bektas Veli, Faculty of Engineering and
Architecture, Department of Biosystem Nevşehir, Turkey, ORCIDs: Prof. Dr. Halil
İbrahim OGUZ https://orcid.org/0000-0003-22137449, hioguz64@gmail.com
2
Cukurova University, Faculty of Agriculture Department of Horticulture Adana,
Turkey, ORCIDs: İlbilge OGUZ, https://orcid.org/0000-0002-7198-4014,
204 | W a l n u t

As is known, after understanding the importance of walnuts in the

nutritional benefits of walnuts for human health, interest in walnut
production has increased rapidly in the world. According to the data of
FAO for 2019, world walnut production is 4.498.442 tons, of which
2.5021.504 tons are produced by China alone. In terms of walnut
production, the USA ranks second with 592.390 tons, Iran ranks third
with 321.074 tons, and Turkey ranks fourth with 225.000 tons. In
recent years, both the production area has been expanding and all
walnut producing countries have been making efforts to increase the
yield per hectare. Besides, the export of shelled walnuts in the world
increased from 272.705 tons to 335.361 tons in 2016. Regarding the
exports of walnuts in shell, the USA alone ranks first 129.827 tons,
Mexico ranks second with 50.029 tons, Chile ranks third with 32.014
tons followed by Ukraine with 28.594 tons, China with 23.148 tons,
Germany with 14.489 tons, Moldova with 11.915 tons and Turkey
with 6.248 tons. Although Turkey is the fourth country in terms of
walnut production in the world, its exports fall far behind. The reason
for this is that the majority of the walnuts produced find buyers in the
domestic market at high prices. Another country that draws attention
to exporting walnuts is Germany. Germany creates awareness by
buying shelled walnuts cheaper than the world walnut market from
countries with low marketing opportunities, packaging them in its own
country and then exporting to the European and world walnut market.
The value of exported walnuts worldwide amounted to 2.246.631 on a
dollar basis. The USA alone meets 37.02% of this value. This is
followed by Mexico City with a share of 21.9%, Chile 9.2%, Germany
 W a l n u t | 205

with 6.2%, China with 5.2%, Ukraine with 3.15%, Moldova with
2.3%. The rest is exported by other countries. Once more, according
to the data of FAO for 2019, Walnut global import increased from
1.389.795 tons in 2016 to 1.722.778 tons in 2020. Germany is the
leading country with 317.843 tons in terms of importing shelled
walnuts. Regarding the walnut in shell imports, Japan ranks second
with 136.993 tons, Spain ranks third with 121.628 tons followed by
Canada with 84.916 tons, South Korea with 82.659 tons, England with
78.509 tons, France with 71.935 tons, Italy with 59.517 tons, the
United Arab Emirates with 43.869 tons and Turkey with 16.615 tons.
Here, there has been a decrease in Turkey's walnut imports in recent
years, which can be expressed as a result of the increase in newly
established walnut plantations in Turkey (FAO, 2019; TUIK, 2020).

In recent years, walnut production areas in Turkey show a rapid

increase with the support and developments in grafting and grafted
sapling production. In addition, some of the important reasons for the
increase in production can be explained by the price stability, the fact
that walnut production is easier than other fruit types, and has fewer
marketing challenges. However, the production target has not yet been
achieved due to problems such as choosing the right variety, choosing
a planting site, preparing the land for planting, planting methods, and
garden management. As it is known, there are many walnut genotypes
that have been propagated by seed, have a very high genetic diversity,
and have a high preferability value in terms of breeding. Considerable
selection studies have been carried out in each walnut production area
206 | W a l n u t

throughout Turkey, and many genotypes which are suitable for

breeding have been selected (Akça, 2001; Şen, 2005, Oğuz and Aşkın,
2007; Keles et al., 2014; Kafkas et al., 2020; Arcan et al., 2021).
During the selection studies carried out after the 1970s, criteria
including late leafing, fruit weight, kernel weight, shell thickness,
resistance to diseases and pests, non-periodicity, lateral bearing habit,
kernel color, and kernels which are easily removed from shell were
emphasized in general. Researchers working on walnut breeding in
Turkey have chosen high-yielding genotypes by making point
selections in many regions of Anatolia for many years. Unfortunately,
some of these selected genotypes could not be propagated by grafting.
However, although these selected genotypes are reproduced and
protected by public projects in orchards established in different
regions, the programmed and interdisciplinary walnut breeding studies
are not sufficient in Turkey. Although there are many researchers
working on walnut breeding, due to the lack of cooperation,
teamwork, and national walnut breeding program, walnut breeding
studies have unfortunately not reached the desired level.

This study was conducted in order to specify in detail the historical

background of walnut breeding studies in the world, breeding studies
by country, outstanding criteria in breeding studies, methods used in
breeding studies, cultivar and rootstock breeding, developments in
breeding methods in recent years and future breeding strategies.
 W a l n u t | 207

2. AN OVERVIEW OF WALNUT BREEDING

So as to define the selective breeding method briefly, it is a breeding

method performed by selecting plants suitable for breeding purposes
and criteria from natural populations which are rich in diversity and
giving them the chance to get more yield. As it is known, the basis of
plant breeding is based on selective breeding. However, this method is
based on finding new promising genotypes by selecting genotypes
suitable for their purpose from existing variations, rather than creating
genetic variation. On the other hand, in other breeding methods,
artificial variability is created through hybridization, in other words,
variations are created so that the selection is made from these
variations. In selection breeding, the hereditary structures of plants are
preserved. Thanks to this feature, selection breeding can influence the
allele frequencies in a population. In other words, the population's
genetic variance can be shifted toward the desired direction. Success
in selection breeding depends on the genetic variability, the source of
variability, and exploitation of variability in the studied populations.
In brief, the selection technique to be used is important. The source of
variation in a population is caused by multiple factors. For example,
one cause is due to environmental conditions (modification), and the
other is due to hereditary structure (idiovariability). The higher
amount of genetic variance in the total, the higher the effect and
accuracy of selection. While the selection criteria to be used in
selective breeding should be considered separately for each ecology,
some characteristics such as lateral bearing trait and yielding,
periodicity, fruit characteristics, resistance to diseases and pests,
208 | W a l n u t

quality, early fruiting, growth strength, and morphological structure of

the tree should be included among the breeding objectives. In line
with these objectives, the main characteristics such as fruit quality,
yielding, tolerance to disease, and pest were emphasized as selection
criteria in the study on the breeding of walnut in the Marmara region
by the selection, which was the first walnut breeding study in Turkey.
At the same time, this study has made a very important contribution to
future studies both as it was the first breeding study conducted and as
it used the selective breeding method (Germain, 2004; Akça, 2005;
Öztürk, 2021).

In general, the aim of walnut breeding is to breed new varieties with

high yield, resistance to diseases and pests, biotic and abiotic stresses.
In 1948, the first breeding study was carried out in France and the
USA using the selection method. High-quality genotypes are selected
from countries such as Iran, China, Afghanistan which are the
homelands of the walnut, and reproduced in countries such as France,
China, Turkey, Iran, Spain, Italy, and the USA, and thus new
variations were obtained by natural hybridization methods. Later,
breeding methods were developed, as a result, phenotyping and new
molecular breeding methods were started to be used. In recent years,
developments in genomics and biotechnology have led to significant
advances in the walnut cultivar and rootstock breeding. In addition, in
the future, skilled biotechnologists can identify and develop desirable
traits for breeding in a suitable gene bank, and long-term germplasm
conservation of biodiversity can be ensured.
 W a l n u t | 209

As a result, the use of molecular breeding and techniques of

biotechnology can play a decisive role in countries such as the USA,
China, France, Iran and Turkey, which carry out walnut breeding
studies. These techniques are high-throughput genotyping platforms,
genomic-based approaches, genome-wide association studies, marker-
assisted selection, genomic selection, and next-generation sequencing
techniques such as genomic editing with the CRISPR-Cas9 system,
bioinformatics tools. Thanks to these techniques, fast and clear results
will be obtained in walnut production, variety and rootstock breeding.
In our recent past and even today, many producers use rootstocks
propagated by seed in walnut production. Walnut producers in many
parts of the world use new varieties developed by leading countries in
walnut production. For example, the Chandler walnut variety is one of
the most produced varieties in the world, for its lateral bearing habit,
productivity and white-colored kernel (Tulecke and McGranahan
1994). Franquette is a late leafing variety. This variety is widely
produced in the world as well. However, although it was a common
variety especially in France, it is now being replaced by Fernor and
Chandler varieties (Germain 1999; Vahdati, et al., 2019). It is always
difficult to achieve specific goals in plant breeding. Yet, the main goal
for all kinds of products is to provide high yield and quality.
Regarding walnut breeding, first thing is to focus on criteria such as
nut size, kernel color, kernel weight, a kernel that is easily removed
from the shell, and shell thickness. In addition to these, the
morphological development of the trees, lateral bearing habit as the
major criterion of yield were accepted as the dominant criteria in the
210 | W a l n u t

breeding of commercial varieties such as Chandler in the middle of

the twentieth century. Besides criteria such as resistance to diseases
(blight, anthracnose) and late leafing were emphasized as well. Some
characteristics of walnuts also vary depending on the ecological and
climatic conditions in which they are produced. For example, in
countries such as Turkey, France and Iran, which are adversely
affected by climatic conditions, late leafing is an important breeding
criterion due to late spring frosts (Avanzato et al. 2014; Bernard et al.
2018). In addition, late leafing has gained importance in resistance to
walnut blight (Xanthomonas arboricola pv. juglandis), which causes
widespread damage, especially in spring during high rainfall.
Earliness in walnut production has been accepted as an important
criterion to avoid autumn rains and therefore to determine the harvest
time correctly (Akca and Ozongun 2004; Bernard et al. 2018;
Ebrahimi et al. 2015; Germain 1989; Leslie and McGranahan 2014).
Moreover, it is believed that criteria such as biotic and abiotic stresses,
tolerance to drought, resistance to diseases and pests, tolerance to
climate changes and global warming will be regarded as goals in
walnut breeding in the future (Vahdati, et al., 2019).

3. WALNUT BREEDING PROGRAMS WORLDWIDE

3.1. Variety Breeding
The history of walnut variety breeding programs throughout the world
is based on the recent past. Variety breeding has started with the
genetic development of walnuts and the with the grafting and
propagation of walnut genotypes, which are considered superior
 W a l n u t | 211

within the natural population by farmers. Squirrels and crows have

had a huge impact on increasing variation in natural populations of
walnuts. Since these animals forgot most of the seeds they buried in
the ground to store food in the winter, they caused many new walnut
variations to be formed by the germination of these seeds in the spring
(Akça, 2001; Şen, 2005; Vahdati and Rezaee, 2014). In order to
summarize the developments in walnut variety breeding, it will be
sufficient to examine it in 4 stages.

The walnut breeding studies should be listed generally in the time

order; from 1700-1948, 1948-1979, 1979-2009 and 2009 to the
present. The first studies in walnut variety breeding started in France
with the selection and grafting of superior genotypes and their
distribution to the producers as seedlings. By this means, varieties
such as Franquette, Mayette, Crone, Grandjean and Parisienne were
obtained. In the United States, the first breeding studies were carried
out with this method as well. The source of walnut breeding material
in the USA consists mostly of Juglans regia L. seeds originating from
Iran and Afghanistan.

The first breeding and selection studies in the USA were made by
Felix Gillet in Northern California between 1835-1908. Then these
studies were conducted by Josef Sexton (1842–1917), a walnut grower
in Southern California between 1842–1917. Eureka, Waterloo, Poe
and Hartley American walnut varieties were selected by these
researchers. Moreover, California farmer George Payne selected the
Payne variety and produced it as an important walnut variety in
212 | W a l n u t

California for many years. In addition, the Payne variety has been an
important genetic resource in American walnut breeding (Ramos
1997; Tulecke and McGranahan 1994). At the same time, it is claimed
that the source of the Eureka walnut variety in the USA originates
from Iran and Afghanistan. Walnut breeding studies in the USA and
France accelerated the hybrid studies, which is the second stage in the
world walnut breeding. In particular, crossing studies have been
initiated between genotypes propagated with seeds imported from
natural walnut populations originating from the Silk Road. In these
hybrid studies, lateral bearing trait has been regarded as the dominant
criterion. Eugene F. Serr and Harold I. Forde carried out this breeding
phase from 1948 to 1979 and as a result of the breeding studies,
walnut varieties such as Midland, Vina, Pioneer, Pedro, Gustine,
Lompoc, Amigo, Chico, Tehama, Serr, Chandler, Howard and
Sunland were developed. Among these cultivars, Chandler, Serr and
Howard cultivars became the most popular walnut cultivars in the
world. Especially the Chandler variety constitutes 72% of the walnut
orchards in California, while the Sert variety constitutes 12%. In these
years, the targeted breeding criteria were late leafing, lateral bearing
habit, earliness, strong tree structure, kernel quality and tolerance to
diseases. Studies have shown that the parents and ancestors of Ser and
Forde cultivars are Payne cultivars (Ramos 1997; Tulecke and
McGranahan 1994; Vahdati and Rezaee 2014; Bernard et al. 2018).

In France, Eric Germain obtained 28 crosses (1900 intraspecific

hybrids) between French and Californian cultivars at INRA in the
 W a l n u t | 213

second stage of the walnut breeding program. The hybrids obtained

here were evaluated and as a result, Lara cultivar and Franquette
cultivar were obtained. Later, Fernor variety with late leafing was
developed in 1987. Here, again, late leafing and lateral bearing criteria
were taken into account for breeding purposes (Bernard et al., 2018;
Germain 1999; Ramos and Doyle 1984). The third and fourth walnut
breeding stages in the world were carried out in Iran between 2009
and 2010, as well as in the USA and France between 1979 and 2009,
and 2009 until today (Hassani et al. 2013). Regarding the fourth stage,
criteria such as late leafing, lateral bearing trait and yielding were
emphasized. Germplasm evaluation and hybridization studies are still
ongoing. Currently, there are four walnut varieties as Persia, Hazar,
Chaldoran and Alvand (Vahdati and Rezaee, 2014).

In addition to these, in Iran, germplasm evaluation, hybridization and

molecular breeding studies continue under the management of
Kourosh Vahdati at Tehran University. In these studies, varieties such
as Kerman, Ilam, Fars, Qazvin, Alborz, Yazd, Kohgilooye and Boyer-
Ahmad, Mazandaran have been developed. Criteria such as lateral
bearing habit, early harvest, dwarfing, drought and stress tolerance are
emphasized in these breeding studies, (Karimi et al. 2014; Vahdati and
Mohseniazar 2016; Vahdati and Rezaee 2014; Vahdati et al. 2015).

Variety breeding studies in walnut are based on germplasm evaluation

and hybrid breeding studies. Among these studies, germplasm
evaluation studies are mostly carried out in Xinjiang province, where
germplasm resources are abundant. About 80% of Chinese walnut
214 | W a l n u t

cultivars were selected from Xinjiang germplasm and originated in

Xinjiang province. The first walnut cultivar breeding studies in China
were initiated in the early 1960s at the Liaoning Economic Forest
Institute and the Chinese Academy of Forestry. However, existing
walnut cultivars were later developed after uniform requirements for
breeding criteria were established. Significant progress has been made
in walnut breeding in China since 1980. Between 1979 and 2006, 26
new walnut varieties were bred. Baokexiang, Beijing 861, Jinglong 1,
Jinglong 2, Lipin 1, Lipin 2, Lubo, Xifu 1, Xilin 1 and Xinzaofeng
were among those varieties. All of these varieties have been
developed through selection. In addition to these, Liaoning 1-8,
Xiangling, Fenghui and Zhonglin varieties were also created as a
result of controlled crosses. They reported that the most important
walnut varieties developed as a result of controlled crosses were
Xinjiang walnuts. Moreover, they reported that around 20 new walnut
cultivars were added as a result of controlled crossing studies in the
fourth breeding stage (Chen et al. 2014; Zhang et al. 2013a; Zhang et
al. 2013b).

Most of the walnut varieties native to Turkey bear fruits on terminal

buds. For this reason, in Turkey, breeding criteria are based on late
leafing and fruiting on lateral branches. The walnut breeding program
in Turkey is based on germplasm assessment and selection of superior
genotypes. Although Turkey is the homeland of walnuts, the majority
of walnut orchards consisted of genotypes propagated by seeds until
 W a l n u t | 215

recent years. Nevertheless, in the last 20 years, newly established

commercial gardens have been established with standard varieties.

The first walnut breeding program in Turkey was initiated by Hayati

Olez in 1971 in the Marmara Region. Later, selection breeding studies
were conducted by many researchers in almost every region of
Anatolia (Şen, in 1980 in North East Anatolia and Eastern Black Sea
Region; Akça, in 1993 Gürün; Beyhan, in 1993 Darende; Akça and C.
Ayhan in 1996; Yarlgaç, in 1997 Gevas: Oğuz, 1998 in Ermenek;
Aşkın and Gün, in 1995 Çameli and Bozkurt; Yaviç, in 2000
Bahçesaray). At the same time, important genotypes have been
identified in terms of breeding. In Turkish breeding studies, varieties
such as Akça 1, Bilecik, Bursa 95, Kaplan-86, Şebin, Şen 2, Tokat 1,
Yalova 1, Yalova 3, Yalova 4, Yavuz 1 were created through
selection. (Akça, 1993; Akça and Ayhan, 1996; Akça and Özongun,
2004; Akça 2005a; ; Akça 2005b; Akça and Polat 2007; Askın and
Gün 1995; Beyhan, 1993; Oguz, 1998; Ölez, 1971; Şen, 1980;
Yarlgaç, 1997; Yavic, 2000). In addition to germplasm evaluation and
selection studies, some controlled crosses between domestic and
foreign varieties have been carried out in the walnut breeding program
in Turkey since 2008. With these controlled crosses, approximately
1340 hybrid genotypes which are under evaluation were produced
(Akça et al. 2016). Moreover, new Turkish walnut cultivars as Maraş
18, Sutyemez 1 and Kaman 1 were developed by means of germplasm
evaluation in 2009 and 2010. Besides new varieties such as Diriliş,
July 15, Maraş 12 and Bayrak are Turkish walnut varieties that have
216 | W a l n u t

been introduced recently (Ozcan et al. 2017). The aim of the Turkish
walnut breeding program is to develop new genotypes that are tolerant
to salt, lime, drought and disease stresses and have characteristics of
late leafing, lateral fruitfulness, early harvest (Ertürk and Akça 2014).

In addition to the countries mentioned above, walnut breeding studies

are also carried out in countries such as Spain, Germany (Bollersen
2017), Georgia, Italy, Hungary, Greece, Romania, Ukraine, Serbia and
Azerbaijan. In some of these countries, the walnut breeding program
has started directly from the second stage. In these countries, genetic
diversity and hybridization-based breeding studies are carried out
within the framework of crossbreeding between French and California
walnut varieties regarding the dominant criteria of breeding studies
and as a result, local varieties were introduced (Avanzato et al. 2014;
Vahdati et al. 2015).

3.2. Rootstock Breeding

As in all fruit growing, rootstock use is very important in determining

productivity. Rootstocks affect the morphological and physiological
development of the grafted culture plant including its adaptation to
different soil conditions, resistance to diseases and pests, leafing time,
late or early maturity, fruit yield and quality, tree growth. Previously,
Northern California black walnut seedlings (J. hindsii) and Jugrans
regia L. walnut seedlings were used as rootstocks in traditional walnut
cultivation in the United States. Juglans regia L. is still widely used as
a rootstock in some countries all around the world. Paradox (Juglans
 W a l n u t | 217

hindsii x Juglans regia hybrid) and Royal (J. hindsii x J. nigra)

rootstocks have been used for a long time in America. Up to 80% of
walnut orchards in California is home to Paradox, since it is a
rootstock that grows strong and is resistant to some diseases. Paradox
is a rootstock stronger, more resistant to Phytophthora species and
more tolerant to soil salinity than J. regia (Forde 1975; McGranahan
and Catlin 1987; Baumgartner et al. 2013). Disease and pest factors
such as Amillaria root disease, gall mite and Phytophthora cause
significant economic losses in walnut production (McGranahan and
Leslie 1991). On the other hand, regarding Paradox rootstock is
tolerant to blackline (CLR) disease, breeders have changed their
direction to breeding rootstocks resistant to both blackline (CLR)
disease and soil-borne diseases. Thereafter, at the end of these
breeding studies, Vlach, RX1 and VX211 rootstocks were developed.
Regarding their tolerance to Phytophthora and nematode respectively,
RX1 (J. microcarpa x J. regia hybrid) and VX211 (J. hindsii x J. regia
hybrid) rootstocks were commercially introduced (Leslie and
McGranahan 2014).

Rootstock breeding studies have been carried out in China, which is

one of the leading walnut producing countries in the world, and
rootstocks such as Jin RS-1, Jin RS-2, Jin RS-3 have been created. It
has been reported that these rootstocks have high resistance to cold,
diseases and pests. It has been stated that they are suitable rootstocks
especially for northern regions of China that are exposed to frost
(Bernard et al. 2018; Zhang et al. 2013b). Furthermore, studies on
218 | W a l n u t

rootstock breeding are still ongoing in countries such as Turkey and

Iran regarding the criteria such as dwarfing, tolerance to salt and
drought stress and resistance to soil-borne diseases such as
Agrobacterium, Phytophthora and Armillaria.

4. GERMPLASM, BIODIVERSITY AND THEIR

CONSERVATION IN WALNUT BREEDING

Like other fruit species, it requires a very long time to breed a new
variety of walnut. In recent years, biotechnological developments have
provided significant gains in terms of the breeding cycle and
biodiversity in walnut breeding. These developments have accelerated
the process and especially helped farmers gain time (Van Nocker and
Gardiner, 2014). Besides, biotechnological studies both ease the
process to create a gene bank consisting of superior genotypes and
save a significant amount of time in the selection of the desired
superior genotypes in breeding studies. As is known, conservation of
biodiversity is a global problem since the lands where walnut genetic
resources are located are under great threat with commercial or
economic activities such as hydroelectric power plants, urbanization
areas, timber trade. Therefore, the only way for conserving germplasm
resources is the creation of new clonal gardens by identifying superior
genotypes in the field and it is an urgent precaution in order not to lose
our resources. In particular, genotypes obtained from selection studies
conducted in Turkey from past to present should be protected by
establishing gene centers in 3 or 4 different regions of Turkey and
granting these genotypes selected in these centers. In other words,
 W a l n u t | 219

classical biodiversity conservation methods are not sufficient to solve

this problem. For this reason, new biotechnological methods such as
tissue culture, plant cell culture, anther culture, embryo culture are
very applicable and useful new techniques in preserving gene
resources. Thus, modern biotechnological applications can provide
significant convenience in the protection of gene resources in the
world (Ogbu 2014; Pathak and Abido 2014). Since the first walnut
orchards established in the world consisted of seedlings propagated by
seeds, a high degree of genetic diversity has increased in the world.

4.1. Cultivar Characterization and Phylogeny

As it is known, newly bred varieties in France and California have

been an important resource in the establishment of new gardens and
performing breeding studies all over the world (Germain 1999;
Tulecke and McGranahan 1994). Although the French Fernor variety
is preferred in cold regions, the Chandler variety is still preferred as
the favorite walnut variety throughout the world. The Chandler variety
was patented and marketed in 1979 by the University of California at
Davis. Chandler is a hybrid of Pedro and UC 56-224 and is preferable
due to its lateral bearing trait, thin shell, high walnut quality, a kernel
that can be removed easily from its shell and late leafing
characteristics (Vahdati, et al., 2019).

4.2. Molecular Breeding

Next-generation sequencing (NGS) techniques, bioinformatics tools,

high-throughput genotyping platform, genomics-based approaches
220 | W a l n u t

such as genome-wide association studies (GWAS), marker-assisted

selection studies (MAS), genomic selection, and genomic editing
using the CRISPR-Cas9 system provide conveniences and new
expansions in walnut breeding. The biggest problem in walnut
breeding is the high levels of heterozygosity in the long juvenile phase
(Vahdati, et al., 2019). It takes at least 15-20 years to breed new
varieties with classical breeding methods. For this reason, we have to
try new technologies that will help us get results in a shorter time.

4.3. Molecular Markers in Fruit and Nut Breeding

Many studies in fruit breeding have described genetic differences in

fruits. Although morphological markers are existed for describing
these differences, there is a need for indicators to detect genetic
diversity more accurately and clearly. Molecular markers are widely
used in cultivar breeding to analyze genetic relationships between
wild and cultivated individuals and related species, to identify cross-
species hybrids, and to analyze germplasm variability.

4.4. Molecular Marker Systems

Researchers benefited greatly from isoenzyme markers in genetic

studies in the past years. Isoenzymes have been commonly used in
walnut breeding for interspecific hybrid identification, genetic
diversity or relationship analysis, and determination of genotype
origin (McGranahan et al. 1986; Cheng and Yang 1987; Aletà et al.
1990; Fornari et al. 2001; Busov et al. 2002; Ninot and Aletà et al.
2003; Vyas et al. 2003). Marker techniques such as restriction
 W a l n u t | 221

fragment length polymorphisms (RFLPs) and robust DNA markers

with higher rates of polymorphism are applied in walnut breeding.
Among these techniques, restriction fragment length polymorphisms
(RFLPs), random amplified polymorphic DNAs (RAPDs), amplified
fragment length polymorphisms (AFLPs) and simple sequence repeats
(SSRs) techniques are still adopted today as they are cheap and fast
methods in breeding studies. These techniques are still used in walnut
breeding because they provide significant convenience in the
identification of walnut germplasm, evaluation of genetic diversity,
and uniformity and stability of varieties (Vahdati, et al., 2019).

5. Overview of the Diversity and Fingerprinting Techniques in

Walnut

The methods that have been used in walnut breeding so far can be
categorized as follows;

a) Hybridization-based markers

The Restriction Fragment Length Polymorphism (RFLP) technique

was the first to use isoenzyme RFLP markers to identify the origin of
somatic embryos derived from ovule tissues. This method is adopted
in walnut breeding for genetic mapping and determination of genetic
diversity. While using this technique, large amount of pure DNA, low
level of polymorphism and radioactivity complexity are also used so
as to contribute to the development of various alternative technologies
(Bernard et al. 2018).
222 | W a l n u t

b) PCR-Based Markers for Walnut Genome Profiling

In walnut breeding, Random Amplified Polymorphic DNA (RAPD)

markers are widely used in genetic diversity and mapping studies
including amplification of genomic DNA in the polymerase chain
reaction. It is preferred by breeders due to the low cost and fast results
(Welsh and McClelland 1990; Williams et al. 1990) It is possible to
divide PCR-based markers used in walnut breeding into 5 groups.

1) Random Amplified Polymorphic DNA (RAPD)

2) Sequence Characterized Amplified Region (SCAR)
3) Inter-Simple Sequence Repeat (ISSR)
4) Amplified Fragment Length Polymorphism (AFLP)
5) Simple Sequence Repeat (SSR) or Microsatellite Technique

In addition to the breeding methods mentioned above, the new

breeding techniques listed below are used in the walnut cultivar and
rootstock breeding. In this section, the details of the techniques are
not given but only their names are mentioned in the order (Vahdati, et
al., 2019).

c) High-Throughput SNP Assays in Walnut

d) Nuclear Ribosomal Internal Transcribed Spacer (ITS)
e) Emerging Marker Technology: Genotyping by Sequencing
(GBS)
f) Physical Mapping
g) Genetic Mapping and QTL Detection
h) Next-Generation Mapping
 W a l n u t | 223

ı) Comparative Mapping
i) Walnut Genome Sequencing
j) Genomics-Assisted Breeding in Walnut
k) Application of Functional Genomics in Genomics-Assisted
Breeding
l) Genetic Engineering
m) Mutation Breeding
n) Hybridization

CONCLUSION

In recent years, after understanding the importance and economic

value of walnuts in human health, the breeding varieties with high
quality have increased. In order to set an example of the dominant
breeding factors in walnut breeding; the criteria includes earliness,
white-colored kernels which are removed easily from the shell, high
yield, lateral bearing habit, productivity, dwarfing, late flowering,
tolerance to salt and drought, resistance to diseases and pests such as
Agrobacterium, Phytophthora, nematodes, Aceria avanensis and
Armillaria. Breeders use breeding methods such as germplasm
assessment, selection, hybridization, genetic engineering, mutation
breeding, genome sequencing, bioinformatics, marker-assisted
selection, haploid and polyploid induction. In recent years, global
climate change has led to significant changes in walnut production and
breeding. Regarding climate change, new strategies have been
recently developed in terms of walnut breeding. Accordingly, the
224 | W a l n u t

breeding programs and methods recommended to be used are as

follows.

The new walnut breeding strategies should include criteria such as low
chilling requirements, early harvest and late leafing characteristics;
maintaining and preserving genetic diversity for breeding new
varieties and rootstocks; simultaneous use of transcriptomics,
metabolomics and proteomics techniques to understand drought and
salinity tolerance characteristics in walnuts; genomic selection; high-
throughput phenotyping combined with genome-wide association
studies in order to map genetic information associated with complex
traits of walnut; production of haploid, double-haploid and wide
hybrid plants to enhance genome assembly quality and focusing on the
use of CRISPR-Cas9 systems in new walnut breeding studies
(Vahdati et al, 2019).
 W a l n u t | 225

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 W a l n u t | 231

Part XI
--------------------------------
Advances in Walnut Breeding
Assoc. Prof. Murat GUNEY1

Walnuts (Juglans regia L.) belong to the family Juglandaceae. In

addition to the genus Juglans L., the family includes Carya Nutt.
(pecans and hickories), Pterocarya Kunth. (wingnuts), Platycarya
Sieb. & Zucc., Engelhardia Lesche., Alfaroa Standl., and Oremunnea
Oerst. The genus Juglans consist of approximately 20 species having
32 chromosomes. Juglans is comprised of a single dichogamous
monoecious species J. regia L. Juglans cinsi içerisinde çoğunlukla
Juglans regia’nın kültürü ve ticareti yapılmaktadır (Woodworth,
1930); (Manning, 1978); (Şen 1986).

Juglans regia species, which has a wide natural distribution area in the
temperate climate zone around the world, was taken to geographical
regions outside its natural area as a result of various migrations,
economic and social relations between tribes and states, and thus
gained new growing areas. Due to long cultivation history of walnut,
it is the most developed horticultural plant and different walnut
species is widely cultivated. Today, walnut has become a type of fruit
grown in many parts of the world, except in tropical and very cold
1
Department of Horticulture, Faculty of Agriculture, Yozgat Bozok University,
Yozgat, Turkey ORCID: https://orcid.org/0000-0003-2882-8347
murat.guney@yobu.edu.tr
232 | W a l n u t

regions (Gunn et al., 2010); (Aradhya et al., 2017). A large area of the
central Asia Mountain is estimated to be the origin of Juglans regia
and the introduction of this precious nut to European agriculture and
commerce was occurred by the ancient Greeks. However, Juglans
regia grows wildly or semi-cultivated plant in a broad area from
southeastern Europe and the Caucasus to Turkey and Iran. More later,
Juglans regia has been introduced into North America, thereafter, it
has been referred to as the English walnut to be distinguished from the
American black walnut (Leslie and McGranahan, 1998).

Turkey is one of the homelands of J. regia and has an important

position in the production of walnut in world. The Anatolian region of
Turkey has a wide range of climate from the mild and humid Black
Sea region to the continental climate of hot dry summers and cool
winters. In Turkey, walnut trees are characterized as border and shade
trees in flat lands and as erosion tree on sloping lands and as a flood-
preventing tree in the valleys. Although walnut trees grow across all
these regions, the area of native forest is mainly limited to central,
north and north-east Turkey (Davis, 1982). Akça and Şen (1994)
reported that more than 10,000 walnut trees are naturally grown in
Turkey.

Turkey as one of the homelands of walnut, ranks among the first

producing countries of walnut (Bükücü and Sütyemez, 2016). As in
many of the other countries, walnut has been produced with seeds for
thousands of years in our country. (Çeri, 2021). Even today, walnut
 W a l n u t | 233

types offered to the market are far from the standard due to the
cultivation of walnut from seed.

In foreign trades, markets have become more selective in quality,

therefore, the demand for quality shelled-walnuts with improved
quality of kernels has increased. However, this demand could not be
met due to the fact that Turkey do not have a certain standard in
walnut cultivation leading Turkey to be lowered in world exports rank
(Sandal et al., 2018).

Due to a very long juvenile period of the walnut tree, breeding studies
take many years. Therefore, there are a limited number of cultivars
within this species and present cultivars have emerged both by natural
selection and hybridization (Ölez, 1971); (Şen 1980); (Sütyemez and
Kaşka, 2005); (Bükücü et al., 2020b); (Sütyemez et al., 2021a).

Breeding objectives

Turkey which is located in the natural distribution area of walnuts, is

one of the oldest walnut-producing countries in the world. Since
walnut has been propagated by seeds for thousands of years, walnut
population has been formed with very different characteristics. The
walnut population in Turkey is an extremely important resource for
breeding programs (Pereira et al., 2007).

Despite other applications of walnut tree in industry and folk

medicine, all walnut species are commonly produced for their edible
kernels. However, kernels wrapped thick hard shells are dissuasive
234 | W a l n u t

factor in commercial production, and the species with thin shells are
mostly preferred for cultivation (Beer et al., 2008); (McGranahan and
Leslie, 1990); (Feng et al., 2018).

Walnut is a monoecious tree in which the tree has both clusters of

male (catkins) and female flowers (pistillate) in different places of the
tree and are pollinated by wind (Şen 1986); (Akça and Ozongun,
2004); (Sütyemez et al., 2021b). Walnut trees also exhibit both
protandry (male maturing earlier than female) and protogyny (female
maturing earlier than male) (Woodworth, 1930); (Forde and Griggs,
1972). Therefore, dichogamy promotes out crossing. Wind pollination
and bloom overlap are vital for walnut production (Lloyd and Webb,
1986). If the pistillate flower are pollinated successfully, the hull of
walnuts separates from the shell at maturity (Manning, 1978). The
male clusters containing 10-100 male flowers (catkins) are formed on
lateral buds of the previous year shoots of the development period
while female flowers with a number of 1-12 are formed at the ends of
the current shoots (Şen, 2009).

In breeding programs of walnut, some preferred characteristics such as

late leafing, flowering on the terminal and lateral branches, high fruit
yield, large and relatively smooth fruit with 50% kernel, and moderate
disease and pest resistance are considered for selected cultivars (Acar
and Kazankaya, 2021).
 W a l n u t | 235

Use of molecular markers

The characterization of genetic resources, population genetics, genetic

mapping, and development of markers related to important traits can
be studied in plants using DNA-based molecular marker techniques
(Semagn et al., 2006); (Topcu et al., 2015); (Ikhsan et al., 2016);
(Dang et al., 2016); (Eser et al., 2019).

Especially in perennial plants with a very long juvenile period from

germination to the flowering of hybrid seed, early selection after
germination of the hybrid seeds will significantly reduce the duration,
cost, and workforce of the breeding programs as well as increase the
effectiveness of these programs (Güney et al., 2021a, b). Besides
cultivation from seed, walnut has also long juvenile period and
breeding programs take a lot of time compared to other plant species
(Germain, 1997); (Chen et al., 2018); (Sütyemez et al., 2021b).
Therefore, molecular studies on walnuts are an indispensable part of
breeding programs (Bernard et al., 2018); (Güney et al., 2021b).
Because markers that can be developed related to different traits will
provide early selection in breeding programs (Moose and Mumm,
2008).

Walnut varieties cultivated all over the world have emerged from
chance seedlings or hybridization breeding programs. Characterization
of these varieties is very important for breeders. In addition, it is very
important to determine the variety’s trueness-to-name in a short time
in fruit cultivation. However, the morphological, physiological and
236 | W a l n u t

biochemical methods used to characterize genetic diversity are time

consuming and influenced by the environment. Contrary, molecular
markers are applied for determination of genetic relationships within
plant populations with higher reliability (Güney et al., 2018); (Güney
et al., 2019); (Güney et al., 2021a, b). Moreover, it has been possible
to characterize genetic materials from different ecologies through the
development of DNA marker techniques. Walnut cultivars and
genotypes found in genetic resources were identified using different
molecular marker techniques such as AFLP (Bayazit et al., 2007),
RAPD (Ahmed et al., 2012), ISSR (Potter et al., 2002), and SSR
(Woeste et al., 2002); (Güney et al., 2021b).

In molecular breeding, molecular markers are used in conjunction

with linkage maps and genomics to select plants with desirable traits
based on the genetic assays (Vinod, 2006); (Güney, 2016). In order to
create an ideal genetic map, first of all, a reference genetic map
created with common markers is needed (Winter et al., 2000); (Güney
et al., 2016); (Güney, 2016); (Kefayati et al., 2019). This can then
easily lead to the development of new genetic maps in different
populations; placement of important characters on link groups with
QTL (Quantitative Trait Loci) analysis in these different genetic maps,
allowing to identify markers for important characters and
characterization and cloning of important genes. Therefore, the
construction of reference genetic maps in walnut are very important
for breeding programs. (Woodworth, 1930); (Fjellstrom and Parfitt,
1994); (Woeste et al., 1996); (Malvolti et al., 2001); (Luo et al., 2015);
 W a l n u t | 237

(Zhu et al., 2015). Constructed genetic map is the preliminary step for
many important downstream studies such as QTL (Kefayati et al.,
2019); (Bükücü et al., 2020b).

Breeding achievement in Turkey

Walnut trees can grow naturally throughout the Turkey. All pioneer
countries in walnut-growing are interested in breeding new cultivars.
Therefore, numerous breeding programs in walnut-growing are done
worldwide, and Turkey is one of the countries with major cross-
breeding programs (Ölez, 1971); (Şen 1980); (Sütyemez and Kaşka,
2005). Owing to propagation with seed not grafting since recent years,
a rich source of walnut population are available in Turkey. There may
be superior varieties with high quality and productivity among this
rich walnut population. Recently, high-quality and productive walnut
varieties that are well adapted to environmental conditions are
selected via characterization studies and grafted on walnut seedlings
leading to establish dense gardens (Çeri, 2021).

In Turkey, nongrafted walnut seedlings had been used in new walnut

plantations until the beginning of 1970s and commercial walnut
orchards were established with grafted trees approximately 50 years
ago. This has provided the opportunity to select the best genotypes
from natural populations for good yield and nut quality characteristics
(Orman et al., 2020); (Sütyemez et al., 2018); (Sütyemez et al., 2019);
(Sütyemez et al., 2021a).
238 | W a l n u t

Altınova-1, Altınova-2, Bilecik, Franquette, Gültekin-1, Kaplan-86,

Yalova-3, Yalova-4, Şebin, Şen-1, Şen-2, Tokat-1, Yalova-1, Yalova-
2, Yavuz-1, Maraş 18, Sütyemez 1, Kaman 1, Chandler, Oğuzlar 77,
Hartley, Pedro, Midland, Fernor, Fernette, Balaban, Sölöz, Yabani
ceviz, Niksar 1, Akça, Diriliş, 15 Temmuz, Maraş 12, Howard,
Bayrak, Adilcevaz 13, Kazankaya, Maraş 12, Chandler, Potamia
Erdin, Kozdere, Zengibar, Yivlik77, Efsus 46, Bahri Koz, Tulare,
Lara, Cisco, Kurtuluş 100, Bertiz Hilali, Ede, Helete Güneşi, İstiklal
100, KSÜ 46, Cerit Yıldızı and Hilal are registered varieties in Turkey
and the information about these varieties are available in Republic of
Turkey Ministry of Agriculture and Forestry website (Anonim,
2021a).

Future trends and conclusion

Since a large part of walnut production in Turkey is obtained from

seed propagated trees, standardization in production could not be
achieved (Yarılgaç, 1997); (Şen, 2009); (Muradoğlu et al., 2017). The
fact that its timber is valuable has also been a factor in the prevalence
of walnut cultivation in Anatolia (Sandal et al., 2018). Especially the
use of old trees by cutting them in lumbering has significantly affected
this gene richness in walnuts. In addition, gardening has been
limitedly started with grafted walnut seedlings since the 1970s
(Orman et al., 2020). Recently, It is pleasing that the production of
walnut seedlings has increased with the obtained standard varieties.
However, to achieve the desired level in both production and export of
walnut, it is necessary to product walnut commercially in accordance
 W a l n u t | 239

with the standard cultivation technique (Şen, 2005); (Sandal et al.,

2018); (Varol et al., 2020).

The main desired characteristics sought in a walnut variety selected

from a breeding program are yielding, good fruit characteristics,
fruiting on the lateral branches, late leafing and early harvest, as well
as resistance to diseases and pests. In terms of fruit characteristics,
varieties with a high yield rate, light-colored interior, thin but robust
hard-shelled, high-fat and unsaturated fatty acids beside non-rough
hard shell are preferred (Şen, 1980); (Yarılgaç, 1997); (Akça and
Köroğlu, 2005); (Akça, 2009); (Bayazit, 2011); (Anonim, 2021b);
(Arcan et al., 2021).

In a breeding program, it is necessary to wait for many years from

germination of the hybrid seed to bloom and bear fruit to select
desired individuals with characteristics such as fruiting on the side
branches, harvest date, and fruit characteristics (Ölez, 1971); (Şen
1980); (Yarılgaç, 1997); (Sütyemez and Kaşka, 2005); (Bayazit,
2011); (Akça, 2016); (Bükücü et al., 2020b); (Sütyemez et al., 2021a,
b). However, it is possible to overcome such problems in perennial
fruit species only by taking advantage of the opportunities provided by
biotechnology. This may be possible by utilizing molecular marker
systems and molecular breeding (Güney et al., 2021a, b). Developing
the DNA markers associated with important characters make it
possible to select superior individuals by marker-based selection
without waiting for many years in terms of these characteristics, and it
will be possible to work with purpose-built materials in a shorter time,
240 | W a l n u t

with less cost and less labor (Kefayati et al., 2019); (Bükücü et al.,
2020b). This will increase the effectiveness of the variety breeding
program and will enable the emergence of alternative varieties in a
shorter time and with less cost according to the market conditions and
consumer needs. One of the most important tools in achieving
breeding varieties with better yield and fruit characteristics is the
creation of accurate and high-density genetic maps.

Consequently, the starting and supporting studies on molecular

breeding in a strategic product such as walnut will lead to a rapid
progress in its breeding and to establish dense walnut orchards with
superior varieties besides meet the quality food needs of a growing
population.
 W a l n u t | 241

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 W a l n u t | 249

Part XII
--------------------------------

Conventional and Molecular Breeding in

Walnut
Dr. Hayat TOPCU1

1. INTRODUCTION

Fruits such as walnuts and walnuts are one of the oldest food sources
for birds and most living creatures (Woodroof, 1967). Walnuts are
classified in the subclass Rosidae, in the genus Juglans spp. (Germain
et al. 1999). Juglans regia L. is a widely distributed monoecious tree
species belonging to the order Fagales and the family Juglandaceae.
This family consists of 11 genera and about 50 species, of which
Carya (hickory tree), Pterocarya (wing nut tree) and Juglans are its
main members (Rehder, 1947). Here are more than 20 species in the
genus Juglans, and they are separated into four divisions:
Trachycaryon (butternut, a single species, J. cinerea), Cardiocaryon
(walnut), Rhysocaryon (black walnut, especially J. nigra), and
Dioscaryon, comprises just J. regia (Manning 1978). The
chromosome number of all species of the genus Juglans is 2n = 2x =
32 and has a diploid structure (Woodworth 1930). The approximate

1 1
Tekirdag Namık Kemal University, Agricultural Biotechnology Tekirdag,
Turkey, ORCIDs: https://orcid.org/0000-0003-3108-4393, hayattopcu@nku.edu.tr
250 | W a l n u t

genome size of the walnut is 1315 Mbp (1.35 pg; monoploid genome
size (1C DNA) = 657.80 Mbp. (Sarikhani Khorami et al. 2018).

The distribution areas of J. regia in the world are widely found in

mainly temperate areas of Europe, Asia, North and South America,
Australia and New Zealand and South Africa (Aradhya et al. 2006).
The Greek philosopher Theophrastus mentioned in 370 B.C. that
walnuts and hazelnuts were found in the Macedonian region (Jaynes,
1969). It has been determined that Persian walnuts (Juglans regia L.)
were found in the garden of King Solomon and there were pictures of
almonds for decoration in the pictures on the candlesticks in the
Temple (Moldenke and Moldenke 1952). Today it is cultivated for
fruit and wood products in Asia, Europe and the Americas (Aradhya et
al., 2017; Bernard et al., 2018). J. regia is a monoecious and wind-
pollinated species. North and South America are known as distribution
centers of J. regia (Aradhya et al., 2007). It is the just species in the
genus Juglans regia L. Juglans, which is widely cultivated in Europe.
Turkey is one of the origins of Juglans regia L (Fao, 2021). Anatolian
walnut, which has a large natural spreading area in the world, has been
taken to different places by various migrations and trade caravans, and
today it can be grown almost all over the world, except for the tropics.
Walnuts are wind-pollinated and have monoecious characteristics, that
is, male and female inflorescences are on the same tree, but in
different places (Sen 1985).
 W a l n u t | 251

Walnuts are one of the healthiest hard-shelled fruits. As such, we can

consider walnuts as foods enriched with many nutrients that have
health benefits. It is also one of the foods with the lowest glycemic
index (Şen, 2017). Generally in walnuts; It is 3.5% water, 15-30%
protein, 55-77% oil, 1.5-3% ash. It is rich in mineral substances such
as Ca, P, Mg, Fe, Na, K. It also contains vitamins A, B1, B2, B6, C
(Bayazıt and Sümbül, 2012). Benefits of walnut ın human health due
to the effect of omega 3 fatty acids It has been determined that it
reduces the risk of coronary heart disease, reduces triglyceride and
cholesterol levels, prevents blood clotting, prevents the development
of inflammatory diseases (Banel and Hu 2009). Due to the high
content of silver and selenium, it has a positive effect on intelligence.
Walnuts can be consumed in many different ways. It is used as a
snack, in the pastry and biscuit industry, in the perfume industry, in
the production of jam, halva, paint, tannin, in the plastic and rubber
industry, as oil, in the pharmaceutical industry, in the production of
sausage and fruit pulp (Sütyemez, 2014). Juglans regia is the only
species whose fruit is grown for consumption. Diversity within this
species is low and varieties are mostly from natural selection (Şen,
2017).

Although the high economic worth of the walnut and its deep-rooted
history, reproduction is limited, especially in regions that are the gene
center of the product, compared to other warm regions. The first
walnut breeding plans rely on history the middle of the twentieth
century. (Bernard et al. 2018). The genetic development of the walnut
252 | W a l n u t

was previously carried out by selecting individuals with superior

characteristics from natural populations. The main goal is to increase
the yield and quality (large fruit, high kernel weight, light colored
kernel) in walnuts, as in other fruit types.

With the acceleration of developments in plant breeding, food outturn

was also positively affected and played an important role in human
nutrition (Tester and Langridge 2010). This has also reduced
variability in agricultural crops around the world and contributed to
genetic makeup against adverse environmental conditions (Keneni et
al. 2012). It is therefore significant to recognize the effects of modern
plant breeding on genetic variation. At the same time, the skillful
command of this genetic variation will provide valuable assistance to
producers. In walnut production, adverse environmental conditions
(such as drought, salt and spring frosts) negatively affect walnut yield
in many regions(Cochard et al., 2002; Lotfi et al., 2009a, b;
Khodadadi et al., 2016; Jinagool et al., 2018; Knipfer et al., 2018; Liu
et al., 2019). One of the very significant environmental factors
limiting walnut productivity in arid and semi-arid regions where
walnuts are grown is thirst (Aleta et al., 2009; Vahdati et al., 2009;
Famula et al., 2019). In addition, increased drought due to global
climate change increases severe drought circumstances in walnut-
growing regions (Lotfi et al., 2010; Karimi et al., 2018). Drought,
especially during the growing period, can seriously affect walnut
development and become one of the most important factors restricts
product (Vahdati et al., 2009; Liu et al., 2019). Another important
 W a l n u t | 253

factor affecting the yield in walnuts is that dichogamy is very

common. The fact that male and female flowers open and become
active at different times is a factor that negatively affects pollination-
fertilization success and reduces yield (Sütyemez, 2014).

Walnut has been studied a lot all over the world because it is a
valuable plant in terms of both economic value and nutrition. With the
rapidly developing technology, traditional breeding studies have
begun to give way to molecular breeding studies. In this section,
traditional and molecular breeding methods, advantages and
disadvantages of walnut will be explained.

2. CONVENTIONAL BREEDING IN WALNUT

For an economical and standard production, the products obtained

from seeds, that is, the trees grown from seeds, cause very important
time and economic losses due to quality, yield and standard
differences. In order to eliminate this negative situation of the walnut
potential, walnut trees grown from seed should be removed and
gardens should be established with grafted saplings of certain
varieties. In addition to a very important time loss, this will cause a
loss of yield (production) with the removal of many yielding trees.
Plants that occur in propagation with seeds do not carry almost any
characteristic of the mother plant (they show genetic variation). For
this reason, seed propagation in walnuts is not a preferred propagation
method, except for plants obtained by rootstock or seed and breeding
studies (Sütyemez, 2014)
254 | W a l n u t

The beginning of agriculture dates back about 10,000 years, and since
this time people have begun to adapt plants for their own purposes. In
addition, naturally occurring beneficial traits were often transferred to
different plants by human hands, as opposed to natural selection
(Evenson end Gollin 2003). Genetic variation is studied from several
aspects like molecular, phenotype and biochemical traits (Govindaraj
et al., 2015; Guney et al., 2021a). Genetic variation in walnuts is
determined by using phenotypic and biochemical prosses by
determining the information in the gene and selecting superior
genotypes with covetable characteristics (Bhat and Kudesia, 2011,
Guney et al., 2021b).

Genetic variation is generally determined using morphological

markers. Germplasm characterization and identification of varieties as
the first step in genetic research, breeding programs and selection
studies form the basis (Smith and Smith 1989). Toward this end, the
International Union for the Conservation of New Plant Varieties
(UPOV) determines principles for defining the diversity, purity, and
constancy in the establishment of novel cultivars for most plants.
Therefore, morphological research guides the selection of plants
suitable for certain growing situations (Solar and Stampar 2011). In J.
regia, first morphological research are related to the identification of
the association between fruits with different plant characteristics
(Sholokhov 1974; Komanich 1980; Şen 1985; Sharma 1996).
Although there was an inverse relationship between yield and
dichogamy (Kornienko 1974; Majacka 1971), no distinction could be
 W a l n u t | 255

determined between yield and fruit quality and protogynous and

protandry characteristics. (Akça and Sen 1997). In a subsequent study,
a correlation was found between fruit internal weight and flowering
shape (Eskandari et al. 2005). Additionally, Amiri et al.(2010)
determined that there is a powerful correlation among lateral branch
yield and fruit yield in Iranian cultivars. On the other hand, in a study
conducted in Slovenia, walnuts with high minor yield were found to
be more sensitive to blight and cold (Solar et al. 2001) and sensitive to
walnut blight was found to be associated with the date of defoliation
(Abedi and Parvaneh 2016). Also, a negative relationship was noted
between yield and tree height by Forde and McGranahan (1996), and
while there was a positive correlation across altitude and frost
resistance, a negative correlation was found between altitude and yield
(Atefi, 1990). In countries such as Iran, it has been determined that
morphological studies and selection studies in natural populations will
help breeding programs (Ghasemi et al. 2012). The characteristics of
these natural populations inclusive genotypes, leafing date, side
branch yield, dichogamy were determined phenologically and
phonologically (Ghasemi et al. 2012; Ebrahimi et al. 2015), bud burst
(Arzani et al. 2008), tolerance to late spring frosts (Mahmoodi et al.
2016), fruit yield, fruit maturity, and fruit features (Haghjooyan et al.
2005; Arzani et al. 2008; Ahandani et al. 2014; Khadivi-Khub et al.
2015). In addition, the vigor of dwarf seedlings to increase planting
frequency in Iran was also investigated (Rezaee et al. 2009). It was
determined that there was a favorable correlation across the
parameters such as seedling diameter and seedling size, number of
256 | W a l n u t

nodes and seedlings and length of internodes, proposing that

measuring of seedling height alone is enough for recognition of dwarf
genotypes, also, parallel researchs were applied in Turkey (Bayazit
2012). Excellent genotypes were chosen for yield, nut features, and
cold resistance (Aslantaş, 2006) and other hopeful genotypes were
chosen for late flowering, walnut blight and anthracnose resistant
(Yarilgac et al. 2001; Asma 2012; Fikret Balta et al. 2007; Karadag
and Akça 2011). Assessment of pomological and phenological
characteristics of natural populations in the Dibra area in Albania
determined a high variability in fruit weight (Zeneli et al. 2005).
Walnut varieties have been assessment in many such studies in Serbia
(Miletic et al. 2003) and additionally some foreign variety in the
climatic circumstances of South Bulgaria (Gandev and Dzhuvinov
2015). In the study conducted in India, genetic and geographical
diversity were examined using 15 fruits (Sharma and Sharma 2001).
In a research conducted in a garden created from seeds under the same
conditions, it was determined that the population created with the
genotypes selected in Italy to conserve genetic resources had as much
mutable as the variation in a larger population (Ducci et al. 2010). In
France, 2450 hybrids from 22 crosses across French and California
cultivars were sourched. The leafing dates of the same trees at the age
of 1 and a few years later were examined and showed that the feature
related to the leafing date can be determined when the plants are at an
early age (Germain 1990). In another study, proportion of apomixis in
walnuts was examined and differences were determined between
 W a l n u t | 257

varieties grown in many European and Mediterranean (Asadian and

Pieber 2005).

The Šeinovo cultivar and the Rasna selection were used as the results
of the examination of 20 walnut genotypes selected from Montenegro
over a three-year period. According to the international walnut
descriptor, the most significant biological and pomological features
were examined. Basic criteria in selection studies: late vegetation
onset, early vegetation end date, kernel internal ratio, easy separation
of the fruit from the skin, peel roughness, full kernel yield, delicious
fruit, light colored kernel and chemical composition of the fruit. It has
been determined that walnut has a successful adaptation in this region
and is well adapted to the agro-ecological conditions in this region
(Jacimovic et al. 2020).

2.1. Mistakes Made in Conventional Walnut Breeding

Traditional breeding practices are basically based on selection of

individuals with superior characteristics, namely sectioning. However,
it should not be forgotten that traditional breeding practices can be
done correctly and that these selected individuals will contribute to
modern breeding programs. Errors made in traditional breeding
practices should be well known and applied correctly (Sütyemez,
2000; Şen,1985 ) The main mistakes made are:

• Establishment of new gardens in unsuitable climate and soil

conditions
• Using plants that are healthy and not true to their name,
258 | W a l n u t

• Producers who do not have the necessary knowledge in terms of

fertilization biology, use more or less pollinator varieties or
plant wrong pollinator varieties.
• Failure to plan the trees to be planted during planting,
• Not being done in accordance with the planting technique.
• Not choosing a pollinator based on flowering characteristics of
the main variety

Walnut growers have long set up new orchards with the seeds of the
fruits they have selected, resulting in high genetic variety in walnuts
around the world. Following improvement of inoculation techniques
and walnut breeding in France and California, walnut variety were
presented. Also, outside of France and California, new walnut
cultivars have been presented using local genetic variation and
hybridization in other countries as well. Some French/Californian
cultivars are produced global. Franquette is the first of these
varieties(Germain 1999; Tulecke and McGranahan 1994).

Due to the fact that it has a long youth sterility, the reproduction and
fruiting period of walnuts is long. For the use of walnuts in breeding,
their varieties must be known. On the other hand, the morphological,
biochemical and physiological practies applied in the identification of
varieties are methods that require a long time and are affected with
environmental conditions (Topçu et al. 2015). All these features are
affected by three situations: genetic substructure, environment, and
interplay across the two; it is of highest significance to explained the
amount of add of each of these ingredients.
 W a l n u t | 259

3. MODERN BREEDİNG İN WALNUT

The products obtained from breeding programs are highly dependent

on the existence of genetic variation, either spontaneously or by
external intervention. In order to increase and maintain food
production, plant breeders and geneticists are constantly working to
develop new strategies and to create a food source with plants adapted
to infertile soils and tolerant to adverse environmental conditions. In
conventional breeding, the transfer of genetic information from one
species to another is done by backcrossing over several generations.
(Al-Khayri et al. 2019). With the improvement of molecular tools
along with new technologies, it has become easier to determine
whether a plant has a trait by using molecular markers, and the
breeding period has been shortened (Arzani et al.,2008; van Nocker
and Gardiner, 2014). With the sustained development of plant
biotechnology, genomic information and molecular markers. They
have become excellent tools in the formation, analysis and
identification of genetic change for the improving of new varieties
(Bernard et al. 2018).

The genetic development of walnuts began with the selection of

superior trees in their main origins. Selection and grafting methods,
among the methods used in traditional breeding studies, first started in
France (Vahdati et al. 2019). The first walnut breeding program using
hybrid plants began in the USA in 1948 with the introduction of
french varieties and using local genotypes selected from seeds
obtained from centers of origin (Iran, Afghanistan, China). Currently,
260 | W a l n u t

both conventional methods with phenotypic evaluation and molecular

initiatives are employ in breeding studies conducted in the USA,
France, China, Iran, Spain and Italy. With the recent developments in
biotechnology and genomics and variety development studies,
breeding programs are also accelerating. Together with these
developments, the research, identification and protection of biological
diversity will enable the creation of a gene bank with the desired
characteristics and the more accurate and faster work in the future by
biotechnological means (Khorami et al., 2018).

4. MOLECULAR BREEDİNG İN WALNUT

The latest technologies used in breeding studies in plants using

biotechnological methods: next generation sequencing (NGS)
techniques, bioinformatics tools, high-throughput genotyping
platforms and genome-wide association studies (GWAS), marker
assisted selection (MAS), genomic selection (GS), and CRISPR- Cas9
ıt includes genome editing with the system. We explain the
application of molecular breeding and biotechnological methods used
in walnut breeding ( Iwata et al. 2016; Laurens et al. 2018).

It is the long-lasting juvenile phase and highly heterozygous nature

that affects the genetic development of walnuts In order to cope with
the problems in traditional breeding methods, the use of new genomic
technologies in molecular breeding methods in walnut has gained
importance. Molecular marker systems used in walnut breeding and
all nuclear and chloroplast genome sequence information, QTL
 W a l n u t | 261

mapping, GWAS and GS, MAS studies will all be explained under the
name of molecular breeding methods.

4.1. Markers in Walnut Breeding

With the development of enzyme-based markers through isoenzyme

electrophoresis, a major breakthrough in the late 1980s, the weak
genetic information in plants has begun to come to light (Hunter and
Markert 1957). Isozyme markers were widely used molecular markers
in genetic studies until PCR-based markers were developed. Isozyme
markers have different uses in walnut genetic analysis, such as
identification of cross-species, genetic diversity, and determination of
the evolutionary process of the genotype (Aletà et al. 1990; Arulsekar
et al. 1986; Busov et al. 2002; Cheng and Yang 1987; Fornari et al.
2001; Malvolti et al. 1993, 1994; McGranahan et al. 1986; Ninot and
Aletà 2003; Solar et al. 1994; Vyas et al. 2003).

The isozyme gene phosphoglucomutase found in J. regia was first

identified and characterized by Arulsekar et al. (1986). In subsequent
studies, hybrids make between Pterocarya spp and J. regia were
defined by isozyme differenties (McGranahan et al. 1986) and the
source of varied walnut types in China and Tibet was investigated
(Cheng and Yang 1987). Malvolti et al. (1993) also studied genetic
diversity in J. regia populations in Italy and In a study comparing
pollen enzymes and leaves in Slovenia, it was determined that there
was more variability in pollen enzymes than in leaves (Solar et al.
1994). Furthermore, a study of leaf and fruit morphology and isozyme
262 | W a l n u t

variation in populations of J. regia from central Italy demonstrated an

special relation among genotype and leaf morphology and among leaf
morphology and fruit morphology. In these two studies, it determines
that there is a causal relationship between genotype and phenotype
(Malvolti et al. 1994). In many different studies, isozymes were utilize
to determine genetic variation / correlation in J. regia / J. nigra
analysis (Fornari et al. 2001; Ninot and Aletà 2003; Busov et al.
2002), or for cultivar determination (Aletà et al. 1990; Vyas et al.
2003).

4.2. Hybridization-Based Markers

4.2.1. Restriction Fragment Length Polymorphism (RFLP)

Molecular identification methods create a precious and effective

technique to scan germplasm collections and ease the utilization of
these source by breeders and investigators (Mason et al. 2015). Two
methods was used to define the source of the somatic embryos of the
ovule in the J. regia (isosyme and RFLP) (Botstein et al. 1980; Aly et
al. 1992). Parentage testing was performed quickly on plants
propagated in vitro. Leter, a California group carried out three studies
in 1994 utilizating with RFLPs. First utilization, the double-group
method with arithmetic mean (UPGMA) cluster analysis (Nei, 1972)
describe that the California germplasm was interrelated that of France
and Iran and had less resemblance to Nepal, China, and Japan
(Fjellstrom et al. 1994). [J. Hindsii × J. regia] × J. regia was
performed to interspecies backcross analysis, generating the first
linkage map of 12 linkage groups (LGs) on 1660 cM in length.
 W a l n u t | 263

(Fjellstrom and Parfitt 1994a). The taxonomic correlation between

varied Juglans species were studied (Fjellstrom and Parfitt 1994b) and
it was determined that J. cinerea does not belong to the Cardiocaryon
division, as stated in the literature, but should belong to the
Trachycaryon division (Manning 1978).

The biggest disadvantages of the RFLP method are the low rate of
polymorphism, the difficulty of using it (Wani et al. 2010), deficiency
of automation and the need for the more quantit of pure DNA
(Schlötterer 2004). Therefore, novel system rely on polymerase chain
reaction (PCR) were advanced.

4.3. PCR-Based Markers for Walnut

4.3.1. Random Amplified Polymorphic DNA (RAPD)

A working group in California found RAPD' markers using a

population of hybrids. [J. hindsii × J. regia] × J. regia and a revised
walnut genetic map including previously published (by Fjellstrom and
Parfitt, 1994a) RFLP markers. This study determined the number of
LG groups as 15, not 12, but a total of 16 LG groups, which should be
in walnut, could not be established (Woeste et al. 1996b). Other
studies by the same team defined and characterized a RAPD marker
related to a hypersensitivity reply to cherry leaf curl virus, black streak
disease in walnuts. (Woeste et al. 1996a). In other walnut hybrid
population {[J. hindsii × J. regia] × J. regia} during the transfer of
genetic information to hybrid plants, a little correlation was
determined among genetic and morphological interval (Woeste et al.
264 | W a l n u t

1998), Morfolojiye dayalı seleksiyon, bir türden diğerine genetik

bilginin aktarılması sırasında ebeveyn genomunun korunmasında
etkili bir yöntem olmadığı ileri sürülmüştür. RAPD markers have also
been used in plants used in a breeding program at the University of
California, including BPayne, BMeylan, and BFranquette, to study
genetic relationships between J. regia cultivars. (Nicese et al. 1998).

4.3.2. Sequence Characterized Amplified Region (SCAR)

RAPD markers are less reliable in plant breeding due to their low
repeatable, but they are simple to improve and easy to use. However,
it has the possibility to convert RAPD amplicons into sequence-
characterized amplified region (SCAR) markers to improve their
reusability. Several recent research have defined molecular markers
related to economically significant features in walnuts, inclusive a
RAPD marker associated with earliness (Keqiang et al. 2002), a
SCAR marker linked to hell thickness (Li et al. 2007) and a SCAR
marker associated with earliness (Li et al. 2010).

4.3.3. Inter-Simple Sequence Repeat (ISSR)

ISSRs are markers with a single long oligonucleotide primer stable at

both at the tip with a simple sequence repeat (Godwin et al. 1997).
ISSR markers have used in walnut genetic diversity experiments,
phylogenetic analyzes and many other genetic experiments
(Christopoulos et al. 2010; Ji et al. 2014; Pollegioni et al. 2003; Potter
et al. 2002), genetic mapping creating (Malvolti et al. 2001) and
examination to define the provenance of a genotype (Malvolti et al.
 W a l n u t | 265

2010). Altogether, the RAPD and ISSR markers have been frequently
employed by scientists working in walnuts for the description of the
regional Juglans regia germplasm from Turkish, Iranian, Romanian,
Greek, and Chinese. (Fatahi et al. 2010; Christopoulos et al. 2010; Pop
et al. 2010; Qianwen et al. 2010; Erturk and Dalkilic 2011; Ji et al.
2014;).

4.3.4. Amplified Fragment Length Polymorphism (AFLP)

Amplified fragment length polymorphism markers (AFLPs) in walnut

(Vos et al., 1995) have been applied for different purposes in many
genetic studies. AFLP markers, like RAPDs, are often sequenced
using fragments to create SCAR markers (Lecouls et al. 2004). AFLPs
were search to examine the genetic diversity of Xanthomonas
arboricola pv juglandis, most significant diseases of J. regia reason
walnut blight (Loreti et al. 2001). In this study, 66 isolates of X.
arboricola pv juglandis from varied countries were describe. The
same AFLP markers were application to determine the varied in host-
associated carob moth (Ectomyelois ceratoniae) populations in
pomegranates, pistachio and walnuts in Iran (Mozaffarian et al. 2008).
Sütyemez (2006) used AFLP markers for the first time to study
genetic variation in walnuts. AFLP markers are used to determine
superior genotypes in walnut genetic studies (Kafkas et al. 2005), to
reveal genetic variation (Ali et al. 2016; Bayazıt et al. 2007; Qing Guo
et al. 2010), to determine the evolutionary process (Wang et al. 2010)
and for the identification of walnut variety (He et al. 2010; Ma et al.
2011; Xu et al. 2012). A group of researchers used AFLP markers to
266 | W a l n u t

identify genetic correlations in wild J. regia and J. sigillata

populations of economic value from Sichuan province in China (Chen
et al. 2008, 2009).

4.3.5. Simple Sequence Repeat (SSR) or Microsatellite

Known simple sequence repeats (SSRs, Microsatellites) (Morgante

and Olivieri 1993) are much stronger and enlightening markers for
fingerprinting analysis and determination of genetic variety, genetic
mapping, population structure and marker assisted selection (MAS)
in plants due to their abundance, dominance and high repetitivable and
high polymorphism in the genome (Wani et al. 2010). SSRs have the
very commonly utilization markers for marker assisted breeding for
fruit trees and crop improvement in many plants over for 10 years
(Bernard et al. 2018; Singh et al. 2008).
Due to the unique properties of SSR markers, walnuts have become
the preferred markers for many utilization in genetic research and
breeding. In walnut cultivation, SSRs are employed to assess the
genetic variety of the resulting property before selecting parent
property that will aid maximal this variation for subsequently ideal
integrations. SSRs have been used in walnut breeding genetic studies
for many diverse goals: Fingerprint clones with quality timber in
Juglans nigra (Woeste et al. 2002), determination of genetic variety
(Gunn et al. 2010; Victory et al. 2006; Wang et al. 2008 Pop et al.
2013) F1 recognition (Pollegioni et al. 2008) and parental recognition
(Pollegioni et al. 2009; Robichaud et al. 2006).
 W a l n u t | 267

Genetic variety was determined in J. regia and J. sigillata populations

using SSR primers developed from J. nigra, and the UPGMA
dendrogram analysis indicated a different geographic trend from the
known taxonomic sorting (Wang et al. 2008). In various studies that
studied the genetic variation of J. regia and J. sigillata in Yunnan,
China, using SSR markers, humans effects and role in biodiversity of
walnut were investigated (Gunn et al. 2010). These markers
determined that the two species were not distinguishable, and the
researchers reached two different conclusions: that J. regia and J.
sigillata could be new species or different species in the gene flow (Qi
et al. 2011).

Other Juglans species, like native populations of J. mandshurica in

northern and northeastern China, were examined using SSRs (Bai et
al. 2010). Results, comprising cpDNA fragments, determined that two
unrelated sites were preserved in northern China the last ice age, in
contrast to many another moderate forest trees that were taken to
southern China. Novel ESTs from J. regia were studied in J. nigra
and Carya spp strains and the high polymorphism display of these
EST markers was found to be transferable across the two strains.
(Zhang et al. 2013). In conclusion, naturally occurring individuals of
J. nigra, J. major and J. microcarpa in Texas were examined using
SSR markers and it was determined that interspecies hybridity
happened in these trees (Grauke et al. 2012). In J. regia, an significant
Italian variety, Sorrento, DNA fingerprinting analyzes were
determined using SSRs (Foroni et al. 2005, 2007), In addition to
268 | W a l n u t

different Italian walnut sources, SSR markers were employ to

determine and identify other varieties obtained by selection studies
(Pollegioni et al. 2011). In subsequent studies, SSR markers were
employed to examinate genetic differences using Iranian genotypes
(Karimi et al. 2010; Mohsenipoor et al. 2010; Ebrahimi et al. 2011;
Mahmoodi et al. 2013; Najafi et al. 2014). Spanish and US cultivars
were determined to differ from California cultivars using SSR markers
(Ruiz- Garcia et al. 2011). In addition to examinate utilization SSRs,
differences and genetic correlations between Indian genotypes have
been investigated (Noor Shah et al. 2016), between trees from cold
fields of the USA and Europe(Ebrahimi et al. 2017), and between
genetic resource in China (Han et al. 2016; Wang et al. 2016) and
Romania (Pop et al. 2013).

The recent years, three recent papers have been published. First, a
high-throughput multiplex PCR protocol was developed using 11 SSR
markers improved in J. nigra to examine the differences in Croatian
genetic sources(Ćelepirović et al. 2016). Another study concerned
populations of J. regias naturally growing in the Italian Alps with
genetic variation (Vischi et al. 2017). Bacterial artificial chromosome
(BAC) end sequences in J. regia were employed to develop novel
SSR markers (Wu et al. 2012; Ikhsan et al. 2016), SSR markers have
been developed to increase the number of specific alleles from
genomic DNA libraries with repeat regions like 'BAAC' or
'BAAG'(Topçu et al. 2015), and EST sequences for the transcriptomes
of different tissue samples (Dang et al. 2016). Polymorphic SSR
 W a l n u t | 269

markers were developed from Class I repeats from Chandler's DNA

sequences. Of the 16 ceviche varieties (8 Turkish, 3 French and 5 US),
800 SSRs were designed and tested, 88 (11%) non-band producing,
161 (20.1%) monomorphic and 551 (68.9%) polymorphic (Eser et al.
2018).

The phenological and morphological properties of the material

"Esterhazy II" in the Hungarian gene center were compared with those
of "Milotai 10" and "Chandler". Viewed characters; budding,
flowering time, variety of polygamy, ripening time, kernel and nut
characteristics. SSRs were used in fingerprint analysis to identify the
same varieties and to determine the association of the analyzed
“Esterhazy II” genotype with different Hungarian cultivars. As a
result, it was determined that the genotype known as "Esterhazy II"
could be observed in several different cultivars (Bujdoso et al. 2021).
All these used markers could potentially be utilization for germplasm
characterization and generation of connectivity maps in walnuts

4.4. High-Throughput SNP Assays in Walnut

Single nucleotide polymorphisms (SNPs) (Wang et al., 1998) have

many superiors over previously found markers because they are
Genotyping by Sequencing (GBS) markers for automated high-
throughput analysis in the genome in large quantities and currently at
moderate cost (Schlötterer 2004). The first utilize of SNP markers to
identify J. regia variants (Ciarmiello et al., 2011) used the 5.8S coding
region of rDNA-repeat (ribosomal DNA) the operon segment
270 | W a l n u t

containing and duplicated spacer regions (ITS1 and ITS2) the first and
second. 244 SNPs and one short insertion-deletion (InDel) were
obtained by aligning the sequences. It was determined that these
regions could be used in phylogenetic analyzes to characterize of J.
regia. It also allowed the development of a genome-wide
infrastructure from coded sequences to discover SNPs. 22.799 SNPs
were discovered at Chandler and 6000 SNPs were selected to
determine the genetic makeup of a mapping population (You et al.
2012). Subsequently, 48,165 SNPs and 1037 InDels were identfied in
J. regia by local genome pyrosequencing (Liao et al. 2014). It has
been stated that these SNP markers will be of great benefit for genetic
variation analysis, determination of nucleotide differences in the
genome, formation of genetic maps and another breeding practices
like marker assisted selection. The causative agent of thousand
cankers disease in the USA, Geosmithia morbida, was also examined
utilizing SNP markers of 209 isolates in a population genetic research
(Zerillo et al. 2014). Eventually, a study with SNPs evaluated syntenic
across walnut and some other plants. These plants contained three
long-lived perennials (Vitis vinifera, Populus trichocarpa, and Malus
domestica) and three short-lived plants (Cucumis sativus, Medicago
truncatula, and Fragaria vesca). As a result, the walnut genome was
found to be closer to the genome of three of the woody perennial
plants (Luo et al. 2015).

Recent progresses in next-generation sequencing tools and the

sustained reduce in expense have conculuded in the huge lineage of
 W a l n u t | 271

sequence information sets, leting the fast exploration of single

nucleotide polymorphisms (SNPs). SNPs deliver along the genome
and are extencively employed in animal and human genetic study,
however their utilization in plants, and especially in walnut, is in the
early phases. So far, the last presence of genomic and transcriptomic
databases has accomplish feasible the exploration of SNPs in silico,
utilization bioinformatics mediums. A walnut genome sequence (cv.
Chandler v1.0) has recently been discovered (Martínez-García et al.
2016; presenting at https://www.hardwoodgenomics.org/english-
walnut-genome), 27 genomes of most significant constitutive in the
walnut recovery program at the UC Davis have been resequenced
and,at last, a new Axiom® Walnut700K SNP array has been planned
by UC Davis for walnut changeability, linkage mapping and related
mapping analysis (Marrano et al. 2019; Neale et al. 2017). The
attendance of a high-density genotyping sequence in walnut lights
novel chances to perform GWAS and genomic choice in walnut-
breeding performs and different walnut populations (Bernard et al.
2018; Marrano et al. 2019; Neale et al. 2017).

4.5. Physical Mapping in Walnut

The physical map demonstrates the physical interval across loci. The
copy of exogenous DNA in bacterial artificial chromosomes (BACs)
is a novel process for genome investigation (Choi and Wing 2000). To
create physical map in walnut, it was amplified and isolated in vitro
from Persian walnut (Chandler), and bacterial artificial chromosome
(BAC) two libraries were generated from the isolated DNA and
272 | W a l n u t

digested with HindIII or MboI endonucleases. Bases of 135 kb and

120 kb in length were added to the DNAs cut with HindIII and MboI
endonucleases, respectively. A whole of 129,024 copies, 64,512 per
BAC library, were prepared at 336 plates (Wu et al. 2012). Presuming
walnut genome length of near 606 Mb, the two generated BAC
libraries expressed almost 27x genome equal. In research,
fluorescence-based upon BAC identifying individuals and BAC-end
sequencing were applied utilization a high-throughput BAC DNA
identifying individuals technique (Luo et al. 2003). A total of 52,840
BAC copies off the (Chandler) HindIII and MboI libraries from
Iranian walnut were sequenced and 48,218 walnut BESs were
uploaded to GenBank (Wu et al. 2012).

As a result of Blast2GO, 1330 matchless GO terms were identified in

6396 BESs. Dispersion of GO terms in the class of biological way,
molecular process, and cellular ingredient indicated that the walnut
genes contain a wide field of functional sections and biological
transaction. Thanks to putting in order of BES with ESTs and all
walnut genome shotgun sequences. Nearly 4000 SNPs were explored
and to create genetic mapp in individuals of cvs. Chandler × Idaho,
which finally caused anchoring of BAC contigs upon a linkage map.
Physical mapping in walnut yielded two markers containing the LB1
locus related to the lateral bed. Lately, a 2-year program at the
University of California, Davis was begun to explore the initiative
mutation at the LB1 loci, improve an estimated SNP marker, and
apply the marker in walnut genetic recovery. Over the first stage of
 W a l n u t | 273

this program, on 700 progenies off an inbred cv Chandler population

were defined that bear recombination in the LB1 area. Front outcomes
from this research put forward three LB1 nominee gene (Dvorak et al.
2015).

The study was applied to appraise 25 excellent walnut varieties in

terms of phenotypic and cytological features. To this end, 560 walnut
varieties in southwest Iran were examined according to UPOV and
IPGRI descriptor. Later a 2-year first evaluation, 25 excellent
genotypes were chosen for phenotypic and genome dimensions
evaluated. Flow cytometry was utilized to evaluation the genome size
of the chosen excellent varieties. High genetic variety existed in the
walnut population gathered off the southwest of Iran. The chosen
excellent cultivars had very yielded high lateral branch yield, shell
thickness, high kernel ratio and seed weight, and a light interior color
that could be easily separated from the Shell. Therefore, genome size
can be take into account as a powerful and precious tool to forecast
nut size and kernel and nut weight (Sarikhani Khorami et al. 2018).

4.6. Genetic Maps and QTL in Walnut

Genetic map: the basis of distinction of markers in the course of

meiosis, indicating the location and relative genetic intervals among
markers throughout the chromosomes occurs chromosome
recombination (Paterson 1996). Locating genes or QTLs related to
relevant traits in genetic maps is the first step. Genetic maps are
important for breeding studies and are of great benefit for marker-
274 | W a l n u t

assisted selection (MAS) (Semagn et al. 2006). Intraspecies and

interspecies populations were established to set up genetic maps and
to identify QTL regions in walnuts.

The initial genetic map in walnut was composed with the utilization of
RFLP markers. Offspring of 63 individuals from a Next-Generation
Mapping between different species backcross of [J. hindsii × J. regia]
×J. regia was examined. These thriving in a J. regia Hartley orchard
open-pollinated male-sterile J. hindsii × J. regia trees were formed
from backcross plant (Paradox Mom) Forty-two RFLP markers were
located in the 12 LG groups, and the genome size was predicted at
1660 cM, inclusive the contain of the walnut genetic map with 66
RAPDs. (Fjellstrom and Parfitt 1994a). A new and more advanced
genetic map was created that includes these RAPDs and previously
identified RFLP markers. This new map consists of 15 LG groups and
one hundred and seven markers (Woeste et al. 1996b). The
inbreeding cross of J. regia was investigate with both RAPDs and
isozymes. A population of 81 F1s from the J. regia BLara 480^ and J.
regia Chandler 1036^ cross was used with 120 RAPDs and four
isozymes. (Malvolti et al. 2001). Parental maps were composed with
LG groups 11 in the female map and 10 in the male map.

Due to the lack of existing information in the walnut literature, it was

targeted to create the first SSR-based genetic linkage map with the
'Chandler' × 'Kaplan 86' F1 crosses and to determine the QTL for
leafing time in walnuts. Of the 1437 SSR primer pairs, 386 SSR
markers were located in 16 LGs. The overall length of the consensus
 W a l n u t | 275

map was 1568.2 cM, with a mean length of 98.0 cM. The Chandler
linkage map contained 279 SSR markers with a total length of 1285.8
cM, while the Kaplan-86 genetic map included 273 SSRs with a total
length of 1574.4 cM. As a result, it was published as a linkage map
that was created based on the SSR marker, with medium density and
could be considered as a basis for genetic and molecular investigations
in J.regia and other species. A QTL region was determined for
foliation time, which express 52.0-68.8% of the phenotypic diversity
in both parental and consensus maps. It has been stated that this
defined QTL region can be used in walnut breeding studies in the
coming ( Kefayati et al. 2019).

4.7. Next-generation mapping

A 6K SNP Select Infinium BeadChip was created (You et al. 2012)

and utilizated, in the last four years, to genotype an F1 offspring of
425 plants from an intraspecific cross between ‘Chandler’ and ‘Idaho’
(J. regia) Number of all SNP markers placed on the map 1525 SNP in
16 LGs and the length of the genetic map was determined as 1049.5
cM (Luo et al. 2015). The available genetic map was employed to
establish a walnut physical map by 15,203 exonic BAC data. The
physical map is near to the approximative length of genome, 606 Mb,
by a predicted total size of 736.1 Mb and symbolists the general of the
genome. (Luo et al. 2015). From the mapping study, two markers
were also found to locate the LB1 gene including in lateral bearing
phenotype on LG11. (Dvorak et al. 2015). As a result, an F1
population of 84 individuals was generated with an in-species cross
276 | W a l n u t

across ‘Yuan Lin’ and ‘BQing Lin’ (J. regia). 2577 specific locus
amplified fragment (SLAF) markers were made use of in three types
(just 2300 SNPs (89.25% ), 87 InDels, 190 SNPs and InDel). These
markers were located on 16 LGs and the size of the resulting map was
determined as 2457.82 cM. (Zhu et al. 2015). In addition, a QTL
region related to anthracnose (Colletotrichum gloeosporioides)
resistance, which is also a significant trait in Chinese plant varieties,
was determined in LG14. QTLs with 10 markers between 165.51 and
176.33 cM and LODs between 3.22 and 4.04 were determined in
LG14. Utilizing 14 polymorphic SSR markers, genetic structure and
distinction were appraised in the Iranian walnut germplasm collection
consisting of 204 individuals from the USDA germplasm collection
and a total of 399 individuals from 62 elite germplasms often utilized
in the walnut breeding program at UC Davis. This study supplies
precious knowledge on genetic variety and applies to resequence to
determine SNPs in the walnut genome and be assisted to define
diverse genotypes in these populations. In addition, molecular
variance (AMOVA) analysis disclosed ~87% of the diversity into
populations, by just 13% of important genetic change take into
account for diversity across groups (Dvorak et al. 2008).

In another study, single nucleotide polymorphisms (SNPs) were

developed with a comparison of diverse resources of data knowledge;
BAC end sequences, SOLiD shotgun genome data and RNAseq from
different walnut materials. These collations detected ~6000 SNPs that
were operates by Illumina to create an Infinium array. This SNPs were
 W a l n u t | 277

employed to compose the genetic map with examining 352 offspring

off a cv. Chandler × cv. Idaho progeny and these map have been
utilized to sequence any phenotypic feature to linkage groups (Dvorak
et al. 2011). Lateral bedding in walnut genetic breeding is one of the
main determinants of output and one of the most significant targets in
breeding. Martínez-García et al. (2014) in a project to develop a high-
throughput genotyping platform for lateral transport (KASPTM
genotyping) used molecular markers developed by Dvorak et al.
(2011). Allele-specific PCR (KASP) genotyping method was
employed in the research owing to its capability in genotypic
thousands of plants utilizing a little series markers with cheap and
loud correctness.

A genome-wide association study (GWAS) was made to identify

markers associated with leaf budding time and flowering characters.
The data of 13 distinct characters were recorded serial with 3 years of
data. The relationship between the variation in 188 walnut In the
study, using the DART-seq method for investigating the genetic
constitution with 14,761 SNP and 18,758 DArt markers, the
determination of the markers related to these characters was studied.
In conclusion, each of the 16 QTL regions of Major impacts was
determined to be related to at least two phenotypic traits. Of this QTL,
QTL05 had the most related features (seven). In the study, leaf
budding time and flowering characteristics were determined in
Juglans regia L. using the GWAS method, and it has a powerfull
278 | W a l n u t

possible to be practical effectively in walnut breeding studies (Bükücü

et al. 2020).

4.8. Walnut Genome Sequencing

With the development of high-throughput sequencing tools, rapid

progress has been made in the area of chloroplast genetics and
genomics. Today, the chloroplast genomes of approximately 800 plant
genomes have sequenced and their information has been uploaded to
the NCBI gene bank (Daniell et al. 2016). The knowledge obtained
from the chloroplast genome has advanced his perspective on all
issues related to the plant genome. Besides all this, the sequencing of
the chloroplast genome has enabled the identification of not only
sequences but also structural differences across plants and plant
species (Daniell et al. 2016; Hu et al. 2016, 2017b). Sequences of the
chloroplast genome contain important information to figure out the
adaptation of plants to adverse environmental conditions and to assist
in the reproduction of close related plant species (Daniell et al. 2016).
The first chloroplast genome sequencing study in walnut was done by
Liu et al (2012) carried out by. Illumina MiSeq tools was used for
sequencing and assembled using SPAdes and CpGAVAS programm,
respectively. As a result of sequencing, the long of the chloroplast was
found to be 160,367 bp, while the GC content was determined as
36.11%. In addition, they determined that 12 protein-coding genes, 14
tRNA and 8 rRNA genes were transcribed in the inverted repeat (IR)
regions from the study. In a later study, Hu et al. (2016) Phylogenetic
study of the walnut chloroplast genome by 11 chloroplast genomes off
 W a l n u t | 279

different species disclosed that the walnut is close assotiations with

the Fagaceae family and the Populus genus in by comparison other
species.

In recent years, chloroplast genomes of 5 species of juglans have been

sequenced to discover structural models of all chloroplast genomes to
define SSRs and to explore divergence hotspots and kinship
relationships. Phylogenetic analysis results strongly assistanced that 5
walnut species were divided into Juglans/Dioscaryon and
Cardiocaryon 2 divisions as previously determined. (Hu et al. 2017a,
b). In another study, wholl chloroplast genomes and 2 core DNA areas
of 10 symbolize taxonomic group of Juglans were utilize for kinship
relationships of the genus Juglans. It disclosed that 10 chloroplast
genomes have 112 genes, containing 78 protein-coding genes, 30
tRNA and 4 rRNA genes. Additionally, Juglans is divided into 3 arms
according to 2 nuclear DNA areas; Juglans, Cardiocaryon and
Rhysocaryon. (Dong et al. 2017).

The first draft genome of Juglans regia (Chandler) was performed

utilizing the Illumina sequencing tools and 500 million reads were
performed at 120x. The core genome of J. regia is 667 Mbp in long by
N50 scaffold size 464,955 bp, 221,640 contigs and 37% GC
ingredient. Nearly 1.2 million SNPs were explored in the outline
consensus sequence genome of J. regia (Martínez-García et al. 2016).
The genome assembly was made utilizing two diverse tools:
SOAPdenovo2 (Luo et al. 2012) and MaSuRCA (Zimin et al. 2013).
The discovery of the provincial walnut genome sequence (cv.
280 | W a l n u t

Chandler v1.0) supported subsequent walnut genome studies.

However, this first sequencing study was performed with short-read
sequencing.

The ultimate sequencing and optical mapping attempt have utilized to

tide over the restrictions of these technologies and develop the
property of the initial genome assembly. Initially, a another genome
sequence of Chandler was get utilized in the Oxford Nanopore
MinION sequencing tools, outcoming in approximately 7 million
reads and 35X genome comprise. The enhanced high-quality walnut
genome (cv. Chandler v2.0) will supply a precious genomic means for
walnut genetics and genome examinations (Neale et al. 2017). The
genomes of plants are generally formed by crossing two different
haplotypes in a heterozygous diploid. To conduct research on this,
Zhu et al. (2019) conducted a genome study by obtaining an occurring
between different species crosses of Juglans microcarpa and J. regia
cv Serr. Bu çalışmada, bu stratejinin, farklı türler arasında
melezlemelerin olduğu veya oluşturulabileceği herhangi bir
heterozigot ağaçta etkin bir şekilde uygulanabileceği gösterilmiştir.

Using the previously published walnut reference genome (Chandler

v2.0), Oxford Nanopore long-read sequencing was are assembled with
chromosome conformation capture (Hi-C) methods and assembled on
a new chromosome basis. Compared to the old genome, the new
splicing was found to have an 84-fold rising in the read at N50, with
16 chromosomal pseudomolecules and representing 95% of the total
length. Single-molecule real-time sequencing utilizing whole
 W a l n u t | 281

transcripts efficiency 37,554 gene patterns with upwards mean gene

long than former gene annotations. And then trialed the possible effect
of the novel chromosome-level genome on diverse fields of walnut
examinations.It was observed that the proteome from Chandler v2.0
available fewer artifacts on male flower growth than the prior to the
reference genome, making possible the recognition of a novel possible
pollen allergen. Generally, Chandler v2.0 will make the service a
worthy source to preferable figure out walnut genetics (Marrano et al.
2020).

Complete structural and functional genome annotations of 6 Juglans

species and 1 group from the Juglandaceae family, generated utilizing
the BRAKER2 semi-supervised gene platform, were studied. For
every annotation, gene give notice were sequence employ19 tissue-
private J. regia transcriptomes arranged to the genomes. Researchers
used this high-quality data to appraise the evolution of genes inside
Juglans and between Juglans and Eurosid species. Researchers studied
to determine remarkable conjunctions in some gene families in J.
hindsii, containing disease resistance-associated with wall-associated
kinase, Catharanthus roseus receptor-like kinase and another included
in abiotic stress reply. Eventually, he approved an old all-genome
copy that get placed in a joint ancestor of Juglandaceae utilization area
replacement compare analysis (Trouern-Trend et al. 2020).
282 | W a l n u t

4.9. Walnut Genome Resequencing

To determine allelic variants in J. regia genetic variation was

performed using sequencing and resequencing methods. In a genome
resequencing study menagemented at UC Davis, they aimed to
explore whole genome sequence changes in 27 participants in walnut
breeding programs. These genomic knowledge will be an significant
source for forward genetic and population examination to determine
alleles related with phenotypes in the walnut germplasm (Marrano et
al. 2019).

4.9.1. SNP exploration and investigate the genetic

constitution with SNP: After resequencing the walnut genome, the
BWA-MEN toolkit was utilizing to align the reads to the SNP and
reference genome, resulting in 17,800,528 SNPs, of which 609,658
SNPs were employed for the last sequence project process. (Marrano
et al. 2019).

4.9.2. SNP Genotyping This new Axiom® Walnut700K, It was

applied to a total of 1284 tree investigate the genetic constitution of in
the walnut breeding study at UC Davis, as well as 95 walnut tree
populations gathered from distinct areas of Iran (Arab et al. 2019;
Marrano et al. 2019). The outcomes indicated that a great part of
SNPs, not only for UC Davis but also for Persian walnut trees, join the
group of polyHigh Resolution (PHR) polymorphisms. The results
demonstrated that the Axiom® Walnut700K SNP sequence is an
 W a l n u t | 283

effective genomic instrument for GWAS examination and walnut

genetics studies (Marrano et al. 2019; Neale et al. 2017).

4.10. Genomics-Assisted Breeding in Walnut

The development of next-generation sequencing technics has enabled

the exploration of SNPs in fruits and vegetables, eased genome-wide
association studies (GWAS), and make possible marker-assisted
selection (MAS) and genomic selection (GS) Genome-wide
association studies (GWAS) happened crucial for the finding of
aplicant areas related to easy and complicated characters. Genomic
choosing moves forward and chooses genotypes that are forecasted to
be excellent rated on their genomic prediction reproductive values.
(Iwata et al. 2016; Laurens et al. 2018; Ru et al. 2015; van Nocker and
Gardiner 2014). They hinder the traditional genetic development of
fruit and nut trees due to their long juvenile sterility and heterozygotic
nature. Additionally, trees are also influenced by biotic and abiotic
conditions that make genetic development difficult (Rikkerink et al.
2007). By speeding up the reproductive period, shortening the juvenile
infertility cycle, and providing early selection with methods such as
GWAS and GS, genetic development can be accelerated. Therefore,
linked to the genomic paths are of great importance in increasing the
yield of genetic progress in fruit and nut trees (Ru et al. 2015).
284 | W a l n u t

4.11. Functional Genomics: Transcriptomics

Various genomic tools are utilized to investigate the transcriptome in

the course of the progress of plants or as a rely of harsh environmental
conditions. cDNA-amplified fragment length polymorphism (AFLP)
study by Bâaziz et al (2012) examined walnut leaves held in the light
and in the dark to define first molecular incidents in the course of
light-induced leaf hydraulic conductivity (Kleaf). The results of the
examination indicated that some of the transcript-derived fragments
(TDFs) acquired through cDNA-AFLP coincide to 57.9% of protein
yields and genes included in general metabolism, respectively.

The early structure of walnut ESTs in Juglans regia off seed coat
matters by Muir et al (2004) was delivered to the NCBI data bank
(https://www.ncbi.nlm.nih. gov). Other study, functional genomic
analysis to define the genes included in coactions across the walnut
root and the nematode was made at UC Davis. In this study, in firstly,
13,559 ESTs were created with data cDNA libraries supplied from
Pratylenchus vulnus and infected and uninfected walnut (J. hindsii ×
J. regia) leaves and roots (Britton et al. 2007). Secondly, it is made to
define and approve nematode and walnut genes related to the infection
study of gene expression across uninfected and infected plants.
Consequently, functional analysis of nematode genes was made
utilizing RNA interference in vitro and Medicago root analysis
(Britton et al. 2009). Besides in this study, 2733 P. vulnus genes and
8622 walnut genes were sequenced and exhibited valued knowledge
 W a l n u t | 285

on the ways included in the interplay across P. vulnus and a sensitive

walnut rootstock

Zhang et al. (2010) acquired a sum of 5025 ESTs for walnut off the
NCBI databank and utilizing to examine SSR motifs by the SSR
Hunter program. Afterward, a sum of 123 primer pairs was developed
of the non-redundant SSR-including unigenes. The efficacy of
candidate markers was researched by composing seven DNA pools
from walnut genotypes attained from varied regions. The results
disclosed that 41 SSR primer pairs with high polymorphism of the
amplified yields may be utilized for coming genetic investigating in
walnut.

In other study, 5213 EST data of walnut (Juglans regia) in NCBI were
employed for improving walnut EST-SSRs, and 207 SSRs were
getting from the EST sets. Total 7262 unigenes were getting from
13,559 ESTs received off the NCBI data bank and 309 EST-SSR
primers were casually arranged. Consequently, 13 very polymorphic
EST-SSR marker was employed for genetic investigation in Juglans
regia, J. nigra, Carya cathayensis, C. dabieshanensis and be in danger
species Annamocarya sinensis (Zhang et al. 2013). Furthermore, 40
polymorphic EST-SSRs were improve for J.regia by Zhao et al.
(2015). Latterly a few transcriptomes investigate have performed in
walnut which comprises expression of the transcription factor gene
JrCBF contained in cold reluctance contraption (Xu et al. 2014).
Additionally, expression of a great family of NBSLRR reluctance
genes in J. regia comprised in plant-microbe co-action (Chakraborty
286 | W a l n u t

et al. 2016) and transcriptome examine of buds, leaves, female flowers

and male flowers in J. regia was utilized to define novel EST-SSRs
(Dang et al. 2016). Furthermore, Li et al. (2018) performed a
comparison transcriptome examination of genes inclusive in
anthocyanin biosynthesis in leaf and peel color change in red and
green J. regia.

Phenylalanine ammonia lyase (PAL) is initial enzyme in the

phenylpropanoid pathway, critical in plant progress, conformation
and improving, however, few studies have recorded on the PAL gene
family in walnuts. In this study, researchers record genome-wide
investigation of J. regia PAL genes and examine their phylogeny,
replication, microRNA and transcriptional expression. Totally 12 PAL
genes were determined in the widespread walnut and group into two
subfamilies relyed on phylogenetic investigation. This widespread
walnut PALs are deployed on eight distinct pseudo-chromosomes.
Seven of the 12 PALs were exclusive determined in J. regia, and five
PALs gene were defined to be nearly related to the woody plant
Populus trichocarpa. In additional to the expression types of four
PALs gene indicated that they had up expression in female and male
flowers. The miRNA ath-miR830-5p regulates two genes, such that
they have down expression in the male and female flowers of the
widespreat walnut. Results of research, supplies beneficial knowledge
for upwards expression in the task of PAL genes in widespread walnut
and Juglans (Yan et al. 2019).
 W a l n u t | 287

In examination, well-analysed the evolutionary advancement of the

plants of the Juglandaceae are established related on both the core
RAD-Seq and the entire chloroplast genome seq. Results assistance
the Juglandoideae study of geometric properties (Hicoreae,
(Platycaryeae, Juglandeae)) at the popülation level. Inside of
Juglandeae, a incompatible location of Pterocarya was determined
across nuclear and plastid genome information, and a more probably
topology (nuclear), was debated relayed on proof off molecular
information and fossil reports. Relied on cautiously chosen fossil
adjustments, the remove times of surviving progeny were predicted
and they were confirmed well on fossil records (particularly regarding
Juglans and Pterocarya). Four departments into Juglans were
vigorously assistanced by the core information. Into Juglans, the
incompatible location of J. hopeiensis was obtained across the nuclear
and plastid genomes. in this study indicates the potency of planning to
unify separate biogeographic examination for illuminating the
evolutionary date of Juglandaceae (Mu et al. 2020).

In this study produced 240,179,782 reads from 11 walnut leaves by

cDNA data. The reads supply a whole de novo transcriptome
installing. Fifteen diverse transcriptome assemblies were established
from 5 distinct reputed assemblers employ in academic literature by
distinct k-mer longs (Bridger, BinPacker, SOAPdenovo-Trans, Trinity
and SPAdes), additionally two combining perspectives
(EvidentialGene and Transfuse). Relied on the 4 feature measures of
assembly, the outcomes showed an yield in the continuum of
288 | W a l n u t

combining the assemblies then being created with de novo assemblers.

Eventually Evidential- Gene was realized as of the first assembler for
the de novo installation of the leaf transcriptome in walnut. Between
totally number of 183,191 transcripts which were created with
EvidentialGene, there were 109,413 transcripts talent of protein
potency (59.72%) and 104,926 were realized as ORFs (57.27%). Also,
6,591 of the foretelled peptide datas included marker peptides, as
92,704 included across a cell membrane areas. Compared the
consubstantiate transcripts with transcripts of the walnut and
published genome installation for the ‘Chandler’ plant variety utilizing
the BLAST algorithm to cause determine a totally of 27,304 and
19,178 homologue transcripts, respectively. De novo transcriptomes in
walnut leaves may be improved for the forthcoming examination in
convenient genomics and genetic investigation of walnuts (Sadat-
Hossein et al. 2020).

İn order to figure out the molecular treatment fundamental the

advocating of walnut to C. gloeosporioides, they have been utilizing
RNA-seq. Totally, 21,798 in a varied way stated genes (DEGs) and
1929 a varied way stated proteins (DEPs) were determined in F26 vs.
F423 at five-time periods, and DEGs and DEPs expressions were
effectively upward of in the first infection phase. Determined 2
modules' important associations with disease reluctance and 9 hub
genes in the transcription statement gene systems. Gene Ontology and
Kyoto Encyclopedia of Genes and Genomes analysis of the DEGs and
DEPs disclosed that very genes were most connected to immune
 W a l n u t | 289

reply, plant hormone signaling transduction, and second metabolites,

and many DEPs were contained in carbon metabolism and
photosynthesis. As a result, assistances brighten the molecular reply of
walnut fruit to C. gloeosporioides and supply a foundation for the
genetic progress of walnut disease reluctance (Fang et al. 2021).

5. RESULTS

Walnut is one of the hard-shelled fruits that have been very popular in
recent years due to its high nutritional content, important economic
value, and valuable timber. The significant improvement in walnut
growing and the promotion of property and efficient diversity has
caused a rising up in its breeding. High output and features have every
time been fundamental walnut growing targets. High yield on lateral
branches, excess nut weight, large fruit, the high kernel rate, delay
leafing, early harvest, and light kernel color are the main quality
characteristics targeted in walnut. Manufacturers have utilized
different methods to succeed in these targets, like germplasm
evaluation and choosing, cross, genetic engineering, variation
breeding, genome sequencing, marker-assisted selection, haploid, and
polyploid causations, proteomics, and metabolomics. Now walnut
growing methods center on combining molecular breeding (utilizing
genomic, transcriptomic, proteomic and metabolomics knowledge)
into conventional breeding methods. Climate condition change and
global heating may seriously affect global walnut generations. For this
reason, walnut growing methods, particularly in the important and
best active territories, are struggling to create walnut varieties rapidly
290 | W a l n u t

and effectively that protect and continuously develop walnut yield and
property. According to this, utilizing of CRISPR-Cas9 methods for
goal-genome regulation, have generations of different chromosome
number plants to increase genome installation feature, joining high-
throughput the set of observable characteristics of an individual with
GWAS and genomic choosing, utilizing of transcriptomics,
metabolomics and proteomics for comprehension to hard
environmental conditions resistance operation in walnut, continuous
assessment of genetic variation to obtain and improve novel walnut
variety and rootstocks and the introduction of varieties that are
resistant to adverse environmental conditions and of high quality
value are priorities for future walnut breeding programmes.
 W a l n u t | 291

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Part XIII
--------------------------------

Secondary Metabolites in Walnut

Dr. Mozhgan ZARIFIKHSOSROSHAHI1*,

Assoc. Prof. Murat GUNEY2,

Prof. Dr. Ebru KAFKAS3

Introduction

The importance of walnut in the human diet

Secondary metabolites, especially phenolic compounds, are of

particular importance in walnut trees because of their significant role
in physiological processes. The nutritional content of walnut has made
it the most preferred food in a healthy diet. The walnut fruit contains a
high rate of fat and protein. On the other hand, almost all parts of the
tree have been used in folk medicine since the ancient era.

Walnut (Juglans regia L) fruit is among the strategic products in

human nutrition due to being a rich source of phytochemical

1*
Department of Horticulture, Faculty of Agriculture, Cukurova University, Adana,
Turkey, ORCID:: https://orcid.org/0000-0001-5491-1430, mn_zarifi@yahoo.com,
2
Department of Horticulture, Faculty of Agriculture, Yozgat Bozok University,
Yozgat, Turkey, ORCID: Assoc. Prof. Murat GUNEY: https://orcid.org/0000-0003-
2882-8347, murat.guney@yobu.edu.tr
3*
Department of Horticulture, Faculty of Agriculture, Cukurova University, Adana,
Turkey, ORCID:: https://orcid.org/0000-0003-3412-5971,
ebruyasakafkas@gmail.com
312 | W a l n u t

compounds. Walnut (Juglans regia L.) is a member of the relatively

small family Juglandaceae, a set of economically very important tree
species (Guney et al., 2021a, b). The importance of walnut
consumption becomes more prominent in the prevention of
cardiovascular diseases, lowering cholesterol and triglyceride levels, a
rich source of silver and selenium which are important for child
nutrition (Ros, 2010); (Tindall et al., 2019); (Nguyen and Vu, 2021).

Walnut fruits are a type of hard-shelled fruit that has been consumed
for centuries due to their rich oil content ranging from 52-70%.
Walnut kernel contains a high rate of monounsaturated fatty acid
content such as oleic acid, which is of great importance for human
health and nutrition, and essential polyunsaturated fatty acids such as
linoleic and linolenic acid. The high content of these essential causes
fatty walnut fruits to have weak oxidative stability, however, the
ability of walnuts to prevent heart disease and reduce blood
cholesterol levels is due to their oleic acid, linoleic acid, and linolenic
acid content (Arcan et al., 2021); (Martinez et al., 2006). The
derivatives of vitamin E, Tocopherols and tocotrienols, act as
antioxidants and have a wide range of physiological, biological, and
biochemical functions (Bozkurt and Ergun, 2021); (Okatan et al.,
2021) (Kafkas et al., 2020). Furthermore, the most important
biochemical function of tocopherols is to prevent the peroxidation of
polyunsaturated fatty acids. Alpha-tocopherol is known as the most
necessary form of vitamin E for the human body and its deficiency is
very common in modern nutrition. Vitamin E, known as antioxidant
 W a l n u t | 313

polyphenol, has been reported to prevent heart and cancer diseases

(Kodad et al., 2014).

Secondary metabolites and their classifications

Constant and inevitable changes in the ecosystem require changes in

biological and physiological processes to increase the adaptation of
plants to these changes and to cope with the existing stresses.

The plants apply various mechanisms of adaptation to the abiotic,

biotic, and anthropogenic factors by synthesizing numerous organic
compounds. The organic compounds Synthesized in plants are divided
into three main groups. The first group, primary metabolites such as
carbohydrates, proteins, and fats, is directly involved in normal plant
growth, development, and reproduction. The second one,
phytohormones, is chemical messengers responsible for coordinating
cellular activities and affect physiological processes occurring
naturally at very low concentrations in plants. phytohormones are
(Gundesli et al., 2020a,b,c); (Su et al., 2017). Contrary to primary
metabolites, secondary metabolites as the other group of plant
phytochemicals, do not participate directly in the developmental or
reproductive stages of a plant and the absence of secondary
metabolites does not lead to immediate cell death but may influence
the survival of the plant and its fertility in a long time. These
metabolites are countable for various physiological and ecological
functions such as regulating of plant growth and developmental
processes such as attracting insects for pollination by color and
314 | W a l n u t

volatiles, defense system against pathogens antifeedants and

allelochemicals against herbivores, innate immunity, response to
environmental stresses such as temperature, water deficiency,
ultraviolet radiation, elicitors, and acting as signals for symbiosis
between plants and microorganisms (Piasecka et al., 2015); (Yang et
al., 2018); (Guerrieri et al., 2019); (Isah, 2019); (Bodoira and Maestri,
2020); (Corso et al., 2020); (Yuan and Grotewold, 2020); (Revutska et
al., 2021); (Pang et al., 2021). Secondary metabolites also are applied
in the pharmaceutical and food industries and play significant roles in
the human health diet (Ergun and Zarifikhosroshahi, 2020); (Ullrich et
al., 2019); (Fakhri et al., 2020).

Secondary metabolites are derived from the biosynthesis pathways of

primary metabolites and are considered as their byproducts. Although
primary metabolites are synthesized in the cells of all plant species,
secondary metabolites may be characterized by specific species.
However, despite a huge variety of known secondary metabolites in
plants, the exact functions of many are still unknown (Kosmacz et al.,
2020). Secondary metabolites are mainly divided into three classes of
terpenoids (≈25,000 compounds), alkaloids (≈12,000 compounds),
and phenolics (≈8,000 compounds) (Croteau 2000). However,
xanthones, quinones, glycosides, steroids, lignans, coumarins, etc are
among known plant secondary metabolites. Terpenoids are the most
structurally diverse natural plant compounds. These compounds are
commercially important due to their wide range of applications in
industrial products, including seasonings, drugs, perfumes,
 W a l n u t | 315

insecticides, and antimicrobials. Terpenoids are used in industry for

their unique properties as biological materials for the production of
heavy tires, shock absorbers, and latex products such as surgical
gloves (Kim et al., 2011).

Alkaloids are compounds that have at least one nitrogen atom in a

heterocyclic ring. These secondary metabolites play a predominantly
defensive role and are sometimes used as sources for nitrogen storage
in plants. These compounds have a bitter taste and have a toxic effect
on the cell membrane system, especially nerve cells. They act as an
insecticide and are very important in the pharmaceutical industry.
Extensive studies have shown the diverse biological activity of indole
alkaloids, from antibacterial activity to anti-inflammatory activity and
anti-tumor activity. In addition, indole alkaloids from the marine
environment are a promising and active group of biomolecules that
cover biological, cytotoxic, antiviral, antiparasitic, and anti-
inflammatory activity (Tadeusz, 2007).

Flavonoids cause color in flowers, fruits, and sometimes leaves. They

are also effective in the pollination and fertility of plants due to their
ability to attract insects. Flavonoids increase resistance to plant
pathogens and are also strong absorbers of ultraviolet light (340-250
nm). In the structure of sexual and vegetative organs, pollen grains
have a positive effect on the action of genes and enzymes. It also
chelates some metal ions such as iron and copper. Flavonoids prevent
oxidation by inhibiting catalyzed elements. Other properties of these
316 | W a l n u t

materials include waste collection properties. Flavonoids are inhibitors

of cancer cells that exert this effect through gene expression, boosting
the immune system (antioxidant), antiviral, antibacterial, anti-
inflammatory, anti-inflammatory, anti-allergic, anti-mutation,
permeability, and capillary fragility (Nijveldt et al., 2001).

Methods for identification of secondary metabolites

Considering the value of secondary metabolites, a variety of

laboratory methods have been introduced to study the structure of
these compounds. Many of these methods are costly and time-
consuming if done for the first time. Therefore, one of the advantages
of identifying the structure of secondary metabolites is the possibility
of creating virtual libraries of information from these compounds.
Hence, compounds can be studied with more cost-effective devices.

Identification of the chemical structure also provides an opportunity to

study the ligand-enzyme properties in vitro. The most widely used
methods include gas chromatography-mass spectrometry (GC/MS),
liquid chromatography-MS (LC-MS), capillary electrophoresis-MS
(CE-MS), nuclear magnetic resonance spectroscopy (NMR), Fourier
transform-near-infrared (FT-NIR) spectroscopy, MS imaging (MSI),
and live single-cell-MS (LSC-MS) (Pang et al., 2021);
(Zarifikhosroshahi and Ergun, 2021).
 W a l n u t | 317

In mass spectrometry, one or more atomic ions are separated based on

the mass-to-charge ratio (m/z) and measuring m/z and the frequency
of ions in the gas phase. More precisely, mass spectrometry examines
the mass-to-charge ratio of molecules using electric and magnetic
fields. This method is one of the most widely used methods in
identifying metabolites. The main advantage of this technique is its
high sensitivity. In addition, combining the MS method with
chromatographic separation increases the accuracy and high ability to
separate and identify compounds in the existing metabolite (Krug and
Müller, 2014). The two combined methods GC-MS and LC-MS are
the most widely used. The GC-MS consists of a combination of gas
chromatography (to separate gas mixture components) and mass
spectrometer (to identify components) and is a widely used method for
the separation and identification of volatiles. The LC-MS method is
also one of the most widely used methods in studies related to
metabolites in plants. The main method for ionization in this
technique is API, which includes two methods EI and APCI (Dunn,
2008).

Nuclear magnetic resonance spectroscopy is another popular method

in metabolic studies. This method determines the atomic status of
compounds, and is also able to detect metabolites that are not
detectable by methods such as MS. NMR spectroscopy can provide
accurate information on the amounts and identities of metabolites
present in the extract as well as in vivo conditions (Sekiyama et al.,
2010). High-pressure liquid chromatography equipped with a UV
318 | W a l n u t

detector is also a prevalent method for the identification of phenolic

compounds and flavonoids (Ergun, 2021 a,b).

Detected secondary metabolites in different parts of the walnut

tree

Based on the previous studies, bioactive compounds such as

polyphenols and sterols contribute to the health-promoting properties
of walnuts (Ni et al., 2021).

Phenolic compounds

Phenolics are organic compounds formed by the bonding of hydroxyl

groups and aromatic rings. Phenolic compounds have been classified
into different sub-classes such as phenolic acids, flavonoids, stilbenes,
coumarins, and tannins. Walnuts are a rich source of phenolic acids,
flavonoids, ellagitannins, and gallotannins. Phenolic compounds are
involved in the regulation of physiological pathways in the growth and
development of plants. Health-promoting effects of walnut extremely
are due to polyphenol content.

The main component of phenolics is trihydroxy benzoic acid (gallic

acid) (Nieto et al., 2020). By hydrolyzation of gallic acid,
ellagitannins and gallotannins are produced. Ellagitannins are more
stable than gallotannins due to covalent and hydrogen bonds in
ellagitannins within carbonyl and hydroxyl groups. Ellagitannins such
as casuarictin, gitannins, pedunculagin and tellimagrandins are the
main group of phenolics in walnut kernels (Anderson et al., 2001);
 W a l n u t | 319

(Fukuda et al., 2003); (Cerdá et al., 2005). Ellagitannins are esters of

hexahydroxydiphenic acid and a polyol such as glucose and can be
hydrolyzed by acids or bases to break down into ellagic acid (Gómez-
Caravaca et al., 2008); (García-Villalba et al., 2015). Besides ellagic
acid which is found both in the form of Ellagitannins and free form,
gallic acid, syringic acid, vanillic acid, p-coumaric acid, and ferulic
acid, were also found abundantly in walnuts. Although glycosylated
forms of phenolic acids are also found in walnut, they are not
desirable compared to free forms because the researches proved that
the addition of glucoside decreases the biological and pharmacological
activities of phenolic acids (Kumar et al., 2013).

Catechins, procyanidins, epicatechin gallate, quercetin, quercetin- 3-β-

D-glucoside, and rutin are also flavonoids that have been detected in
different walnut species. Walnut kernels are also rich in tannins.
Tannins are divided into two classes, condensed tannins
(proanthocyanidins) and hydrolyzable tannins (Ossipov et al., 2003).
Condensed tannins are polymeric flavonoids composed of at least two
linked catechin units (Khanbabaee and Ree, 2001). Despite the low
percentage of the pellicle (Figure 1) compared to the total mass,
walnut pellicle contains more tannin in walnut (Alasalvar and Shahidi,
2010). Walnut green husk contains a large amount of quinines
(Maleita et al., 2017); (Meshkini and Tahmasbi, 2017). Most quinones
have an unsaturated cyclic dione structure in the molecule, and
anthraquinone and its derivatives are particularly important in
traditional Chinese medicine (Beiki et al., 2018). The main quantity of
320 | W a l n u t

phenolic compounds of walnuts is mostly presented in the hull

(Fukuda and Yoshida, 2003). Pellicles commonly have a higher
amount of phenolics compared to the kernel without pellicles. The
flavanols levels were 77 times more in the pellicle compared to kernel
without pellicle (Shen et al., 2021).

Thirty-four phenolic acids were detected by Shen et al., (2021) in

kernel and pellicles of walnut. The walnut pellicles contained high
levels of ellagic acid, catechin, (-)-epicatechin gallate, and catechin
gallate. However, ellagic acid was identified as the main phenolic
compound in kernels without pellicles. Shen et al., (2021) also
detected ferulic acid, chlorogenic acid, caffeic acid, and
protocatechuic acid, kaempferol, luteolin, and vitexin in the walnut
kernel. Hydrolyzable tannins, phenolic acids, and flavonoids are richly
concentrated in walnut pellicles (Alasalvar and Shahidi, 2010). Zhang
et al., (2020) detected about 32 phenolic compounds in walnut
pellicle of which ellagic acid was the major one.

Ni et al., (2021) reported that walnut kernels contain phenolic acids

including hydroxybenzoic acid, cinnamic acid, coumaric acid, syringic
acid, derivatives and three unknown compounds. These derivatives
comprise syringic acid, gentisic acid, gallic acid, 3-coumaric, 4-
coumaric, and dihydro-p-coumaric acid. Vu et al., (2018) also
detected 16 phenolics in 11 different black walnut cultivars including
phenolic acids, flavonoids, and catechins. Ellagic acid was the
predominant phenolic compound in all cultivars. Despite differences
among cultivars, p-Coumaric acid, quinic acid, Gallic acid, and (−)-
 W a l n u t | 321

Epicatechin gallate were detected in the majority of cultivars.

Significant differences were identified in the quantities of gallic acid,
quinic acid, 1,3,6-trigalloylglucose, catechin, and Penta-O-galloyl-βD-
glucose between the studied black walnuts and English walnut.

Regueiro et al., (2014) detected a wide range of phenolic compounds

(120 compounds) including hydrolyzable and condensed tannins,
flavonoids, and phenolic acids of some which have never been
detected before (stenophyllanin C, malabathrin A, eucalbanin A,
cornusiin B, heterophylliin E, pterocarinin B, reginin A and alienanin
B) in walnut kernel by "electrospray ionization hybrid linear trap
quadrupole-Orbitrap mass spectrometry" (LC–LTQ-Orbitrap). Among
detected phenolics (Chlorogenic acid, Neochlorogenic acid, 3-O-p-
Coumaroylquinic acid, 4-O-p-Coumaroylquinic acid, Coumaric acid
hexoside, Gallic acid, Ethyl gallate from phenolic acids; Catechin,
Epicatechin gallate, Procyanidin dimer, Quercetin, Quercetin
galloylhexoside, Quercetin hexoside, Quercetin pentoside,
Monogalloylglucose, Digalloylglucose, Trigalloylglucose,
Tetragalloylglucose, Ellagic acid, Ellagic acid pentoside, Ellagic acid
hexoside, Glansreginin, Casuariin, Tellimagrandin II, Heterophylliin
D, Pedunculagin, Casuariin, Strictinin, Rugosin, Casuarinin,
Stenophyllanin, Pterocarinin, Platycaryanin A, Euprostin A,
Flavogallonic acid dilactone, Alienanin. Oenothein, Reginin,
Malabathrin from Flavonoids), ellagitannins, ellagic acid, and its
derivatives were the most abundant.
322 | W a l n u t

Figure 1. Different parts of walnut fruits.

For the first time, Medic et al., (2021) identified the phenolic profile
of bark, husks and buds of walnut in six cultivars and detected 29, 38,
and 57 phenolics compounds, respectively. Of 83 total phenolics, 25
naphthoquinones, 15 hydroxycinnamic acids, 8 hydroxybenzoic acids,
13 flavonols, 2 flavones, 1 flavanone, and 19 flavonols were detected.
In all three parts, naphthoquinones were the main phenolic compound.
Juglone is a naphthoquinone that is characterized as the main phenolic
in the species of Juglandaceae family (Figure 2). Naphthoquinones
take part in various oxidative processes, act as links in the
transportation of electrons, and might also play role in defensive
mechanisms in interspecies chemical competition such as allelopathy.
Naphthoquinones also are important in pharmaceutical industries in
 W a l n u t | 323

developing natural base drugs as antibacterial, antifungal, antiviral,

antiparasitic, and antitumor (Pinho et al., 2012).

Figure 2. The sturucture of Juglone

Soto-Madrid et al., (2021) also detected 9 polyphenols including

Gallic acid, Protocatechuic acid, Catechin, Caffeic acid, Ferulic acid,
Polydatin, Hesperetin, Resveratrol, Quercetin, Myricetin, Kaempferol,
Hesperidin, Juglone in the husk of walnut. Previously, Shi et al.,
(2017) also have been reported that among different phenolic
compounds detected in walnut husk, (gallic acid, chlorogenic acid,
catechin, caffeic acid, vanillic acid, syringic acid, epicatechin, ferulic
acid, syringaldehyde, rutin, ellagic acid, quercetin, myricetin, and
juglone) juglone was the predominant phenolics.

Gutiérrez et al., (2018) identified hydroxycinnamic acid, chlorogenic

acid, caffeic, ferulic, p-coumaric and sinapic acids, cis and trans-
mono-caffeoylquinic, dicaffeoylquinic, mono-feruloylquinic and cis
and trans-mono-p-coumaroylquinic acid isomers by Ultra High-
Pressure Liquid Chromatography.
324 | W a l n u t

Phenolic data in the aforementioned studies have demonstrated that

phenolic contents differ among walnut cultivars. In other words, the
variations in phenolic compositions are ascribable to genetic traits.
There is increasing evidence to suggest that levels of phenolics in
walnuts may also be affected by agronomic practices, environmental
and climatic conditions and geographic factors (Nguyen and Vu,
2021); (Toide and Tajima, 2015); (Fuentealba et al., 2017); (Cohen et
al., 1996); (Lynch et al., 2016).

Phytosterols

Phytosterols are a type of lipid molecule that possess structural and

functional similarities to cholesterol. phytosterols are common in nut
kernels. Weihrauch and Gardner (1978) reported the presence of three
phytosterols (β–sitosterol, campesterol, and stigmasterol) in the kernel
of walnut for the first time. Later, various types of phytosterols were
identified in walnut. Thirteen phytosterols were detected in walnut
kernels by Abdallah et al., (2015) in Tunisia. Among reported
phytosterols, β–sitosterol was the major one with 63 – 89% of the total
sterol levels quantified in walnuts (Phillips et al., 2005); (Robbins et
al., 2011); (Vu et al., 2019); (Abdallah et al., 2015).

Studies proved that Phytosterols as well as β–sitosterol has the ability

to lower cholesterol level in the human body. Beta-sitosterol has also
been shown to improve Benign Prostatic Hyperplasia (Pizzorno and
Murray, 2020). Significant variations in individual and total
phytosterol levels of walnut were reported in previous studies. These
 W a l n u t | 325

differences were attributed to genetic factors, geographic origin,

environmental factors, and seasonal variations (Gong et al., 2017);
(Gong et al., 2017); (Crews et al., 2005); (Rabadán et al., 2018);
(Rabadán et al., 2019). The differences in extraction methods, harvest
time, and horticultural characteristics are among other factors
affecting phytosterols types and quantities in walnut (Nguyen and Vu,
2021).

Tocopherols

Tocopherols are another group of secondary metabolites that occur

abundantly in the walnut kernel. Tocopherols are liposoluble, natural
compounds with high antioxidant activities which comprise α, β, γ,
and δ tocopherols. Tocopherols donate the hydrogen of the hydroxyl
group to the lipid peroxyl radical. Among different tocopherols, α-
tocopherol is the most important one due to vitamin E activity
(Beyhan et al., 2016).

Studies showed four different isomers of tocopherols ( α-, β-, γ- and δ)

detected in walnut oils. Among detected tocopherols in walnut kernel
oil, γ-Tocopherol is the predominant form (Robbins et al., 2011);
(Beyhan et al., 2016); (Arcan et al., 2021). However notable variations
are reported in tocopherol contents of walnut kernels by researchers.
Tocopherol levels were extremely affected by cultivars, genetic and
environmental factors, and harvest time (Özrenk et al., 2012); (Beyhan
et al., 2016).
326 | W a l n u t

Tocotrienols, lipid-soluble compounds, are also other secondary

metabolites detected in walnuts (Li et al., 2006); (Matthäus et al.,
2018); (Robbins et al., 2011); (Amaral et al., 2005a, b). Tocotrienols
are also from the vitamin E family with antioxidant capacity. The
difference between tocotrienols and tocopherols is related to their
chemical structure in which tocotrienols have unsaturated isoprenoid
side chains with three carbon-carbon double bonds versus saturated
side chains for tocopherols (Kamal-Eldin and Appelqvist, 1996).
However, limited information about the presence of these compounds
in walnuts is available.

Plant Hormones

Phytohormones also as a group of secondary metabolites are involved

in the regulation of the metabolic activities within cells and the
development of the plant. The regulators of plant growth and
development play a key role in managing the stress signals and
biochemical and physiological pathways (Hasanuzzaman et al., 2020).
Melatonin is not only a phytohormone, it is released by the pineal
gland in the human body at night and controls the sleep-wake cycle
(Auld et al., 2017). Melatonin is also involved in various functions in
improving the performance of the mitochondrial electron transport
chain and antioxidant activity (Ahmad et al., 2020); (Jaferi and
shahsavar, 2021). Reiter et al., (2005) reported that walnuts are a good
source of melatonin and when eaten the concentrations of blood
melatonin increased which was associated with the increased
antioxidative capacity of the blood. Later, several studies identified
 W a l n u t | 327

the amount of melatonin in walnut (Reiter et al. 2005); (Tapia et al.,

2013); (Pycia et al., 2019); (Paroni et al., 2019); (Wang et al., 2020);
(Verede et al., 2021). It is reported that the content of melatonin
differs extremely in some nuts studied in different researches (Reiter
et al., 2005); (Tapia et al., 2013); (Kocadağlı et al., 2014); (Paroni et
al., 2019). These differences may due to factors such as the cultivar,
variety, harvesting time, and the fruit maturity (Kocadağlı et al., 2014)
besides the status of nuts consumption (Chang et al., 2016); (Paroni et
al., 2019) and extraction methods (Verede et al, 2021).
328 | W a l n u t

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 W a l n u t | 339

Part XIV
--------------------------------

Fatty Acids Composition of Walnuts

Dr. Yılmaz Uğur1, Prof. Dr. Selim Erdoğan2

1. Introduction

Lipids are compounds found in plant and animal cells that are
insoluble in water but soluble in nonpolar solvents such as
chloroform, ether, and hexane (Korkmaz et al., 2012). They form our
body's largest source of energy. All animal and plant foods contain
different amounts of oil. One of the food groups that should be
consumed daily to provide adequate and balanced nutrition is oilseeds.
Oilseeds have considerably rich content in terms of B vitamins, oil,
protein, and minerals. Vegetable products such as walnuts, pistachios,
peanuts, almonds, and pumpkin seeds are taken part in the group of oil
seeds (Ayaz, 2008; Gundesli et al., 2021).

Walnuts are prevalently consumed due to their substantial nutritional

components (Esen, 2013). As well as consuming directly as nuts, it is
an indispensable component of many products in the food sector, too.
It is used in the manufacture of foods such as many desserts, cakes,

1*
Inonu University, Health Services Vocational School, Department of Pharmacy
Services, Malatya, Turkey, ORCIDs:, https://orcid.org/0000-0002-9040-4249,
yilmaz.ugur@inonu.edu.tr
2
Inonu University, Faculty of Pharmacy, Malatya, Turkey, ORCIDs:
https://orcid.org/0000-0002-9169-9771, selim.erdogan@inonu.edu.tr
340 | W a l n u t

and cookies, especially baklava and kadayif. Walnut is also an

essential material for the pharmaceutical, cosmetic, and chocolate
industries (Yan et al., 2021; Demirağ, 2019; Guney,et al., 2021). In
addition to many components, fatty acids are also effective in the
demand for walnuts as a nutritional source. The high level of
polyunsaturated fatty acids in walnut oil has made it a valuable and
preferred food. The major fatty acids found in walnut oil are oleic,
linoleic and linolenic acids. These polyunsaturated fatty acids are
more desirable because of their health benefits (Uzun et al., 2021;
Cunnane et al., 1993; Rabrenovic et al., 2011; Demirag, 2019; Kafkas
et al., 2020).

Studies have revealed that walnut consumption reduces plasma

cholesterol levels. This is thought to be due to the presence of linoleic
and linolenic acids, which are essential fatty acids in the diet, in
suitable proportions in walnut oil (Yildiz et al., 2021; Sabate et al.,
1993; Abbey et al., 1994; Rabrenovic et al., 2011). Linolenic and
linoleic acid, which are essential fatty acids that are not produced by
the body but must be taken through food, is also known as omega-3
and omega-6 fatty acids (Harris et al., 2007).

The positive effects of omega-3 fatty acids on health were noticed

firstly in studies on the Inuit people of Greenland. Despite their
traditional diet with high-fat, Inuits have been observed to be resistant
to heart, rheumatism, asthma, and many diseases common in industrial
countries. It has been suggested that the reason for this is the
widespread consumption of fish meat containing unsaturated fatty
 W a l n u t | 341

acids and the oils of marine mammals (Dyerberg et al., 1975;

Anonymous, 2021a). Omega-3 fatty acid consumption is important in
the prevention of prostate, breast, lung, and intestinal cancers and both
the prevention and treatment of cardiovascular diseases, hypertension,
rheumatoid arthritis, osteoporosis, diabetes, asthma, Alzheimer's,
depression, and schizophrenia. In addition, it has been reported that it
has positive effects on the development of intelligence in the early
period, the strengthening of the immune system, nervous system
development, and brain functions (Stevens et al., 1995; Simopoulos,
1991; Tatar et al., 2001; Ceylan et al., 1999; Çabuk et al., 1999;
Gundesli et al., 2019).

Walnut oil contains higher omega-3 and omega-6 polyunsaturated

fatty acids than other nuts (Yildiz et al., 2021, Amaral et al., 2003). In
studies conducted on walnuts, the presence of other components
besides mono and polyunsaturated fatty acids, which are essential for
human health, has been determined, and their impacts on health have
been the subject of many research. In this article, studies investigating
the fatty acid composition of walnut oil and its effects on health have
been compiled.

2. Fatty Acids
Fatty acids are hydrocarbons having long chains of lipid-carboxylic
acid found in oils and cell membranes as a component of
phospholipids and glycolipids (Anonymous, 2021b). They are
represented by the formula R–COOH. The "R" can be a saturated or
unsaturated long hydrocarbon chain containing one or more carbon-
 342 | W a l n u t

carbon double bonds. Fatty acids differ according to chain length and
the presence, number, and position of double bonds in the
hydrocarbon chain.

The systematic nomenclature of fatty acids recommended by the

International Union of Pure and Applied Chemistry (IUPAC) is done
by adding the suffix -oic to the end of the hydrocarbon chain name.
The IUPAC system names fatty acids just on the basis of the number
of carbon atoms, and the number and position of unsaturated fatty
acids relative to the carboxyl group. Although the IUPAC
nomenclature is technically clear, fatty acid names are long and
therefore, for convenience, trivial names are frequently used in
scientific articles (Anonymous, 2018). The systematic names,
common names, and chemical structure of some common fatty acids
are presented in Table 1.

Table 1. Some common fatty acids

Systematic Trivial
Abbreviation Chemical structure
Name Name

C12:0 Dodecanoic acid Lauric acid CH3(CH2)10COOH

Tetradecanoic Myristic
C14:0 CH3(CH2)12COOH
acid acid

Hexadecanoic Palmitic
C16:0 CH3(CH2)14COOH
acid acid

Octadecanoic Stearic
C18:0 CH3(CH2)16COOH
acid acid

Arachidic
C20:0 Eicosanoic acid CH3(CH2)18COOH
acid
 W a l n u t | 343

Tetracosanoic Lignoceric
C24:0 CH3(CH2)22COOH
acid acid

9-cis-
Palmitoleic
C16:1 Hexadecenoic CH3(CH2)5CH=CH(CH2)7COOH
acid
acid

cis-9-
C18:1 Octadecenoic Oleic acid CH3(CH2)7CH=CH(CH2)7COOH
acid

cis,cis-9,12-
Linoleic
C18:2 Octadecadienoic CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH
acid
acid

cis,cis,cis-
α- CH3CH2CH=CHCH2CH=CHCH2CH=CH
9,12,15-
C18:3 Linolenic
Octadecatrienoic
acid (CH2)7COOH
acid

Structural variation among fatty acids gives rise to functional

differences that result in different impacts upon metabolism, cell and
tissue responses (Burdge and Calder, 2015). Structurally, the fatty
acids are of two kinds as saturated and unsaturated (Figure 1).
Unsaturated fatty acids have mono- and polyunsaturated types, too.
Fatty acids that aliphatic carbon chains are fully saturated with
hydrogen atoms contain only C-C single, but contain no C=C double
bonds are identified as saturated fatty acids. Fatty acids containing
C=C double bonds are referred to as unsaturated fatty acids. If they
contain only one C=C double bond, they are monounsaturated fatty
acids. If they contain more than one C=C double bond, they are then
called polyunsaturated fatty acids.
344 | W a l n u t

Polyunsaturated fatty acids from nutritional perspectives are

categorized as essential and nonessential types. The fatty acids, which
mammals cannot synthesize in their body, are known as essential fatty
acids; they must be obtained from the exogenous dietary sources.
Omega-6 linoleic acid, omega-3 α-linolenic acid omega-6 arachidonic
acid are essential fatty acids. Fatty acids that mammals can synthesize
in their bodies are called nonessential fatty acids. The polyunsaturated
fatty acids that have a C=C double bond between the 6th and 7th
carbon position counting from the terminal methyl end are called
omega-6 and those with the double bond between the 3th and 4th
carbon are called omega-3 polyunsaturated fatty acids (Hashimoto and
Hossain, 2018). The classification of the most common fatty acids and
their sources are presented in Table 2.

Figure 1. The Structural Configurations of The Saturated and Unsaturated Fatty

Acids (Anonymous, 2021c)

Table 2. Saturated and Unsaturated Fatty Acids and Their Typical Sources
(Doğan and Akgül, 2005; Chisholm et al., 1998; Geng et al., 2021; Kaflas et al.,
 W a l n u t | 345

2020; Uzun et al., 2021; Ogungbenle and Anisulowo, 2014; Maguire et al., 2004;
Nogales-Bueno et al., 2021; Amaral et al., 2003; Savage et al., 1999; Arcan et al.,
2021)

Series Trivial Name Abbrev Typical sources

Butyric acid C4:0 Dairy fat
Caproic acid C6:0 Dairy fat
Dairy fat, coconut and palm
Caprylic acid C8:0
kernel oils
Coconut and palm kernel
Lauric acid C12:0
oils
Dairy fat, coconut, palm
Saturated Fatty Myristic acid C:14:
and walnut kernel oils
Acids
Most fats and oils (included
Palmitic acid C16:0
walnut oil)
Most fats and oils (included
Stearic acid C18:0
walnut oil)
Arachidic acid C20:0 Peanut oil, walnut oil
Behenic acid C22:0 Peanut oil, walnut oil
Lignoceric acid C24:0 Peanut oil
Marine oils, macadamia oil,
Palmitoleic acid C16:1 most animal and vegetable
oils (included walnut oil)
All fats and oils, especially
olive oil, canola oil and
Oleic acid C18:1
Monounsaturated high-oleic sunflower and
Fatty Acids safflower oil, walnut oil
Most vegetable oils
cis-vaccenic acid C18:1
(included walnut oil)
Gadoleic acid C20:1 Marine oils
Mustard seed oil, high
Erucic acid C22:1
erucic rapeseed oil
346 | W a l n u t

Nervonic acid C24:1 Marine oils

Flaxseed oil, walnut oil,
α-linolenic acid 18:3ω-3 perilla oil, canola oil,
soybean oil
Fish, especially oily fish
Eicosapentaenoic
20:5ω-3 (salmon, herring, anchovy,
Polyunsaturated acid (EPA)
smelt and mackerel)
Fatty Acids
Fish, especially oily fish
(Omega-3) Docosapentaenoic
22:5ω-3 (salmon, herring, anchovy,
acid (DPA)
smelt and mackerel)
Fish, especially oily fish
Docosahexaenoic
22:6ω-3 (salmon, herring, anchovy,
acid (DHA)
smelt and mackerel)
Most vegetable oils
Linoleic acid 18:2ω-6
(included walnut oil)
Polyunsaturated
Evening primrose, borage
Fatty Acids Ɣ-linolenic acid 18:3ω-6
and blackcurrant seed oils
(Omega-6)
Animal fats, liver, egg
Arachidonic acid 20:4ω-6
lipids, fish

3. Effects of Fatty Acids and Walnut on Health and Nutrition

Fatty acids are considered the crucial nutrients that affect growth and
development and nutrition-related chronic disease later in life
(Anonymous, 2018). They are major components of cell membrane
structure, modulate gene transcription, function as cytokine
precursors, and serve as energy sources in complex, interconnected
systems (Norris and Milton, 2013). That is, fatty acids are required in
human nutrition as a source of energy, and for metabolic and
structural activities. The most common dietary fatty acids have been
 W a l n u t | 347

subdivided into three classes as saturated, monounsaturated, and

polyunsaturated fatty acids.

The energy provided by saturated fatty acids is equivalent to the

calorie given by other fatty acids, but they cause fat accumulation and
gaining weight in the body. It is stated that tm he consumption of
saturated fat should be reduced to minimize the risk of cardiovascular
diseases. Saturated fatty acids prevent the clearance of LDL in the
blood and increase the rate of fat in the blood. As a result, they can
cause atherosclerosis by forming deposits in the vessels. It is also
stated that it increases the level of LDL cholesterol and leads to the
formation of insulin resistance, thus increasing the tendency to
diabetes (Çakmakçı and Tahmas-Kahyaoğlu, 2012; Siri-Tarino et al.,
2010). The effects of monounsaturated fatty acids on LDL cholesterol
and triglycerides are neutral, but they have an increaser effect on the
high-density lipoprotein (HDL) cholesterol. A significant reduction in
the level of LDL cholesterol can be achieved by consuming
polyunsaturated fats. There are two main groups of polyunsaturated
fatty acids; Omega-3 and omega-6 fatty acids. Omega-3 fatty acids are
found in oily marine fish as well as the oils of flaxseed, canola, soy,
walnut, and hazelnut. Omega-3 fatty acids are found in most vegetable
oils, including walnuts. Omega-3 fatty acids reduce blood triglyceride
levels by reducing the production of LDL cholesterol. Due to the
heart-protective effect of omega-3 fatty acids, people who consume
these oils have a decrease in deaths due to coronary heart disease
(Samur, 2012; Siri-Tarino et al., 2010; Maguire et al., 2004).
348 | W a l n u t

Among polyunsaturated fatty acids, arachidonic acid, α-linolenic acid,

and linoleic acid are significant and essential fatty acids in terms of
health and nutrition by playing substantial roles in biochemical and
physiological events in the body. Omega fatty acids, which are a type
of polyunsaturated fatty acid, have been associated with various health
benefits like the treatment of rheumatoid arthritis and coronary heart
disease, improving blood pressure control, and preserving kidney
function. The effects of omega-3 fatty acids on various disorders such
as cancer, edema, rheumatoid arthritis, cardiovascular, are closely
related to their metabolism. Therefore for the protection against
metabolic diseases and disorders, the inclusion of omega-3 fatty acids
in the diet has been widely accepted as one of the keystones of a
healthy lifestyle and nutrition (Gogus and Smith, 2010; Rennie et al.,
2003; Holm et al., 2001; Çakmakçı and Tahmas-Kahyaoğlu, 2012).

Essential fatty acids, such as omega-3 and omega-6 fatty acids, serve
crucial cellular functions. For example, during pregnancy and
lactation, omega-3 essential fatty acids are accepted as structural
components for the development of the brain and central nervous
system. They are a necessary part of the human diet because the body
can't synthesize these molecules. Essential fatty acids may affect the
prevalence and severity of cardiovascular disease, diabetes, cancer,
and age-related functional decline (White, 2009; Anonymous, 2018).

Walnut is considered important nutrition with benefits for human

health because walnut kernels are rich in protein, oil, unsaturated fatty
acids, vitami ns, minerals, essential fatty acids, and other nutrients.
 W a l n u t | 349

Walnut oil is a good source of omega-3 and omega-6 polyunsaturated

fatty acids, which are reported to have beneficial effects on blood
lipids, by lowering blood cholesterol, decreasing the rate of serum
concentrations of LDL, and increasing HDL. Compared with other
nuts, walnuts have the lowest ratio of saturated fatty acids to total fatty
acids (Geng et al., 2021; Nogales-Bueno et al., 2021; Kafkas et al.,
2020; Li et al., 2007). The major fatty acids found in walnut oil are
oleic acid, linoleic acid, and α-linolenic acid (Rabrenovic et al., 2011).

In the study carried out by Nergiz-Ünal et al. (2013), it was remarked

that the atherosclerotic plaque development in the aortic arch of mice
fed with walnuts was a 55% reduction. Plasma levels of triglycerides,
cholesterol, and prothrombin also lowered by 36%, 23%, and 21%,
respectively, compared to the control diet. In addition, the
accumulation of lipids in the liver was decreased, while plasma
antioxidant capacity was increased.

In a study in hypercholesterolemic patients, it was assessed the effects

on serum lipids and cardiovascular risk of replacing 40% of the fat in
a normal diet with olive oil, walnuts, or almonds. In the 18
participants who joined the study, LDL-cholesterol was reduced from
baseline by 7.3%, 10.8%, and 13.4% after the olive oil, walnut, and
almond diets, respectively. Total cholesterol and LDL/HDL ratios
decreased in parallel (Damasceno et al., 2011).

Torabian et al. (2010) investigated the effect on the total cholesterol

and triglyceride levels of long-term walnut consumption in 87 subjects
350 | W a l n u t

with normal to moderate high plasma total cholesterol. The results

showed that including walnuts as part of a normal diet favorably
altered the plasma lipid profile. The lipid-lowering effects of walnuts
were more evident among subjects with high plasma total cholesterol.

The fact that walnut is nutritionally valuable food comes from their
rich composition, especially their polyunsaturated fatty acid profile.

4. Analysis of Fatty Acids

The determination of fatty acids in oil-containing samples is carried

out in three stages (Anonymous, 2018):

4.1. Extraction of oil from samples: Samples are ground before

the solvent extraction to produce a more homogeneous sample and to
increase the surface area of lipid exposed to the solvent. For polar
lipids such as glycolipids or phospholipids, it is suitable for polar
organic solvents such as alcohols. On the other hand, it is suitable the
nonpolar solvents such as hexane, ethyl ether, petroleum ether, and
pentane for nonpolar lipids. In samples such as walnuts, peanuts,
almonds, peanuts, and hazelnuts, the soxhlet method is generally used
(Hewavitharana et al., 2020; Servaes et al., 2015; Señoráns and Luna,
2012; Min and Ellefson, 2010; Thiex et al., 2003).

4.2. Preparation of ester derivatives of fatty acids: The fatty acid

composition of samples is determined as the methyl esters of fatty
acids by GC. The conversion process of fatty acids into fatty acids
methyl esters is called derivatization. Acid and basic derivatization
 W a l n u t | 351

methods are used in this process. The commonly used acid

derivatization reagents are hydrochloric acid, acetyl chloride, sulfuric
acid, and boron trifluoride. In basic derivatization methods, reagents
such as sodium methoxide, sodium bisulfate, and potassium hydroxide
are used. There are also other methods used reagents such as
trimethylsulfonium hydroxide and pentafluorobenzyl bromide (Avci et
al., 2018; Asperger et al., 2001; Ichihara and Fukubayashi, 2010;
Aldai et al., 2005; Ostermann et al., 2014; Hewavitharana et al.,
2020).

4.3. Analysis by gas chromatography (GC): Analyses of fatty

acids methyl esters are performed by GC having a flame ionization
detector. For analyses, it is preferred bonded polar capillary columns
(Anonymous, 2018).

5. Fatty Acids in Wlanut Oil

Many studies have been conducted to determine the fatty acid

composition of different walnut cultivars/genotypes. The results
compiled from these studies are given in Table 3. Palmitic acid
(C16:0),
352 | W a l n u t

Tablo 3. Fatty Acid Composition of Different Walnut Cultivars/Genotypes

Fatty Acids References

Cultivar
Origin
Genotype C14: C16: C16:1ω C18: C18:1ω C18:1ω C18:2ω C20: C18:3ω C20:1ω C18:3ω C21:
0 0 7 0 9 7 6 0 6 9 3 0

Uşak Yildiz et al.,

Uşak 0,03 6,36 0,05 2,62 17,58 59,32 0,13 13,75
Turkey 2021

Adilcevaz 4,30 1,00 29,20 55,80 9,90

Van
Ahlat Lake 5,10 1,00 29,80 52,00 12,70
Turkey Batun et al.,
2017
Edremit 5,90 2,40 29,80 52,90 9,70

Çatak 5,70 1,60 24,30 55,50 13,10

Kayser
Uzun et al.,
KW1-50 i 5,71 2,12 25,44 54,40 12,30
2021
Turkey
Dogan and
W1-4 Turkey 0,13 5,73 2,24 23,91 52,13 15,94
Akgul, 2005
Malaty
Gerçekçioğlu et
44HEK a 5,57 2,87 33,63 47,12 10,17
al., 2020
Turkey

Kaman-2 6,30 2,60 20,50 55,50 14,80

Kırşehi
Özcan et al.,
Kaman-5 r 6,50 2,60 26,40 53,60 14,30
2010
Turkey
Büyük
6,30 2,50 22,20 49,70 14,50
Oba
Kaplan-
7,21 4,49 28,01 50,31 9,75
86
Malatya-1 6,98 3,22 19,33 59,89 9,97

Şebin 7,13 3,82 34,01 46,55 8,44

Şen 1 7,24 3,72 27,49 51,52 9,96

Hatay Bayazıt and
Turkey Sümbül, 2012
Tokat 1 7,70 3,67 21,09 56,45 11,00

KR 2 7,28 3,73 34,03 45,30 9,66

77H1 8,77 3,74 22,45 55,23 9,79

65/4 7,14 4,11 36,76 41,55 10,10

Kaplan-
0,07 6,22 0,04 3,25 19,07 58,64 12,70
86
Bilecik 0,06 6,53 0,04 3,17 21,95 56,64 11,63
Adana
Yalova-1 0,05 6,07 0,04 2,25 18,82 62,51 10,26 Ada et al., 2021
Turkey
Yalova-3 0,06 6,21 0,09 2,51 25,51 52,13 11,81

Yalova-4 0,06 6,84 1,96 27,09 54,22 9,83

 W a l n u t | 353

Fatty Acids References

Cultivar Orig
Genotype in C14: C16: C16: C18: C18: C18: C18: C20: C18: C20: C18: C21:
0 0 1ω7 0 1ω9 1ω7 2ω6 0 3ω6 1ω9 3ω3 0

Chandler Kahram 0,04 7,63 1,77

Arcan.,
anmaraş
2021
Kaplan-86 Turkey 0,62 7,44 1,92

Bilecik 0,02 5,82 0,10 3,82 13,80 62,92 0,12 13,16

Chandler 0,02 5,92 0,12 3,41 14,47 62,82 0,10 12,92

Hartley 0,02 6,16 0,11 3,59 12,95 64,56 0,12 12,12

Howard 0,02 6,13 0,11 3,44 14,36 62,20 0,12 13,26

Maraş-12 0,02 6,57 0,13 3,70 21,02 59,62 0,11 8,55

Adana Kafkas et
Turkey al., 2017
Maraş-18 0,02 6,98 0,13 3,59 27,57 53,42 0,10 7,83

Midland 0,02 6,41 0,12 3,86 17,63 61,22 0,11 10,33

Pedro 0,02 6,72 0,11 3,97 14,77 61,89 0,13 12,17

Şen 0,03 6,58 0,12 3,23 26,86 53,24 0,11 9,50

Serr 0,02 6,57 0,12 3,88 14,94 61,37 0,10 12,74

Franquette 0,03 7,48 0,05 2,43 16,99 1,53 59,22 0,07 0,06 0,19 11,69

Marbot 0,02 7,14 0,08 2,77 16,51 1,26 58,90 0,08 0,07 0,19 12,74

Mayette 0,03 7,00 0,08 2,55 18,09 1,21 57,46 0,07 0,05 0,19 12,98
Amaral et
Portugal
al., 2003
Melanaise 0,03 7,02 0,07 2,65 14,49 1,24 61,31 0,06 0,04 0,17 12,51

Lara 0,03 6,94 0,06 2,22 14,26 1,29 62,50 0,06 0,05 0,18 12,16

Parisienne 0,03 6,32 0,07 2,41 17,45 1,11 62,45 0,07 0,03 0,22 9,64

Chandler 5,96 0,05 2,28 12,98 1,08 62,22 0,17 0,14 14,23 0,57

Franquette 7,17 0,07 2,40 14,84 1,47 58,64 0,18 0,14 14,12 0,54
Nogales-
Howard Spain 6,88 0,05 2,25 12,43 0,99 60,36 0,14 0,12 16,92 0,56 Bueno et
al., 2021
Lara 6,76 0,06 2,38 12,16 1,01 63,74 0,18 0,17 12,61 0,58

Tulare 6,65 0,06 2,47 12,27 0,93 62,27 0,09 0,10 14,60 0,26

Sampion 7,10 0,40 1,60 19,00 60,90 11,00

Jupiter 7,00 0,10 1,80 22,90 58,10 9,90

Rabrenovic
Sejnovo Serbia 6,70 1,70 16,20 63,30 0,80 11,20
et al., 2011

Elit 7,10 0,30 2,20 21,60 58,80 9,90

G-139 7,70 0,40 1,60 19,80 57,20 13,60

354 | W a l n u t

Fatty Acids References

Cultivar
Origin
Genotype C14: C16: C16: C18: C18:1ω C18: C18:2ω C20: C18: C20: C18: C21:
0 0 1ω7 0 9 1ω7 6 0 3ω6 1ω9 3ω3 0
Petrović-
Serbia Serbia 7,03 0,11 2,75 14,47 1,34 63,15 11,15 Oggiano et
al., 2020
Esterhazy 7,47 1,63 17,44 0,68 58,83 0,12 13,54

G139 6,65 1,40 16,46 0,71 61,98 0,14 12,71

Europe
and
G120 7,73 2,05 19,58 0,69 57,09 0,12 12,45
United
Stadtes
Tehama 7,61 1,35 19,54 0,81 57,88 0,14 12,38

Vina 6,46 1,43 17,94 0,68 58,03 0,11 15,07

Rex 6,59 0,07 12,66 0,81 62,48 0,11 16,17

Dublin's Savage et
7,76 0,08 18,95 0,85 57,01 0,12 13,10 al., 1999
Glory
Meyric 7,30 0,08 18,09 0,85 58,43 0,11 13,31

Stanley 6,72 0,08 20,36 0,63 59,24 0,11 11,18

New
Zealand
McKinster 6,22 0,06 18,71 0,77 61,31 0,06 10,65

150 7,15 0,06 17,39 0,74 60,45 0,12 12,65

151 6,75 0,06 16,20 0,71 61,72 0,14 12,71

153 6,84 0,06 14,35 0,58 61,64 0,10 15,21

Ivanhoe 0,04 7,86 0,24 2,43 16,95 59,23 13,24

Franquette 0,06 7,83 0,08 2,04 14,17 64,26 11,57

Howard 0,04 5,74 0,06 2,59 11,36 64,34 15,87

Durham 0,03 6,87 0,13 2,39 20,65 60,23 9,70

Earliest 0,04 6,95 0,13 2,40 17,49 63,11 9,88

Solano 0,02 9,49 0,14 2,27 10,85 61,50 0,08 15,65

Hartley 0,06 7,09 0,13 2,50 13,74 65,26 0,12 11,10

Kafkas et
USA
R. al., 2020
0,03 8,03 0,13 2,27 18,34 61,69 0,06 9,46
Livermore

Chandler 0,02 6,46 0,07 2,92 17,48 58,96 0,06 14,03

95-014-3 0,02 7,16 0,46 2,40 15,99 60,83 13,13

03-001-
0,05 6,73 0,24 2,45 14,31 60,00 0,08 16,14
2357
R.
0,01 6,59 0,12 2,15 14,78 64,31 0,02 12,01
Livermore

Chenier 6,95 0,08 3,00 19,21 61,67 9,08

Sinensis#5 7,16 0,14 2,44 15,71 66,07 0,08 8,41

 W a l n u t | 355

Cultivar Fatty Acids References

Genotyp Origin
C14: C16: C16: C18: C18:1 C18: C18:2ω C20: C18: C20: C18:3 C21:
e
0 0 1ω7 0 ω9 1ω7 6 0 3ω6 1ω9 ω3 0
Ogungbenl
Aramok 12,1 e and
Nigeria 0,08 0,36 5,08 72,87 0,10 9,09
o-Ekiti 9 Anisulowo,
2014
New Chisholm et
NI 7,70 2,20 20,80 58,00 11,10
Zealand al., 1998
Maguire et
Cork Ireland 0,13 6,70 0,23 2,27 21,00 57,46 0,08 11,58
al., 2004
Combe 0,03 5,87 0,06 3,24 15,73 57,29 0,11 15,75 0,02
Li et al.,
Canada
2007
Lake 0,03 5,59 0,05 2,83 16,39 60,96 0,05 12,11 0,02

Min. value 0,01 4,30 0,04 0,06 10,85 0,58 41,55 0,02 0,03 0,06 7,83 0,02

Max. value 0,62 12,19 0,46 5,08 36,76 1,53 72,87 0,80 0,18 0,22 16,92 0,58

Avg. value 0,05 6,85 0,13 2,40 9,46 0,96 58,64 0,11 0,10 0,14 12,04 0,36

stearic acid (C18:0), oleic acid (C18:1ω9), linoleic acid (C18:2ω6),

and α-linolenic (18:3ω3) are seen to be dominant fatty acids in
walnuts grown in Turkey's ecology. Walnuts grown in European
ecology contain cis-vaccenic acid (C18:1ω7) around a 1% rate in
addition to these fatty acids. According to the data in Table 3, it is
seen that the walnut genotype grown in Nigeria contains the highest
levels of C16:0, C18:0, and C18:2ω6. It can be said that growing
conditions and cultivar characteristics are effective in the variation
between the results. For example, it has ranged from C16:0 between
4.30% and 12.19%, C18:0 0.06% and 50.8%, C18:1ω9 10.85% and
36.76%, and C18:2ω6 41.55% and 72.87% (Tablo 3).

The total of unsaturated fatty acids (average %81,47) in walnut oil has
a higher value than the total of saturated fatty acids (average %9,76).
Containing high levels of alpha-linolenic and linoleic acids, which are
essential fatty acids known as omega-3 and omega-6, make walnut an
indispensable food. As shown in Table 3, the average levels of omega-
356 | W a l n u t

3 (C18:3ω3) and omega-6 (C18:2ω6) fatty acids are 12.04% and

58.64%, respectively. Compared with most other nuts, walnuts are
highly enriched in omega-6 and omega-3 polyunsaturated fatty acids,
which are essential dietary fatty acids. Essential fatty acids also make
substantial contributions to the prevention of cardiovascular diseases.
It has importance to include walnuts in the normal diet, which is
similar to the shape of the human brain and is thought to contribute
greatly to brain development.

6. Conclusion

The importance of natural nutritional supplements in the protection of

our health has increased in recent years, with scientific studies proving
the effectiveness of some foods in the prevention and treatment of
diseases naturally. This situation has increased the demand for natural
health products.

Walnut, which is in the group of nuts and contains a high level of oil,
is considered a functional food due to its nutritious and beneficial
effects on health. It contains abundantly the essential fatty acids,
which are effective in the prevention of many diseases, especially
cardiovascular diseases, and in brain development. It should not be an
exaggeration to say that one of the elements of a healthy and balanced
diet is walnuts.
 W a l n u t | 357

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Tatar, O., Hışıl, Y., Dönmez, M. (2001). Determination of omega-3 fatty acids in
some fish eggs. Dünya Gıda Dergisi 10 (6): 61-64.
Thiex, N.J., Anderson, S., Gildemeister, B. (2003). Collaborators: Crude Fat,
Hexanes Extraction, in Feed, Cereal Grain, and Forage
(Randall/Soxtec/Submersion Method): Collaborative Study, Journal of
AOAC International 86 (5): 899–908.
Torabian, S., Haddad, E., Cordero-MacIntyre, Z., Tanzman, J., Fernandez, M.L.,
Sabate, J. (2010). Long-term walnut supplementation without dietary advice
induces favorable serum lipid changes in free-living individuals. European
Journal of Clinical Nutrition 64 (3): 274–279.
Uzun, A., Kaplan, M., Pinar, H., Paris, K. (2021). Oil contents and fatty acid
composition of walnut genotypes selected from Central Anatolia region and
364 | W a l n u t

assessments through GT biplot analysis. Bulgarian Chemical

Communications 53 (3): 279-286
White, B. (2009). Dietary fatty acids. American Family Physician 80 (4): 345–350.
Yan, S., Wang, X., Yang, C., Wang, J., Wang, Y., Wu, B., Mohammad P…Zheng, J.
(2021). Insights into walnut lipid metabolism from metabolome and
transcriptome analysis. Frontiers in Genetics 12: 715731.
Yildiz, E., Pinar, H., Uzun, A., Yaman, M., Sumbul, A., Ercisli, S. (2021).
Identification of genetic diversity among Juglans regia L. genotypes using
molecular, morphological, and fatty acid data. Genetic Resources and Crop
Evolution 68: 1425–1437.
 W a l n u t | 365

Part XV
--------------------------------

Nature's Miracle Walnut and Human Health

Assoc. Prof. Muhammet Ali Gundesli1

INTRODUCTION

The history of using plants to treat human health and many diseases is
as old as human history; such that it has a history of several thousand
years in many countrie. In the past centuries, natural remedies,
especially medicinal plants, were accepted as the basis of treatment. In
the last decade, it has been shown that there is a great tendency and
willingness to accept natural treatments in developed and developing
countries (Delaviz et al., 2017). The use of plants in traditional
medicine for many diseases has become widespread in the world. In
many studies conducted over the last 30 years; They found that there
is a relationship between people's eating habits and diseases. While
investigating the relationships between some well-known diseases and
nutrition, the most questioned food component/food item was fats.

1
Department of Plant and Animal Production, Nurdagı Vocational School,
Gaziantep University, Gaziantep, Turkey, ORCIDs: Assoc. Prof. Muhammet Ali
GUNDESLİ, https://orcid.org/0000-0002-7068-8248, maligun46@gantep.edu.tr
366 | W a l n u t

Walnut, a member of the Juglandaceae plant family, is one of the most

valuable and common tree nuts worldwide due to its rich nutritional
content. Thanks to its high adaptability to different climatic conditions
that walnuts are widely cultivated, especially in regions with semi-
tropical or temperate climates (Kafkas et al., 2020). The walnut which
has a history of thousands of years, is the oldest tree known to man
and is used in the treatment of different diseases. Almost all parts of
this plant such as bark, kernel (seed), flower, leaf, green bark, septum,
oil have unique food, cosmetic pharmaceutical industries and
medicinal properties (Figure 1 and 2) (Vadivel et al., 2012; Gupta et
al., 2019). Therefore, the different tissues of the walnut tree, besides
the antioxidant and antimicrobial effects of the different compounds
they contain, as well as the inflammatory antihistamine, antiseptic,
antiulcer, antinociceptive, antidiabetic, antiasthmatic, antifertility,
central nervous system, wound healing, lipolytic, insecticide,
larvicide, and human health effects. contains many features (Zang et
al., 2018; Salejda, et al., 2016; Trandafir, et al., 2016; Zarghami
Moghaddam et al, 2017; Raja et al., 2017; Jahanban-Esfahlan et al.,
2019). Furthermore, Walnut (Juglans regia L.) has long been
consumed as a highly nutritious food in many parts of the world and is
an important component of the Mediterranean diet. Walnuts are
widely available in our diet and have long been known for their
nutritious properties. Walnuts are rich in unsaturated fatty acids
(omega 3), fiber, protein sources, vitamins (vitamin A, vitamin E,
vitamin K, vitamin B1, vitamin B2, vitamin B6, folic acid and niacin),
various minerals (copper, manganese, calcium, phosphorus, zinc,
 W a l n u t | 367

magnesium, sodium, potassium, selenium, iron) iodine and

polyphenols that It is a good diet and antioxidant source containing
(Sen, 2011; Şen ve Karadeniz, 2015; Chatrabnous ve ark., 2018;
Gulsoy et al., 2021). In recent years, in many studies conducted in
different organs, various studies have been carried out on the
determination and determination of biochemical, phytochemical and
antioxidant properties of walnuts and their contributions to human
nutrition and health (Akca, 2001; Simsek and Gülsoy, 2016; Binici et
al., 2021. It has been determined that these substances in walnuts have
protective and curative effects in many diseases as a result of both
epidemiological and clinical studies in terms of human health. these
diseases are diabetes, cancer, cardiovascular diseases, inflammation
and neurodegenerative effects that cognitive function, heart health,
cancer, diabetes, weight, gut health, and reproductive health. Its many
disease-promoting properties and potential health effects has been
attributed to the presence of many different bioactive nutrients,
including mainly unsaturated fatty acids, fiber, protein, phenolic
compounds, tocopherols and many minerals. This review aimed to
explore potential roles in various health outcomes, including heart
health, diabetes, cancer, cognitive function, brain, and obesity
(weight)

Health Benefits of Walnut

The benefits of walnuts, which are easily accessible, can be stored for
a long time, can be added to foods in different ways and are easy to
add to the daily diet, are endless. Walnut, which is a part of our daily
368 | W a l n u t

life, benefits human health in many ways when consumed regularly

and consciously. Thanks to the many bioactive compounds it contains,
walnuts are becoming more popular with each passing day and are
more and more involved in human nutrition, thanks to their nutritional
value and strong antioxidant level, their important role against
oxidative stress and the degenerative effects of free radicals. Therefore
many researchers have attached great importance to determining the
bioactive compound content of walnut and other plant groups,
evaluating the antioxidant activity, and the importance of these
substances in terms of human health

Walnut and Heart Health

Cardiovascular diseases are one of the most common diseases and the
most important causes of death worldwide, and still a danger to
humans. Heart disease - or cardiovascular disease - is a broad term for
chronic conditions related to the heart and blood vessels. Among these
terms, tachycardia, heart attack, heart failure, arrhythmia, heart valve
disease and heart failure are the most known ones. High LDL (bad
cholesterol) is the main cause of heart disease (Zambon, et al., 2000;
Anderson et al., 2001; Spaccarotella, et al., 2008; Guasch-Ferré, et al.,
2013 ). Epidemiological and clinical studies conducted in recent years
have shown that there are strong relationships in reducing
cardiovascular diseases and increasing the antioxidant defense system,
especially thanks to the characteristic lipid profile of walnuts, which is
one of the nut groups. Omega 3 fatty acids are seen as benefits for
improving heart health. Thanks to the omega 3 fatty acids in walnuts,
 W a l n u t | 369

it helps to protect heart health. In addition, thanks to gamma-

tocopherol, a type of vitamin E, and other elements, the elements play
an important role in cleaning the environment of the heart, especially
cholesterol, and reducing the risk of cardiovascular disease by
reducing insulin resistance, cholesterol concentrations, lipid
peroxidation and oxidative (Rim et al., 1993; Kris-Etherton, et al.,
1999; Anderson, et al., 2001; Albert, 2002; Spaccarotella, et al., 2008;
Banel et al., 2009; Ros, 2009; Bernsteine, et al., 2010; Kendall et al.,
2010; Ros, 2010; Sabate et al., 2010; Estruch et al., 2013; Guasch-
Ferré, et al., 2013; Aune et al., 2016; Ros ve ark., 2018). However,
some researchers have reported in recent studies on walnuts that
ellagitannins in walnuts have protective effects in relation to
cardiovascular diseases (Papoutsi et al., 2008; Spaccarotella et al.,
2008; Larrosa et al., 2010; Vadivel et al., 2012; Nergiz-Unal et al.,
2013; Sánchez-González, et al., 2017). There is growing evidence and
studies are ongoing regarding the role of walnut consumption in
reducing the risk of coronary heart disease.

Walnut and Cancer

Nowadays, while cancer continues to be one of the most important

health problems all over the world, the rapid increase of this disease,
which results in death in the world, is very important for human
health. (Anand et al., 2008; Globocan, 2012; Cevik and Pirinçci,
2017). It is not correct to say that walnuts cure cancer, but there are
opinions that they can eliminate the factors that cause cancer or reduce
the risk of developing the disease. Some researchers have widely
370 | W a l n u t

investigated the effect of polyphenols in walnuts and especially

ellagitannins on the initiation, development, progression and
prevention of cancer and found positive results (Owen et al., 2000;
Clemons, et al., 2001; Corona et al., 2007; Spaccarotella et al., 2008;
Khan and Mukhtar, 2013; Reiter et al., 2013; Hardman, 2014;
Sanchez-Gonz alez et al., 2015). According to different sources, the
relationship between cancer and nutrition varies between 10-70%,
with an average of 35% (Cevik and Pircisci, 2017). In recent years,
researchers have tried to find out the relationship between nutrition
and cancer and which foods have protective effects against cancer,
and studies are still ongoing. Some studies have reported that walnuts
have the highest antioxidant activity and can play a beneficial role in
the prevention of cancer and are associated with a decrease in deaths
due to this disease. (Thiebau et al., 2009; Heuvel et al., 2012; Guasch-
Ferré et al., 2013; Aune et al., 2016; Ozkan and Celik, 2016; Fang et
al., 2021). A study led by Mantzoros 2015 showed that a diet
containing walnuts could slow colorectal tumor growth by exerting
beneficial effects on cancer genes. This is the first study to evaluate
whether walnut consumption causes changes on micro-ribonucleic
acids (mRNA), which are defined as nucleotides that play a role in
gene expression. The researcher stated that the walnut-containing diet
contains fatty acids that protect against colon tumor due to the direct
effects or the additive effect or synergistic effects of other multiple
components in walnuts. The investigator found that the total amount
of omega-3 fatty acids, including plant-based alpha-linolenic acid
(ALA), was 10 times higher in tumor tissues of mice fed a walnut-
 W a l n u t | 371

containing diet compared to mice fed a control diet, particularly the

smaller tumor size had higher omega-3 fatty acids in tumor tissues.
shows that it is related to the percentage of fatty acids; This suggests
that ALA may provide a protective benefit. Tumor growth rate was
also observed significantly slower in the walnut group compared to
the control group (Mantzoros, 2015). In another study, Ros et al.,
(2018), found that walnuts contain substances such as some
polyphenols, phytomelatonin, ellagitannins and urolithin that enable
them to have anti-cancer effects. In addition, it states that it reduces
substances that cause cancer, thanks to an important form of vitamin E
in its content. Walnuts in a study investigating the effects of gamma-
tocopherol on factors related to prostate and vascular health,
especially in elderly men, it was reported that walnuts may have an
effect on prostate health due to the antioxidant vitamin E content
(Spaccarotella et al., 2008). In some studies on breast cancer, which is
common in women, it has been stated that walnut consumption may
suppress the growth and survival of breast cancer in humans (Brennan
et al., 2010; Nagel et al., 2012; Kim et al., 2014; Hardman et al.,
2019). It is also known that omega-3 fatty acids containing alpha-
linolenic acid have a protective effect on different types of cancer
such as bladder, breast and endometrial cancer. In a study by of
common breast cancer in women, it was noted that walnut
consumption may suppress the growth and survival of breast cancer in
humans. In walnut oil, besides alpha-linolenic acid; There are also
other fatty acids that may have anticarcinogenic effects, such as oleic,
linoleic, palmitic and stearic acid (Batirel et al., 2018). However, it is
372 | W a l n u t

seen that more research needs to be done in order to better understand

the effect levels of cancer and nutrition (walnut) in humans.

Brain and Walnut

Population growth in the world is increasing and increasing life

expectancy is causing a large population to age. It is seen that this
demographic change has increased the incidence of neurodegenerative
diseases in terms of human health. Neurodegenerative diseases are
defined as the damage of nerve cells or the failure of their functions to
fulfill their functions and deterioration (Marx, 2006; Poulose, et al.,
2014; Jack ve ark., 2015). Although the central nervous system is
particularly vulnerable, it also affects other organ systems (heart
disease, cancer, arthritis, diabetes and age-related disorders).
Therefore, systemic protection from oxidative stress and inflammation
may affect the brain not only from their direct effects, but also in the
functioning and protection of other organs. In many of these
neurodegenerative diseases, people continue their negative and
irregular life activities (such as lifestyle, unconscious and unhealthy
diet, lack of sports, smoking). One of the most effective and cheapest
ways to prevent this situation can be obtained with a healthy diet with
antioxidant and anti-inflammatory effects. Walnut (Juglans regia L.),
which is the most important type of nuts, has been determined in
many studies to be rich in high amounts of nutrients and bioactive
compounds, including Omega-3, polyphenolics, polyunsaturated fatty
acids (alpha-linolenic acid, linoleic acid (Pereira, et al., 2008; Shukitt-
Hale, et al., 2008; Willis, et al., 2009a,b,c,d; Poulose, et al., 2014;
 W a l n u t | 373

Câmara, et al., 2016; Pribis, 2016). In various clinical studies, PUFAs

and other phytochemical compounds play an important role in brain
health and healthy neuronal processes. The important alpha-linolenic
acid, which is responsible for the modulation of membrane stability,
neuroplasticity, signal transmission rate, and serotonin and dopamine
concentrations found in walnuts, has been shown to directly affect the
physiology of the brain by affecting different parts of the brain.
Alpha-linolenic acid is also a precursor of a fatty acid that regulates
serotonin and dopamine density. (Pereira et al., 2008; Heinrichs, 2010;
Poulose ve ark., 2014; Arab ve Ang, 2015; Jack et al., 2015; Pribis,
2016). Pribis, (2016) reported that people showed positive effects on
mood (anger, anxiety, fear, depression and confusion) and reduced
these problems. The result, there is growing evidence that walnuts
have beneficial effects on the brain and mood, thanks to the many
bioactive compounds they contain.

Diabetes and Walnut

Diabetes is one of the most common and rapidly increasing diseases in

the world, which is at the forefront among the diseases of the age,
which is the primary cause of the formation of many different
important diseases. Type 2 diabetes is the most common type of
diabetes, which has many different types. This disease is basically
defined as an imbalance between the body's blood sugar and the
hormone insulin, which ensures the proper use of this sugar in the
cells. Therefore, it is of great importance to keep the blood sugar in
the body under control in order to prevent Diabetes (Pan, et al., 2013;
374 | W a l n u t

Nijike et al., 2015; Fang et al., 2016; Ros, et al., 2018; Cole and
Florez, 2020). In order for people's vital activities to continue without
any problems, and especially for the prevention and protection of
diabetes, people should pay attention to their lifestyle and proper
eating habits. Walnuts contain high amounts of dietary fiber,
antioxidants and phytosterols. Compared to other nuts containing high
amount of monounsaturated fat, walnuts are very rich in
Polyunsaturated fatty acids (PUFAs) compared to other types.
(Maguire et al., 2004; Chen and Blumberg, 2008; Anonim, 2021). In
particular, walnuts, due to their fatty acid compositions (linoleic acid
and α-linolenic acid), have been shown to positively affect insulin
resistance and type 2 diabetes risk by increasing circulating PUFA
concentrations (Riserus, 2008; Riserus, 2009; Zibaeenezhad ve ark.,
2016Arab ve ark., 2018; Liu ve ark., 2020 ). Walnut have
monounsaturated fatty acids, protein and fiber and are low in
carbohydrates. This means they help fill us up while keeping blood
sugar low. Some epidomological studies have found that the effects of
walnut consumption on insulin secretion (Estruch et al., 2006) and
glucose homeostasis (Jenkins et al., 208), causing an inverse
relationship between them, are associated with a lower risk of type 2
diabetes (Pan et al., 2013). A recent study published in Circulation
Research found a lower risk of heart disease and death in people with
Type 2 diabetes who ate nuts. In addition, in many studies, walnuts
have been shown to improve good cholesterol and reduce bad
cholesterol.
 W a l n u t | 375

Walnut ve Diet quality (Obesity)

According to the World Health Organization (DSO), obesity is an

important disease and a public health problem that is difficult to treat
and occurs as a result of an increase in tissue as a result of excessive
accumulation of fat in the body. The reason that makes obesity
important is not only about appearance, but also it is known to be
associated with many diseases (diabetes, cardiovascular disease,
cancers, musculoskeletal disorders). Today, Obesity is increasing day
by day both in the developed and developing world (Koruk and Şahin,
2005; Brennan, et al., 2010; Çayır et al., 2011; Zileli et al., 2016;
Njike, et al. , 2017; Liu et al., 2019; Fang et al., 2020). Nutritional
habits of people have an important role in the development of obesity.
It is an important component of hard-shelled foods, especially walnut
diet. Walnut, "Obesity’’ is known to be associated with various
diseases, and some studies have also stated that it has the potential to
prevent obesity and has an effect. (Estruch et al., 2018; Pal, 2020).
Brennan et al., (2010) reported that walnuts good fat (ALA/omega 3),
fiber and protein, especially pulp, can help in weight management,
especially providing satiety and reducing calorie consumption. In
another study, it was seen that it may have a different fatty acid
metabolism in terms of polyunsaturated fatty acids and saturated fatty
acids, so it will affect weight loss (Fang et al., 2020). In another study,
Katz et al., (2012) has reported that A meta-analysis investigating the
effect of walnut consumption on blood lipids found that during short-
term trials, walnut-enriched diets significantly reduced total
376 | W a l n u t

cholesterol and low-density lipoprotein cholesterol (LDL-C)

compared with control diets. As a result, one of the most important
strategies to reduce the incidence of obesity in many epidemiological
studies is to address an optimal energy balance. For this reason,
although walnut is a fatty acid-rich food, it has been shown that there
is a relationship between daily and regular walnut consumption and
weight loss. (Brennan et al., 2010; Jackson and Hu, 2014; Njike ve
ark., 2015; Njike, et al., 2017; Rock, et al., 2017; Bitok, et al., 2018;
Fang, et al., 2020)

CONCLUSION

Walnut, which is the most important type of hard-shelled fruit, has a

very important place as a support in nutrition and medical treatments
in terms of many bioactive compounds it contains. In this study, it
gives brief and important information about walnuts and human
health. In recent years, some studies on walnuts and their bioactive
components and diseases have been examined. Studies have shown
that walnuts are rich in unsaturated fatty acids such as linoleic acid
and Omega 3, protein, vitamins and minerals, as well as bioactive
substances such as melatonin, ellagic acid, vitamin E, carotenoids and
polyphenols, which have antioxidant effects. Many epidemiological
and clinical studies conducted in recent years have shown that many
epidemiological and clinical studies conducted regularly in recent
years have shown that regular walnut consumption is associated with
coronary heart disease, brain, hypertension, diabetes, weight gain over
time, obesity, visceral obesity, metabolic Syndrome has shown to have
 W a l n u t | 377

a beneficial effect on many health outcomes, such as cancer. It has

been shown that walnut consumption has an important place in
nutrition and prevention of diseases such as coronary heart disease,
brain, hypertension, diabetes, weight gain over time, obesity, cancer.
In conclusion, walnut is an optimal healthy food for a balanced and
healthy diet. For this reason, it is necessary to raise awareness about
many beneficial bioactive compounds in walnuts and their benefits,
scientific research on their benefits to human health should be
increased and studies on different diseases should continue. For a long
health life, walnuts should be included in people's healthy and regular
nutrition programs.

Figure 1. Evaluation of different organs of walnut

378 | W a l n u t

Figure 2. Walnut bioactive compounds and health

 W a l n u t | 379

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