Emir. J. Food Agric. 2014.

26 (6): xxx-xxx
doi: 10.9755/ejfa.v26i5.15722
http://www.ejfa.info/

REVIEW ARTICLE

Biotechnology of mulberry (Morus L.) - An appraisal

K. Vijayan*, P. Jayarama Raju, A. Tikader and B. Saratchnadra

Central Silk Board, BTM Layout, Madiwala, Bangalore, Karnataka 560068, India

Abstract

Mulberry (Morus sp.) is one of the economically important trees present in Asian countries. It is cultivated for
leaf, which is used for feeding the caterpillars of a silk producing insect (Bombyx mori L.). It also adds value
through edible fruits, timber and several pharmaceutically important chemicals. As there is limits on
improvement of mulberry through conventional breeding, due to high heterozygosity and long generation
period, attempts have recently been made to complement conventional breeding with modern biotechnological
tools such as plant tissue culture, recombinant DNA technology and molecular markers to make mulberry
genetic improvement easier, speedier and more efficient and effective. The techniques of tissue culture has
grown considerably in mulberry and encompassed areas including micropropagation, plant regeneration from
leaf discs, and screening for stress tolerance. Recently, genetic engineering was adopted to enhance drought and
salt tolerance in mulberry. Molecular markers such as RPAD, ISSR and SSR have also been used in mulberry
for molecular characterization of germplasm, biodiversity analysis, genetic mapping and identification of
molecular markers for some traits. However, there are still a number of issues such as resistance to fungal and
bacterial diseases, infestation of pests and insects, and tolerance to drought and salinity, which are to be sorted
out to make mulberry cultivation sustainable to meet the demand of the growing silk industry. This review
describes the developments of tissue culture, transgenesis and molecular markers in mulberry and highlights the
constraints and future prospects.

Key words: Cryopreservation, Gene transfer, Molecular markers, Haploids, Tissue culture, Triploids

Introduction hypoglycaemia, and avulsed teeth (Lee et al., 2011)

Mulberry (Morus) of the family Moraceae is an and it is a good source of phenolic acids and
economically important tree grown commercially in flavonoids (Arfan et al., 2012). Further, mulberry
India, China and several other Asian countries to trees have become an integral part of the
feed the caterpillars of the silk producing landscaping in a number of countries (Tipton,
Lepidopteran insect Bombyx mori L. (Vijayan et al., 1994). Traditionally, mulberry (Morus) was placed
2011a; Vijayan et al., 2012). Its leaf is also used for in the tribe Moreae of the family Moraceae under
feeding cattle, goat and other animals as it is highly the order Urticales (Takhatajan, 1980), however,
nutritious and palatable to herbivorous animals based on molecular evidence, the angiosperm
(Benavides et al., 1994), besides having several phylogenetic group (APG II, 2003) placed
medicinal properties including antioxidant (Yen et Moraceae in the order Rosales. More than 68
al., 1996) and hypoglycaemial ones (Kelkar et al., species of Morus have been widely recognized
1996). It is also grown for fruit, which is used for (Datta, 2000), of which M. alba, M. latifolia, M.
human consumption, production of jam, jelly, mutlicaulis are grown for leaves while M. nigra is
marmalade, frozen desserts, pulp, juice, paste, ice grown for fruit and M. serrata for timber (Vijayan
cream, and wine (Koyuncu, 2004). Mulberry fruit is et al., 2011a). Different cytomorphs such as
a good medicine for dysentery, constipation, diploids (Morus alba; 2x, 2n=28), triploids (M.
alba, M. indica; 3x, 3n=42), tetraploids (M.
Received 30 June 2013; Revised 18 December 2013; Accepted laevigata, M. cathayana, and M. boninensis; 4x,
22 December 2013; Published Online 15 January 2014 4n=56), hexaploids (M. serrata and M. tiliaefolia;
*Corresponding Author
6x, 6n=84;), octaploids (M. cathayana; 8x,
8n=112), and docosaploids (M. nigra; 22x,
K. Vijayan 22n=308) are available in mulberry, though
Central Silk Board, BTM Layout, Madiwala, Bangalore,
Karnataka 560068, India diploids and triploids dominate mostly (Vijayan et
al., 2012). It is believed that mulberry originated in
Email: kvijayan01@yahoo.com
the northern hemisphere, particularly in the

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Himalayan foothills and spread to the tropics of Micropropagation

southern hemisphere (Benavides et al., 1994). At Mulberry can be vegetatively propagated
present, mulberry is growing in all regions between through stem cuttings, grafting or budding.
50oN Lat. and 10oS Lat. from sea level to altitudes However, success of these methods depends on a
as high as 4000 m (Tikader and Dandin, 2005), number of factors such as genetic makeup of the
which include Asia, Europe, North and South plant, age and physiological conditions of the
America, and Africa. parental cutting, climatic conditions and others.
Additionally, newly developed mulberry varieties
Need of biotechnological tools in mulberry
cannot immediately be propagated through stem
The main focus of mulberry breeding is to
cuttings as at least 6-7 month maturity is required to
improve leaf productivity as it alone contributes
make the cuttings from the parental plant (Kapur et
more than 38.2% to the sericulture productivity
al., 2001). Micropropagation (Figure 1), on the other
(Banejee, 1998). However, it is not easy to improve
hand, allows multiplication of the plant in a short
the leaf productivity as it is a multifactorial trait
period under the controlled conditions. Further, in
determined by a number of associated characters
conventional method of propagation through stem
such as plant height, number of branches, leaf
cuttings, each stem cutting produces only one plant,
retention capacity, nodal length, leaf size and
whereas in micropropagation thousands of plants can
weight, total biomass etc. (Doss et al., 2011). High
be produced from a single plant piece Moreover
heterozygosity and inbreeding depression hinder
micropropagation can provide plantlets throughout
the development of inbreds, hence, directional
the year irrespective of seasonal variations,; It is,
breeding failed to make much progress. Therefore,
thus, an efficient and cost effective tool for rapid
the heterozygous parents are used to generate F1
multiplication of mulberry in a relatively shorter
progenies, which are then subjected to different
time and space. Micropropagation also facilitates
evaluation and selection procedures to identify the
production of virus-free plants from the apical
best one. This type of breeding system bars the
meristematic tissues. However, success of
possibility of introgressing genes of desirable traits
micropropagation is dependent on a number of
from wild relatives/species due to genetic drag and
factors among them genetic makeup, age and origin,
subsequent difficulty in eliminating the undesirable
physiological and pathological conditions of the
traits that come along with it. Under such
explants, media composition and culture conditions
circumstances, the feasible means of improving
are considered key factors. Ohyama (1970) initiated
specific traits without disturbing the current trait
mulberry micropropagation by regenerating whole
combinations is adoption of biotechnological tools
plants from axillary buds of M. alba. Later, a number
like transgenesis, which enable introduction/over
of investigators used different media and explants as
expression of desirable genes (Vijayan et al.,
evident from Table 1. Shoot tips and dormant
2011a), or knocking out undesirable genes (RNA
axillary buds were found suitable for mulberry
interference) (Vijayan et al., 2011b). Mulberry,
micropropagation. A number of factors significantly
being a tree with high heterozygozity, poses
aeffect microprogation in mulberry, among them
difficulties on improving traits of economic
composition of media is an important factor.. Among
importance through conventional breeding and
the different media compositions and hormones
selection. Environmentally less influenced and
tested, MS (Murashige and Skoog, 1962) medium
developmentally stable molecular markers provide
containing 2 mg L-1 6-benzylamino purine (BAP) is
reliable tools for the breeders to characterize the
the best for shoot multiplication (Lalitha et al.,
germplasm and to select parents and offsprings
2013). However, it is also important to note that 6-
through marker assisted selection. Thus, it would be
benzyladenine (BA) above 2 mg L-1 is inhibitory for
prudent to use biotechnological tools to harness the
shoot initiation and multiplication (Bhau and
vast benefit mulberry offers to mankind.
Wakhlu, 2003). Among the sugars, sucrose is the
Tissue culture in mulberry best (Bhau and Wakhlu, 2003). Vijayan et al. (2000)
Tissue culture technique in mulberry has found that 3% glucose is better for shoot formation
developed and ramified into different areas such as from leaves. Among different pH levels, 5.6-5.8 is
micropropagation, callus culture, organogenesis, optimum for shoot multiplication (Enomoto, 1987).
screening of genotypes for stress tolerance, Agar in the culture medium also plays important role
induction of polyploids, cryopreservation, in the success of micropropagation. Among various
transgenesis and others. Following are some of the concentrations, 0.8% agar is the best and above 1.0%
significant contributions tissue culture made in found reducing shoot growth (Pattnaik and Chand,
mulberry. 1997; Thomas, 2003).

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Table 1. Investigations on in vitro plant regeneration in mulberry (Morus spp.) during the last one dcade.
Sl. Species Tissue Mode of Medium + PGR Medium + PGR References
No. regeneration (mgL-1) for shoot (mgL-1) for
growth Root growth
1 M. alba Shoot tips, Micropropagation MS + BA (2) + MS + NAA (1) Anis et al., 2003
Nodal NAA (0.2)
segments
2 M alba Nodal Micropropagation MS + BA (1.5) MS + NAA (0.5-1.0)/ Bhau and
segment, IBA (0.5-1.0) Wakhlu, 2003
Shoot tips
3 M. alba Cotyledons Micropropagation MS+ BA (1.2) MS+IBA / NAA Thomas, 2003
(0.25)
4 M. alaba Leaves Micropropagation MS + BAP (0.5 and IAA (0.1 and 1), IBA Chitra and
1) or TDZ (.5 and 1) (0.1 and 1, NAA Padmaja, 2005
(0.1 and 1) or 2,4-D
(0.1 and 1)
5 M.alba Nodal Micropropagation MS + BA (2.5)+ MS + IBA (1.0) Ahmed et al.,
segments GA3 (0.3) 2007; Kashyap
and Sharma, 2006
6 Morus Side bud Organogenesis MS +IAA ( 0.2) MS + IAA (0.1)+ BA Ji et al., 2008
spp +BA (3.0) ( 0.5)
7 M. alba Nodal Oraganogenesis MS + MS + Balakrishnan et
segments BAP (5.0) + KIN IBA (0.5) al., 2009
(1.10)
8 Morus Nodal Oraganogenesis MS + BAP (2.00)+ MS +NAA (0.50) Rao et al.,
spp. explants IAA (0.15) 2010a,b.
9 M. nigra Nodal Micropropagation MS+ BAP (1.5) + MS + BA (1.0) + Zaki et al., 2011
segments NAA (0.5) NAA (2.0).
10 M. alba Nodal Micropropagation MS + BAP (2.0) MS +NAA (1.0), Chattopadhyay et
segments al., 2011
11 M. alba Nodal Micropropagation MS +BAP (1.0) + MS + Sanjeevan et al.,
segments NAA (0.25)+ GA3 IBA (0.5) 2011
(0.5 ).
12 M. Nodal Micropropagation MS + IBA (2.0-0.4) + Akram and Aftab,
macroura segments activated charcoal 2012
(0.1%)
13 M. indica Nodal Micropropagation BA (2.0) and GA ½ MS + IBA (2.0) Lalitha et al.,
segments (0.5). 2013
AE: von Arnold and Eriksson (1981) medium, MS: Murashige Skoog (1962) medium, 4PU: [NL(2-chloro-4-
pyridyl)Nlphenyl-urea], 2,4-D: 2,4-dichlorophenoxyacetic acid, 2,4,5 T: 2,4,5-trichlorophenoxyacetic acid, BA: 6-
benzyladenine, BAP: 6-benzylamino purine, IAA: indole-3-acetic acid, IBA: indole-3-butyric acid, IPA: Indole-3-
proponic acid, GA3: gibberellic acid, Kn: Kinetin, NAA: α-naphthaleneacetic acid, TDZ: thidiazuron.

Rooting in mulberry is achieved mostly in half- initiates within 12-18 days of the transfer of
strength MS medium as it is found better than full explants to the media (Hossain et al., 1992; Pattnaik
length (Table 1). Among the plant growth and Chand, 1997). In vitro plantlets need to be
hormones, 0.5 mg L-1 NAA is best for M. alba, M. hardened in the greenhouse before transfer to the
indica, M. multicaulis and M. latifolia as it gives field. Since the newly developed plantlets are
more than 95% rooting (Vijayan et al., 2003), grown in a pathogen free atmosphere in the
though indole-3-butyric acid (IBA) is the best auxin laboratory, sudden exposure to outside conditions
for M. nigra (Rao and Bapat, 1993). Vijayan et al. may put them into the risk of getting affected by
(1998) observed that higher concentrations of most pests and diseases. In order to avoid/reduce such
of the auxins (>1.0 mg L-1) were inhibitory for root risks, hardening is to be done carefully. The
formation in mulberry and there was a strong medium to which the plant is transferred should be
interactive relationships among genotype, hormone sterile, hence, autoclaved vermi-compost is
type and its concentrations. Generally, rooting considered as one of the most suitable substratum

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for the initial stages of 2-3 weeks (Yadav et al., (Vijayan et al., 1998). Subculturing at an interval of
1990). After the initial hardening, the plants are 20-25 days favors better callus growth and
transferred to earthenware pots of appropriate size lessening of phenol oxidation. Once the friable
containing garden soil composed of 50% soil and callus is transferred onto a medium supplemented
50% compost. The pots are kept under shade for with higher concentration of cytokinin and less
another 2-3 weeks, before transferring to the field auxin, shoot buds start develop. BAP is the most
(Chattopadhya et al., 2011; Vijayan et al., 2011b). preferred cytokinin for shoot regeneration (Narayan
Biological hardening is an alternative method for et al., 1989). Indirect organogenesis often creates
hardening micropropagated plants and in this variations among the regenerated plants, a
technique plants are challenged with biotic stress phenomenon called somaclonal variation, which
caused by soil microbes. Many fungi like vasicular often facilitates isolation of useful plants. Narayan
arbuscular mycorrhiza (VAM), Trichoderma, et al. (1989) isolated such a variant (SV1) from
Piriformospora indica, Azotobactor chroococcum plantlets developed from a productive variety S1.
etc. are used for biological hardening (Kashyap and SV1 was found have better leaf yield (34,938
Sharma, 2006). kg/ha) than S1 (28,048 kg/ha). Direct
organogenesis from explants especially from
Organogenesis in mulberry
cotyledons and leaf segments has great potential for
Organogenesis is a complex phenomenon
transgenesis. In mulberry, direct plant regeneration
involving de novo formation of organs. Successful
from leaf explants was first reported by Kim et al.
organogenesis depends on a number of factors
(1985) and later by Yamanouchi et al. (1999).
which include appropriate selection of explants, age
Vijayan et al. (2000) obtained shoots from leaves
of the explants, media compositions, specific
on MS medium supplemented with BAP 2 mg L-1
growth regulators, genotype, sources of
and glucose as the sugar source (Figure 2).
carbohydrate, gelling agent, and other physical
Subsequently, Bhatnagar et al., (2001) achieved
factors including light, temperature, humidity and
50% regeneration from hypocotyl and 70% from
other factors. Depending on these factors plant
cotyledon using thidiazuron TDZ (0.5 mg L-1). The
regeneration may occur either directly or indirectly
effectiveness of TDZ on direct shoot induction
(Jain and Datta, 1992). In direct organogenesis,
from leaf explants was further confirmed by Chitra
plants develop directly from the explants without
and Padmaja (2005) and Raghunath et al. (2013).
formation intermediate callus while in indirect
Currently, it is possible to regenerate plantlets
organogenesis plant develops via callus formation.
directly from most of the (Table 2).
Again, callus induction depends on a number of
factors such as nature of explants, genotype, Somatic embryogenesis
medium and its composition. A variety of explants Somatic embryogenesis provides a valuable
has been tested to initiate callus formation in tool to enhance the pace of genetic improvement of
mulberry (Table 1). Cambial regions (Narasimhan commercial crop species (Stasolla and Yeung,
et al. 1970), hypocotyls segments (Shajahan et al., 2003). Several investigating groups attempted
1997), cotyledons (Thomas, 2003), stem segments induction of somatic embryos in mulberry but the
(Vijayan et al., 1998), and young leaves (Chitra and rate of success is less. Shajahan et al. (1995)
Padmaja, 2005) have been tested and it is found obtained heart shaped embryos from M. alba
that internodal segment from young shoot is the hypocotyl segments cultured on MS medium
best explant for callus initiation in mulberry supplemented with 2,4 D (0.45-4.52 µM) and BAP
(Vijayan et al., 1998) and MS medium (Murashige (2.2 µM ). Agarwal (2002) and Agarwal et al.
and Skoog 1962) is most commonly used for callus (2004) could obtain primary and secondary somatic
induction in mulberry (Vijayan et al., 2011b). embryoids by culturing zygotic embryos on MS
Similarly, 2,4-D is the hormone best suited for medium containing 0.05 mg L-1 2,4-D + 0.1 mg L-1
callus induction. Addition of Kn, IAA and NAA BAP and 6% sucrose. However, due to the
facilitates better proliferation and friability of difficulty in hormonally controlling the formation
callus. Supplementation of coconut water (150 ml of adventitious shoots and roots in mulberry,
L-1) and casein acid hydrolysate (100 mg L-1) somatic embryogenesis has not been developed as it
enhanced callus induction from foliar explants is in many other crop plants. Thus, concerted
(Susheelamma et al., 1996). Multiplication of callus efforts are needed to make somatic embryogenesis
through repeated subculturing makes the callus successful in mulberry.
more friable and responsive to shoot formation

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Figure 1. Micropropagation in mulberry. A-nodal explant, B-shoot from the explant, C-multiple shoots growing from
explant, D-rooting of the micropropagated plant, E- hardening of the micropropagated plant.

Table 2. Direct plant regeneration from mulberry leaf, cotyledon and hypocotyls explants
(Adapted from Vijayan et al., 2011c).
Explant Medium +PGR Species References
Leaf MS+ BA , MS+ BA+ NAA Morus sp. Machii, 1999
MS + BA + BR M. alba Lou et al., 1996
MS + TDZ + TIBA M. alba Adachi et al., 1999
MS + BA M. indica Vijayan et al., 2000
MS+TDZ + IAA + AgNO3 M. indica Raghunath et al., 2009

Mod. MS + TDZ M. alba; Yamanouchi et al., 1999

M. latifolia
Mod- M. indica Das et al., 2011
MS+IAA+TDZ+AgNO3
MS + TDZ + IAA + AgNO3. M. indica Raghunath et al., 2013
Cotyledon/ hypocotyls MS + IAA + BAP M. indica Kathiravan et al., 1997
region
MS+TDZ M. alba Sawagichi et al., 1997

MS+TDZ+IAA+AgNO3 M. indica Raghunath et al., 2009

Mod- M. indica Das et al., 2011
MS+IAA+TDZ+AgNO3
AgNO3- Silver nitrate; BA-Benzyl adinine; BR- Brassinosteroid; IAA-Indole acetic acid; MS-Murashige and Skoog
(1962) medium; NAA-α-Napthalene acetic acid; TDZ- Thidiazuron.

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Figure 2. Screening of germplasm for salt tolerance using axillary bud growth in different concentrations of NaCl. 1-
0.0%, 2- 0.25%, 3- 0.5%, 4- 0.75% and 5- 1.0% NaCl.

Haploid production Protoplast isolation, culture and regeneration of

Haploid plants being gametophytic in origin plantlets
possess only half the normal number of Somatic hybridization through protoplast fusion
chromosomes as present in the parent. They can be has opened a new avenue for developing new
used to produce homozygous lines, which are characteristics, which are not possible through
invaluable for any breeding programmes especially conventional breeding (Johnson and Veilleuz,
for tree crops with longer generation cycle and high 2010). There are only a few reports dealing with
heterozygosity. Since the first successful report on plant regeneration from protoplasts in mulberry
regeneration of haploid plants from pollen grains of (Tewary and Sita, 1992; Umate et al., 2005). A
the cultured anthers of datura (Guha and combination of 2% cellulase, 1% macerozyme and
Maheshwari, 1964), this technique has been 0.5% macerase is found optimal for better isolation
extensively used in most of the agriculturally of viable protoplast. Protoplast fusion in mulberry
important plant species. However, only limited was successfully achieved using chemical fusogen
success could be obtained on tree species. In (Onishi and Kiyama, 1987) and electro-fusion
Mulberry, though anther culture was first attempted (Onishi and Tanabe, 1989). Although protoplast
by Shoukang et al. (1987) and later by Katagiri isolation and regeneration was achieved,
(1989), Sethi et al. (1992) and Chakraborti et al. development of somatic hybrids in mulberry could
(1999), till date no plants could be regenerated. not be achieved. Hence, efforts in this end need to
Nonetheless, Thomas et al. (1999) regenerated be continued.
gynogenic haploids by culturing immature female
Other applications of tissue culture in mulberry
catkins on MS medium supplemented with BA (2.0
Screening for stress tolerance
mg L-1) and 2,4-D (1.0 mg L-1) for the first three
Since salt tolerance in plants is a complex
weeks, and the remaining period with 2,4-D (1.0
phenomenon involving morphological,
mg L-1), glycine (88.8 mg L-1) and proline (15 mg
physiological and biochemical processes, screening
L-1). However, no further report on haploidy is
of genotypes for salt tolerance need to be done in
available in mulberry, though doubled haploidy is
such conditions where the influence of external
of much use in mulberry breeding.
factors is minimal (Vijayan et al., 2011c).
Maintenance of uniformity of salinity across the

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field and seasons is difficult, terefore, screening of Induction of tetraploidy

the plants under in vitro is considered as an ideal In general the mulberry is propagated through
option, where most of the environmental conditions vegetative means. Hence, sterile high yielding
can be controlled. Vijayan et al. (2003) using varieties/cultivars do not pose any problems for
axillary buds of 63 mulberry germplasm accessions their true to type multiplication. Triploidy in
maintained at the Central Sericultural Research and mulberry is considered as the optimum level of
Training Institute, Berhampore, West Bengal, India ploidy because triploids show several advantages
isolated salt tolerant genotypes by surface over plants of other ploids. Triploids are superior in
sterilizing the nodal explants and culturing on MS leaf yield, stress resistance and chemical
medium (Murashige and Skoog, 1962) components of the leaf (Yang and Yang, 1989).
supplemented with 2 mg L-1 BAP, 30 mg L-1 Considering these advantages, tetraploids are
sucrose and 0.0% to 1.0% NaCl (Figure 3). developed from diploids by colchicine treatment of
Genotypes showing early sprouting and better the growing shoots. In this method, small cotton
growth rate in 1.0 % NaCl were selected as salt pads soaked with 1.0-2.0% colchicine solution is
tolerant. Out of the 63 accessions tested, 16 applied over the growing buds for 2-3 consecutive
sprouted in 1.0% NaCl, and 13 of them survived till days. Though this method is easier to apply, it
30 days and these genotypes were considered as suffers from quick drying of the cotton pad,
salt tolerant. For rooting assessment, in vitro excessive loss of colchicines and difficulty in
grown shoot apices (1-2 cm in length) were maintaining the uniform concentration of the
cultured on MS medium containing 2.6 M NAA, colchicines solution. Application of colchicine in
30 g l-1 sucrose and 0.1, 0.2, 0.3 and 0.4% NaCl. vitro solves most of these problems and also makes
Out of 63 genotypes tested, only five genotypes the system more economic. Chakraborti et al.
could develop roots at 0.2% NaCl. Five genotypes, (1998) cultured apical buds of field grown mulberry
which showed better root and shoot growth in both on MS medium supplemented with BAP (2 mg L-1)
experiments, were further tested in pot culture and four concentrations of colchicine (0.0, 0.05, 0.1
under different salt levels to confirm the efficacy of and 0.2%). It was observed that the optimal level of
the in vitro screening. The study while confirming colchicine was 0.1% as it produced 39.4 ± 4.8%
the higher salt tolerance of the selected genotypes tetraploids. Higher concentrations of colchicine
also demonstrated the efficacy of in vitro reduced the survivability of the buds and low
screening to isolate salt tolerant genotypes in concentration reduced the percentage of tetraploid
mulberry (Vijayan et al., 2003). Later, Ahamad et formation. It is further observed that in vitro
al. (2007) used this technique to investigate the application of colchicine was 80.8% more efficient
effect of NaHCO3 on various characters of than the cotton pad method. Additionally, in vitro
mulberry. This method is more economical, application of colchicine is more cost effective as
efficient and less time consuming for screening the same medium can be used for at least 4 repeated
large number of mulberry accessions for salt treatments without reducing the efficiency of the
tolerance. conchicine to induce tetraploidy.
Another method of getting triploidy in
mulberry is to culture the endosperm because in
angiosperm, endosperm is a triploid tissue formed
via double fertilization (Bhojwani and Razdan,
1996). In mulberry, Thomas et al. (2000), for the
first time, successfully developed triploids from
endosperm of the variety S36. Endosperm tissue
isolated from young fruits was cultured on MS
medium supplemented with 2,4-D (5 µM) and
different concentration of BA, Kn, TDZ, IBA,
NAA, gibberellic acid (GA3), along with tomato
juice (TJ), yeast extract (YE), casein hydrolysate
(CH) and coconut milk (CM). Shoot buds
developed when the callus was subcultured on a
Figure 3. Direct plantlets regeneration from Leaf medium containing a cytokinin or a combination of
explants. A.shoot formation from leaf, B-root formation cytokinin and NAA. The best result for callus
from the shoot, C-whole plant regenerated from leaf. induction (70-72%) was obtained on MS medium

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with BAP (5 mM), NAA (1 µM) and CM (15%) or of sodium alginate were used, the best results were
YE (1000 mg/l). The maximum number of shoot obtained at 4% alginate (Kamareddi et al., 2013).
formation was on the medium containing TDZ (1 Although somatic embryos could not be developed
µM), or BA (5 µM) and NAA (1 µM). On easily in mulberry, dormant axillary buds proved to
cytological investigation, it was found that all the be an ideal material for the synthesis of synthetic
ten plants examined were triploids with 42 seed, it can be used for cryopreservation of
chromosomes. This clearly indicated the efficiency germplasm accessions.
of the protocol and it could be used for developing
Cryopreservation of germplasm
more triploids in mulberry.
The high heterozygosity hinders conservation
Synthetic seeds of mulberry germplasm through seeds as the
Synthetic seeds are the encapsulated somatic progenies from such seeds are heterogenous in
embryos, which functionally mimic zygotic seeds nature and getting true to the parental type is
and can develop into seedlings under sterile difficult. Thus, mulberry germplasm is conserved
conditions. In a broader sense, it would also refer to as ex situ germplasms, which is laborious, needs
encapsulated buds or any other form of meristems, huge resources, and is in a risk of destruction by
which can develop into plants. In mulberry, natural calamities, pests and diseases (Vijayan et
synthetic seeds are produced mostly by al., 2011d). Thus, safe alternative methods with
encapsulating the apical/axillary buds or somatic economically viability need to be explored.
embryos with 3-5% sodium alginate and 100mM Cryopreservation is one such alternative wherein
calcium chloride solution as complexing agent plant materials are stored at ultra-low temperatures
(Kamareddi et al., 2013). Sodium alginate solution (-196°C) in liquid nitrogen. At this temperature all
is mixed with culture medium containing all the metabolic activities of the cell including
necessary ingredients essential for proper growth. divisions remain arrested; hence, the material
Patnaik et al. (1995) successfully developed this remains unaltered for long period. Two different
technology for artificial seed sysnthesis in cryopreservation techniques in vogue are the
mulberry. However, adoption of this technology for classical one and the modern vitrification
mulberry propagation was limited to a few species (Engelmann, 2000). In classical cryopreservation
of M. indica (Bapat and Rao, 1990; Patnaik et al., technique, the plant material is cool down slowly
1995). Researchers have explored the possibility of at a controlled rate of 0.1-4°C/ min to about -40ºC
using in vitro derived vegetative propagules for and rapidly immersed in liquid nitrogen. In
synthetic seed production since it was found vitrification, plant material is physically or
difficult to develop somatic embryos in mulberry osmotically dehydrated and is subsequently
(Pattnaik and Chand, 2000; Kavyashree et al., subjected to ultra-rapid freezing resulting in
2004). Shoot cultures established from axillary vitrification of intracellular solutes, i.e. formation
buds on Linsmaier and Skoog’s basal medium of an amorphous glassy structure without
(LSBM) supplemented with BAP (4 mg L-1) and occurrence of ice crystals. Although different plant
TIBA (1 mg L-1) used for encapsulation. Sodium materials are used for cryopreservation,, the most
alginate and carboxy methylcellulose were added in appropriate material for cryopreservation of
the range of 2, 4, 6 and 8% (w/v) to liquid initiation mulberry is winter buds (Niino, 1995). Genetic
medium separately. For complexation, 25 x 103µM, constitution of the plant material also influences
5 x 104 µM, 75 x 103 µM and 10 x 104 µM calcium significantly the success and duration of the
chloride solutions were prepared using distilled preservation (Rao et al., 2007). Cryopreservation
water. Gel complexation was done by mixing the techniques have been standardized for M. indica,
axillary buds with hydrogels, dropping these into M. alba, M. latfolia, M. cathayana, M. laevigata,
different concentrations of calcium chloride M. nigra, M. australis, M. bombycis, M. sinensis, M
solution and incubating in orbital shaker for multicaulis and M. rotundiloba species (Rao et al.,
different time intervals (20, 30, 40, 50 and 60 min) 2007, 2009). The general procedure for
to obtain uniform beads. The sodium alginate and cryopreservation of shoot tips is that the shoot
carboxy methylcellulose embedded axillary buds segments are pre-frozen at -3°C for 10 days, -5°C
were collected using a sterilized tea strainer and for three days, -10°C for one day and -20°C for one
rinsed 2-3 times in sterile water to remove traces of day and immersed into liquid nitrogen. The
calcium chloride. The synthetic seeds were tested cryopreserved material is brought back to the
for their conversion potential under in vitro and ex ambient temperature via thawing. During this
vitro conditions. Although different concentrations process, the material is warmed either slowly at 0oC

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in air for more than 3 h or rapidly in agitated water found that when mature nodal plants are used for
at 37oC for 5 min. After thawing, surface-sterilize micropropagation, fungal growth takes place after
the bud with 70% ethanol for 1 min and sodium 20-30 days from the lenticels and scale leaves. In
hypochlorite solution (0.5% effective chlorine order to avoid this contamination the young shoots
concentration) for 20-30 minute and rinse it should be subcultured as soon as they emerge from
thoroughly with sterile distilled water for three to the buds of the explants collected from the field.
four times before culturing on MS medium
Problems in callus culture
containing all necessary supplements. Niino et al.
Oraganogenesis in mulberry is also not devoid
(2006) obtained 65% survival of the buds
of problems as often excessive blackening of the
cryopreserved at -135oC for 5 years. The Surviving
callus occurs due to exudation and oxidation of the
buds resume growth within 5 days and develop
phenol contents. The oxidative products of phenolic
shoots without intermediary callus formation. It is
compounds lower the pH of the culture medium,
also found that partial dehydration of the bud up to
thereby affecting the callus growth. This problem
38.5 % before pre-freezing at -20 °C improves the
can be limited to an extent by the addition of
recovery rate. Encapsulation of winter-hardened
activated charcoal (Mhatre et al., 1985) and or
shoot tips with calcium alginate coating was also
silver nitrate (Chakraborti et al., 1999) in the
tested successfully (Padro et al., 2012). It is
culture medium. Regular subculturing at an interval
concluded from different experiments that dormant
of 20-25 days is another way of controlling this
buds of mulberry can be cryopreserved for 11 years
problem (Narayan et al., 1989; Vijayan et al.,
without reducing the viability of the buds (Fukui et
1998). Another major problem is the formation of
al., 2011).
roots from the callus before the shoot formation.
Problems associated in mulberry tissue culture Once the root formation starts shoot formation
Problems in bud culture becomes difficult from the same callus. This
High rate of contamination is one of the major serious problem should be controlled by hormonal
problems that limit the use of mature field grown manipulation such that the effect of cytokinin
stem cutting for in vitro studies, especially for should not be less than that of auxin in the initial
flowering and in vitro fertilization (Vijayan et al., stages of organogenesis.
2011b). Since the field grown plants are often
Problems associated with acclimatization and
heavily loaded with various epiphytic and
field transfer
endophytic microbes, it is very difficult to remove
During in vitro culture, plantlets grow under
them, especially from the lenticels. Further, it is
control conditions such as humidity, irradiance,
also noted that occurrence of microbial
temperature and other factors. The plantlets
contamination depends to a great extent on
cultured in vitro in a water-saturated atmosphere
genotype, maturity of the explants and seasons.
generally wilt rapidly when transferred to normal
During rainy season, heavy bacterial infection was
greenhouse or field conditions and also are highly
observed. Patnaik and Chand (1997) reported that
prone to microbial attacks, if adequate care is not
an initial thorough washing of field grown nodal
taken. One of the major reasons for wilting of the
explants with running water for 1-2 h followed by
transferred plant is the rapid loss of water from the
treating it with 5 % (v/v) liquid detergent and 7 %
leaves due to poorly developed stomata and
sodium hyphochlorite for 8-10 minutes are essential
epicuticular wax (Santamaria et al., 1993). Poor
before doing the normal surface sterilization with
contact of roots with the soil also limits water up
0.1% aqueous (w/v) mercuric chloride to contain
take (Fila et al., 1998). Although it is quite common
contaminations. Narayan et al. (1989) washed the
that leaves with normal stomatal structure replaces
field grown nodal explants under running tap water,
the older ones, it is very common that plantlets die
then immersed in 0.1% (w/v) carbendazim for 20
during the acclimatization process (Huylenbroeck
min and washed thoroughly with distilled water
et al., 1996). To minimise such casualties, it is
before treating with 0.2% (w/v) cetavlon for 10
essential to provide with a regulated temperature,
min, and with 0.1% (w/v) aqueous mercuric
humidity, irradiance, CO2 concentration and air
chloride solution for 5 min for effective surface
flow rate during acclimatization (Bolar et al.,
sterilization. Chitra and Padmaja (2002) washed the
1998). Initially the in vitro developed plants can be
nodal explants first in running tap water for 30 min
kept inside the culture room under sterile
and surface sterilized with 70% alcohol for 1 min
conditions. Then, gradually it can be shifted to
followed by 0.1% mercuric chloride (HgCl2) for 15
green house where humidity and temperature are
min under sterile conditions. Vijayan et al. (2011b)

426
K. Vijayan et al.

under control. After a couple of months the grown cultivars, land races and species, identification of
plants can be shifted to shady places in the field, markers associated with economically important
thereafter it can be transplanted to field. traits, and developing fingerprints of germplasm
accessions as detailed hereunder.
Genetic engineering in mulberry
Although conventional plant breeding has
contributed significantly by developing several high Table 3. High yielding mulberry varieties developed for
yielding mulberry varieties (Table 3), high commercial cultivation in India.
heterozygozity and long generation cycles and
inbreeding depression prevent introgression of traits Variety Regions/Zones Cultural
from wild species and relatives. Transgenesis is one condition
such technique that enables direct transfer of genes
V1 Karnataka, Andhra Pradesh, Irrigated
of interest. Out of the two popular gene transfer Tamil Nadu
techniques viz., particle bombardment and
Agrobacterium tumefaciens-mediated Anantha Karnataka, Andhra Pradesh, Irrigated
transformation, the latter received much attention in Tamil Nadu
mulberry due to the easiness and efficacy. Vishala All sericulture regions of Irrigated
Bhatnagar et al. (2003) generated transgenic India
mulberry expressing GUS genes. In the process, it
is found that A. tumefaciens strain LBA4404 was S1635 Karnataka, Andhra Pradesh, Irrigated
Tamil Nadu, Maharashtra,
more infective than the other strains. Nearly 90- West Bengal, Uttar Pradesh,
100% transformation was obtained with LBA4404, Bihar, Assam and other
70-75% with GV2260 and 25-35% with A281. states in Central India
Among the plasmids, pBI121 and pBI101:Act1
were most efficient (100%). Subsequently, several DD Karnataka, Andhra Pradesh, Irrigated
transgenic mulberry plants overexpressing desired Tamil Nadu, Maharashtra
and Kerala
genes were developed (Table 4), including glycine
gene AlaBlb, the oryza cystatin gene OC and the S36 Karnataka, Andhra Pradesh, Irrigated
barley HVA1 gene (Wang et al., 2003; Lal et al., Tamil Nadu and Kerala
2008; Checker et al., 2012). The transgenic S1 West Bengal, Uttar Pradesh, Irrigated
mulberry over-expressing HVA1 from barley Bihar, Assam and Central , Rain-
showed comparatively better growth under abiotic India fed
stresses (Lal et al., 2008; Checker, 2012). The
transgenic plants with Osmotin gene under the S799 Sericultural regions in Irrigated
constitutive expression of the CaMV 35S promoter central India
and a stress-inducible promoter rd29A also S13 Red soil of Karnataka, Rain-fed
expressed high salt, drought and cold stress Andhra Pradesh, Tamil
tolerance (Das et al., 2011). Thus, transgenesis in Nadu
mulberry seems to have come of the age. In view of S34 Black soils of Karnataka, Rain-fed
the above developments, attention should be Andhra Pradesh, Tamil
directed to some of the burning issues of mulberry Nadu
cultivation. Major diseases like powdery mildew
(Phyllactinia corylea) and bacterial blight BC259 Hilly Eastern areas of India Rain-fed
(Xanthomona campestris cv. Mori) are causing a S146 Central India, Doon valley, Rain-fed
crop loss of 20-30% in the field (Philip et al., Himachal Pradesh and Hilly
1994). Therefore, sincere and serious efforts should Eastern areas of India
be made to develop disease resistant transgenic
Tr-10 Central India, West North Rain-fed
plants against these diseases. Jammu, Doon valley,
Molecular markers Himachal Pradesh and Hilly
From the research during the last couple of Eastern areas of India
decades (Table 5) clearly demonstrates that four Chak Western North, Jammu and Rain-fed
major types of molecular markers have been used majra Himachal Pradesh
in mulberry for various purposes which includes
Chinese Western North, Jammu and Rain-fed
elucidation of genetic diversity among germplasm
white Western Himachal Pradesh
accessions, relationship among populations,

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Random amplified polymorphic DNA (RAPD) Naik, et al. 2013), to identify molecular markers
Random amplified polymorphic DNA markers associated with sex characteristics (Figure 4) in
uses arbitrary short oligomers (usually 10 bases) to mulberry (Vijayan et al., 2009), to identify mutants
PCR amplify the genomic DNA (Williams et al., (Anilkumar et al., 2012) and to develop linkage
1990) and the variation in the band pattern is due to maps in mulberry (Venkateswarlu et al., 2006). The
base pair substitutions modifying the primer advantages of RAPD are requirement of small
binding sites (Moeller and Schaal, 1999). In amount of template DNA, low cost of development,
mulberry, RAPD was the first molecular marker easiness in use and the major disadvantages are the
used for genetic diversity analysis. RAPD has been poor reproducibility (Rafalski, 1997),
used by many investigators to work out genetic underestimation of genetic distances between
diversity among the cultivars (Awasthi et al., 2004; distantly related individuals (Powell et al., 1996)
Ozrenk et al., 2010; Chikkaswamy and Prasad, and are unable to distinguish homozygous from
2012; Chikkaswamy et al., 2012; Ipek et al., 2012; heterozygous ones.

Figure 4. PCR products showing the marker OPY-15.1200 associated with male sex in mulberry
(Adapted from Vijayan et al., 2009).

Table 4. Transgenesis in mulberry (Morus spp.). (Adapted from Vijayan et al., 2011c).
Gene Expression profile Reference
WAP21* Cold tolerance Ukaji et al. (2001)
GUS GUS incorporated into the protoplast through Sugimura et al. (1999)
elctroporation
Agrobacterium rizhgenes Hairy roots Oka and Tewary, 2000
COR Cold tolerance Ukaji et al., 2001
GUS GUS activity Bhatnagr and Khurana, 2003
AlaBlb Salinity tolerance Wang et al., 2003
OC Insect resistance Wang et al., 2003
SHN 1 Drought tolerance Aharoni et al., 2004
GUS Efficacy of different transformation technique Agarwal and Kanwar, 2007
HVA1 Drought and salinity stress Lal et al., 2008
Checker et al., 2012
bch1 High-temperature tolerances Das, 2009
bch Drought and salinity stress Khurana, 2010
NHX Drought and salinity stress Khurana, 2010
Osmotin Drought and salinity stress Das et al., 2011
HAL3a, dehydrin Abiotic stress Das et al., 2013
*AlaBlb- Soyabean glycine gene; bch-L inhibitor 2-aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid; COR-
cold on regulation; GUS- β-Glucuronidase; HVA1-Hevea braziliensis abiotic stress gene;NHX- Na+/H+
exchanger OC- osteocalcin Osmotin-osmotic stress induced gene; SHN 1-schnurri from Drosophila
melanogaster; WAP21- water allocation plan.

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Table 5. Summary of molecular marker applications in mulberry (Morus spp.).

Molecular marker Work/Achievement Reference
RAPD* Initiated use of molecular markers in the systematic of Morus Xiang et al., 1995
RAPD RAPD was successfully tested in mulberry Chengfu et al., 1996
RAPD Genetic identities and relationships of mulberry cultivated species Feng et al., 1996
(Morus L.) were worked out.
RAPD Genetic identities and relationships of mulberry cultivated species Lichuan et al., 1996
(Morus L.) worked out.
RAPD Relationship among cultivating species of Morus studied. Feng et al., 1997
RAPD Relationship between parents and their hybrids were worked out. Lou et al., 1998
RAPD Polymorphism among polyploidy mulberry was studied. Zhang et al., 1998
AFLP Elucidated the interrelationships of different Morus species Sharma et al., 2000
RAPD Proved that RAPD is capable of distinguishing mulberry genotypes Bhattacharya and
Ranade , 2001
AFLP Identified the genetic back ground of polyploids Wang and Yu, 2001
RAPD Germplasm characterization was done. Zhao and Pan, 2002
RAPD DNA finger printing of Mysore local and V1 cultivars were done. Naik et al., 2002
ISSR Genetic relationship between Japanese and Indian mulberry genotypes Vijayan, 2003
ISSR Relationship among a few selected cultivars was studied. Vijayan et al., 2003
SSR Six new microsatellite markers were developed from Morus indica a Aggarwal et al., 2004b
used in germplasm charterization.
ISSR & RAPD Genetic diversity and relationships in mulberry Awasthi et al., 2004
RAPD Itraspecific variability in M. laevigata was estimated. Chatterjee et al., 2004
ISSR Genetic diversity among wild populations of the species M. serrata Vijayan et al., 2004a
ISSR & RAPD Phylogentic relationship among five Indian species of mulberry has Vijayan et al., 2004b
been worked out.
AFLP Differentiated introduced accessions from native accessions Botton et al., 2005
rbcL-accD Phylogenetic relationships among the species of Moraceae. Matsuda et al., 2005
ISSR Genetic relationships among 34 Indigenous mulberry genotypes of Vijayan et al., 2005
India
SSR Developed 10 microsatellite primers pairs and tested in 27 mulberry Zhao et al., 2005a
accessions, which could differentiate wild mulberry accessions from
cultivated ones.
ITS & trnL-F Phylogenetic relationship among 13 mulberry Zhao et al., 2005b
genotypes belonging to nine species was developed.
RAPD Identified duplicates in the germplasm collections Naik and Dandin,
2006
RAPD, ISSR Developed a genetic linkage frame work for mulberry genome Venkateswarlu et al.,
&SSR 2006
ISSR Genetic diversity of cultivars from Kerala and markers for Vijayan et al., 2006a
economically important traits.
ISSR Genetic relationship between wild and cultivating species of mulberry Vijayan et al., 2006b
ISSR ISSR markers were used to estimate the genetic variation between Zhao et al., 2006a
fengwei sang and its sports
ISSR ISSR markers were used to assess the relationship between wild and Zhao et al., 2006b
cultivated species
ISSR Worked out the genetic diversity in the wild populations of mulberry Zhao et al., 2006c
from the western part of northern China
ISSR Genetic variability in the mulberry genetic resources of India and Kar et al., 2007
identification of markers associated with biochemical characters.
RAPD Genetic variations and relationships among 15 mulberry genotypes Orhan et al., 2007
from Turkey were worked out.
ISSR& SSR Genetic diversity of 27 mulberry accessions including 19 cultivated Zhao et al., 2007
and 8 wild accessions studied
AFLP Characterized 43 accessions of mulberry from Turkey Kafkas et al., 2008
ISSR Genetic diversity among ecotypes of mulberry in China was studied. Zhao et al., 2008
RAPD Studied the inter and intra-specific hybrids of mulberry Tikader and Dandin,
2008
AFLP Genetic diversity among cultivars of Hunan province Huang et al., 2009
ISSR& RAPD Molecular markers identified for sprouting and sex expression. Vijayan et al., 2009

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Table 5. Contd..
SRAP Genetic relationship among germplasm accessions Zhao et al., 2009
RAPD Worked out the relationship among 47 Ozrenk et al., 2010
mulberry genotypes.
ISSR Lin et al., 2011
RAPD Used for estimating the genetic diversity of EMS treated mulberry Anilkumar et al., 2012
genotypes.
RAPD & ISSR Twenty different varieties of mulberry were assayed with selected Chikkaswamy and
RAPD and ISSR genetic markers. In the present investigation, the Prasad, 2012
selected mulberry varieties were amplified with 16 RAPD and 8 ISSR
primer
RAPD & ISSR Sixteen decamer RAPD primers and 8 ISSR primers were used to Chikkaswamy et al.,
estimate the genetic diversity among 20 genotypes of Mulberry. 2012
RAPD &ISSR Genetic diversity among mulberry growing in different agroclimatic Ipek et al., 2012
conditions in Turkey was estimated.
RAPD Genetic diversity among mulberry accessions collected from south Naik et al., 2013
India.
SSR, RAPD, Testing the quality of genomic DNA extracted with a new protocol Anuradha et al., 2013
ISSR
SSR Genetic diversity between and within two mulberry species widely Wangari et al., 2013
grown in Kenya
SSR Genetic diversity among mulberry genotypes maintained in temperate Wani et al., 2013
climatic conditions in India
*RAPD- Random amplified Polymorphic DNA; ISSR- Intersimple sequence repeats; AFLP-Amplified fragment length
polymorphism; SSR-Simple sequence repeat; ITS-Internal transcribed Spacer; trnL-F-

Table 6. Microsatellite primers available for Morus spp.

Primer sequences Microstaellite motif Reference
F: CGGTCACGCCTTCTTCTCC (CA)30 Zhao et al., 2005a
R: TGACCGAGAAATGAGGAAGGAG
F: GCTTCGATCAATCTAGCTTCCC (CA)49 Zhao et al., 2005a
R: GCAAACTACGCCACCCCG
F: CGAGGGAGGGATGAGGAGC (TG)27 Zhao et al., 2005a
R: CACATTCATGCACCCTCCTATA
F: TCCAGCAAAGATGTGACAAAAGTT (CA)5CC Zhao et al., 2005a
R: TTGCCTTCCCGATTATGCTG (CA)27
F: ACTCAAAATGAAGGAAAAGGAATTATAC (TG)18 Zhao et al., 2005a
R: TTTACTTAAATCCCAGCCACA
F: AGAACCCTTCCGCCCTATG (CA)56 Zhao et al., 2005a
R: CCTTGGCGTAGGCAAAGTTG
F: TACAGGGCTCGGGCAAATG (CA)26 Zhao et al., 2005a
R: TGATCCGAAGCTTGGGGTCT
F: TCTTCGCCCGTTGTTCGC (CA)27 Zhao et al., 2005a
R: AGCAATTTTCTTCAACTCACCTTCT
F: TTCTGTCGTGTCCTCCGTCAA (TG)18 Zhao et al., 2005a
R: TGAGAACATACACTAATAGGTGAAAAC
F: CCCTTTCATCGCCTCCTCC (CA)14ACAA (CA)12 Zhao et al., 2005a
R: CTCTGCCCATTCAGTAGCGG
F: GCCGTGTACCAGTGGAGTTTGCA (GTT)6 + (GTT)4 Aggarwal et al., 2004
R: TGACCGTTTCTTCCACTTTACCTAATG
F: CGTGGGGCTTAGGCTGAGTAGAGG (GTT)11 Aggarwal et al., 2004
R: CACCACCACTACTTCTCTTCTTCCAG
F: GGGTTGGGTAGATGGGCTTATGTTA (GA)33 Aggarwal et al., 2004
R: CCCTATTAACTTTTTGGTCACCTCTA
F: GGTCAAGCGCTCCAGAGAAAAG (GAA)6 Aggarwal et al., 2004
R: GGTGCAGAGGATGAAAGATGAGGT
F: CCCCCTGCAATGCCCTCTTTC (CCA)8 Aggarwal et al., 2004
R: TGGGCGAGGCAGGGAAGATTC

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K. Vijayan et al.

Inter simple sequence repeats (ISSR) The study revealed that M. laevigata can be
Inter simple sequence repeat (ISSR) markers considered as a separate species while the other
amplify DNA segments between two identical four species may be grouped together and treated as
microsatellite repeat regions oriented in opposite sub-species. Subsequently, Vijayan et al. (2005)
direction by primers of 16-25 bp long designed demonstrated the admixturing of the mulberry
from the microsatellite core regions bordering genetic pool of the eastern India and the southern
them. The primers either anchore at 3′ or 5′ end India using 34 mulberry cultivars collected from
with 1 to 4 degenerate bases extended into the different regions of India. In China, Zhao et al.
flanking sequences (Zetkiewicz et al., 1994) or to (2007) used ISSR along with RAPD to estimate the
remain unanchored (Gupta et al., 1994). Usually di- genetic diversity of 27 mulberry accessions.
nucleotide repeats anchored either at 3′ or 5′ end Similarly, genetic diversity of 73 local mulberry
reveal high polymorphism (Joshi et al., 2000). varieties from Shanxi Province were assessed with
Polymorphism occurs whenever one genome 15 primers and with the help of stepwise clustering
misses the sequence repeat or has a deletion or and random methods and the modified heuristic
insertion or translocation between the repeats. ISSR algorithm, 21 core collections were constructed and
markers are generally dominant markers following the ratio of core collection was 28.77% (Lin et al.,
Mendelian inheritance (Ratnaparkhe et al., 1998), 2011). Recently, Chikkaswamy et al. (2012) used
however, incidence of segregation as co-dominant ISSR along with RAPD to estimate the genetic
markers also been reported (Sankar and Moore, diversity of 20 mulberry varieties. Along with
2001). ISSR markers have higher reproducibility RAPD, it has also been used to develop linkage
than RAPD markers (Moreno et al., 1998). Since map of mulberry (Venkateswarlu et al., 2006).
the development of ISSR markers does not need
Amplified fragment length polymorphism
prior knowledge of the genome to be analysed, it
(AFLP)
has been used initially to assess the genetic
Amplified fragment length polymorphism
diversity of mulberry. Vijayan and Chatterjee
(AFLP) is a combination of RFLP and polymerase
(2003) used 13 ISSR primers from University of
chain reaction (PCR) techniques wherein the speed
British Columbia, Canada (set # 9) to estimate
of PCR combines with the precision of RFLP (Vos
diversity among 11 indigenous cultivars. The study
et al., 1995) though it requires only a small amount
showed that AG, TG and AC repeat primers
of DNA, it can be readily automatable. AFLP is
generated excellent band profiles but primers of AT
more robust, reliable and reproducible than RAPD
repeats failed to amplify even at a low annealing
and ISSR (Jones et al., 1997). The process of AFLP
temperature. The important tri and tetra nucleotide
is initiated by extraction of DNA and subsequent
repeat primers generated excellent amplification are
digestion with rare and frequent cutter restriction
those from ACC, ATG, AGC, GAA, GATA and
enzymes like Eco-R1 and Mse-I. The digested
CCCT. The penta-nucleotide repeats GGAGA and
fragments are then ligated to double-stranded
GGGGT also amplified well. Awasthi et al. (2004)
oligonucleotide adapters and PCR amplified with
used ISSR in combination with RAPD to
primers that bind to the adapter sequence,
characterize 15 germplasm accessions and proved
restriction site sequence and adjacent selective
the efficiency of ISSR once again. Later, Vijayan
base(s). The products of the PCR amplification are
(2004) worked out the genetic relationships
subsequently run on a polyacrylamide gel to resolve
between Japanese and Indian species (Figure 5).
it. Because of the use of two restriction enzymes
ISSR has also been used to estimate the
with different properties, a large number of DNA
biodiversity of wild populations of mulberry
fragments suitable for PCR amplification are
(Vijayan et al., 2004a). Populations of M. serrata
generated. Since poly acrylamide gel
present in Uttaranchal (29o22′- 30o45′ N latitude
electrophoresis can resolve fragments differing in
and 75o52′- 80o12′ E longitude) and Himachal
length even by a single base pair (Miyashita et al.,
Pradesh (30o30′ - 30o54′ N latitude and 77o06′ -
1999), a large number of fragments are resolved in
77o40′ E longitude) were assessed and conservation
the gel. Like RAPD, AFLP also not required any
strategies were formulated. Likewise, the
prior information on the target genome (Vos et al.,
phylogenetic relationship among nineteen
1995) and the markers are dominant in nature
genotypes belonging to five mulberry species viz.,
(Powell et al., 1996). AFLP was first used in
M. latifolia, M. bombycis, M. alba, M. laevigata
mulberry to study the genetic diversity of 45
and M. indica were also worked out using both
mulberry accessions from different eco-geographic
RAPD and ISSR markers (Vijayan et al., 2004b).

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regions of Japan and other parts of the world. Five hybridization is possible, and due to continuous
primer combinations were used and an average of variation of most phenotypic characteristics, the
110 AFLP markers was generated by each primer taxonomy of the genus Morus, especially for M.
pair. The size of the bands varied from 35bp to 500 alba, M. latifolia and M. bombycis species, is not
bp. The polymorphism ranged from 69.7 to 82.3% well defined. Later, Kafkas et al. (2008)
across all the genotypes. The UPGMA-dendrogram characterized 43 mulberry accessions from different
grouped the accessions into four major clusters regions of Turkey using fluorescent dye amplified
agreeing with the genetic relatedness established fragment length polymorphism (AFLP) markers
among them by conventional methods (Sharma et and capillary electrophoresis. Unweighted pair-
al., 2000). Further, it is concluded that mulberry group method of arithmetic mean (UPGMA)
cultivars are naturalized because they have been clustering grouped the accessions according to the
established, adapted and persisted in areas far away species they belong. Furthermore, the study also
from their origin, making their classification very clearly brought out the ability of AFLP markers to
difficult and unreliable when based solely on identify the accessions of M. nigra, M. rubra, and
morpho-phenological traits (Sharma et al., 2000). M. alba without any ambiguity. These studies
Later, it was used to estimate the genetic variability demonstrated unequivocally, the resolving power of
within as well as among different mulberry species. AFLP, which can be used for identifying genotypes
Botton et al. (2005) assessed 48 accessions belong for conserving genetic resources, eliminating
to three species collected from Japan, Brazil, South duplicate accessions from germplasm collections
Caucasus, Middle East, Philipines, and Italy but and monitoring erosion of genetic diversity within
were maintained in Italy. From the study it is the populations.
concluded that since spontaneous and artificial

Figure 5. PCR fingerprint of the eighteen mulberry genotypes from Japan (1-9) and India (10-18). (A.) with the ISSR
primer UBC-881. (B.) with the RAPD primer OPY-16. M1- M. acidosa, M2- M. bombycis, M3- M. latifolia, M4-M.
latifolia,M5- M. bombycis, M6-M latifolia, M7-M. bombycis, M8- M. alba, M9- M. latifolia, M10- M. indica, M11- M.
alba, M12- M.alba, M13- M. spp, M14-M. alba, M15- M.alba, M16- M. indica, M17- M.indica, M18- M. Indica
(Adapted from Vijayan, 2003).

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QTLs for enabling marker assisted selection

Simple sequence repeats (SSR)
breeding in mulberry.
Simple sequence repeats (SSR) or
microsatellites or short tandem repeats (STR) or Conclusions and directions for future research
simple sequence length polymorphism (SSLP) are Biotechnology of mulberry has advanced far
tandem repeats of short (2-6 base pair) DNA and wide in areas like tissue culture and molecular
fragments present throughout the genome (Litt and biology and contributed to micropropagation of
Luty, 1989). Variations at SSR loci are generated hard to root genotypes, isolation of somaclonal
through (a) replication slippage (b) unequal variants, screening of germplasm for tolerance to
crossing-over and (c) genetic recombination. abiotic stresses, induction of polyploids,
Among them, replication slippage is considered to production of synthetic seeds, and cryopreservation
be a major factor affecting the repeat number for of genetic resources, development of transgenic
short tandem repeat sequences, whereas unequal plants, characterization of germplasm accessions
crossing-over is thought to result in a very large and identification of markers associated with
number of alleles for long tandem repeat arrays economically important traits. However, there is
(Huang et al., 2002). SSR markers are co-dominant, much more to do than what has been accomplished.
stable, robust and are highly reproducible. Inbred lines are urgently required for elucidating
However, the major disadvantage of SSR is the the genetic basis of most of the economically
need for prior information on the target genome to important characters in mulberry. Considering the
develop suitable primer sets. With the introduction difficulty to develop inbreds through conventional
of next generation sequencing techniques the cost breeding, developing the same through doubled
of sequencing has come down considerably. This haploidy should be attempted. The development of
facilitates development of SSR primers much a reproducible system for the production of doubled
cheaper than earlier. Further, it is interesting to note haploids, either using anther cultures, microspore
that due to, synteny of genomic regions; primers cultures and/or cultivation of ovary segments
developed in a closely related species can be used containing unfertilized ovules, need to be
to amplify microsatellite loci of other species developed. Deeper insight into each particular step
(Hormaza, 2002). In mulberry, the first attempt to in the process of haploid plant production can help
isolate microsatellite markers was made by to develop more sophisticated and more successful
Aggarwal et al. (2004), wherein six primer sets protocols for rapid application of the gametic
were developed from the genomic DNA of M. embryogenesis. Although a few transgenic plants
indica (Table 6). The markers developed by these harbouring some of the desired genes have been
primer sets produced high polymorphism when developed, regeneration of plants from leaf disc of
tested on a set of 43 elite genotypes including 13 most of the high yielding varieties is still remain as
related Morus species. The markers could easily the major bottle neck. Thus, efforts need to be made
differentiate the species. Later, Zhao et al. (2005a) to develop easy protocol for these mulberry
developed another 10 primer sets (Table 6) and varieties so that genes of special interest can be
validated their suitability by testing in 27 mulberry inserted into their genome easily. Regarding the
accessions. Using these SSR markers, Wangari et al molecular marker systems, only a few SSR primers
(2013) and Wani et al. (2013) studied the genetic are still available for use. These few primers are not
diversity among mulberry genotypes present in enough to make saturated linkage maps to identify
Kenya and India respectively. Likewise, these SSR QTLs tightly linked to economically important
markers have also been used in constructing a traits. Thus, it is important to develop large
linkage map along with RAPD and ISSR markers numbers of SSR and SNP (Single Nucleotide
(Venkateswarlu et al., 2006). Similarly, Anuradha Polymorphism) markers for wider use of these
et al. (2013) used SSR primers along with RAPD marker systems. In this context it is heartening to
and ISSR to test the quality of mulberry genomic note that the first draft sequence of mulberry
DNA extracted with a new protocol. Although SSR genome has just been published (He et al., 2013),
markers have several advantages over other which will facilitate development of more
dominant marker systems, they have not yet been information on mulberry genome to enable fast
exploited widely in mulberry. Hence, attempts improvement of this very important crop plants of
should be made to develop more number of SSR Asia
markers so as to utilize them in identification of
Acknowledgement

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http://www.ejfa.info/

The authors express their deep gratitude to Dr. Anis, M., M. Faisal and S. K. Singh. 2003.
S. Gandhi Doss, CSR&TI, Mysore for providing Micropropagation of mulberry (Morus alaba
photographs of micropropagation, to Dr. D. S. L) through in vitro culture of shoot tip and
Mohan Jain, Dept of Agricultural Science, nodal explants. Plant tissue culture. 13:47-51.
University of Helsinki, Finland and Dr. A.J.
Anuradha, J.H., K. Vijayan, C. V. Nair and A.
Cheruth, United Arab Emiratus University, UAE
Manjula. 2013. A novel and efficient protocol
for language correction.
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