ORIGINAL RESEARCH ARTICLE

OPEN ACCESS
Development, evaluation and release of biofortified rice varieties
Neeraja CN*, Madhu Babu P, Sanjeeva Rao D, Surekha K, Subba Rao LV and Ravindra Babu V
ICAR-Indian Institute of Rice Research (IIRR), Hyderabad.
*Corresponding author email: cnneeraja@gmail.com
Received: 10th Feb 2019; Accepted: 22nd May 2019

Abstract
Polished rice is a poor source of micronutrients and was reported to be responsible for malnutrition in the developing
countries where rice is the major energy source. Biofortification for zinc (Zn) in polished rice is a promising and
cost effective approach for the development of Zn-dense rice to alleviate micronutrient malnutrition. Several donors,
especially landraces with >50 ppm in brown rice and >35 ppm in polished rice were identified and used in the development
of breeding lines with high Zn and yield. The developed lines were evaluated under ICAR - All India Coordinated Rice
Improvement Project for their high yield and Zn across the locations and across the years and promising lines were
identified and released as varieties. Five varieties since 2015 were released through Central Varietal Release Committee
for high Zn in polished rice in India.
Keywords: Biofortified rice varieties, high zinc, polished grains, development and release

Introduction Of these, biofortification, an approach of the development

of micronutrient-dense staple food crops to alleviate
Rice (Oryza sativa L.) is the staple food crop of 50% of micronutrient malnutrition is targeted, sustainable and
the world and a major energy source especially in India. cost effective, hence most preferred. Since 2000, several
Polished rice, the most preferred form for consumption, is attempts are being made in rice for Zn biofortification
a poor source of micronutrients and the excess dependence through conventional breeding approaches across the
on polished rice was reported to be responsible for world. Using the donors for high Zn content, several
malnutrition whose daily caloric intake is mainly confined breeding lines with high Zn are being developed and
to rice (Juliano, 1993; Bouis and Welch, 2010). Polished evaluated under HarvestPlus, international and national
grains of most of the rice varieties have 12-14 ppm of Zn, programs. As a proof of concept of combining high Zn
thus providing only one fifth of daily recommended Zn with high yield, breeding lines with high Zn in polished
requirement of ~15 mg (though varies across sex and age) rice were developed through conventional breeding
(Promo-u-thai et al., 2010). with funding received from HarvestPlus, Department of
Biotechnology (DBT), and Indian Council of Agricultural
Dietary deficiency of Zn is a substantial global public
Research (ICAR), Government of India. These breeding
health and nutritional problem with one third of the world
lines were evaluated in India through national trials on
population at risk due to low dietary intake of Zn (Krishna
Biofortification in All India Coordinated Rice Improvement
swamy, 1998; www.zinc.org/health/ ). A breeding target
Project since 2013 (www.harvestplus.org ; www.irri.org ;
of 28 ppm was set in polished rice based on the nutrient
http://www.icar-iirr.org/). The entries of the trial constitute
needs, daily food intake, retention and bioavailability the biofortified breeding lines developed from all over the
analyses in order to meet at least 25% of the estimated country. The present study summarizes the observations
average Zn requirement for overcoming the most severe and the lessons learnt during the course of development of
Zn deficiency (www.harvestplus.org ). Enhancing the Zn varieties with high Zn in polished rice and its release since
content of polished rice has lot of potential to address wide 2013 in the Indian situation.
spread Zn deficiency problem responsible for malnutrition
in developing countries. Different strategies, such as Materials and Methods
biofortification, foliar or soil application of Zn fertilizers Identification of donors: Around 100 genotypes
have been suggested and demonstrated to increase the Zn comprising landraces, breeding lines and farmer varieties
content of cereals (Nestel et al., 2006; Swamy et al., 2016).

24 H Journal of Rice Research 2019, Vol 12, No. 1

were evaluated across four locations viz., rice experimental approaches to address Zn deficiency in the human diet,
fields at Hyderabad (IIRR), Bengaluru (UAS), Chinsurah biofortification is the most feasible, sustainable and
(Rice Research Station) and Coimbatore (TNAU) during economical approach. Several biofortified rice varieties
wet season 2012. The soil properties of the plots of the four have been released in Bangladesh and India and are being
locations were given in Table 1. evaluated in Philippines and Indonesia (Swamy et al.,
2016).
Development of segregating material and selection
of promising lines: The identified promising donors were Rice grain Zn concentration is affected by a large number
crossed with rice varieties known for their yield, quality of plant and environmental factors (Welch and Graham,
and adoption by the farmers. The favorable recombinants 2002). Zn reported to be poorly available under the
for yield and high Zn in polished rice were selected among irrigated conditions (Fageria, 2013). The Zn content in
the segregants, multiplied and submitted for evaluation. the grain appears to be significantly affected by the pH,
organic matter content and available Zn levels of native
Evaluation through ICAR - All India Coordinated
soil. However, genotypic variation was observed to be the
Rice Improvement Project (AICRIP), ICAR-
most significant factor to affect grain Zn content (Chandel
IIRR, Hyderabad: The nominated promising lines for
et al., 2010). In the present study, the genotypes were
yield and Zn were evaluated at 10 to 20 locations all over
evaluated in four locations with differential soil profiles
the country with two check varieties (control) viz., IR64
(Table 1). The Zn found to be on higher side in the plots
and BPT5204 for yield and high Zn viz., Kalanamak and
owing to the application of Zn fertilizer as part of the
Chittimuthyalu in polished rice. The seed samples are
package of practices in rice experimental fields. However,
being sent to ICAR-IIRR for estimation of Zn in polished
Zn deficiency is reported to be widespread in soils and
rice. The lines screened as Initial varietal trial (IVT),
crops of India and is one of the significantly depleted
advanced varietal trial 1 (AVT 1) and advanced varietal
mineral nutrients under intensively cropping systems
trial 2 (AVT 2) for three years and the consistent lines were
(Takkar, 1997). On the basis of the analysis of about 65,000
tested for 12 quality parameters for their suitability (http://
soil samples, it has been found that about 51.2% of Indian
www.icar-iirr.org/).
soils are deficient in Zn. The critical levels of DTPA- Zn
The varieties with recommended level of Zn, yield and for cereal crops were found to vary among the soil types
desirable quality parameters were released as varieties and crops (Singh et al., 2015). Thus, farmers need to be
through Central Varietal Release Committee (CVRC). As a sensitized regarding package of practices for growing the
case study, the evaluation data of AICRIP- Biofortification biofortified varieties.
2015-17 was presented.
Wide genetic variation was observed among the evaluated
Estimation of Zn: The seed of each plant was harvested genotypes for grain Zn content in brown and polished rice.
and divided into three parts to be analyzed as three The rice land races or traditional varieties are considered
replicates. The seeds were dehusked using JLGJ4.5 to have high nutritive and therapeutic value (Deb et al.,
testing rice husker (Jingjian Huayuan International Trade 2015). Extensive genetic variability for grain Zn content
Co., Ltd) sponsored by HarvestPlus and polisher (Krishi has been earlier reported in rice wild accession and
International India Ltd.) with non-ferrous and non-Zn landraces (Anandan et al., 2011; Anuradha et al., 2012;
components. Each sample of brown and polished rice (5 Nachimuthu et al., 2014; Huang et al., 2016). Many
g) was subjected to energy dispersive X-ray fluorescent landraces were identified with >50 ppm in brown rice and
spectrophotometer (ED-XRF) (OXFORD Instruments >35 ppm in polished rice and have been successfully used
X-Supreme 8000) at ICAR-IIRR as per standardized or being used in breeding programs (Table 2A and 2B).
protocols (Sanjeeva Rao et al., 2014). The screening of landraces for high nutrient content should
be a continuous process for identification of new donors
Results and Discussion
for high Zn in polished rice.
Zn deficiency is a major public health and nutritional
Across the locations, the genotypes found highly variable
issue affecting mostly the developing countries whose
in their grain Zn content and only one genotype was found
staple diet is polished rice (Krishnaswami, 1998; Juliano,
to be in the top ten genotypes. Surprisingly, the extent of
1993). The objective of Zn biofortification is to increase
polishing also showed lot of variation across the locations
Zn concentration in polished rice and of the various

Journal of Rice Research 2019, Vol 12, No. 1 H 25

and genotypes (Table 3). The genotypes with high Zn quality parameters and filling of the grain on polishing.
content in brown rice were found not to necessarily contain The rice grain
high Zn content in polished rice, suggesting the effect of

Table 1. Soil properties of experimental plots of the four locations during wet season 2012
Hyderabad (HYD) Chinsurah (CHN) Coimbatore (CBT) Bengaluru (BLR)
Trait
Before After Before After Before After Before After
Planting Harvesting Planting Harvesting Planting Harvesting Planting Harvesting
pH 8.52 8.47 6.04 7.12 7.82 7.87 6.02 5.99
E.C (dS m-1) 0.92 0.71 0.14 0.19 0.64 0.47 0.25 0.27
O.C (%) 0.56 (M) 0.70 (M) 0.75 (M) 0.72 (M) 0.73 (M) 0.67 (M) 0.24 (L) 0.23 (L)
N (kg/ha) 230 (L) 187 (L) 164 (L) 183 (L) 195 (L) 227 (L) 181 (L) 183 (L)
P2O5 (kg/ha) 81 (H) 107 (H) 39 (M) 41 (M) 100 (H) 84 (H) 9 (L) 14 (L)
K2O (kg/ha) 616 (H) 641 (H) 458 (H) 410 (H) 957 (H) 926 (H) 359 (H) 336 (H)
Fe (ppm) 3.48 (L) 2.30 (L) 56.50 (H) 31.92 (H) 0.74 (L) 0.94 (L) 28.82 (H) 30.50 (H)
Zn (ppm) 3.66 (H) 3.61 (H) 1.61 (H) 1.28 (H) 3.61 (H) 3.50 (H) 1.55 (H) 1.00 (H)

quality is highly influenced by environmental factors like Combining the yield and high Zn in polished rice is a major
temperature, soil moisture, crop management and post- challenge owing to the dilution effect of the micronutrient
harvest practices. Before releasing of a biofortified variety, content (Impa et al., 2013). Earlier focus of the rice
multi-locational evaluation should be compulsorily made. breeding programs was the production of high yielding
varieties to ensure the food security, thus breeding for high
Around 20% average loss of Zn is observed in the polished
micronutrient contents was not a priority objective (Graham
rice samples in comparison to brown rice across the
and Welch, 1996). With growing awareness of importance
locations, the percentage loss of Zn content on polishing
of biofortification, especially of Zn, breeding for high Zn
found to be broadly varying from 1.2 to 59.5. Among the
in polished rice is one of the important objectives in global
genotypes, the range of percentage reduction was too wide,
rice breeding programs.
only two genotypes showed <10 % across the locations.

Table 2A. Zn content in the brown rice of the top ten genotypes in the four locations estimated through ED-XRF
Location HYD BLR CHN CBT
Name Zn (ppm) Name Zn (ppm) Name Zn (ppm) Name Zn (ppm)
GMP 22* 51.9 GMP 33 39.5 GMP 23* 39.4 GMP 14* 24.5
GMP 23* 48.5 GMP 45 39.3 GMP 24** 37.9 GMP 24** 22.2
GMP 35* 45.2 GMP 31 38.9 GMP 20* 30.0 GMP 12* 21.0
GMP 24** 45.1 GMP 43* 38.2 GMP 22* 29.9 GMP 22* 21.0
GMP 25 44.8 GMP 24** 35.6 GMP 38 29.6 GMP 11* 19.3
GMP 39* 43.3 GMP 40* 34.3 GMP 36* 28.9 GMP 43* 19.1
GMP 28* 41.8 GMP 28* 34.1 GMP 8 26.3 GMP 44 20.9
GMP 32 41.6 GMP 29 33.9 GMP 39* 26.2 GMP 13 16.8
GMP 40* 40.0 GMP 5 33.8 GMP 11* 25.8 GMP 27 16.4
GMP 14* 39.6 GMP 1 32.7 GMP 35* 25.6 GMP 23* 16.3
* top genotype in more than one location, ** top genotype in four locations

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Table 2B. Zn content in the polished rice of the top ten genotypes in the four locations estimated through ED-XRF
Location HYD BLR CHN CBT
Name Zn (ppm) Name Zn (ppm) Name Zn (ppm) Name Zn (ppm)
GMP 24* 42.7 GMP 45* 36.1 GMP 20 28.7 GMP 24* 21.3
GMP 23* 42.0 GMP 33 35.1 GMP 24* 27.9 GMP 44* 18.1
GMP 25 38.5 GMP 31 32.9 GMP 23* 27.0 GMP 14 17.3
GMP 28* 38.3 GMP 40 32.2 GMP 39* 24.7 GMP 43* 17.1
GMP 22* 36.6 GMP 43* 32.1 GMP 43* 23.7 GMP 27 14.5
GMP 39* 34.5 GMP 28* 32.0 GMP 45* 23.0 GMP 12 14.1
GMP 44* 34.1 GMP 29 31.7 GMP 35* 22.6 GMP 23* 13.9
GMP 35* 34.1 GMP 1 31.6 GMP 21 22.5 GMP 22* 13.6
GMP 37 33.6 GMP 26 30.8 GMP 41 22.5 GMP 6 13.3
* top genotype in more than one location

Table 3. Percentage reduction of Zn content in polished rice to brown rice

Location HYD BLR CHN CBT
Genotype
Range 3.7 - 39.6 1.2 - 56.5 2.2 – 59.5 2.8 – 43.2 Mean Range
GMP 1 16.4 3.4 25.0 43.2 22.0 3.4 – 43.2
GMP 2 18.1 11.2 24.0 15.1 17.1 11.2 – 24.2
GMP 3 21.9 3.5 14.3 20.3 15.0 3.5 – 21.9
GMP 4 7.8 14.0 18.8 22.9 15.9 7.8 - 22.9
GMP 5 14.8 15.4 6.9 16.3 13.3 6.9 – 16.3
GMP 6 22.8 6.1 4.8 7.0 10.2 7.0 - 22.8
GMP 7 33.7 3.3 33.8 21.4 22.3 3.3 – 33.8
GMP8 35.2 3.4 23.6 28.7 22.7 3.4 – 35.2
GMP 9 15.3 12.1 22.8 13.8 16.0 12.1 – 22.8
GMP 10 27.7 10.0 8.7 14.1 15.1 8.7 – 27.7
GMP 11 21.0 13.7 17.4 34.2 21.6 13.7 – 34.2
GMP 12 27.4 27.2 24.4 32.9 28.0 24.4 – 32.9
GMP 13 7.2 12.7 31.6 38.7 22.5 7.2 – 38.7
GMP 14 39.6 12.5 21.7 29.4 25.8 12.5 – 39.6
GMP 15 9.7 4.8 7.1 2.8 6.1 2.8 – 9.7
GMP 16 7.3 1.2 14.7 31.3 13.6 1.2 – 31.3
GMP 17 19.3 5.3 5.1 25.2 13.8 5.1 – 25.2
GMP 18 19.2 2.6 4.5 30.4 14.2 2.6 – 30.4
GMP 19 3.7 3.1 29.5 28.4 16.2 3.1 – 29.5
GMP 20 22.4 10.7 4.3 26.4 16.0 4.3 – 26.4
GMP 21 27.5 9.6 2.2 36.7 19.0 2.2 – 36.7
GMP 22 29.5 6.2 49.5 35.2 30.1 6.2 – 49.5
GMP 23 13.4 9.5 31.5 14.7 17.3 9.5 – 31.5
GMP 24 5.3 16.0 26.4 4.1 12.9 4.1 – 26.4
GMP 25 14.1 12.4 18.4 3.2 12.0 3.2 – 18.4
GMP 26 16.1 5.5 26.9 7.0 13.9 5.5 – 26.9
GMP 27 21.6 6.7 28.9 11.6 17.2 6.7 – 28.9
GMP 28 8.4 6.2 9.0 6.7 7.5 6.2 – 9.0

Journal of Rice Research 2019, Vol 12, No. 1 H 27

 Location HYD BLR CHN CBT
Genotype
Range 3.7 - 39.6 1.2 - 56.5 2.2 – 59.5 2.8 – 43.2 Mean Range
GMP 29 13.1 6.5 36.4 19.1 18.8 6.5 – 36.4
GMP 30 10.5 11.7 40.7 24.1 21.8 10.5 – 40.7
GMP 31 23.5 15.4 26.7 14.7 20.0 15.4 – 26.7
GMP 32 23.8 1.4 13.6 20.0 14.4 1.4 – 23.8
GMP 33 15.1 11.1 11.5 35.1 18.2 11.1 – 35.1
GMP 34 26.8 4.1 25.6 26.7 20.8 4.1 – 26.8
GMP 35 24.6 56.5 11.7 23.0 28.9 11.7 – 56.5
GMP 36 25.8 11.5 57.8 20.8 29.0 11.5 – 578
GMP 37 14.1 20.8 27.8 18.8 20.4 14.1 – 27.8
GMP 38 15.6 3.8 59.5 6.0 21.2 3.8 – 59.5
Mean 20.0 15.0 30.5 21.0 21.6

Several combination of crosses were developed using AICRIP during 2013; however the threshold value has been
promising donors for Zn in polished rice and popular raised to 24 ppm. The recommended dietary allowance
rice varieties. In a cross combination of a land race donor (RDA) of Zn for human population in the age group of
and a popular variety, out of the selections made in fixed 25-50 years is 12-15 mg, respectively (FAO/WHO, 2000).
lines, only one line could show high yield (>20 g single In India, the average daily intake of rice is around 220 g
plant yield) and high Zn (> 28 ppm) (Figure 1). Similarly, and the polished rice with 28 ppm Zn would give 6.16 ppm
though a few, promising lines were selected from all cross Zn which is just 50% of RDA (Sanjeeva Rao et al., 2014).
combinations and are being evaluated.
With funding from HarvestPlus, DBT and ICAR, several
institutions have developed breeding lines for high Zn in
rice. Based on the need for the evaluation of the breeding
lines, a trial was constituted for biofortification during
2013 in ICAR – AICRIP and till date, several lines have
been screened at multi-locations. Around 50 % of the
nominated lines are advanced to AVT 1 based on their yield
and Zn content. Approximately half of the AVT 1 lines
were advanced to AVT 2 and based on the stringent yield,
Zn and quality parameters, and the varieties are released
(Table 5 and Table 6a & 6b).
Table 5. The number of breeding lines nominated under
AICRIP
Year IVT AVT1 AVT2
2013 29/9
Figure 1.Common breeding line between the RILs for high 2014 45/11 6/3
yield and high Zn
2015 45/26 30/14 4/2
The evaluation constitutes the comparison of biofortified 2016 32/10 12/11 12/3
lines with existing varieties for yield (IR64) and Zn content 2017 31/3 12/2 11/1
(landraces with high zinc) as check varieties. Initially,
182/59 60/30 27/6
a concentration of 20 ppm was set as threshold value in

28 H Journal of Rice Research 2019, Vol 12, No. 1

Table 6a.An example data set for yield and Zn content Zn (Gangashetty et al., 2013; Sathisha, 2013) in a set of
2017 2016 2015 landraces. However, from our experience in developing
Mean Zn Mean Zn Mean Zn the breeding lines for high Zn and yield and from the
Entry details
yield (ppm) yield (ppm) yield (ppm) nominations to AICRP-rice, we suggest the feasibility of
(20) (17) (14) combining the yield and high Zn.
IET 25477 4791 25.5 4360 28.3 3620 28.4
IET 25475 5102 25.3 4478 27.7 3881 27.6 Invitro bioavailability studies of a released variety viz., DRR
IET 25443 4728 25.0 4660 26.5 3629 24.4 Dhan 45 showed that the Zn content and bioavailability
IR 64 5941 18.5 5044 19.3 3933 17.2
was 50% more than the control IR64 variety. The coupled
Chittimuthyalu 4597 24 3185 24.8 2804 21.5
in vitro digestion/Caco-2 cell model studies with extrinsic
Table 6b.An example data set for quality parameters 65
Zn isotopic labeling demonstrated higher absorption of
2017 2016 Zn from DRR Dhan 45 in intestinal cells (Neeraja and
Entry details
HRR AC GT HRR AC GC Voleti, 2019).
IET 25477 51.4 24.2 71 52 24.58 59 Thus, with consorted efforts of various disciplines of plant
IET 25475 57 27.57 23 50.8 25.93 53 breeding, soil science, biochemistry and plant physiology,
IET 25443 61.5 25.63 22 56.3 25.96 22 the biofortified rice varieties have been developed. An
IR 64 na na na 52 23.29 55 evaluation system for biofortified varieties has been
Chittimuthyalu 65.7 23.52 22 60.8 23.4 22 evolved by ICAR- IIRR through AICRIP for their release.
However, the impact of the biofortified rice varieties
HRR: head rice recovery, AC: amylose content, GC: Gel consistency
in addressing malnutrition of the nation appears to be a
Table 7.Summary of five released varieties with high Zn long way to go, owing to its adoption by the growers and
III year II year I year consumers.
Mean Zn Mean Zn Mean Zn Policy recommendations
Variety
yield (ppm) yield (ppm) yield (ppm)
(20) (17) (14) • Proof of concept for the development of rice biofortified
IET 25477 4791 25.5 4360 28.3 3620 28.4 varieties with high yield and Zn
(Zinco Rice)
West Bengal, Chattisgarh and Odisha IGKV Raipur
• Combination of genetic and agronomic strategies for
IET 24760 4804 22.84 4693 20.0 4214 24.0 biofortified varieties
Surabhi (NSL) • Multi-location evaluation is compulsory for stable
Maharashtra and Gujarat
biofortified varieties
Nuziveedu Seeds Limited
IET 24555 5008 20.91 4989 19.8 6202 na • Clinical trials as proof of concept for inclusion of high
(DRR Dhan 48) Zn rice in public distribution system and mid-day meal
Andhra Pradesh, Telangana, Tamil Nadu, Karnataka and Kerala. schemes.
(IET 24557 4562 26.13 5079 20.6 6373 na
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