Zinc biofortification in rice

Rice (Oryza sativa L.) is major staple food in the world (especially in South and South East Asian countries).
Important staple foods for more than half of the world’s population (IRRI, 2006)
Source of livelihoods and economies of several billion people.
On a global basis, rice varieties provide 21% and 15% per capita of dietary energy and protein, respectively.
About 50% world’s populations depends on rice as their main source of nutrition.
However, rice is a poor source of micronutrients.
Micronutrients deficiency is a global problem contributing to world’s malnutrition and a major public health problem in many countries, especially in regions where people rely on monotonous diets of cereal-based food, as the Zn level or content in the grains of staple crops, such as cereals and legumes, is generally low.
Increasing the Zn content in the grains of these crops is considered a sustainable way to alleviate human Zn deficiency.
Zn deficiency being an important nutrient constraint, any approach to improve Zn uptake and its transport to grains has significant practical relevance.
The concentration and bioavailability of Zn in rice is very low and its consumption alone cannot meet the recommended daily allowance.
To address this problem, a agronomic and genetic approach called Biofortification which aims at enrichment of foodstuffs with vital micronutrients have been evolved and pursed as a potent strategy, internationally.

1. Zinc Biofortification in Rice SPEAKER ZINZALA VIVEK N . Ph.D. (Crop Physiology ) Dept. of GPB , N.M.C.A . ,NAU, Navsari

2. SEMINAR CONTANT Introduction Rice and its importance Role of Zinc What is Biofortification ? Methods of Biofortification Review of Research Conclusion 2

3. Introduction Rice ( Oryza sativa L.) is major staple food in the world (especially in South and South East Asian countries). Important staple foods for more than half of the world’s population (IRRI, 2006) Source of livelihoods and economies of several billion people. On a global basis, rice varieties provide 21% and 15% per capita of dietary energy and protein, respectively. About 50% world’s populations depends on rice as their main source of nutrition. However , rice is a poor source of micronutrients. Micronutrients deficiency is a global problem contributing to world’s malnutrition and a major public health problem in many countries, especially in regions where people rely on monotonous diets of cereal-based food, as the Zn level or content in the grains of staple crops, such as cereals and legumes, is generally low. Increasing the Zn content in the grains of these crops is considered a sustainable way to alleviate human Zn deficiency. Zn deficiency being an important nutrient constraint, any approach to improve Zn uptake and its transport to grains has significant practical relevance. The concentration and bioavailability of Zn in rice is very low and its consumption alone cannot meet the recommended daily allowance. To address this problem, a agronomic and genetic approach called Biofortification which aims at enrichment of foodstuffs with vital micronutrients have been evolved and pursed as a potent strategy, internationally . 3

4. Classification of rice Common Name : - Rice Scientific Name : - Oryza sativa L. Family : - Poaceae Chromosome no. : - 2n = 24 Genome size : - 430 Mb Indica :- It is tropical rice grown in India, awnless or short awn, late in maturity, long stem. Japonica : - Temperate and sub tropical rice, grown in Japan, early maturity, photosynthetically very active, short stem. Javanica :- wild form of rice, grown in Indonesia. 4

5. Nutritional values Nutritional value per 100 g Energy : 1,527 kJ (365 kcal) Sugars : 0.12 g Protein : 7.12 g Dietary fiber : 1.3 g Thiamine : 0.0701 mg Riboflavin : 0.0149 mg (1%) Zinc : 1.09 mg Calcium : 28 mg Iron : 0.80 mg Magnesium : 25 mg 5 Source: USDA Nutrient database

6. Season Crop season Local name Sowing time Harvest time Kharif Aus(W.B, Bihar) May-June Sept-Oct. Rabi Aman or Aghani June-July Nov-Dec. Summer or Spring Dalua(Orissa),Boro (W.B) Nov-Dec. March-April 6

7. Year 2015-16 2016-17 Area (Million ha ) 159.44 160.82 Production (Million metric tons) 472.96 486.78 Productivity (Metric tons per hectare) 4.42 4.52 7 Table 1: Area , production and productivity of Rice in WORLD Source : www.fao.org Table 2: Area, Production and productivity of Rice in India Year Area ( million hectares) Production (Million metric tons) Productivity (Metric tons per hectare ) 2015-16 43.50 104.41 3.60 2016-17 43.19 110.15 3.83

8. Fig.1: State wise Production of Rice in India 8 Source : Visualize.data.gov.in

9. Fig. 2: Which state is biggest rice producer ? 9 Source: https ://www.mapsofindia.com/answers/india/state-biggest-rice-producer/

10. Fig. 3: Rice exports by the five major exporters 10 Source: www.fao.org/economic/RMM

11. Zn is an important micronutrient for plant growth. Essentiality of Zn was discovered by- A.L. Sommer and C.P. Lipman In plant, Zn content varies from- 27 ppm to 100 ppm In Soil, Zn content in indian soils varies from: Arid/semi-arid climate - 20-89 mg kg -1 Humid/sub-humid tropics - 22-74 mg kg -1 Vertisols - 69-76 mg kg -1 Oxisols (coarse textured ) - 24-30 mg kg -1 Katyal and Vlek , 1985 Zinc 11

12. Table 3: Recommended Dietary Allowances ( RDAs) for Zinc Age Male Female Pregnancy Lactation 0–6 months 2 mg 2 mg 7–12 months 3 mg 3 mg 1–3 years 3 mg 3 mg 4–8 years 5 mg 5 mg 9–13 years 8 mg 8 mg 14–18 years 11 mg 9 mg 12 mg 13 mg 19+ years 11 mg 8 mg 11 mg 12 mg 12 Zn Supplemented by media

13. Role of Zinc in Plant system 13

14. Importance of Zn Critical in tissue growth Wound healing Immune system function Bone mineralization Proper thyroid function Cognitive functions Fetal growth and sperm production Essential for cell division, synthesis of DNA and proteins 14

15. 15 Alloway , 2008 Fig. 4:

16. Fig. 5: Zinc Deficiency in India The average level of Zn deficiency in Indian soils is 50 % and is projected to increase to 63% by 2025. 16 M.V. Singh, 2000

17. Zinc deficiency symptoms 17 zinc deficiency in different crops Khaira disease in Rice White bud of maize Little leaf of cotton Mottled leaf of citrus or frenching of citrus Interveinal chlorosis Reduction in the size of the young leaves In acute deficiency, younger leaves show necrosis and dead spots Dicot plants show, short internodes ( rossetting ) and decrease in leaf expansion (Little leaf) Premature leaves drop Bud fall off Seed formation is less Fruits are deformed associated with yield reduction.

18. Factors affecting availability of Zinc in soil 18

19. Deficiency of Zn in Human 19 Poor Neurological Function Weak immunity Diarrhea Leaky gut Acne or rashes Infection in children Complication in Pregnancy Wound healing Thinning hair

20. What is Biofortification? Biofortification  is the process by which the nutritional quality of food crops is improved through agronomic practices, conventional plant breeding, or modern biotechnology . Biofortification differs from ordinary fortification because it focuses on making plant foods more nutritious as the plants are growing 20 Greek word “ bios ” means “life” Latin word “ forticare ” means “make strong” Make life stronger

21. Poverty (poor people rarely have access to commercially fortified foods). Among micronutrients iron , zinc etc., remain significant problems in developing country populations. Diverse diet, comprising fruits, vegetables and animal products, in terms of energy requirement and micronutrient needs. Why Biofortification ? Why Rice Biofortification ? Staple food for 2.4 billion poor people No. of rice consumer rise by 70% in three decades. Per capita consumption of rice is high as 2014 kg/year The Rice endosperm (starchy & most edible part of rice seed) is deficient in many nutrients including vitamins, proteins, micronutrients, etc. Zinc located in Aleurone layer lost during milling and polishing Unprocessed rice becomes rancid i.e. smelly or unpleasant in taste Thus even small increase in nutritive value of rice matter a lot more 21

22. Micronutrients biofortified into Rice Zinc Iron Biofortification Promoters Biofortification requires a multidisciplinary research approach 22

23. Approaches for Biofortification in Rice Convetional breeding Transgenic plant strategy Iron & Phytate content & Zinc, Vit. A, Protein etc … Increased Bioavailability of nutrients 23 Agronomic strategy

24. Fig. 6: Biofortification is a Food-based Approach 24 Source : http :// faostat.fao.org/site/368/DesktopDefault.aspx?PageID=368#ancor Six crops account for 57% of energy and 49% of protein “ consumed ” by populations living in least developed countries (FAO food balance sheets)

25. Biofortified Crops Improve Food and Nutrition Security 25 Increase foods available in homes Better agronomic characteristics Greater: yields, resistance to pests, tolerance to stresses Higher nutritional concentration More: iron, zinc, beta-carotene and/or tryptophan and lysine Increase the intake of these nutrients Improve nutrition security Improve food security

26. How to overcome Zn deficiency 26

27. Case studies 27

28. Managing Zinc deficiency Through Agronomic Approach Sources Zinc content Zinc sulphate heptahydrate 21-23 % Zinc sulphate monohyadrate 33-36% Zinc oxysulphate 40-55% Zinc oxide 55-70 % Zinc nitrate 22% Zn-EDTA 12-14 % Zn- HEDTA 9 % 28 Soil application of Zinc fertilizer Foliar Spray

29. Transport of zinc in plant 29

30. Table 4: Zn content and uptake at different stages of rice cultivar of Barh Avroadhi Treatment No. Treatments Zn content (ppm) Zn uptake (g ha -1 ) Tillering stage Panicle initiation stage Flowering stage T1 No Zinc 29 22 21 128 T2 ZnSO 4 @ 15 kg ha -1 (B) 32 25 24 148 T3 ZnSO 4 @ 30 kg ha -1 (B) 34 30 28 178 T4 ZnSO 4 @ 45 kg ha -1 (B) 37 35 34 214 T5 ZnSO 4 @ 0.25% (FS) 31 24 23 135 T6 ZnSO 4 @ 0.50% (FS) 33 28 27 160 T7 ZnSO 4 @ 0.75% (FS) 35 32 33 198 T8 Farmer Practice (B) 31 24 23 135 C.D. (P=0.05) 07 04 06 17 30 Kumar and Kumar (2009) Bahraich , U.P. B- Basal FS- Foliar Spray

31. Table 5: Effect of soil application of different sources of Zn on Zn content of grain and straw (mg kg -1 ) of rice cultivar IET 4094 (mean data of 2 years) Treatments Grain Straw T1 Control 11.06h 10.61g T2 Zn 10 kg ha -1 as ZnSO 4 at basal 12.77g 12.29f T3 Zn 10 kg ha -1 as ZnSO 4 in two splits 13.75f 13.48e T4 Zn 20 kg ha -1 as ZnSO 4 at basal 17.18d 16.35c T5 Zn 20 kg ha -1 as ZnSO 4 in two splits 18.33c 17.21b T6 Zn 0.5 kg ha -1 as Zn-EDTA at basal 15.18e 14.44d T7 Zn 1.0 kg ha -1 as Zn-EDTA at basal 19.26a 18.28a T8 Zn 1.0 kg ha -1 as Zn-EDTA in two splits 18.76b 17.99a 31 Naik and Das (2010) West Bengal, India

32. Table 6: Concentration and uptake of Zn in the shoot and grain of lowland rice (BRS Jaburu ) Treatment s Shoot Grain Zn rate (mg kg −1 ) Zn conc. (mg kg −1 ) Zn Uptake (mg pot −1 ) Zn conc. (mg kg −1 ) Uptake (mg pot −1 ) T1 0 (Control) 53 4.02 40.67 0.88 T2 5 72 6.78 37.33 1.42 T3 10 67 6.16 38 1.49 T4 20 71 6.82 38.33 1.6 T5 40 75.33 7.33 37 1.45 T6 80 90.33 7.99 38.67 1.55 T7 120 213.33 20.01 45.33 1.77 F-test ∗∗ ∗∗ NS ∗∗ CV(%) 8 8 11 12 32 Goias , Brazil Fageria et al . (2011)

33. Table 7: Effect of Zn fertilization on Zn concentrations in rice grain and straw and their uptake by aromatic hybrid rice-PRH 10 Treatment Zn concentration (mg kg -1 DM) in straw Zn concentration (mg kg -1 DM) in grain Total Zn uptake in grain + straw (g ha -1 ) 2007 2008 2007 2008 2007 2008 T1 control 124.6 125.7 14.9 15.0 1,124.0 1,093.5 T2 only N 143.3 144.6 16.9 17.1 1,653.1 1,662.3 T3 2% ZEU* (ZnSO 4 .7H 2 O) 176.9 178.4 22.9 23.1 2,219.3 2,290.3 T4 2% ZEU ( ZnO ) 163.9 165.3 19.9 20.1 1,977.7 2,061.7 T5 5 kg Zn ha -1 (ZnSO 4 .7H 2 O) 160.6 162.0 21.0 21.2 1,934.2 1,980.4 T6 5 kg Zn ha -1 ( ZnO ) 151.1 152.4 19.1 19.2 1,754.6 1,824.8 T7 CMCU** 143.7 142.5 17.0 16.9 1,675.1 1,669.7 SEm ± 0.56 0.56 0.08 0.08 22.1 37.6 CD (P=0.05) 1.60 1.60 0.24 0.24 63.4 107.8 33 Jat et al . (2011) New Delhi , India ZEU* = Zn-enriched urea; CMCV** = Coating material coated urea; DM = Dry matter;

34. Fig. 7: Effect of zinc fertilization on A) grain Zn content, B) Straw Zn content, C) Grain Zn uptake, D) Straw Zn uptake of rice variety ADT 43 34 Muthukumararaja and Sriramachandrasekharan (2012 ) Tamilnadu , India

35. Table 8: Zinc fertilizer application on some qualities parameters in rice genotypes Treatment Zinc content in grain (mg kg -1 ) Zinc content in straw (mg kg -1 ) Zinc uptake in grain (kg ha -1 ) Zinc uptake in straw (kg ha -1 ) Zinc fertilizer T1 40 kg Zn ha -1 27.25 a 9.62 a 13.48 a 7.12 a T2 20 kg Zn ha -1 23.67 ab 8.52 a 11.58 ab 6.63 ab T3 Control 18.25 b 6.48 b 9.65 b 5.48 b Genotypes Sang Tarom 27.56 a 9.88 a 14.67 a 7.40 a Mahalli Tarom 23.33 b 8.19 b 9.26 b 5.86 b Neda 20.00 c 7.28 b 13.30 a 6.40 b Shiroodi 21.33 bc 7.48 b 9.05 b 5.98 b 35 Mazandaran , Iran Yadi et al . (2012)

36. Fig. 8: Zinc (Zn) concentration in paddy ( A) of cultivar CNT 1 when foliar application with 0.5% zinc sulfate (ZnSO 4 ) was applied at different stages of plant growth. 36 Boonchuay et al . (2013) Nil - no foliar; PI - panicle initiation; BO - booting; WAF - weeks after flowering. Chiang, Thailand

37. Fig . 9: Zinc (Zn) concentration in husk (B) of cultivar CNT 1 when foliar application with 0.5% zinc sulfate (ZnSO 4 ) was applied at different stages of plant growth. 37 Chiang, Thailand Boonchuay et al . (2013) Nil - no foliar; PI - panicle initiation; BO - booting; WAF - weeks after flowering.

38. Fig . 10: Zinc (Zn) concentration in brown rice ( Oryza sativa L .) (C) of cultivar CNT 1 when foliar application with 0.5% zinc sulfate (ZnSO 4 ) was applied at different stages of plant growth. 38 Nil - no foliar; PI - panicle initiation; BO - booting; WAF - weeks after flowering. Chiang, Thailand Boonchuay et al . (2013)

39. Fig . 11: Effect of different levels of Zinc treatments on rice straw, leaf and stem zinc content. 39 Kabeya and Shankar (2013) High Zn groups: IR20, BPT5204, IR73898 Low Zn groups : Thanu , IET17913, IR59656 Bangalore, India

40. Table 9: Effect of different levels treatment of Zn on some growth characteristics Treat- ments Zinc groups Plant height (cm) No of effective tiller per plant SPAD IR20 BPT5204 IR73898 IR20 BPT5204 IR73898 IR20 BPT5204 IR73898 zero zinc High Zn groups 104.33c 85.67c 111.33c 14.67a 16.67b 15.00b 45.21b 50.99a 48.68b 20kg ZnSO 4 ha -1 115.00b 121.33b 127.67b 18.00a 22.00a 19.00b 53.08a 52.88a 52.26ab 30kg ZnSO 4 ha -1 125.00a 129.00a 145.00a 19.33a 25.67a 28.00a 57.81a 54.47a 54.92a Thanu IET17913 IR59656 Thanu IET17913 IR59656 Thanu IET17913 IR59656 zero zinc Low Zn groups 93.00c 68.00c 114.33c 15.67b 11.67b 14.00a 45.2b 40.12c 45.60a 20kg ZnSO 4 ha -1 122.00b 78.00b 120.67b 18.33ab 15.00ab 15.33a 54.71a 54.75a 48.80a 30kg ZnSO 4 ha -1 138.00a 125.50a 133.00a 23.00a 20.00a 19.67a 48.99b 47.20b 45.09a 40 Bangalore, India Kabeya and Shankar (2013)

41. Fig. 12: Effects of chelated and mineral zinc on the Zn concentration of rice leaves (ppm) 41 Javid et al . (2014) Faisalabad , Pakistan

42. Table 10: Yields of grain and straw and zinc content as affected by zinc of rice variety Ranjit (mean data of 2 years) Treatments ZnSO 4 (kg ha -1 ) Grain yield (t ha -1 ) % response Straw yield (t ha -1 ) % response Zinc in grain (mg kg -1 ) Zinc in straw (mg kg -1 ) T1 26.41 55.08 17.4 32.5 T2 5 35.51 34.45 57.13 3.72 20.4 33.8 T3 10 35.68 35.1 58.68 6.53 18.2 40.7 T4 15 41.60 57.51 63.06 14.48 18.3 42.2 T5 20 42.61 61.34 65.24 18.44 20.1 39.3 T6 25 45.92 73.87 66.40 20.55 21.7 45.6 T7 30 43.74 65.61 63.07 14.51 20.8 45.4 S.Em 0.69 1.24 0.49 1.5 CD (0.05%) 1.50 2.70 1.06 3.27 42 Kandali et al . (2015) Jorhat , Assam

43. Table 11: Effect of zinc on zinc uptake in grain and straw of rice variety Ranjit (mean data of 2 years) Treatment ZnSO 4 (kg ha -1 ) Zinc uptake in grain (g ha -1 ) Zinc uptake in straw (g ha -1 ) T1 45.8 179.3 T2 5 72.5 193.2 T3 10 64.8 238.5 T4 15 75.9 266.5 T5 20 87.1 256.8 T6 25 97.5 311.7 T7 30 88.6 286.5 S.Em 3.31 10.26 CD (0.05%) 7.21 22.36 43 Kandali et al . (2015) Jorhat , Assam

44. Table 12: Effect of various zinc treatments on yield attributes of aromatic rice variety P usa Basmati 1 Treatments Plant height (cm) Tillers m -2 Panicle length (cm) Grains panicle -1 1,000-grain weight (g) T1 104 310 24 85 21.1 T2 5 kg Zn ha -1 (soil) 107 326 26 91 22.2 T3 1 kg Zn ha -1 (foliar) 105 318 25 88 22.0 T4 5 kg Zn ha -1 (soil) + 1 kg Zn ha -1 (foliar) 108 342 27 94 22.7 T5 2.83 kg Zn ha -1 through Zn-coated urea (soil) 107 328 26 91 22.3 SEm ± 2.12 3.51 0.79 1.03 0.38 LSD ( p = 0.05) NS 9.95 NS 2.93 NS 44 Shivay et al . (2015 ) New Delhi, India

45. Table 13: Effect of various zinc treatments on zinc concentrations in kernel, husk , straw and their uptake in aromatic rice variety Pusa Basmati 1 Treatments Zn concentration in kernel (mg kg -1 rice kernel) Zn concentration in husk (mg kg -1 rice husk) Zn concentration in straw (mg kg -1 rice straw) Zn uptake in Kernel (g ha -1 ) Zn uptake in husk (g ha -1 ) Zn uptake in straw (g ha -1 ) Total Zn uptake in crop (g ha -1 ) T1 Check 20.0 125.0 91.0 48.0 147.5 618.8 814.3 T2 5 kg Zn ha -1 (soil) 21.3 130.0 100.0 56.0 169.0 745.0 970.0 T3 1 kg Zn ha -1 (foliar) 22.0 147.0 102.0 56.1 183.8 739.5 979.4 T4 5 kg Zn ha -1 (soil) + 1 kg Zn ha -1 (foliar) 25.0 175.0 107.0 75.7 262.5 868.8 1207.0 T5 2.83 kg Zn ha -1 through Zn-coated urea (soil) 23.8 170.0 105.0 65.5 229.5 801.2 1096.2 SEm ± 0.30 1.46 0.98 1.07 2.41 7.95 9.27 LSD ( p = 0.05) 0.86 4.13 2.78 3.02 6.82 22.55 26.26 45 Shivay et al . (2015 ) New Delhi, India

46. Managing Zinc deficiency Through Genetics Approach 46 Mutation breeding approach Molecular breeding approach Transgenic breeding approaches

47. Using the mutagen NaN 3 developing mutant rice varieties biofortified with iron (Fe) and zinc (Zn) is an important strategy to alleviate nutritional deficiencies in developing countries. Using the materials and method 47 CS-1: Comparisons and selection of rice mutants with high iron and zinc contents in their polished grains that were mutated from the indica type cultivar IR64 Jeng et al . (2012) Taichung, Taiwan

48. Materials and methods 48 Polished rice Zn Fe IR-64 16 (mg kg -1 ) 3.9 (mg kg -1 ) mutants 15.36 to 28.95 (mg kg -1 ) 0.91 to 28.10 (mg kg -1 ) Sodium azide (NaN 3 )

49. Fig. 13: Frequency distribution of Fe and Zn in mutants 49

50. Table 14: Micro-minerals in the polished rice grains of selected NaN 3 -induced mutants Micronutrient Mean (mg kg -1 ) Range (mg kg -1 ) Fe 4.02 0.11-28.10 Zn 15.6 8.37-28.95 Mn 8.12 4.56-25.72 Cu 2.97 0.06-10.06 50 Mutants Fe (mg kg -1 ) Zn (mg kg -1 ) Mn (mg kg -1 ) Cu(mg kg -1 ) M-IR-75 28.10 15.36 7.28 3.74 M-IR-58 27.26 17.44 8.24 6.07 M-IR-180 0.91 26.58 9.32 1.77 M-IR-49 3.48 28.95 6.91 4.05 M-IR-175 13.36 26.16 9.78 5.59 IR-64 3.90 16.00 8.00 2.85

51. Table 15: Grain yields of selected mutants grown in different crop seasons Mutant Grain weight (g 1000 grains -1 ) Grain yield (ton ha -1 ) 2010 autumn 2011 spring 2010 autumn 2011 spring M-IR-75 28.00 ± 1.89 28.31 ± 1.38 6.49 ± 2.71 10.81 ± 3.06 M-IR-58 27.41 ± 2.04 28.32 ± 1.56 2.14 ± 0.76 5.81 ± 0.62 M-IR-180 26.49 ± 2.16 27.24 ± 0.27 5.58 ± 0.16 9.95 ± 1.25 M-IR-49 27.26 ± 1.78 27.43 ± 2.56 4.20 ± 2.47 5.01 ± 1.75 M-IR-175 28.64 ± 1.41 27.90 ± 0.16 4.21 ± 0.54 7.00 ± 1.80 IR64 26.21 ± 0.24 27.41 ± 1.00 5.66 ± 0.83 8.87 ± 0.40 LSD0.05 3.02 2.69 0.74 3.37 51

52. Conclusion Two selected mutants, M-IR-75 and M-IR-58, accumulated considerably higher levels of Fe in their polished grains than the wild type cultivar IR64. Additionally , three selected mutants , M-IR-80, M-IR-49 and M-IR-75, accumulated more Zn than the wild type cultivar IR64. Thus , the mutant M-IR-75 can be recommended to rice farmers for producing polished Fe-rich rice grains in order to alleviate anemia caused by Fe deficiency in areas where polished rice is consumed as a staple food. Moreover, the high-Fe (M-IR-75 and M-IR-58) and high-Zn (M-IR-180, M-IR-49 and M-IR-175) mutants can be used as genetic resources for rice improvement programs. 52

53. CS-2: Identification of putative candidate gene markers for grain zinc content using recombinant inbred lines (RIL) population of IRRI38 X Jeerigesanna Identifying the target quantitative trait loci (QTL) genes to estimate grain zinc content using candidate gene markers Plant material One hundred sixty RILs derived from IRRI38 X Jeerigesanna 53 Aims Gande et al . (2014) Karnataka, India

54. Molecular analysis of RILs using candidate gene designing of candidate gene primers, the gene sequence information was downloaded from National Centre for Biotechnology Information (NCBI) and primers were designed using primer-3 tool. 24 candidate gene markers used among 11 markers showed highly polymorphism. 54

55. Genes Chr. No Source Annealing temp Exp Product size OsYSL2a 2 NCBI NA 1010 OsYSL2b 2 NCBI 60.0 980 OsVIT1 NA Chandel et al. 2011 63.0 980 OsNAAT1 2 Chandel et al. 2011 58.0 700 OsNAC 3 Chandel et al. 2011 58.0 600 OsZIP1a 3 NCBI 59.0 883 OsZIP1b 3 NCBI 59.5 561 OsZIP3a 4 NCBI 59.5 1131 OSZIP3bII 4 NCBI 59.5 1104 OsZIP3b 4 NCBI 61.5 370 OsZIP7c 5 NCBI NA 940 OsZIP7d 5 NCBI 57.0 1032 55 Table 16: Candidate gene primers were designed using NCBI and Primer-3 tool. Genes Chr. No Source Annealing temp Exp Product size OsZIP7e 5 NCBI 49.5 940 OsZIP8a 7 NCBI 67.0 880 OsZIP8b 7 NCBI 52.0 1064 OsZIP8c 7 NCBI 52.0 927 OsZIP4a 8 NCBI 60.0 641 OsZIP4b 8 NCBI 61.5 673 OsZIP4d 8 NCBI 60.0 963 OsZIP4c 8 NCBI 56.0 1000 OsNAC5a 11 NCBI 53.0 916 OsNAC5b 11 NCBI 55.0 885 OsNAC5c 11 NCBI 53.0 600 OsNRAMP7 12 Chandel et al. 2011 60.0 2000 RM263 2 Gramene 55.0 199 RM152 8 Gramene 56.0 151 RM21 11 Gramene 55.0 157

56. Single marker analysis SMA was done with t-test and regression analysis using SPSS 16.0 (SPSS Inc.) to find the association between molecular markers and grain zinc content. Polymorphic candidate markers which showed significant association with grain zinc content Single marker analysis revealed that out of 11 polymorphic markers, four ( OsNAC , OsZIP8a, OsZIP8c and OsZIP4) showed association with a phenotypic variation of 4.5, 19.0, 5.1 and 10.2%, respectively (Table 17) among the RIL population. 56

57. Table 17: Single marker analysis (SMA) showing P and R 2 values of candidate gene and SSR markers in RILs of IRRI38 X Jeerigesanna for grain zinc content. S/N Marker P R 2 (%) Mean Difference Estimated effect 1 OsZIP3b 0.34 2.1 1.2 4.2 2 RM263 0.59 0.7 1.8 1.4 3 RM21 0.28 1.6 0.6 3.2 4 RM152 0.98 00 0.4 0.0 5 OsNAC 0.03* 4.5 1.7 9.0 6 OsZIP3bII 0.41 0.4 0.3 0.8 7 OsZIP8a 0.00** 19 3.9 38.0 8 OsZIP8c 0.02* 5.1 1.6 10.2 9 OsVIT1 0.73 0.4 0.2 0.8 10 OsZIP4b 0.00** 10 2.5 20.4 11 OsZIP7e 0.71 0.4 1.8 0.8 Mean 23.7ppm SD 3.37 57 P, Significance; R 2 , percentage of phenotype variability.

58. Fig. 14: Polymorphic candidate gene 58 L, 100 bp ladder; P1, IRRI38; P2, Jeerigesanna

59. Validation of putative markers is used to confirm the reproducibility of usefulness in marker aided breeding program. Validation of four candidate gene markers with 96 germplasm accessions showed significant association for three markers (OSZIP8a, OsNAC and OsZIP4b) with a phenotypic variation of 11.0, 5.8 and 4.8% respectively. 59 S/N Marker P R 2 (%) Mean Difference Estimated effect 1 OsNAC 0.02 5.8 4.2 22 2 OsZIP8a 0.01 11 2.4 2.8 3 OsZIP8c 0.51 1.4 1.8 11.6 4 OsZIP4b 0.03 4.8 3.2 9.6 Mean 29.35 SD 6.26 P, Significance; R 2 , percentage of phenotype variability.

60. Conclusion The present study revealed that RILs having high grain zinc content with high genetic variability. Single marker analysis showed four candidates gene markers with a significant phenotypic variation among the RIL population . Three putative candidate gene markers (OsZIP8a, OsNAC and OsZIP4b) with a phenotypic variation of 11.0 , 5.8 and 4.8% were found. These putative markers can be used in biofortification programs by breeders and bio-technologists. 60

61. CS-3: Biofortified indica rice attains iron and zinc nutrition dietary targets in the field 61 Trijatmiko et al . (2016) Manila , Philippines popular rice varieties, IR 64, low iron (Fe) 2 μg g −1 and zinc (Zn) with 16 μg g −1 . selected 1689 transgenic events, in cultivar, IR 64, field evaluated in two countries and reported that increased 15 μg g −1 Fe and 45.7 μg g −1 Zn in polished grain .

62. 62 Fig. 15: Strategy for the development of biofortified high-iron rice and the Fe concentration achieved in T 2 polished seeds.

63. Fig. 16: Expression of transgenes in the representative events. 63 Root Leaves

64. Fig. 17: Field trials for evaluation of target trait and agronomic characters of two lead events. 64

65. Fig. 18: Characterization of lead events . 65 Analysis of Fe and Zn content in Endosperm visualized by X-ray fluorescence imaging.

66. Released zinc biofortified variety 66 Variety Zn GNR-4 15 ppm GNR-8 21.8 ppm DRR dhan-45 22.6 ppm BRRI dhan-62 19 to 20 ppm BRRI dhan-64 25 ppm BRRI dhan-72 23 ppm BRRI dhan-84  27.6 ppm

67. Conclusion Zn application significantly increase yield and yield attributes of rice crop also content and uptake of zinc was also increased significantly with increasing levels of zinc. Application of zinc fertilizers offers a rapid solution for increase productivity and Zn concentration in grain and straw of rice. Soil or foliar applications of Zn may also increase grain zinc concentration and thus contribute to grain nutritional quality for human beings. Biofortification of the zinc content using conventional breeding and biotechnological methods can enhance the nutrient content in grains of rice. Using QTL and mutation breeding, OsZIP8a, OsNAC and OsZIP4b are genes identified for high Zn uptake and transfer of these genes into the cultivar will boost the Zn content in rice grain . Biofortification is one of the best methods to alleviate malnutrition and development of new cultivars with elevated concentrations of Zn using conventional and biotechnological approaches. 67

68. Thank you Food is the moral right of all who are born into this world -- Borlaug Nutritious food is the moral right of all who are born into this world