Micronutrient malnutrition is widespread, especially in poor populations across the globe where daily caloric intake is confined mainly to staple cereals. Rice, which is a staple food for over half of the world's population, is low in bioavailable micronutrients required for the daily diet. Improvements of the plant-based diets are therefore critical and of high economic value in order to achieve a healthy nutrition of a large segment of the human population. Rice grain biofortification has emerged as a strategic priority for alleviation of micronutrient malnutrition

1. Presented By,
SATHISHA T N
Depart. of Genetics and Plant Breeding,
UAS Dharwad
1

2. Content
• Introduction
• Merits of Biofortification of rice
• Biofortification of Fe and Zn
Conventional approaches
Molecular approaches
• Biofrtification of Pro vit- A
• Conclusion
2

3. 3

4. 4

5. 5
Vitamin A deficiency
– 250,000 children per year go blind
– 124 million children worldwide are
deficient in vitamin A
Iron deficiency
– More than 1.6 billion people ,in world
deficient of iron
– approximately 25 % of population
– Developing country 50 % of anemia
Zinc deficiency
– More than 400,000 children die due
to zinc deficency
– Most of the poor in Asia suffer

6. Anemia Prevalence and Number of Individuals
Affected
WHO
Region
Preschool Age Children Pregnant Women Non-pregnant women
Prevalence
(%)
No.
affected
(mill.)
Prevalence
(%)
No.
affected
(mill.)
Prevalence
(%)
No.
affected
(mill.)
Africa 68 84 57 17 48 70
Americas 29 23 24 4 18 39
South-East
Asia
66 115 48 18 46 182
Europe 22 11 25 3 19 41
Eastern
Mediterran
ean
47 1 44 7 32 40
Western
Pacific
23 27 31 8 22 97
Global 47 293 42 56 30 468
Source: Worldwide Prevalence of Anaemia 1993-2005, World Health Organization, 2008
6

7. Zinc Deficiency Among Children Under Age 5
Region Prevalence(%) Deaths(‘000) DALYs lost(‘000)
East Asia & Pacific 7 15 1,004
East Europe & Central
Asia
10 4 149
Latin America &
Caribbean
33 15 587
Middle East & North
Africa
46 94 3,290
South Asia 79 252 8,510
Sub-Saharan Africa 50 400 14,094
High Income Countries 5 0 2
7

8. 8

9. 9

10. Advantages of Biofortification
• Targets the poor: eat high levels of food staples
• Rural-based: complements fortification and supplementation
• Cost-effective: research at a central location can be multiplied
across countries and time
• Sustainable: investments are front-loaded, low recurrent costs
Greek word “bios” means “life”
Latin word “fortificare” means “make strong”
MAKE LIFE STRONG!
“Bio-fortification is the enrichment of staple food crops with essential
micronutrients”.
Sally Brooks,
Biofortification

11. 11

12. CONVENTIONAL APPROACHES
12

13. Nutrient content of rice
Source: FAO Rice Factsheet, 2004
13
Fe and Zn distribution in rice grain

14. Screening of germplasm for high iron and
zinc content
14

15. Genetic variation in Iron and zinc content in IRRI
Gregorio et al., 2000
Total 1138 12.2 (6.3-24.4) 25.3 (23.5-58.4)
15

16. Fe and Zn contents in the four rice varietal groups
Lee et al.,(2008) 16

17. 17

18. Iron and zinc content of some selected varieties
Gregorio et al., 2000
18

19. 19

20. Gregorio et al., 2000
Iron content of selected variety after polishing
20

21. Effect of milling on iron concentration in rice
21

22. Sl
No
Name Grain
type
Fe(mg/ 100g) content in
polished rice
0 % 5% 10%
1 MSE-9 LB 3.44 1.24 1.08
2 Kalanamak SB 3.40 1.21 1.09
3 Kanachana MS 2.04 1.28 0.66
4 Karjat- 4 MS 2.56 2.06 1.90
5 Chittimutyalu SB 2.49 1.40 0.98
6 Udayagiri SB 3.01 0.95 0.90
7 Jyothi LB 1.98 1.49 0.40
8 VRM-7 SB 2,28 0.79 0.78
9 Metta Triveni SB 2.61 0.70 0.70
10 Versha SB 3.75 1.12 0.81
Rice varieties with high Fe content in grains
22
DRR Hydrabad

23. Sl
No
Name Grain
type
Zn(mg/ 100g) content in
polished rice
0 % 5% 10%
1 Chittimutyalu SB 3.05 2.57 2.44
2 Poornima SS 3.13 2.78 2.70
3 ADT-43 MS 3.09 2.66 2.09
4 Ranbir Basmati LS 3.09 2.83 2.74
5 Type-3 LB 3.03 2.83 2.65
6 Udayagiri SB 3.01 1.95 1.13
7 Ratna LS 3.27 2.52 2.30
8 Jyothi LB 3.13 2.24 2.06
9 Panta Sugandh
17
LS 3.25 2.47 2.06
10 Kesari MS 3.15 1.99 1.93
Rice varieties with high Zn content in grain in India
DRR Hydrabad
23

24. Fe and Zn content in popular cultivar
24

25. Scientist Material Fe zn
Lee et al., (2008) 246 rice
germplasm
2.02 to 12.0
(mg/Kg)
10.0 to 33.0
(mg/Kg)
Ravindra Babu et al.
(2012)
173 varieties and
21 hybrids
2.4 (PTB-51) to
34.4 (MSE 9)
10.1 (Karjat 3) to
32.7(Ratna)
(mg/kg)
Virk et al.
(2006b,2007)
15 genotypes 7.4 (mg/kg) 23.26 (mg/kg)
Senadhar et al.,
(1998)
939 varieties 7.4-24 (mg/kg)
Prom u Thai et al.,
2007
Australien
varieties
10- 20 (mg/kg)
Prom u Thai and
Rerkasen (2001)
Thai rice varieties 7-22 (mg/kg)
Pintasen et al ., (2007) 17 Thai rice
varieties
10.8-16.2
(mg/kg)
Martinez et al., 2009 5743 milled rice in
CIAT & NARS
5-7 (mg/kg)
EMBRAPA
Traditional variety
(12.6-42.2
ppm)
25

26. Hybridization followed selection
• Popular cultivar 1.3 to 1.5 mg/ 100 g
White x purple
2.1 mg/ 100 g
KDC x Hom Pamah
313-19-1-1
(White color rice)
2.8 mg/ 100 g
O nivar x JaoHom Nin
(JHN)
5 mg/ 100 g rice
26

27. High iron rice
IR 72 x Zawa Bonday
IR 68144-3B-2-2-3
21 ppm
• Early mature
• Tolerance to tungro virus
• No seed dormancy
• Excellent seedling vigour
• 10% below yield than IR-72
27

28. Fe and Zn content in Popular rice hybrids in India
charecter Protien Iron (mg
/100g)
Zinc
(mg/100g)
1. DRRH-2A 9.79 12.58 4.38
2. PA 6129 8.86 8.95 4.26
3. Sahyadri-2 9.45 7.91 3.87
4. Sahyadri-4 8.8 10.48 4.3
5. Pusa RH-10 8.26 4.52 3.75
6. Indirasona 7.64 7.85 3.87
7. GK 5003 7.39 8.15 3.84
8. PSD-3 5.74 7.29 3.99
9. Sahyadri-3 7.67 4.13 3.07
10. PA 6201 7.86 7.76 4.59
11. HSD-1 7.84 6.76 3.17
12. PA 6444 7.15 6.11 3.72
13. Suruchi 7.93 6.87 3.35
14. JKRH-2000 7.42 7.15 3.55
15. US 312 7.47 6.62 3.76
16. CORH-3 8.1 7.37 3.53
17. KRH-2 6.49 4.39 3.43
18. Sahyadri-1 7.74 5.9 3.33
19. PHB-71 8.48 2.89 3.81
20. CRHR-5 8.59 2.96 3.15
21. CRHR-7 8.3 6.93 3.62
Mean 7.95 6.84 3.73 28

29. Correlation among iron and zinc content in rice
grain
(Nagesh et al ., 2012)
29
Phenotypic and Genotypic Correlation coefficient for Iron and Zinc in 48 rice hybrids

30. 30

31. Estimation of heterosis for grain iron and zinc content
6 lines x 8 testers
48 hybrids
SH1- Swarna
SH 2- Chitimutayalu
Nagesh et al., 2012
31

32. 32

33. G x E interaction for iron and zinc
33

34. Grain zinc content (ppm) of promising rice genotypes grown in 3 environments
Virk et al. (2006b,2007)
34

35. Fe concentration in 10 genotypes in 4 location Wet and Dry season
Dry season
Wet season
(Suwarto and Nasrullah 2011) 35

36. Genotype x Environment interaction on iron concentration in rice
Suwarto and Nasrullah 2011
36

37. Mutation breeding
37

38. Rice mutant with high iron and zinc
content
IR-64
Sodium azide (NaN3)
258 M8 generation mutants
Analysed for micronutrient and yield along with check IR-64
Polished rice Fe Zn
IR-64 3.9 (mg / kg) 16 (mg / kg)
mutants 0.91 to 28.10 (mg / kg) 15.36 to 28.95 (mg / kg)
38

39. Frequency distribution of Fe and Zn in mutants
39

40. Principle coordinate analysis for wild type cultivar IR-64 and 254 NaN3 -induce mutants
40

41. Micronutrient Mean (mg/Kg) Range (mg/Kg)
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
Micro-minerals in the polished rice grains of selected NaN3-induced mutants
Mutants Fe (mg / Kg) Zn (mg /Kg) Mn (mg / Kg) Cu(mg / Kg)
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
41

42. Grain yields of selected mutants grown
in different crop seasons
42

43. MOLECULAR APPROACHES TO
IMPROVE GRAIN QUALITY IN RICE
43

44. Molecular Mapping of High Iron and Zinc Rich
Regions in Rice (Oryza sativa L.) Grains Using
Microsatellite Markers
Nagesh et al., 2013
Swarna × Madhukar
44
178 F 2 population
72 SSR marker

45. Parental polymorphism survey between Swarna and
Madhukar
45
SC-120, SC-128
SC-129(33.56 %)

46. Selective genotyping for rapid identification of regions
associated with iron and zinc content
46
Swarna × Madhukar

47. Segregation pattern of SSR primers
Legend: M-100bp DNA marker, P1 – Swarna, P2 – Madhukar
47

48. Single marker analysis for iron and zinc with SC120,
SC128 and SC129
Swarna × Madhukar
178 F2 population
SC120, SC128 and SC129 markers screened
48

49. Identification of QTLs for mineral contents in
rice grain
49

50. Identification of QTL for micronutrient from a
wild rice
Teqing X wild rice (Oryza rufipogon)
3 back cross with Teqing
85 introgression lines (ILs)`
Oliveira et al ., 2008
(179 polymorphic SSR)
50

51. Micronutrient content in parent and
introgression lines grown during 2 season
51

52. Phenotypic variance for micronutrient in
introgression lines (ILs)
52

53. QTLs identified for micronutrient traits in
introgression lines
53
Fe
Fe
Zn
Zn
Zn

54. Mapping QTLs for iron and zinc content in rice
168 RILS
101 SSR + 9 GENE SPECIFIC MARKERS
Anuradha et al. 2012
Madhukar × Swarna
54
Unpolished
rice
Fe (ppm) Zn (ppm)
Madukar 17.3 57.3
Swarna 22.5 27.2
RILs 0.2-224 0.4 to 104
Mean 30.9 48.2

55. Distribution of Fe and Zn concentration in 168 RILs
55

56. 56
(14)
(4)
RM 535
Markers linked to Fe and Zn in Madhukar × Swarna RILs

57. QTL mapping for Fe and Zn concentration
57
Composite interval mapping
13 QTLs

58. Identification of QTL Fe and Zn
58

59. Putative candidate genes for QTLs governing
Fe and Zn
59

60. Bio fortification of rice by transgenic approaches
60

61. Fe accumulation in rice seeds
1. Iron storage protein:
Soybean ferritin gene expression
using endosperm-specific promoters,
Ferritin is a ubiquitous protein stores
about 4,000 Fe atoms in a complex.
2. Iron translocation over production of the natural metal
chelator nicotianamine Os NAS genes,
3. Iron flux into the endosperm by means of iron(II)-nicotiana mine
transporter OsYSL2 expression under the control of an
endosperm-specific promoter and sucrose transporter promoter
61

62. Soybean ferritin chimeric gene in the pGPTV-bar/ Fer vector
PCR analysis showing amplification of 0.78 kb size band of ferritin
gene 62
Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene
Vasconcelos et al.2003
Lane 1, non-transformed control
plant
2, plasmid control
3, blank;
4, 5, 7, 8 and 9, five individual
transformants
6, negative regenerated plant.
M, 1 kb DNA molecular marker.

63. 63

64. Iron detection in transverse sections of rice
endosperm
Non transgenic
IR68144
Transgenic
IR68144
64

65. Iron and zinc concentrations in brown seeds of transgenic To
lines (Fr) and control of IR68144
Average Fe and Zn concentrations in
transgenic (lines 1/4) of IR68144
65

66. Iron biofortification by introduction of multiple
genes by transgenic approaches
66

67. Gene cassette introduced into rice to produce the Fer-NAS-YSL2 lines
Japonica rice cultivar Tsukinohikari
67

68. Detection of transgene insertion in the transgenic lines by PCR
68

69. Ferritin accumulation in T2 brown seeds Western blot analysis.
69

70. Quantitative real-time RT-PCR analysis of OsYSL2 and HvNAS1. (a) OsYSL2
and (b) HvNAS1 expression levels. T2 plants
70

71. Fe concentration of T2 polished seeds
71

72. Fe
Zn
Cd
Metal concentration in T3 polished seeds obtained from the paddy field
72

73. NT= non-transgenic rice;
AN= OsActin1 promoter–HvNAS1 line;
Fer= NAS-YSL2 transgenic lines 15-8, 16-2, 19-2, 19-4, 19-5, and 32-3,
T3 seeds Fe, Zn, Mn, and Cu concentrations in brown seeds
73

74. Plant height and yield data transgenic lines in field
experiment
74

75. The problem with rice:
• Does not express
phytoene synthase in
endosperm
• Does not express
phytoene desaturase
or z-carotene
desaturase
75
Golden Rice

76. Development of golden rice
Fig. 1
Rice grain Golden ricewhite rice
Fig. 2
76

77. Schematic diagram of the gene cassettes in the
two plasmids used to cotransform maize callus
77

78. Psy orthologous gene in different sources
78

79. 79
Schematic diagram of the T-DNAs used to generate
golden rice-2

80. 80
Golden rice-2 lines

81. 81

82. 82

83. 83

84. • Golden rice fulfills the wishes of the GMO opposition
• It was not developed by industry, and industry does not benefit
from it
• It presents a sustainable, cost-free solution, not requiring other
resources
• It is given free of charge and it benefits the poor and
disadvantaged
• It does not create new dependencies on, or advantages for, rich
landowners
• It can be resown every year from saved seed
• It does not reduce agricultural or natural biodiversity
• It does not present any negative impact on the environment or
risk to human health
Overcoming the GMO opposition
84

85. 85

86. Objectives of investigation
1. Screening germplasm / traditional landraces for protein, iron
and zinc content.
2. Evaluating promising germplasm with higher protein,
iron and zinc in different soil types / environments
3. To estimate G X E interaction for protein, iron and zinc
along with yield.
86
Master’s Research
Genetic variation among traditional landraces of rice with
specific reference nutritional quality

87. Diversity in traditional varieties
87
Fe
(mg /Kg)
Zn
(mg / Kg)
Min 19.6 22
Max 46 67
Mean 30 43

88. Entry
No Name
Zinc
(mg/kg)
Iron
(mg/kg)
1 65 Bili Kalavi 46 42.3
2 14 Dodigya 44.4 32.1
3 66 Dodiga 43.9 37.3
4 61 Gopal Dodiga 43.7 44.7
5 4 Ambemohor -1 42.2 37.4
6 19 Hakkalkarisali 40.1 50.2
7 11 Chandibatta 39.5 53.5
8 5 Ambemohor-2 38.2 57.9
9 63 Budda 38.1 43.7
10 9 Belgaum Basmati 37 50.7
11 44 Kumud-1 34.7 55.2
12 121 Antara Sali 28.6 56.5
Top iron and zinc rich lines
88
Sl.No Name Zn(mg/ 100g)
1 Chittimutyalu 3.05
2 ADT-43 3.09
3 Udayagiri 3.01
4 Ratna 3.27
5 Jyothi 3.13
Sl No Name Fe
(mg/ 100g)
1 MSE-9 34.4
2 Kalanamak 34.0
3 Chittimutyalu 24.9
4 Udayagiri 30.1
5 Jyothi 19.8
Checks

89. 89

90. 90