Bio fortification for Enhanced Nutrition in Rice by Conventional and Molecular approaches

Bio fortification for enhanced nutrition in rice:Conventional and molecular approaches

Presented By, SATHISHA T N

Depart. of Genetics and Plant Breeding, UAS Dharwad

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Content

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

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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

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 Mediterranean

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, 20086

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

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Biofortification

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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

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CONVENTIONAL APPROACHES

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Nutrient content of rice

Source: FAO Rice Factsheet, 2004

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Fe and Zn distribution in rice grain

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Screening of germplasm for high iron and zinc content

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)

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Fe and Zn contents in the four rice varietal groups

Lee et al.,(2008)

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Iron and zinc content of some selected varieties

Gregorio et al., 2000

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Gregorio et al., 2000

Iron content of selected variety after polishing

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Effect of milling on iron concentration in rice

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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

22DRR Hydrabad

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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

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Fe and Zn content in popular cultivar

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)

EMBRAPATraditional variety

(12.6-42.2 ppm)

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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

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High iron rice

IR 72 x Zawa Bonday

IR 68144-3B-2-2-321 ppm

• Early mature • Tolerance to tungro virus• No seed dormancy • Excellent seedling vigour• 10% below yield than IR-72

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Fe and Zn content in Popular rice hybrids in Indiacharecter Protien Iron (mg

/100g)Zinc

(mg/100g)

1. DRRH-2A 9.79 12.58 4.382. PA 6129 8.86 8.95 4.263. Sahyadri-2 9.45 7.91 3.874. Sahyadri-4 8.8 10.48 4.35. Pusa RH-10 8.26 4.52 3.756. Indirasona 7.64 7.85 3.877. GK 5003 7.39 8.15 3.848. PSD-3 5.74 7.29 3.999. Sahyadri-3 7.67 4.13 3.0710. PA 6201 7.86 7.76 4.5911. HSD-1 7.84 6.76 3.1712. PA 6444 7.15 6.11 3.7213. Suruchi 7.93 6.87 3.3514. JKRH-2000 7.42 7.15 3.5515. US 312 7.47 6.62 3.7616. CORH-3 8.1 7.37 3.5317. KRH-2 6.49 4.39 3.4318. Sahyadri-1 7.74 5.9 3.3319. PHB-71 8.48 2.89 3.8120. CRHR-5 8.59 2.96 3.1521. CRHR-7 8.3 6.93 3.62Mean 7.95 6.84 3.73

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Correlation among iron and zinc content in rice grain

(Nagesh et al ., 2012)

Phenotypic and Genotypic Correlation coefficient for Iron and Zinc in 48 rice hybrids

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Estimation of heterosis for grain iron and zinc content

6 lines x 8 testers 48 hybrids

SH1- SwarnaSH 2- Chitimutayalu

Nagesh et al., 2012

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G x E interaction for iron and zinc

Grain zinc content (ppm) of promising rice genotypes grown in 3 environments

Virk et al. (2006b,2007)34

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Fe concentration in 10 genotypes in 4 location Wet and Dry season

Dry season

Wet season

(Suwarto and Nasrullah 2011)

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Genotype x Environment interaction on iron concentration in rice

Suwarto and Nasrullah 2011

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Mutation breeding

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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)

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Frequency distribution of Fe and Zn in mutants

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

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Micronutrient Mean (mg/Kg) Range (mg/Kg)

Fe 4.02 0.11-28.10

Zn 15.6 8.37-28.95MN 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

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Grain yields of selected mutants grown in different crop seasons

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MOLECULAR APPROACHES TO IMPROVE GRAIN QUALITY IN RICE

Molecular Mapping of High Iron and Zinc Rich Regions in Rice (Oryza sativa L.) Grains Using Microsatellite

Markers

Nagesh et al., 2013

Swarna × Madhukar

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178 F 2 population

72 SSR marker

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Parental polymorphism survey between Swarna and Madhukar

SC-120, SC-128

SC-129(33.56 %)

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Selective genotyping for rapid identification of regions associated with iron and zinc content

Swarna × Madhukar

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Segregation pattern of SSR primers

Legend: M-100bp DNA marker, P1 – Swarna, P2 – Madhukar

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Single marker analysis for iron and zinc with SC120, SC128 and SC129

Swarna × Madhukar

178 F2 population

SC120, SC128 and SC129 markers screened

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Identification of QTLs for mineral contents in rice grain

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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)

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Micronutrient content in parent and introgression lines grown during 2 season

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Phenotypic variance for micronutrient in introgression lines (ILs)

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QTLs identified for micronutrient traits in introgression lines

Fe

Fe

Zn

Zn

Zn

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Mapping QTLs for iron and zinc content in rice

168 RILS

101 SSR + 9 GENE SPECIFIC MARKERS

Anuradha et al. 2012

Madhukar × Swarna

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

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Distribution of Fe and Zn concentration in 168 RILs

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(14)

(4)RM 535

Markers linked to Fe and Zn in Madhukar × Swarna RILs

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QTL mapping for Fe and Zn concentrationComposite interval mapping

13 QTLs

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Identification of QTL Fe and Zn

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Putative candidate genes for QTLs governing Fe and Zn

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Bio fortification of rice by transgenic approaches

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Fe accumulation in rice seeds1. 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

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Soybean ferritin chimeric gene in the pGPTV-bar/ Fer vector

PCR analysis showing amplification of 0.78 kb size band of ferritin gene

Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene

Vasconcelos et al.2003

Lane 1, non-transformed control plant2, plasmid control3, blank;4, 5, 7, 8 and 9, five individual transformants6, negative regenerated plant. M, 1 kb DNA molecular marker.

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Iron detection in transverse sections of rice endosperm

Non transgenic IR68144

TransgenicIR68144

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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

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Iron biofortification by introduction of multiple genes by transgenic approaches

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Gene cassette introduced into rice to produce the Fer-NAS-YSL2 lines

Japonica rice cultivar Tsukinohikari

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Detection of transgene insertion in the transgenic lines by PCR

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Ferritin accumulation in T2 brown seeds Western blot analysis.

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

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Fe concentration of T2 polished seeds

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FeZn

Cd

Metal concentration in T3 polished seeds obtained from the paddy field

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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

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Plant height and yield data transgenic lines in field experiment

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The problem with rice:• Does not express

phytoene synthase in endosperm

• Does not express phytoene desaturase or z-carotene desaturase

Golden Rice

Development of golden rice

Fig. 1

Rice grain Golden ricewhite rice

Fig. 2

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

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Psy orthologous gene in different sources

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

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Golden rice-2 lines

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• 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

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Objectives of investigation1. 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.

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Master’s Research

Genetic variation among traditional landraces of rice with specific reference nutritional quality

Diversity in traditional varieties

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Fe (mg /Kg)

Zn (mg / Kg)

Min 19.6 22Max 46 67Mean 30 43

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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

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

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Focus on “better” food,not only “more” food

The expected rewards are high…

Thank You90