This document discusses biofortification of rice through conventional breeding and genetic engineering techniques. It provides a brief history of rice hybridization research and development. It then discusses various methods used to biofortify rice with micronutrients like vitamin A, folate, iron, zinc, and lysine. Case studies on developing golden rice enriched with beta-carotene and rice enriched with soy glycinin protein are described. Advantages of biofortified rice in reducing micronutrient deficiencies and disadvantages related to costs and access are noted.

1. Malnutrition?????
-micronutrient deficiency….
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2. BIOFORTIFICATION IN RICE
VIPIN MOHAN
2011-09-112
College of agriculture
Vellayani, tvm
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3. Rice–thegrain
Second in production & consumption
Staple food
90% of world’s rice is produced and consumed in Asia
Wide adaptability
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4. THE rice grain
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5. History
- China initiated research on hybrid1964
- China identified male sterility1970
- 3 line breeding was established1973
- First commercial three-line rice hybrid released
in China1976
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-Photo-Thermo-Sensitive Genic male sterility
(PTGMS) rice genetics and application was
confirmed
1981
- Yield superiority of rice hybrids in the tropics
confirmed (IRRI)
1982
- India and Vietnam started hybrid rice
programs with IRRI
1990s
- First commercial two-line rice hybrid
released in China
1994
- Commercial rice hybrids released in India,
Philippines and Vietnam
1994 -
1998

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8. Biofortification
The development of nutrient dense staple
crops using the best traditional breeding
practices and modern biotechnology
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9. Selective
breeding
Genetic
engineering
High-nutrient
varieties
High-yielding
varieties
Various
techniques
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10. F2
P2
F1
P1
x
Large populations consisting of
thousands of plants
DonorRecipient
Conventional plant breeding
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11. Selective breeding
Through hybridization
Marker assisted selection
Molecular marker indicates the presence or absence of gene at an early stage
Success rate is very low
Long period of time required
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13. Transgenic rice
• First transgenic crop - mid 80’s
• Many crops have already been under cultivation over last
10 years
• Rice - prone to many pest, disease and unpredictable
climate change
– genetic engineering is utilized
• Protocols for rice transformation have been developed
which allow transfer of foreign genes from diverse
biological systems into rice
Prabhu et al (2011)
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Protoplast based
Biolistic method
Agrobacterium-mediated

16. Micronutrients Biofortified into Rice
• Vitamin A (Retinol)
Vitamin B9 (Folic acid/Folate)
Iron
Zinc
Glycinin
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18. Importance
•Give proper vision
•Hematopoiesis
•Immune response
•Skeletal tissue growth
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19. WHO estimates:
 350000 children are becoming blind due to Vitamin A
deficiency yearly
 670000 people die annually
 200000 children with Vitamin A deficiency syndrome (VDS)
 19 million pregnant women
www.irri.org/goldenrice
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20. Ingo Potrykus and Peter Beyer initiated to develop
golden rice (1999)
Fortified with beta-carotene
Beta-carotene gives the distinctive tint
Antonio Alfonso (2013)
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21. In golden rice
• Phytoene synthase - from daffodil (Narcissus pseudonarcissus)
• Lycopene B-cylase - from daffodil (Narcissus pseudonarcissus)
Phytoene desaturase - from the soil bacterium (Erwinia uredovora)
(Portrykus et al., 1999)
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22. The Golden Rice Solution
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
 -carotene
(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
Daffodil gene
Single bacterial gene
Daffodil gene
-Carotene Pathway Genes Added
Vitamin A
Pathway
is complete
and functional
Golden
Rice
(isopentenyl-diphosphate)
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Golden Rice Generations

25. • Daffodils gene is replaced by Maize gene – New
varieties
• " " - 23 times more provitamin A
• Original golden rice - 1.6 micrograms/g rice
• New strain - 37 micrograms
• www.irri.org/goldenrice
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26. Let’s dream for an effectual golden rice that could
eradicate Vitamin A deficiency and easily accessible to
common farmers
If Vitamin A becomes adequate, overall child mortality
can be reduced by 23-24%
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28. Folate (vit B9) Biofortification in rice
Tripartite molecules
Methylation and in DNA biosynthesis
Targeted expression of GTPCH1 and ADCS to increase folic acid
biosynthesis in seeds
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(Storozhenko et al. 2007)
Case study
Folate fortification of rice by metabolic engineering

31. Transformed plants with ADCS has 49 fold increases in levels
of PABA
Transformed plants with GTPCH1 has no increase in levels of
pterin.
But GA transgenic lines give 6 - 38.3 nmol/g (15-100 fold)
folate accumulation.
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32. Folate dietary deficiency results in:
Neural tube defects  E.g.: Spina bifida
Cardio vascular diseases
Different forms of dementia
Megaloblastomic anaemia
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34. Iron (Fe) biofortification of rice
Iron is a redox active
Most widespread micronutrient deficiency worldwide.
Metabolic functions
Blood production
Component of enzymes involved in synthesis of collagen and some
neurotransmitters
Providing a transport medium for electrons within the cells -cytochromes
Structural component of haemoglobin
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35. In the Fe Rice Biofortification process
Three genes were introduced into the japonica rice variety:
a) Ferritin - Enhances iron storage in grains
b) Nicotinamine synthase – Produces nicotinamine which
chelates iron temporarily facilitating its transport in plants
c) Phytase-degrades phytate
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37. Importance of Zn
* Critical in tissue growth
* Wound healing
* Immune system function
* Bone mineralization
* Proper thyroid function
* Coagnitive functions
* Fetal growth and sperm production
* Essential for cell division, synthesis of DNA and proteins
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38. Zn deficiency leads to
Impairment of physical growth,immune system and
learning ability
Fetal brain cell diseases and affects mental development in
pregnant mothers
Hindered normal growth and development in children
Increased risk of infections , DNA damage and cancer
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39. In the Zn biofortification process
Three Nicotinamine synthase genes of the OsNAS family were
introduced
Specific over expression of OsNAS resulted in significant increase in
NA concentration and Zn
OsNAS 2 activation has 20 fold more Nicotinamine and 2.7 fold Zn in
polished rice grains
OsNAS 3 activation was reported reverse signs of iron deficiency
when fed anaemic mice
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41. Glycinin biofortification in rice
Lysine rich globulin protein
Rice seed endosperm – lysine deficient
Glycinin accounts for more than 20% seed dry weight
Five glycinin genes: GY1, GY2, GY3, GY4 & GY5
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42. Effects of lysine deficiency include:
Supplements or food sources should provide 12 mg/kg
body weight of lysine/day
Hair loss or poor growth
Excessive fatigue and mood changes
Loss of appetite
Anaemia
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43. Molecular Approaches
 Modifying the higher protein sequence of a major crop protein to
contain higher content of desired EAA
 Producing a synthetic protein rich in target EAA
 Expressing a heterologous protein with high content of desired EAA
 Manipulating the expression of homologous protein for desired EAA
 By increasing the pool of a specific free EAA through metabolic
engineering
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44. Case study
Accumulation of soybean Glycinin and its assembly with the Glutelins
in rice (Katsube et al. 1999)
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Promoter – GluB - 1
Vector - Agrobacterium tumefaciens
5 % total glycinin protein was obtained

45. Advantages of rice biofortification
Increase in nutritional value
Reduced adult and child micronutrient caused mortality
Reduced dietary deficiency diseases.
Healthier populations with strong and quick immune responses to
infections
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46. Disadvantages of rice biofortification
High production costs
Loss of wild-type rice varieties
Low substantial equivalence
Poor rural population have limited access and resources to purchase
biofortified rice
Genetic engineering methods used may compromise immunity in
humans
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47. but….
• Growth in rice production :
• 2.5 – 3.0 % during 1970 – 80
• 1.5 % during 90’s
• Population growth :
• by 2025 - 8 billion
• Required rice production :
• 40 % more
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(Datta S. K. 2013)

48. References
• Datta S. K. 2013. Rice Improvement through Application of Biotechnological Tools 45p.
• Katsube T., Kurisaka N., Ogawa M., Maruyama N., Ohtsuka R., Utsumi S., and Takaiwa F.(1999).
Accumulation of Soybean Glycinin and Its Assembly with the Glutelins in Rice. J. Pl. Physio. 120:
1063-1073.
• Sun, S.S.M., Liu, Q.Q. (2004). Transgenic approaches to improve the nutritional quality of plant
proteins. In Vitro Cell Development. J. Biol. pl. 40: 155-162.
• Babu, R. V. (2013). Importance and advantages of rice biofortification with iron and zinc. J. SAT
Agric. Res. 11.
• Storozhenko S., Brouwer V. D., Volckaert M., Navarrete O., Blancquaert D., Zhang G. F., Lambert
W., and Straeten D. V. D.(2007). Folate fortification of rice by metabolic engineering. J. Nat. Biotech.
25(11):1277-1279.
• www.irri.org/goldenrice
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