Crop Bio-fortification is the idea of breeding crops to increase their nutritional value. Bio-fortification differs from ordinary fortification because it focuses on making plant foods more nutritious as the plants are growing, rather than having nutrients added to the foods when they are being processed. This is an improvement on ordinary fortification when it comes to providing nutrients for the rural poor, who rarely have access to commercially fortified foods.

1. 1

2. Crop Biofortification Through
Genetic Engineering
Minor Guide
Dr. G. U. Kulkarni
Associate Professor
Dept. of Genetics and Plant Breeding
COA, JAU, Junagadh
Major Guide
Dr. Rukam S. Tomar
Associate Research Scientist
Main Sugarcane Research Station
JAU, Kodinar
Ph.D. STUDENT
Rathod Balaji Ulhas
Reg. No. - 1010119025
Dept. of Biotechnology, JAU, Junagadh.
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3. Contents
• Introduction
• Importance of crop bio-fortification
• Why Need Biofortification
• Strategies for fortification
• 1) Dietary Diversification
• 2) Food Fortification
• 3) Agronomical Practices
• 4) Conventional Breeding
• Studies on Biofortification through genetic engineering for
micronutrient fortification (Fe,Zn,Vitamin-A,Vitamin-C ,E,Oil and
Amino acid)
• Conclusion
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4. 4

5. What is bio-fortification
• Bio-fortification:
• Greek word “bios” means “life” and Latin word “fortificare”
means “make strong”. Food fortification or enrichment is the process of
adding micronutrients (essential trace elements and vitamins) to food.
• Crop bio-fortification:
• Crop Bio-fortification is the idea of breeding crops to increase their
nutritional value.
• Bio-fortification differs from ordinary fortification because it focuses on
making plant foods more nutritious as the plants are growing, rather than
having nutrients added to the foods when they are being processed.
• This is an improvement on ordinary fortification when it comes to providing
nutrients for the rural poor, who rarely have access to commercially fortified
foods.
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6. 6

7. Importance of crop bio-fortification
• Bio-fortification for important crop plants through
biotechnological applications is a cost-effective and sustainable
solution for alleviating VAD, etc.,. Some points present here to
clearly identified role of crop bio fortification …….
• To overcome the malnutritions in human beings
• To increment of nutritional quality in daily diets
• To improvement of plant or crop quality, and increment of
variability in germplasm
www.zymoresearch.com
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8. Why Need Biofortification
 Hunger is the physical sensation of desiring food.
 The term “hidden hunger” has been used to describe the micronutrient
malnutrition inherent in human diets that are adequate in calories but lack
vitamins and/or mineral elements.
 India is one of the countries having problem of malnutrition
 More than 50% of women, 46% of children below 3 years are underweight
and 38% are stunted.
 As per India state hunger index, all the states are with serious to alarming
indices with M.P. most alarming.
Gillespie and haddad 2003,FAO 2006
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9. 9

10. Goal of Biofortification
10

11. STRATEGIES FOR
FORTIFICATION
Dietary Diversification
Food Fortification
Supplementation
Agronomical Practices
Conventional Breeding
Biotechnological Approaches
Genetic Engineering
Genome editing
Aluru et.al. 2008
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12. Dietary Diversification
 It involves the attempt to increase the consumption of grain,
vegetables and suitable fresh fruits.
 But this approach is more complex, involving a number of
factors including
-accessibility, affordability,
-bioavailability and
-change in dietary habits.
Nalubola 2002
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13. Food Fortification
• Addition of one or more essential nutrients to food, for correcting the
deficiency in the population or specific population groups
• For example,
-The iodination of salt and flour and
-Fortification with iron and vitamins in sugar
• Micronutrients fortification during food processing is difficult and most of
the micronutrients are lost during processing for food or feed
-More expensive
-Potentially less affordable by those at the greatest nutritional risk
(Cheng and Hardy 2003).
13

14. Supplementation
• Nutrients are added directly by means of syrup or pills to make up
for the deficiencies in food.
• Most appropriate- during pregnancy or in an acute food shortage
• but it has failed due to
-Lack of adequate
-Infrastructure
-Education in the developing countries.
-Expensive and
-Not a feasible option in poorer countries.
(Karunanandaa et al. 2005).
14

15. Agronomical Practices
• Nutrient management in the field is of high ecological and
economic importance.
• It is currently practical for only some nutrients such as, zinc,
selenium and iodine deficiency, which rises when nitrogen level
increases in soil.
• Not very effective-
-Iron
-Toxic nature elements
-Create negative impact on
the environment
(Zimmermman and Hurrel 2002).
15

16. Conventional Breeding
• Crop breeding for varieties with higher micronutrient content
• Most powerful tools in biofortification of crops, which can reach
the poor in rural areas.
• Use of biotechnological tools-MAS-improve the nutritional value
• Success Example-
-QPM maize (Quality Protein Maize),
-High carotene sweet potato and maize
Limitations-
-Narrow range of the germplasm
-Lack of micronutrients traits in wild species
-Hybridization barriers
-Not possible in vegetatively propagated crop Harjes et al. 2008
16

17. Tabulation of crops, nutrients, research status, and concerned publications on
biofortification through breeding.
17Continued

18. 18

19. Garg et al.2018
19

20. BIOFORTIFICATION
THROUGH GENETIC ENGINEERING
20

21. 21

22. Utilization of different genes for biofortification by Genetic Engineering means
22

23. Iron
incidence of
low-birth
weight babies
increases
Anaemia
Reduced
Work capacity
Reduced
capabilities,
mental
performance
Improper
physical
growth
RDA
for iron is 10 mg for children,
12-18 mg for men and 15-18
mg for women 23

24. soybean
(Kitano-shiki)
• Ferritin gene
Rice
(Kita-ake)
• .
Iron (Fe)
increases
• Threefold (22.5 ug/g
DW)
Iron fortification of rice seed by the soybean
ferritin gene
Goto et al.1999 Nature Biotechnology 24

25. Immunological tissue printing of a seed
from transgenic rice expressing soybean
ferritin cDNA.
Comparison of iron content in transgenic rice seeds expressing soybean ferritin
Metal concentration analysis
25

26. Phaseolus
ferritin
• Phaseolus
vulgaris
Ferritin gene
Rice grain
(Taipei)
Fe increases
• 22.07 μg Fe/g
DW
• Two fold
Genetic engineering approaches to improve the
bioavailability and the level of iron in rice grains
(Lucca et al. 2000). Theoretical Application
Genetics
Graphite furnace atomic absorption
spectroscopy
26

27. OsNAS genes
• Overexpression of
the OsNAS1,2,3
Gene
Rice(Nipponbore)
endosperm
Fe and Zn
• 19 μg/g DW
• Six fold increases
Constitutive Overexpression of the OsNAS Gene Family Reveals
Single-Gene Strategies for Effective Iron- and Zinc-Biofortification of
Rice Endosperm
Johnson et al.2011
ICP-OES
27

28. ZINC
Affect ability to
work and
longevity.
co-factor for
~300 enzymes
leads to
dwarfism and
hypogonadium
more than
1000
transcription
factors
RDA value for zinc is 10 mg for children, 15
mg for men and 12-15 mg for females
(Anonymous 2001). 28

29. Genes from
barley
•HvNAS1 gene
Rice
(Tsukinohikori)
Fe and Zn
•35 μg/g
Overexpression of the Barley Nicotianamine Synthase Gene HvNAS1
Increases Iron and Zinc Concentrations in Rice Grains.
Masuda et al. 2009. Rice
Metal concentration analysis
29

30. Over-expression of OsIRT1 leads to increased iron and zinc accumulations in
rice.
Genes
• OsIRT1
Rice
Zn and Fe
• More iron (112 and 121% in shoots
and roots, respectively) and zinc (136
and 135% in shoots and roots,
respectively)
Lee et al. 2009.
Plant, Cell and
Environment
Atomic absorption
spectrometry
30

31. Vitamin -
A
Diarrhea
diseases
and measles
Night
blindness
Growth
retardation
Damage of
mucous
membrane
Daily per capita availability of vitamin A has been estimated to 600 -1500 μg in
adults (Anonymous 2001).
VAD affects 100-400 million children worldwide and about 20,000-50,000 preschool
children become blind every year.
31

32. Generation of transgenic maize with enhanced provitamin
A content
Bacterial
genes
• crtB and crtI
Maize
(Hi-II)
Vitamin –A
increases
• 34 fold increases
(Aluru et al. 2008)
Journal of
Experimental
Botany
HPLC Analysis
32

33. Genes
(B73)
• maize psy1 gene encoding
phytoene synthase, bacterial
crtI CrtB or CrtI.
Wheat (EM12)
endosperm
Vitamin-A
increses
• 4.96 μg/g DW
• 10.8-fold
Expression of phytoene synthase1 and Carotene Desaturase crtI Genes Result
in an Increase in the Total Carotenoids Content in Transgenic Elite Wheat
(Triticum aestivum L.)
Cong et al.2009. J. Agric. Food Chem.
Semiquantitative RT-PCR
analysis
33

34. Coordinate expression of multiple bacterial carotenoid genes in canola
leading to altered carotenoid production
Bacterial Genes
N.Misawa
• crtB , crtI and crtY
Canola
(Quantum)
Vitamin-A
increase
• 857 μg/g fresh weight β-
carotene
• 50-fold
Ravanello et al. 2003.
Metabolic Engineering
HPLC analysis
34

35. oil
Develops an off
flavor
Protecting brain
cells
Formation of trans
fatty acid and that
is not good for
cardiovascular
Stability of oil
reduces
35

36. Improvement of rice (Oryza sativa L.) seed oil quality through
introduction of a soybean microsomal omega-3 fatty acid desaturase
gene
Soybean
(Bay)
• GmFAD3
Rice
(Reiho)
• r
Alpha –
linolenic acid
• 10 fold increases
(Anai et al. 2003) Plant Cell Rep
Fatty acid analysis
36

37. Pythium
irregulare(fungus)
• PiD6
Brassica juncea
GLA
• 40% of the total seed fatty acids
• Within triacylglycerols, GLA is
more abundant
High-Level Production of Linolenic Acid in Brassica juncea Using a omega 6
Desaturase from Pythium irregulare.
Hong et al. 2002. Plant Physiology
FA analysis
37

38. B.offucinalis
• cDNA of the B. officinalis 6-desaturase gene
Soybean
GLA and STA
• GLA levels ranged from 3.4 in the seed to
approximately 13%, while dual expression up to
28.7%,
• STA levels varied from just under 0.6 to 4.2%
Production of Delta-Linolenic Acid and Stearidonic Acid in Seeds of
Transgenic Soybean.
(Sato et al.
2004) Crop
Science
38

39. Vitamin C
Improves
cardiovascular
and immune
cell function
Role in
physiological
and metabolic
processes
Healthy
immune
system
Collagen,
Carnitine
RDA value is 40-45 mg for children and 45-
60 mg for men and women (Anonymous 2000). 39

40. Engineering increased vitamin C levels in plants by overexpression of
a D-galacturonic acid reductase.
Strawberry
((Fragaria ×
ananassa)
• GalUR
Arabidopsis
thaliana
vitamin C
increses
• Two- to threefold
(Agius et al. 2003) Nature Biotechnology
Ascorbate oxidase assay
40

41. Increasing vitamin C content of plants through enhanced ascorbate
recycling
wheat
• Dehydroascorbatereductase
DHAR
Maize
Ascorbic acid
levels
• 2- to 4-fold and significantly
increased
(chen et al. 2003) PNAS
Ascorbate oxidase assay
41

42. Vitamin -E
Prevent the
breakdown of
body tissue
Neurological
symptoms
Damage to
the retina of
eye
RDA value is 8-15 mg for men and
women (Anonymous 2000).
42

43. Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol
production and increased antioxidant content.
Barley
• HGGT(homogentisic acid
geranylgeranyl transferase)
Corn seeds
Vitamin –E
( tocotrienol
tocopherol )
• six-fold
These results provided insight into the genetic basis for tocotrienol biosynthesis in plants
and demonstrated the ability to enhance the antioxidant content of crops by introduction of
an enzyme that redirects metabolic flux
(Edgar et al. 2003) Nature Biotechnology
HPLC analysis
43

44. Arabidopsis Thaliana
Genes
• [At-VTE3; At-VTE42
• methyl-6-phytyl benzoquinol
methyltransferase genes
Soyabean
Vitamin-E
• 95% -tocopherol, a dramatic
change
• five fold increase
Engineering vitamin E content: from Arabidopsis mutant to
soy oil.
These findings demonstrated the utility of a gene identified in Arabidopsis to alter the
tocopherol composition of commercial seed oils, a result with both nutritional and food
quality implications (Van et al. 2003) Plant Cell
HPLC scan
44

45. Amino
acid
Providing energy
for your body
Almost every
body function
Formulation of
balanced diets
Modulates
neurological and
immunological
functions
Healing and
repair
45

46. The effects of enhanced methionine synthesis in potato
tubers.
Arabidopsis
• Cystathionine γ-synthase
(CgS)
Potato
(Desiree)
Methionine
• 2- to 6-fold increase in the
free methionine content
(Gabor et al. 2008) BMC Plant Biology
GC-MS
46

47. High lysine and high tryptophan transgenic maize resulting from the
reduction of both 19- and 22-kD a-zeins.
Maize
• Transforming maize with
constructs expressing chimeric
double-stranded RNA
Maize
Lysin and
Treptophan
• free amino acid level averaged 5-fold higher than
the levels observed in wild-type controls.
• 10 fold asparagines increases
• Lysine from 2438ppm to 4035ppm
• Tryptophan 598ppm to 877 ppm
(Huang et al. 2006) Plant Molecular Biology
47

48. Next-generation protein-rich potato expressing the seed protein gene
AmA1 is a result of proteome rebalancing in transgenic tuber.
Genes
• AmA1 (Amaranth
Albumin 1)
Potato 7
cultivar
Protein
(amino acid)
• 60% increase in total
protein content.
(Chakraborty et al. 2010) PNAS
2D Electrophoresis Analysis
Shows Increase in Protein
Content
48

49. Histopathological analysis of gut tissues
AmA1 Potato Tubers Are Nontoxic, Nonallergenic, and Safe for Consumption
Comparison of total protein content of wild-
type and AmA1-transgenic tubers
49

50. Tabulation of crops, nutrients, research status, and concerned publications
on biofortification by transgenic means
Continued 50

51. Continued
51

52. Continued
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53. Continued
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56. CONCLUSION
 Further research is needed to understand the mechanisms of
uptake and transport to redirect nutrients for efficient
accumulation in cereal seeds to be successful, biofortification
strategies must combine screening of germplasm for enhanced
micronutrient content with breeding and genetic engineering
strategies to improve the nutritional quality of cereals.
 Much basic research in this area is still required before future
applications can be successful. Because of its social impact, and
public concerns about the genetic engineering of food crops,
biofortification has also become an important topic in the socio-
economic literature. It is worth exploring as it has an immense
potential developing nutritious crops, which will serve as a
promising tool for improved human health.
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