This presentation starts by introducing what biofortification is and sliding on to types of biofortification followed by two scientific case studies.

1. Alleviation of Malnutrition through
Breeding and Bio-engineering
approaches
MANDEEP SINGH
11047

3. Hidden Hunger
Impairs mental and physical development of
children and adolescents:
 Iron
 Zinc
 Vitamin A
 Protein
Lack of critical micronutrients in the diet:
 Lower IQ
 Reduced productivity
 Increased risk of illness
 Stunting
 Blindness

4. Hidden Hunger – Distribution
Severity of the most common micronutrient deficiencies
(vitamin A, iron and zinc)
http://www.harvestplus.org
Alarmingly
High

5. Vitamin and Mineral Nutrition
Information System (VMNIS)
 Globally 20 percent of maternal deaths are attributed to anemia
 58 and 51 percent of children under 5 years old were anemic in 2016 in
Sub-Saharan Africa and South Asia, respectively
 5-20 percent prevalence of night blindness in South East Asia
 Nearly 250 million preschool children are Vitamin A deficient
 Zinc deficiency is variable and ranges from 15-50 percent

7. Biofortification
 Greek word “bios” means “life”
 Latin word “fortificare” means “make strong”
MAKE LIFE STRONG!

8.  Biofortification is the process by which the
nutritional quality of food crops is improved through
agronomic practices, conventional plant breeding,
modern biotechnology
- World Health Organization

9. Success Determined By
 Biofortified crop must be high yielding and profitable to farmer
 Crop must be acceptable to both farmers and consumers
 Amount of staple food actually consumed on daily basis by age and
gender
 Nutrient losses in post harvest phase
 Bioavailability of the nutrient

10. • Iron-biofortification of rice, beans, sweet potato, cassava and legumes;
• Zinc-biofortification of wheat, rice, beans, sweet potato and maize;
• Provitamin A carotenoid-biofortification of sweet potato, maize and
cassava;
• Amino Acid and Protein-biofortification of sourghum and cassava.

11. Appraches for Biofortification
 Agronomic
 Conventional Breeding
 Genetic Modification

12. Agronomic Biofortification
 Cakmak (2008)- Increased uptake and accumulation of Zn in whole wheat by raising zinc
fertilizers addition to soil
 White et al., 2011- increased Zn supply to Peas increases concentration of bioavailable Zn
in Pea seeds

13. Plant Growth Promoting Rhizobacteria (PGPR)
 Supplementary measure
 Tricoderma asperellum, Methylobacterium oryzae, Pseudomonas putida,
Pseudomonas fluroscens, Azospirillum lipoferum
 Cyanobacteria- Anabaena spp., Calothrix spp.
 Sharma et al., 2013 – Enhanced Fe content in Rice
Limitation
 mostly applicable only for increasing Fe content

14. Limitations
 Expensive
 Require regular sprays
 Bulkiness of fertilisers hinders in transportation
 Vitamin A, proteins and Fe fortification is not possible by agronomic
means
 Adverse environmental effect

15. Conventional Breeding
P1 × P2
I I I I I I I I I I I I I I I I
I
I I I I I I I I I I I I I
• Most trusted approach
• Genetic variations available in the crop
gene pool is utilized
• Generally Prebreeding is required
• Pedigree or Backcross method

16. Genotypic variation for Fe
Crop Mean (mg/kg) Range (mg/kg) Samples
Rice 12.2 6.3-24.4 1138
Pearl millet 45.5 30.1-75.7 120
Maize 23.8 9.6-63.2 1814
Cassava 9.6 7.7-12.6 26
Bean 55 34-89 1000
Crop Mean (mg/kg) Range (mg/kg) Samples
Rice 25.4 14-58 1138
Pearl millet 43.9 24.5-64.8 120
Maize 18.5 13-24 1814
Cassava 6.4 4.4-8.6 26
Bean 35 21-54 1050
Genotypic variation for Zn
(White and Broadlay, 2005)

17. Plant Breeding Process
Lines with characteristics of interest for ……………….
Farmer
High yields
Resistance to pests
Stress tolerant
Consumer
Appearance
Taste
Cooking time
Nutritionist
High nutritional
Value
Bioavailability

18. Ideally:
With desirable
characteristics for the
farmer, consumer and
nutritionist
Plant Breeding Process
X X

19. Adapted from: Global Food Security 12 (2017)

20. Success List
 Orange fleshed sweet potato
• β-carotene enriched
• Released in Uganda
• Developed by HarvestPlus
• Two varieties- Ejumula and Kakamega (2004)
• World food prize, 2016
 Quality Protein Maize
• Increased tryptophan and lysine
• ‘opaque-2’ and ‘floury-2’ mutants
• Released in several countries
• In India- Shakti, Shaktimaan 1, Vivek QPM-9, HQPM-7
• World food prize, 2000

21.  Iron Beans
• Fe enriched Phaseolus vulgaris
• Developed by International Centre for Tropical Agriculture (CIAT)
• Vareities- NUA35, NUA56
• Released in Latin America and Central Africa
 Pearlmillet
• Fe enriched Pearlmillet
• Developed by HarvestPlus parterned with Nirmal seeds
• Released in India
• Vareities- Dhanshakti, ICMH 1202 (ICRISAT)
 Zinc Rice
• ‘Brri dhan-62’ – world’s first zinc rice
• Developed by Bangladesh Rice Research Institute

22. Limitations
 Slow and tedious
 Linkage drag and undesirable characters
 Lack of diversity for the target trait
 Generally most of the sources are unadapted
 Polygenic
 Low heritability

23. Breeding if possible, Modifying if
Necessary

24. Genetic Modification
(Maximum researched and minimum utilised)
 When there is limited or no genetic variation in nutrient content is
present
 Unlimited genepool
 No linkage drag
 Simultaneous incorporation of genes involved in
1. Micronutrient concentration
2. Bioavailability
3. Reduction in antinutrients

25. Success List
 Golden rice
• β-carotene enriched
• Ingo Potrykus and Peter Beyer
• Taipei 309- Japonica rice line was used
• Rice endosperm specific promoters
• 1.6 mg/g of rice
Geranylgeranyl diphosphate
phytoene
lycopene
α-carotene β-carotene
psy
crt1
lcyGolden Rice 2- psy gene from maize
37mg/g of rice

26.  Cassava
• β-carotene, Fe and protein enriched
• Developed by BioCassava Plus (BC+) project funded by
Bill and Melinda Gates Foundation
• FEA1 gene from Chlamydomonas reinhardtii for iron
uptake
• β-carotene- 30 fold more than normal
• Fe content- 30 mg/g
Glyceraldehyde-3-phosphate
GGDP
Phytoene
β-carotene
DXS
crtB
Phase II under Dr. Martin Fragene, director BioCassava
Plus at Donald Danforth Centre

27. Garg et al., 2018

28. Utilization of different genes for biofortification by transgenic means
Garg et al., 2018

29. Representation of
reported
biofortified crops
by Transgenic,
Agronomic, and
Breeding means
Frontiers in Nutrition | www.frontiersin.org

30. Case study (Conventional breeding)

31. Materials
 Parental inbreds viz., HKI161, HKI163, HKI193-1,and HKI193-2 of four QPM
hybrids, HQPM1 (HKI193-1× HKI163), HQPM4 (HKI193-2 × HKI161),
HQPM5 (HKI163 × HKI161) and HQPM7 (HKI193-1 × HKI161)
 Donors- HP704-22 and HP704-23
 Recipient inbreds- o2o2 crtRB1+crtRB1+ lcyE+lcyE+
 Donor parents- O2O2 crtRB1crtRB1 lcyElcyE
 Four crosses were made- cross-I (HKI161 × HP704-23), cross-II (HKI163 ×
HP704-22), cross-III (HKI193-1 × HP704- 23), cross-IV (HKI193-2 × HP704-
22)

32. MABB
MARKERS USED
o2 - phi057
crtRB1 - 3′TE InDel
lcyE - 5′TE InDel

33. Results
Morphological characterization of improved lines with their
respective recurrent parents

34. Biochemical analysis of introgressed inbreds

35. Provitamin A concentration in original and reconstituted hybrids
Reconstituted hybrids
were at par in case of
lysine and tryptophan
content but having
higher amount of
provitamin A content
(9.25 to 12.88μg/g)

36. Case study (Transgenic approach)

37. Pathway
 Phytosiderophores - Nicotianamine (NA) (Internal) and 2′-deoxymugenic
acid (DMA) (External)
 Metal transpoters – 1. Fe2+ : OsYSL15
2. Fe2+ and Zn2+ : OsIRT1
3. Zn2+ : OsZIP1
S-adenosylmethionine (SAM)
Nicotianamine (NA)
2′-deoxymugenic acid (DMA)
Nicotianamine synthase (NAS)
Nicotianamine aminotransferase (NAAT)

38. Materials and Methods
 Transgenic plants development expressing both OsNAS1 and HvNAATB
 T2
seeds were grown on medium containing 100 μM FeCl3,200 μM FeCl3
and 300 μM FeCl3 separately along with wild type as control
 T3
seeds were grown on 100 μM FeCl3 and later transferred to 10 μM
CdCl2
 Inductively coupled plasma mass spectrometry (ICP-MS) – for checking
metal content in plant
 HPLC-electrospray ionization (ESI)-time of flight (TOF)-MS – for checking
NA and DMA concentration
 Quantitative RT PCR – for checking endogenous gene expression of
transformed genes

39. Results
NA and DMA levels in roots, leaves and seeds

40. Fe concentration

41. Zn concentration

42. Modulation of metal transporters
(Homeostatic regulation of metal ions)

43. Cadmium concentration

44. Findings in a nutshell
 Increased levels of NA and DMA is positively correlated with levels of Fe and
Zn in rice endosperm
 Fe levels achieved : 22-57 mg/g of dry weight
(1.4-3.7 mg/g in wild type)
 Zn levels achieved : 22-78 mg/g of dry weight
(1.2-4.2 mg/g in wild type)
 Homeostatic regulation is achieved by modulating concentration of metal
transporters to avoid its toxicity
 Cadmium (toxic metal) levels are significantly reduced due to decrease in
cadmium carrier OsLCT1

45. How do we know that biofortification works?
Q#3: Does consumption of biofortified foods improve
micronutrient status of women and children?
Efficacy studies Effectiveness studies
Q#2: Are the micronutrients in the biofortified food crops
bioavailable (absorbed and utilized) when consumed by the
target population group(s)?
Anti-nutrient analysis, In vitro &
animal bioavailability models
Bioavailability studies in
humans
Q#1:Does the biofortified crop contribute >30% EAR* of
provitamin A, iron or zinc to target population?
Post harvest nutrient retention
studies
Background food processing
and dietary intake studies
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46. Who promotes biofotification?
India
Biofortification
Program

47. Conclusion
Although the knowledge gaps and tasks ahead
may seem daunting, investment in
biofortification is a cost-effective approach to
ensure a more nourishing future

48. Thank You
“Health comes from the farm, not the pharmacy”