Speaker : Hirdayesh Anuragi Course No.

: GP-692
Degree : Ph.D., G & PB. Date : 07.04.2018
Adm. No. : 2015A29D Time : 3:00 pm
 OUTLINE

INTRODUCTION

BREEDING FOR BIOFORTIFICATION

FUTURE OF BIOFORTIFICATION

IMPORTANT CASE STUDIES

SUMMARY AND CONCLUSIONS

FUTURE THRUSTS
INTRODUCTION

 Biofortification
 Malnutrition scenario
 Advantages of biofortification
 Limitations of biofortification
 Greek word “bios” means “life”
Latin word “fortificare” means “make strong”
MAKE LIFE STRONG!
 Deliberate increase in bio-availablity of essential vitamins and minerals in
staple crops, through agronomic practices, plant breeding or modern
biotechnology tools ,so as to improve the nutritional quality of the food for
better public health.
 Example: B-carotene bio-fortification in Rice, Zn bio-fortification in wheat,
amino acid in cereals and pulses etc.
 More feasible and cost-effective method of crop based nutrition to the
families which cannot afford/has no access to balanced diet.
 It combines agriculture + health + nutrition sectors working
together for food based nutritional security.
Biofortification
and/or
In the crop/field
Selective breeding Genetic modification (faster!)
-during plant growth

Fortification

In the factory
Food processing
-manually during processing of the crops

Supplementation

On the plate
Dietary supplements
-in case of acute deficiency

“Health comes from the farm, not the pharmacy”

 Lack of critical nutrients in the diet:
 Iron: 79% pre-school children & 56% women anemic
 Zinc: 50% people with diarrhea, poor grwoth & immunity
 Vitamin A: 375,000 children go blind each year

Impairs mental and physical development

WHO (2016)
 WHO (2016)

Severity of the most common micronutrient deficiencies

(vitamin A, iron and zinc)
 3 billion people : micronutrients deficiency
 2 billion population : Zinc deficiency
 1.6 billion population : Iron deficiency
 1 billion people : iodine deficient regions
 400 million people : vitamin A deficiency
 Malnutrition Deaths : > 20 million death/year

 Suffering with malnutrition problem

 >50% of women, 46% of children are underweight
and 38% are stunted
 Acc. to India state hunger index, all the states are
with serious to alarming indices with M.P. most alarmin
 Higher chances to serve the poor people: covers staple crops which are
major source of nutrient for the poorer families.
 One-time investment : only one time initial investment is needed
 Low recurrent costs : cost of seed production in maintenance breeding.
 Sustainable method of fortification: fortification passes from one
generation to next generation.
 Long term nutritional security: Fortification and supplementation are
temporary and short term approaches.
 Reach the most vulnerable people: in case of developing countries.
 Relies on the plant’s biosynthetic (Vit.) or physiological (mineral) capacity:
no effect of policy change or weak funding.
BREEDING FOR BIOFORTIFICATION

 Major targets for biofortification

 Important strategies of biofortification
 Achievements of biofortification
 Challenges of biofortification
Group Targeted crops Targeted nutrients
Rice b-carotene, Zinc and Iron
Wheat Zinc, Iron and Selenium
Cereals Maize Lysine, tryptophan and zinc
Pearl Millet Iron and zinc
Sorghum Iron
Beans Iron
Legumes Mungbean Iron and Zinc
Lentil Iron
Potato Iron

Root crops Cassava b-Carotene

Sweet potato b-carotene

Brassica Low Erucic acid & glucosinolate
Oil Seeds
Soybean Low trypsin inhibitors
 Most common conventionally bred crops: maize, beans, rice, wheat…

Most common micronutrients deficiencies: vitamin A, iron and zinc

www.harvestplus.org
 Consumer’s preference: Breeding targets are set based on demand of the
local consumers

 High yielding: Crop productivity must be maintained /enhanced to

guarantee farmer acceptance

 Effective: Micronutrient enrichment levels must have significant impact on

human health

 Stability: Enriched levels must be relatively stable

 Taste and cooking quality: Consumer acceptance has to be tested

Agronomic
 Immediate results
 Often used as a complement to other strategies Foliar spray
 Negative environmental impact Soil application
 Recurrent costs

Conventional plant breeding Introduction

 One-off cost Selection (Seed bank) +
Hybridization Molecular
 Long-development time breeding
Mutagenesis
 Requires genetic variation
Wide crosses
Genetic engineering
 In absence of genotypic variation
 One-off cost Expression of transgene
expression of trait
 Long-development time
RNAi for expression
 Low public acceptance
QPM Maize : For high Lys & Try content
 opaque-2
 opaque-6
 opaque-7
 opaque-11
 flory
 brittle
Barley: for high Lys and Try
 notch-1
 notch-2
 Hiproly
Commonly used techniques associated with each ‘omics’ discipline, and the importance of
integrating environment, plant tissue, and developmental stage data for crop
biofortification
 GM-derived variety of rice rich in beta-carotene (Vit-A) by Ingo Potrykus & Peter Beyer.
 Golden Rice 1: 1.6 μg/gm of carotenoids
 Golden Rice 2: 23 times more = 37 μg/g (Syngenta, 2005)
 Confirmed bioavailability: 72g/day is enough to avoid VAD
 Studies show no risk to human health IPP
 Presented in 2000 but still not grown
commercially in any country Geranylgeranyl diphosphate

Daffodil gene Phytoene synthase

Complete Vitamin A Pathway

Phytoene

Single bacterial gene; Phytoene desaturase

performs both functions
ξ-carotene desaturase

Lycopene
Daffodil gene Lycopene-beta-cyclase

 -carotene
(vitamin A precursor)
 Glycine

Aspartate Threonine Isoleucine

AK TS
DHDPS
DHDP Aspartate Semialdehyde Ortho-Phospho
(ASD) Homoserine
No. of steps CGS

Methionine
Lysine
SAMS
LKR
S-Adenosile Methionine
TCA cycle (SAM)

For high Lys content For high Met content For high Try content
Bacterial FB insensitive DHDPS expression of CGS FB regulated by Anthranilate synthase

expression of LKR expression of SAMS & TS Bacterial FB insensitive AS

expression of AK Bacterial FB insensitive CGS Expression of gene ‘OASA1D’

expression of AK
 Enhanced fertilizers
 Soil application
 Foliar application

Fe & Zn MA Breeding
Conventional breeding
Biofortification
 Selection  Candidate gene
 Mutation  QTLs

Reduce phytic acid Genetic manipulation

 Mutation  Transgenics for
 Gene silencing -High uptake
-High mobilization
-High storage
Genomics & molecular breeding approaches Potential transporters and their regulatory
for developing calcium biofortified genes during translocation of Ca2+ in
finger millet finger millet.
 Phytic acid,
 Polyphenols, Poor micro nutrients
Availability (Fe & Zn)  Selection
 Tannins
 Mutation

 Gene suppression (RNAi)

 Erucic acid Poor quality  Antisense RNA tech
 Glucosinolate Of Brassica Oil
Crop variety Important feature Released in/by
WB 02 zinc & iron rich variety 2017 (IIWBR)
Wheat
HPBW 01 iron & zinc rich variety 2017 (PAU)
BRRI Dhan 62 World’s 1st Zn-rich variety (22-27 ppm) 2013 (Bangladesh)
DRR Dhan 45 India’s 1st High Zn variety (24.0 ppm) 2016 (IIRR)
Rice
Chhattisgarh Zinc Rice-1 Zn biofortified rice variety (22-24 ppm) 2015 (IGAU)

CRRDhan 310 >10% protein content ICAR-NRRI

Dhanshakti 1st Fe biofortified crop cultivar (71 mg/kg) 2012 (ICRISAT)

Pearl ICMH 1201 High-yielding and high-Fe hybrid 2014 (ICRISAT)

millet HHB 299 Iron & zinc rich hybrid 2017 (CCS HAU)
AHB 1200 Iron rich hybrid 2017 (MKVP)
Sorghum ICSR 14001 50% higher iron and zinc 2014 (ICRISAT)
Lentil Pusa Ageti Masoor Iron rich variety 2017 (IARI)
Pusa Mustard 30 Low erucic acid variety 2013 (IARI)
Mustard
Pusa “00” Mustard 31 Low erucic acid & low glucosinolate variety 2016 (IARI)
ICAR, 2017
ICAR, 2017
ICAR, 2017
ICAR, 2017
ICAR, 2017
ICAR, 2017
ICAR, 2017
ICAR, 2017
ICAR, 2017
FUTURE OF BIOFORTIFICATION

 Future breeding strategies for biofortification

 Target traits for biofortification in future
 Transgenic tools: proven more effective in present and will be dominating in
future for addressing many biofortification breeding challenges.
 Development of Cost-effective markers: many crops have no or very limited
numbers of markers which are not sufficient to carry out suitable research.
 Incorporation of identified QTLs or candidate genes: transfer genes into elite
lines through conventional or modern breeding.
 Dissecting physiological mechanism: in order to manipulate particular stage to
get higher accumulation.
 Utilization of genome sequences for detecting candidate gene: future
strategies will be based on available genome sequences of crops.
 OMICS based breeding: Genomics, transcriptomics, proteomics &
metabolomics may give better understanding of trait behaviour.
 Calcium biofortification (finger millet)

 Ascorbic acid (Vit-C) biofortification

 Vitamin B biofortification (many crops): Vit. B2, B6, B12

 Folate biofortification (Pearl millet)

 Selenium biofortification (wheat): Essential for I metabolism

 Iodine biofortification (Tomato)

 Poor public acceptance: associated with undesirable features (Yellow color in GR)
 Issue of Transgenic: GMOs are not accepted due to social and political issues
 High Initial cost: Costly research in developing and under-developed countries
 Difficult maintenance: due to extremely precautionary regulations
 Lack of sufficient number of markers: slows down the marker based breeding
 Incomplete or no genome sequencing: in many crops
 Less profit margins in production and distribution: less attractive for the private
sectors
 Scarcity of government funds: worsen the biofortification scenario for the public
 Insufficient genetic variability for many trait: additional genetic variability need
to be created
IMPORTANT CASE STUDIES

 QTLs & candidate gene for Fe & Zn in rice

 Gene silencing for low glucosinolate in Brassica
 RNAi mediated silencing of phytic acid in Wheat
Mapping QTLs and candidate genes for iron and zinc concentrations in unpolished
rice of Madhukar × Swarna RILs

Madhukar × Swarna
Fe: 17.3 ppm Fe: 22.5 ppm
Zn: 53.7 ppm Zn: 27.2 ppm

High Fe 168 RILs

Fe: 0.2 ppm to 224 ppm
Zn: 0.4 ppm to 104 ppm

Phenotyping Genotyping
(Fe & Zn conc.) (519 SSRs)
High Zn

Linkage map & QTL analysis

(Mapmaker version 3.0) & (QTL Cartographer 2.5)

7 QTLs for Fe + 7QTLs for Zn

in silico Analysis Candidate genes

Anuradha et al., 2012

 Fe + Zn Fe + Zn

For high Fe + Zn in seeds : OsYSL1 Uptake,

Candidate genes For high Fe : OsMTP1 Transport,
For high Zn : OsARD2, OsIRT1, OsNAS1, OsNAS2 Accumulation
Anuradha et al., 2012
 Targeted silencing of BjMYB28 transcription factor gene directs development of low
glucosinolate lines in oilseed Brassica juncea

Seed Glucosinolates : From (80 to 120 µmol/g dry weight) to (<30 µmol/g DW).

Arabidopsis
BjMYB28-1
BjMYB28-2
AtMYB28 Full length homologs in Varuna (high glucosinolate)
BjMYB28-3

glucosinolate BjMYB28-4
Expression profile across tissues in glucosinolate
contrasting lines
RNAi (ihpRNAi) construct
Differential expression

BjMYB28-3 Potential candidate for development

of low glucosinolate lines

undetectable in the low glucosinolate canola quality line

suggesting its involvement towards controlling glucosinolate variation

Augustine et al. (2014)

 T-DNA construct of BjMYB28(RNAi)

down-regulation
S AS
BjMYB28-3

total seed glucosinolates in T1 seeds

(* represent transgenic lines with single-copy

integration of T-DNA)
Southern Blot analysis of BjMYB28(RNAi) Total glucosinolate in T1 and T2 seeds of
transgenic lines. single-copy transgenic events
Conclusion: BjMYB28 as major transcription factor gene controlling the aliphatic
glucosinolates accumulation in B. juncea. Augustine et al. (2014)
RNAi-Mediated Downregulation of Inositol Pentakisphosphate Kinase (IPK1) in Wheat Grains
Decreases Phytic Acid Levels and Increases Fe and Zn Accumulation

Phytic acid (PA): major anti-nutritional factor reduces the availability of Fe and Zn

Manipulate PA bio-synthetic pathway

functional wheat inositol pentakisphosphate kinase (TaIPK1)

4 non-segregating RNAi lines of Hexaploid wheat

S3-D-6-1 S6-K-3-3 S6-K-6-10 S16-D-9-5

T4 seeds
Homozygous transgenic RNAi lines

decreased transcript of TaIPK1 + 28–56% reduction of the PA

Silencing of IPK1

iron (Fe) and zinc (Zn) content

Aggarwal et al (2018)
 IPK1 in sense & anti-sense Selectable marker
Promotor
orientation
TaIPK1:pMCG161 RNAi construct monocot specific promoter

Transform into Hexalpoid wheat

No Template Control

plasmid pMCG161
Transgene Integration & Segregation Analysis

Control plant
Putative transgenic plants
Stable & Non segregating plants @ T4 stage

Gene Expression Analysis

(Quantitative RT-PCR)

Micronutrient &
Phosphorus (P) Analyses
(ICP-MS)

Morphological Analyses
Aggarwal et al (2018)
 C Transgenic C Transgenic
C Transgenic C Transgenic

C Transgenic

Conclusion: IPK1 is a promising candidate for Developing Low Phytate Crops &
Mineral accumulation in wheat grains.
Aggarwal et al (2018)
SUMMARY & CONCLUSIONS

 Summary and Conclusions

 Future thrusts
 Protein, Minerals and vitamin deficiencies affect over one-half of the world’s population
and despite of major international efforts, nutritional insecurity is still a serious global
concern today.
 Beside fortification and supplementation, biofortification is technically more feasible, cost
effective and most sustainable method of overcoming these deficiencies.
 Exploring the existing genetic variation, mutation, molecular and transgenic plant
breeding have provided the scope to increase the bioavailability of nutrients in staple food
crops.
 Advanced techniques of selection, mutation and gene silencing could help in minimizing
the anti-nutritional factors in order to enhance micronutrient bioavailability.
 Now need to get consumer acceptance, thereby increasing the intake of the target
nutrients. With the advent of good seed systems, the development of markets and
products, and demand creation, this can become a reality
 Genome sequencing and bioinformatics has lead to the identification of many potential
candidate genes which can be utilized in breeding many biofortified lines.
 Provide public health education on the benefits of eating biofortified foods hence will
increase the demand of breeding more biofortified crops.
 Develop better agricultural infrastructure for adoption of new biofortified varieties.
 Collaborative work of public and private agricultural research institutes with
biofortification as their prime breeding objective.
 Varietal release committees should make minimum levels of minerals and vitamins a
requirement for approval for release in addition to regular traits.
 Develop more genetic markers for effective MAS, map based gene cloning, whole genome
fingerprinting, association studies and population based analyses.
 Effective partnership of researchers and entrepreneurs is needed for quicker distribution
of biofortified variety.
 Physiological mechanisms can be manipulated for increasing accumulation of nutrients in
the edible portion of the plant.
 ‘Omics’ aspect of important traits can provide better strategy for biofortification.
 Transgenic if allowed, can offer a tremendous scope for biofortification of many crops.
- Fighting the hidden hunger
SYMBOL OF TRUST