V G SHOBHANA Dr. N SENTHIL KALPANA K. Dr. P NAGARAJAN Dr. M RAVEENDRAN Dr. P BALASUBRAMANIAN

1. BIOFORTIFICATION IN MAIZE • V G SHOBHANA • Dr. N SENTHIL • KALPANA K. • Dr. P NAGARAJAN • Dr. M RAVEENDRAN • Dr. P BALASUBRAMANIAN CENTRE FOR PLANT MOLECULAR BIOLOGY TAMIL NADU AGRICULTURAL UNIVERSITY COIMBATORE – 641 003

2. BIOFORTIFICATION Fortification x Biofortification Methods: • Selective Breeding • Genetic modification The Big Difference!! • Developing world – • Vitamin A, Zinc, Iodine and Iron • Developed world – • Selenium, prostrate cancer The Orange Ribbon Symbol of Malnutrition

3. Importance • Two billion people - currently micronutrient malnourished - increased morbidity and mortality rates, lower worker productivity and high healthcare costs. • Nutritional deficiencies (iron, zinc, vitamin A) - almost two-thirds of the childhood death worldwide. • Major food crops can be enriched(‘biofortified’) with micronutrients using plant breeding and transgenic strategies. • Micronutrient enrichment traits exist within their genomes. • Micronutrient element enrichment of seeds can increase crop yields when sowed to micronutrient-poor soils, assuring their adoption by farmers.

4. The Golden Rice Story

5. Percentage of population affected by under-nutrition by country, according to United Nations statistics

7. Phytic acid (Phytin or Phytate) BIOSYNTHETIC PATHWAY • Myo-inositol-1,2,3,4,5,6-hexakisphosphate or Ins P6. • Is the most abundantmyo-inositol phosphate in plant cells, but its biosynthesis is poorly understood. • Also uncertain is the role of myo-inositol as a precursor of phytic acid biosynthesis. • MW :660.03Formula :C6H18O24P6

8. PHYTIC ACID • Myo-inositol 1,2,3,4,5,6-hexakisphosphate, is abundant component of plant seeds. • Deposited in protein bodies as a mixed salt of mineral cations such as K+, Mg2+, Ca2+, Zn2+, and Fe 3+ (50% to 80% of the phosphorus in seeds). • Phytic acid serves as a major storage form for myo-inositol, phosphorus, and mineral cations for use during seedling growth. Other known role of phytic acid - control of inorganic phosphate (Pi) levels in both developing seeds and seedlings. • In maize kernels, nearly 90% is accumulated in embryo and 10% in aleurone layers (also in rice and barley). • Maize endosperm contains only trace amount of phytic acid.

9. Importance • Monogastric animals digest phytic acid poorly. • Undigested phytic acid is eliminated and is a leading phosphorus pollution source. • Low-phytic acid grain and legume in feed - reduces phosphorus pollution to environment and reduce amount of phosphorus supplementation required in animal feeds (Ertl et al., 1998). • Such grain would also offer more available Fe and Zn for human nutrition (Mendoza et al., 1998).

10. Variability of phytate P in crop plants

11. Biosynthetic pathways of phytate in plants Two types of pathway * Lipid -dependent (hydrolysis of PI(4,5)P2 by phospholipase) * Lipid -independent (sequential phosphorylation of I(3)P or inositol) Paulik et al.,(2005)

12. Analysis of biochemical characters • Phytic acid – Wheeler and Ferrel, 1971 430 genotypes were screened for their phytate content • Low and high maize inbreds were identified • Crossing of low inbred with high inbreds evolved in 50 hybrids • Iron and Zinc – major minerals – screened by Atomic Absorption Spectrophotometer

13. The following strategies were adopted to reduce the phytate • Plants can be transformed for increased phytase production in the seeds. • The transgenic approach will, in the long run, prove to be most versatile and cost-effective. • Mutation breeding for impaired phytic acid biosynthesis has proved to be useful in maize, barley and rice ( Raboy, 2000). • Available low phytate mutant lines can be crossed with locally adopted cultivars and will result in low phytate maize with desired agronomic backgrounds.

14. Genetics • Maize has 10 chromosomes (n=10). • The combined length of the chromosomes is 1500 cM. • "Chromosomal knobs". They are highly repetitive heterochromatic domains that stain darkly. • Barbara McClintock used these knob markers to prove her transposon theory of "jumping genes". • Composition • Figures in grams (g) or milligrams (mg) per 100g of food. • Minerals • Calcium: 9mg • Phosphorus: 290mg • Iron: 2.5mg • Seed (Fresh weight) 361 Calories per 100g • Water: 10.6% Protein: 9.4g • Fat: 4.3g • Carbohydrate: 74.4g • Fiber: 1.8g • Ash: 1.3g • Vitamins • Vit A: 140mg • Thiamine (B1): 0.43mg • Riboflavin (B2): 0.1mg • Niacin: 1.9mg

15. Mutation work - Dr. V Raboy, USDA • Pollen treated M2 progenies - developed by Dr. Raboy – yielded two maize mutants. • lpa 1 and lpa 2 with 60% reduction in the seed phytate levels were produced. • These mutants were widely used in most of the breeding programmes in US. • lpa 1 – 1.1 (mg/g) phytate P in 4.7 (mg/g) total P • lpa 1 – 2.6 (mg/g) phytate P in 4.6 (mg/g) total P • Indian corns have 2.0 – 2.5 (mg/g) phytate P in 4.0 - 4.5 (mg/g) of total P.

16. INBREDS SELECTED FOR MUTATION BASED ON PHYTIC ACID CONTENT

17. INBREDS SELECTED FOR MUTATION BASED ON THE PHYTIC ACID CONTENT

18. Methodology • Low phytic acid donors with lpa1 and lpa2 genes will be used from Victor Raboy, USDA and will be used to develop low phytate maize. • Local inbred lines will be used as recurrent parents. • Identification of closely linked DNA markers with phytate in maize using already available linked markers like umc157 with lpa1 and umc167 with lpa2. • Develop backcross population and marker assisted backcross selection for low phytate maize lines.

19. Expected output • Identification of low phytate genotypes of maize which could be potential donors in breeding for micronutrients. • Molecular markers linked to low phytate will assist in identifying target genes involved in adsorption, transport and unloading of micronutrients in the grain. • Low phytate versions of high yielding maize hybrids in cultivation in India with increased iron and zinc bioavailability and reduced phosphorus pollution in the environment.