biofortification

biofortification for micronutrients

1. Bio-fortification: A promising approach to increase the micronutrient content in vegetable crops Impa H R H-2018-117-M

2. Global Nutrition Report Anemia and Stunting are the two forms of malnutrition burdens being experienced by the country. 37.9 per cent of children under-5 are affected by stunting (low height for age). 20.81 per cent of children under-5 are defined as ' wasted ’ (low weight for height). 51.4 per cent of women in reproductive age were affected by anemia. In India, 17.8 per cent of adult men and 21.6 per cent of adult women are overweight.

3. 2014

4. World status of biofortified crops 150 Biofortified varieties of 10 crops have been released in 30 countries Testing of another 12 crops is being carried out in 25 countries

5. HARVEST PLUS Component of the CGIAR Research Program on Agriculture for Nutrition and Health Launched in 2004, USA Under joint venture of CIAT and IFPRI Funding from the Bill and Melinda Gates Foundation, the UK Department for International Development (DFID) and others.

6. There are 4 primary ways a person can get micronutrients into their system BIOFORTIFICATION SUPPLEMENTATION Taking a vitamin pill FOOD FORTIFICATION Through the addition of micronutrients to staple products DIETARY DIVERSITY eating a balanced diet Biofortified staple foods cannot deliver high level of minerals and vitamins per day as fortified foods, but increase the daily adequacy of micronutrient intakes among individuals throughout the lifecycle. ( Bouis et al ., 2011)

7. BIOFORTIFICATION AND ZERO HUNGER CHALLENGE Second Global Conference on biofortification recommend to the UN to celebrate 2018 - 2020 as the International Year of Biofortified and Underutilized Crops Meet the Zero Hunger Challenge by 2030

8. Recommended dietary allowances for Indians Minerals Daily requirement Functions Deficiency symptoms Calcium 500-600 mg Development of teeth and bones, required for phosphorus absorption. Causes improper blood clotting and osteomalacia . Phosporous 1000 mg Component of nucleic acid and plays vital role in cellular metabolism Causes weight loss and general weakness. Iron 10 mg Formation of haemoglobin and involved in transport of oxygen. Anaemia, pale lips and spoon shaped nails. Sodium 4000-6000 mg To maintain the osmotic balance and keep the cells in proper shape. Weight loss and nervous breakdown. Iodine 0.1-0.2 mg Functioning of thyroid gland and production of thyroxin hormone. Goiter . Osteomalacia Anaemia Goiter NIN, Hyderabad

9. Vitamins Daily requirement Functions Deficiency symptoms Vit A (Retinol) 5000 IU For clear vision and increases resistance to infections Night blindness, xerophthalamia and keratinisation Vit B1 (Thiamine) 1.2 mg For proper utilization of carbhohydrates Beriberi disease Vit B2 (Riboflavin) 1.7 mg Oxidation reaction inside the cell Ulcer in oral cavity. Vit B12 ( Cobalamin ) 2.4 µg Maturation of red cell and proper functioning of CNS Pernicious anaemia Night blindness Xerophthalamia Beriberi Ulcer

10. Vitamins Daily requirement functions Deficiency symptoms Vit C (Ascorbic acid) 60-90 mg For collagen synthesis and calcification of bones and teeth Scurvy and reduced resistance to diseases Vit D 10-20 µg Calcification of bones and teeth Rickets in children and osteomalacia in adults Vit E ( Tocopherol ) 5 mg Promotion of fertility Paralysis of eye muscles Vit K (Anti hemorrhagic vitamin) 0.015 mg Coagulation of blood and secretion of bile juice from liver Unusual bleeding from the gums, nose or gastrointestinal tract Scurvy Rickets

11. What is Biofortification ??? Greek word “ bios ” means “ life ” and Latin word “ fortificare ” means “ make strong ”.  Biofortification is the process of adding nutritional value to the crop. It refers to the nutrient enrichment of crops to address the negative economic and health consequences of vitamin and mineral deficiencies in humans (Prasad et al., 2015).

12. (Winkler , 2011)

13. Success of biofortification depends on… High nutrient density must be combined with high yields and high profitability. Efficacy must be demonstrated: Sufficient nutrients must be retained in processing and cooking and these nutrients must be sufficiently bioavailable . The biofortified crops must be adopted by farmers and consumed by those suffering from micronutrient malnutrition in significant numbers. (Jena et al., 2018)

14. Methods of Biofortification AGRONOMIC BIOFORTIFICATION Conventional breeding GENETIC ENGINEERING BIOFORTIFICATION Gene biofortification

15. AGRONOMIC BIOFORTIFICATION

16. Success of agronomic biofortification depends on The presence and bioavailability of soil nutrients for plant uptake (soil to crop). Nutrient allocation within the plant and re-translocation into the edible part (crop to edible part). Bioavailability of nutrients to the human body (food to human).

17. Soil to crop Bioavailability of micronutrients from soil to crop is influenced by many soil factors (i.e. pH, organic matter content, soil aeration and moisture and interactions with other elements). Addition of P also appears to induce Zn deficiency through dilution effects and interference with Zn translocation from the roots. (Singh et al. 1988)

18. Mobility of minerals Mobility in soil Mobile NO 3 - ,SO 4 2- ,BO 3 2- ,Cl - and Mn 2+ Less mobile NH 4 + ,K + ,Ca 2+ , Mg 2+ and Cu 2+ Immobile H 2 PO 4 - , HPO 4 2- , Fe 2 O 3 and Zn 2+ Degrees of success in agronomic biofortification is directly proportional to mobility of mineral element in soil and plant. (White and Broadley , 2003)

19. Crop to food Bioavailability from crop to economic part is influenced by the crop (variety) – which defines whether micronutrients are (re-)localized into edible parts of the crop. Mobility in plants Highly mobile N, P and K. Moderately mobile Zn Less mobile S, Fe, Mn , Mo and Cl Immobile Ca and B.

20. Food to human Bioavailability of micronutrients in the food for the human body is influenced by many factors that can be either food or host related. (Gibson, 2007) Enhancers like ascorbic acid can increase iron bioavailability, while polyphenols and especially phytate or phytic acid are major inhibitors that form complexes with Fe and Zn and limit uptake in the human body. (Clemens, 2014)

21. Impact of different fertilization techniques The combination of soil and foliar application is often the most effective method. ( Phattarakul et al., 2012; Cakmak , 2010) Foliar pathways are generally more effective in ensuring uptake into the plant because immobilization in the soil is avoided. (Garcia- Banuelos et al., 2014) Foliar fertilization with micronutrients often stimulates more nutrient uptake and efficient allocation in the edible plant parts than soil fertilization for leafy vegetables. (Lawson et al., 2015)

22. Agronomic biofortification has so far been most effective with Zn and Se. ( Cakmak , 2014) Suitable micronutrients – Zinc ( Foliar application of ZnSO 4 ) Iodine (Soil application of iodide or iodate ) Selenium (Soil application as Selenate ) In capsicum, low-moderate (0.25-1.0 mg/L) of KI application improved the fruit quality by enhancing the ascorbic acid and soluble sugar content in addition to increased level of iodine to 350-1330 µg/kg FW. (RDA of iodine is 150 µg/d) (Li et al., 2017)

23. In a glasshouse pot experiment, effect of soil applied zinc (in the form of zinc sulphate) at 0, 5, 10 & 15 mg kg -1 of soil on the growth, yield and biochemical attributes was studied of two tomato cultivars, VCT-1 and Riogrande . Objective : To study the effect of Zn on yield and soluble protein level Zn (mg/kg) of fruit Treatment VCT-1 Riogrande Control 24 16 5 mg Zn/kg 30 24 10 mg Zn/kg 35 29 15 mg Zn/kg 42 32     International Journal of Agriculture and Biology 2012

24. SWOT analysis Comparatively simple method than other methods Suitable for immediate results Success limited to minerals Dependent on several factors Regular application of nutrients Often used as a compliment to other strategies Negative environmental impact Reverse action ( Eg : Se) S W O T Susana et al., 2013

25. CONVENTIONAL BREEDING Precedent plant breeding Yield attributes and resistance breeding Lack of priority on nutritional aspects Present plant breeding Fortification of vitamins, antioxidants and micronutrients in edible parts (Prasad et al ., 2015)

26. GENETIC VARIATION BREEDING PROGRAMME

27. Source of Nutrients Crop Wild species Phytonutrient TOMATO Solanum hirsutum Carotene Solanum peruvianum ß carotene and Vitamin C Solanum pimpinellifolium Lycopene and Vitamin C Solanum cheesmanii ß carotene Solanum hirsutum Solanum peruvianum Solanum pimpinellifolium Solanum cheesmanii

28. Crop Wild species Phytonutrient CASSAVA POTATO Manihot oligantha Lycopene and protein Solanum stoloniferum Ascorbic acid LEGUMES Phaseolus coccineus Iron Phaseolus polyanthus Iron Solanum stoloniferum Manihot oligantha Phaseolus coccineus Phaseolus polyanthus

29. Steps in biofortification by conventional plant breeding Discovery Dissemination Outcome 2. Set nutrient target levels 1.Identify target populations 3. Screen germplasm and gene 4. Breed biofortified crops Development 5. Test performance of new crop varieties 6. Measure nutrient retention in crops/food 7. Evaluate nutrient absorption and impact 8. Develop strategies to disseminate seeds 9. Promote marketing & consumption of biofortified food 10. Improve nutritional status of target populations HarvestPlus

30. Glucoraphanin accumulation in Beneforté ®  broccoli B. oleracea X B. villosa (wild) var italica F 1 ( Super Broccoli ) Accumulates 3 times higher level of glucosinolate – GLUCORAPHANIN. Rich in Vit A, D, Ca and folate . Low in calories. ( Traka et al ., 2013)

31. Golden tomatoes Orange coloured golden tomato lines CLN2366 A and B Contain 10 to 12-times more ß-carotene One golden tomato can provide a person’s full day vitamin-A requirement

32. PANT LOBIA -1 ( Boukar et al. , 2011) Pioneer research on biofortifcation of cow pea has initiated G.B. Pant University of Agriculture and Technology, Pantnagar , India. Variety Iron ( ppm ) Zinc ( ppm ) Year of release Pant Lobia-1 82 40 2008 Pant Lobia-2 100 37 2010 Pant Lobia-3 67 38 2013 Pant Lobia-4 51 36 2014 PANT LOBIA -2

33. SWOT analysis Successful for minerals and vitamins One-off cost Easier distribution Long development time Wide public acceptance Simple legal frame work Requires genetic variation S W O T Susana et al., 2013

34. LACK OF VARIATION LACK OF SEX FORMS GENETIC ENGINEERING MICRONUTRIENTS ANTI NUTRITIONAL FACTORS

35. GENETIC ENGINEERING

36. Targets of transgenes include Redistributing micronutrients between tissues. Increasing the efficiency of biochemical pathways in edible tissues. Even the reconstruction of selected pathways. Some strategies involved in the removal of antinutrients . (Jena et al., 2018)

37. ACHIEVEMENT IN INDIA PUSA BETAKESARI Year of identification : 2015-16. Characteristics : First ever indigenously bred biofortified beta carotene (800-1000 µg/100 g) rich cauliflower variety. Marker assisted backcrossing. 54 th Convocation, IARI - 2016

38. Genetic Engineering S Successful for minerals and vitamins Speed up process of conventional plant breeding W Interactions among transgenes O Fast ‘ omics ’ developments T Low public acceptance High socio-political problems Environmental impact (gene flow) Susana et al., 2013

39. VEGETABLE VARIETY PHYTONUTRIENT AVAILABILTY CARROT Pusa Asita Anthocyanin 339.29mg/100 g Pusa Rudhira Lycopene 10 .7 7. 55mg/100g Pusa Nayanjyoti Beta carotene 7.55 mg/100g 1.44 mg/100g 4.60 mg/100g 4.77 mg/100 g Pusa Vrishti Pusa Yamdagni Pusa Meghali Solan Rachna 5.59 mg/100g Pusa Nayanjyoti Pusa Rudhira Pusa Vrishti Pusa Yamdagni Pusa Asita

40. VEGETABLE VARIETY PHYTONUTRIENT Radish Pusa Jamuni Palam Hriday Anthocyanin Pusa Gulabi Lycopene Cabbage Red Acre Kinner Red Anthocyanin Bitter gourd Pusa Aushadi ß carotene Pusa Vishesh , Pusa Hybrid -2 Iron Pusa Jamuni Kinner Red Pusa Hybrid - 2 Pusa Gulabi

41. Broccoli Palam Vichitra Anthocyanin Tomato Pusa Uphar , Pusa Rohini Pusa Hybrid 2, Pusa Red Plum Vitamin C & Lycopene Paprika KTPL-19 Capsanthin Brinjal Punjab Sadabahar Anthocyanin Tapioca Sree Visakam Beta Carotene Sweet potato Sree Kanaka, Sree Rethna, Sree Vardhini and Bhu Krishna Carotene Punjab Sadabahar Sree Visakam Sree Rethna Pusa Rohini Pusa Hybrid -2 KTPL-19 Pusa Uphar Sree Kanaka Sree Vardhini

42. VEGETABLE VARIETY COMPONENT Pumpkin Pusa Vikas Arka Chandan Vitamin A Water melon Arka Jyoti , Durgapura Lal , Durgapura Kesar Carotene Amaranthus Pusa Lal Chaulai , Pusa Kiran , Arka Arunima Carotenoids Anthocyanin Basella Local Red and Local Green Carotenoids Durgapura Lal Durgapura Kesar Arka jyoti Arka Chandan Pusa Vikas Pusa Lal Chaulai Local Green Local Red Pusa Kiran ( Selvakumar , 2014) ( Praneetha et al., 2017)

43. SUCCESS STORY In Rwanda, iron-depleted university women showed a significant increase in heamoglobin and total body iron after consuming biofortified beans for 4.5 months. ( Haas et al., 2017 ) Location Huye , Rwanda Population Adult female (18-27 years) Study design Randomization Randomized efficacy trial By individual Sample size 195 Intervention Iron biofortified beans Length of feeding 4.5 months

44. Treatment Iron (mg/kg) Biofortified bean 86 Unfortified bean 50 A total of 86 (%) of participants were iron defecient and 36 (%) were anemic (haemoglobin < 120 g/L) at baseline. The intervention group receiving the iron biofortified beans consumed 14.5 mg of iron per day, whereas the control group receiving the conventional beans consumed 8.6 mg of iron per day. Result Treatment Iron content of blood Women who consumed biofortified bean 138 g/L Women who consumed conventional bean 90 g/L

45. CASE STUDIES

46. 1: Amino acid profile in cassava and its interspecific hybrid Common Cassava Poor true protein Interspecific hybrid ICB 300 ( M. esculenta × M. oligantha ) LYSINE METHIONINE TRUE PROTEIN (Nasser and Sousa, 2007) Brazil

47. Material and methods Common Cassava – Named as UnB 1 root (female parent). Manihot oligantha – Protein rich wild species (male parent). Inter – specific hybrid – Named as ICB 300. Amino acid analysis The a nalysis of amino acid compositions were performed by an amino acid analyzer (Hitachi L8500, Tokyo, Japan). The total protein contents of the samples were calculated by summing up the amounts of the amino acids.

48. Amino acid profile in peeled roots of cassava Amino acids UnB 1 root M. oligantha ICB 300 ( Interspecific hybrid) Ala 0.020 0.093 0.098 Arg 0.037 0.261 0.108 Asp 0.016 0.146 0.137 Cys 0.027 0.029 0.025 Glu 0.039 0.222 0.221 Gly 0.012 0.078 0.075 His 0.000 0.038 0.036 Ile 0.008 0.068 0.069 Leu 0.016 0.131 0.127 Lys 0.010 0.098 0.079 Met 0.014 0.041 0.037 Phe 0.016 0.129 0.120 Pro 0.000 0.054 0.066 Ser 0.012 0.088 0.078 Thr 0.008 0.061 0.066 Tyr 0.000 0.000 0.000 Val 0.019 0.115 0.112 Total 0.254 1.654 1.454

49. Result and discussion Among the three samples analyzed in this study, the sample M. oligantha showed the highest amount of protein (1.654 g/100 g sample), followed by the ICB 300 sample (1.454 g/100 g) and UnB 1 root ( 0.254 g/100 g). Both cysteine and cystine are involved in cyanide detoxification. Excessive detoxification may be responsible for the low concentration of sulfur-containing amino acids. ICB- 300

50. 2. Agronomic biofortification of carrot with selenium Greenhouse conditions A completely randomized design was used in a 2x2x2 factorial scheme (with and without Se application, two sources of Se: selenate and selenite , two forms of application of Se: soil and foliar applications). Five replications. Ciência e Agrotecnologia Oliveira et al ., 2018 Objective: To evaluate the effect of different application forms and sources of Se in the growth, production, nutrition, physical-chemical characteristics, content and accumulation of Se in carrots. Mat e rial and Me th o d s Brazil

51. Results

52. The application of Se, both through soil and foliar means, of both sources, increases its content in carrot roots. The most effective form of Se biofortification is foliar application as selenate . By consuming 50g of fresh carrot that has been biofortified with selenate , applied through foliar application, which was the highest content of Se (14 mg kg -1 of dry mass) the daily intake of the element would be of 105 μg . Adult human is expected to ingest at least 70 μg per day, with a maximum tolerable limit of 400 μ g (USDA, 2012). Conclusion

53. 3. Agronomic bio fortification of cowpea with iron International Journal of Chemical Studies Treatments T Control T 1 Foliar spray of FeSO 4 @ 0.5 % at 45 DAS T 2 Foliar spray of FeSO 4 @ 0.5 % at 45 & 60 T 3 Foliar spray of FeSO 4 @ 1.0 % at 45 DAS T 4 Foliar spray of FeSO 4 @ 1.0 % at 45 & 60 T 5 Soil application of FeSO 4 @ 12.5 kg ha -1 T 6 Soil application of FeSO 4 @ 25 kg ha -1 T 7 Soil application of FeSO 4 @ 37.5 kg ha -1 T 8 Soil application of FeSO 4 @ 50 kg ha -1 DAS DAS Budhani et al ., 2018 Gujarat

54. Treatments 2 nd picking 4 th picking 6 th picking Average T – Control 163.33 156.00 143.67 154.33 T 1 - Foliar spray of FeSO 4 @ 0.5 % at 45 DAS 188.00 168.67 150.00 168.67 T 2 - Foliar spray of FeSO 4 @ 0.5 % at 45 & 60 DAS 216.33 210.67 200.00 209.00 T 3 - Foliar spray of FeSO 4 @ 1.0 % at 45 DAS 196.00 182.00 169.00 182.33 T 4 - Foliar spray of FeSO 4 @ 1.0 % at 45 & 60 DAS 233.67 217.00 187.67 212.67 T 5 - Soil application of FeSO 4 @ 12.5kg ha -1 187.00 183.33 173.33 181.00 T 6 - Soil application of FeSO 4 @ 25 kg ha -1 241.67 208.00 192.33 205.00 T 7 - Soil application of FeSO 4 @ 37.5 kg ha -1 237.00 223.00 219.33 226.00 T 8 - Soil application of FeSO 4 @ 50 kg ha -1 243.33 224.67 227.33 232.00 Effect of different treatments on Fe content (mg kg -1 ) periodically in pod of cowpea

55. Fe content in pod increased significantly over control during 2 nd , 4 th as well as 6 th picking. The treatment T-8 (soil application of FeSO 4 @ 50 kg/ha) recorded maximum Fe content in 2 nd (243.33 mg/kg), 4 th (224.67 mg/kg) as well as 6 th picking (227.33 mg/kg) in the pods. Conclusion

56. FUTURE THRUST To reach everyone, biofortification must be integrated in public and private programs. Consumer preference. Improving the efficiency with minerals which are mobilized in the soil.

57. Conclusion Biofortification help in overcoming nutrient deficiency economically, especially in rural areas. Need for collaboration between plant breeders, molecular biologists and nutrition scientists. Promoting large-scale prospective studies on nutritional ascepts , productivity along with consumer preference .