Potential use of High iron and low phytate GM rice and their Bio-safety Assessment

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1 Potential use of High iron and low phytate GM rice and their Bio-safety Assessment Dr. Karabi Datta University of Calcutta, India

2 Background High iron rice and iron bioavailability Micronutrient deficiency is a global concern for human nutrition and health. Rice is primary food for more than half of the world population representing major energy source of millions of people. Designing rice for biofortification /bioavailability and for stable nutrition profile in polished rice by biotechnological transgenic approach may play important role for human health benefits.

3 Presentation Profile Ferritin gene: Rice/Soybean ferritin Introgression of high iron trait in local rice cultivar Improvement of iron/phosphorus bioavailability (reduction of phytic acid level): Embryo/ aleurone specific promoter driven Down regulation of mips/ipk1 by RNAi technology Substantial Equivalence study for biosafety analysis

4 IRON AND ZINC IN RICE CULTIVARS Amount of Iron and zinc estimated with unpolished and milled rice seeds of local rice cultivars through AAS (Atomic absorption spectroscopy) by modified dry ashing method.

5 Iron Biofortification: Ferritin Gene from Rice Schematic vector map showing ferritin gene cloned from Swarna in overexpression pipkb001 vector under the control of glutelin and globulin promoter Semi-quantitative rt-pcr of ferritin gene from the T 3 transgenic seeds. figure shows overexpression of ferritin in lines , , , , and as compared to the non-transgenic control (NT). PCR Screening of transgenic plants with Osfer2 gene specific primer pairs T 3 transgenics in green house Expression analysis through qrt-pcr of T 2 transgenic plants

6 BIOCHEMICAL ANALYSIS: ESTIMATION OF IRON AND ZINC IN TRANSGENICS Amount of iron and zinc were estimated with T 3 transgenic milled rice seeds through AAS (Atomic absorption spectroscopy) by modified dry ashing method.

7 Accumulation of Iron and Zinc in seeds: Ferritin from Rice PS2: 7.6 µg/g : 15.9 µg/g 2.1 fold increase Histochemical localization of iron by Prussian blue staining in transverse section of rice seeds PS2: 22.4 ug/g : 30.7 ug/g 1.4 fold increase Histochemical localization of zinc in seeds by dithiozone staining

8 Seed Germination Agronomic studies

9 Iron Biofortification: Ferritin Gene from Soybean Schematic diagram of expression vector containing Soybean ferritin gene Histochemical localization of zinc in seeds by dithiozone staining (a) (b) Histochemical localization of iron by Prussian blue High iron/zinc GM Rice with X 2.5 more iron in polished seed Analysis of mineral content of brown and milled transgenic and control rice by AAS (a) iron (b) Zinc Vasconcelos M, Datta K et al, Plant Science,

10 Nutrition Rice: Introgression of High Iron Trait into Local Rice Cultivar Female Plant: Swarna Male (Donor) Plant: FR-19-7 (Transgenic IR68144; developed by Datta SK s Group; Vasconcelos et al., 2003)

11 Molecular Characterization of Introgressed Seeds Expression analysis of hybridized seeds Semi quantitative RT-PCR of soybean ferritin gene in hybridized seeds (BC 2 F 4 ) Transcriptional level expression of soybean ferritin gene in hybridized seeds by qpcr (BC 2 F 4 ) Swarna: 6.75µg/g Hybridised: µg/g 2.5 fold increment over Swarna

12 Agronomic Evaluation of Ferritin introgressed lines SSR profiles of Swarna plants, introgressed high iron plants and IR68144 BC 2 F 4 plants are closely related to Swarna and distantly related to IR68144 transgenic line Different agronomical parameters of hybridized rice plants as compared to control Swarna

13 Nutrition Rice Bioavailability : Reduction of Phytic acid Level Besides sequestering inorganic phosphate, phytic acid due to it s six highly negatively charged anion, is a potent chelator of mineral cations Phytic acid strongly binds to mineral cations like Fe 2+, Zn 2+, Ca 2+, Mg 2+ to form a mixed salt called phytate Hence to increase mineral (Fe 2+ ) as well as phosphorus bioavailability the reduction of phytic acid levels in rice seeds is required Phytic acid 1D-myo-inositol 3-phosphate synthase (MIPS) and inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) are two key enzymes in phytic acid biosynthesis. Down regulation of these two enzymes in tissue specific manner by RNAi technology may play important role to reduce phytic acid level in seeds.

14 Reduction of Phytic Acid level : Improvement of Bioavailability Nontransgenic Ole18 Promoter expression Lpt2 Promoter expression RNAi Vector for down regulation of mips gene RNA isolated from Swarna seedlings mips amplified from cdna ipk1 amplified from cdna Rice cultivars used for transformation: Pusasugandh II. RNAi Vector for down regulation of ipk1 gene

15 Molecular Analysis of Transgenic Plants (a) Semi-quantitative RT-PCR of ipk1 gene from the T 3 transgenic shoots and seeds (b) Southern hybridization showing integration of RGA2 intron in transgenic T 3 plants (a) ipk1, (b) mips Semi-quantitative RT-PCR of mips gene from the T 3 transgenic shoots and seeds

16 Low Phytate Rice with Bio-available Nutrients Phytic acid content as determined by HPLC analysis Ali et al, PloSONE (2013); Rice (2013)

17 Related Publications

18 Effects of lipoxygenase enzyme in seed quality deterioration Nutrition Rice: Maintenance of Rice Nutritional Quality During Storage Rice grain deterioration is the common problem of rice storage. LOX activity in rice grain is localized in the bran milling fraction. It catalyzes the addition of molecular oxygen to polyunsaturated fatty acids Linoleic acid (18:2) ROS LOX DNA damage Protein damage Cell membrane Fatty acid oxidation Carotene oxidation Cell death Nutritional loss Physiological causes of seed nutritional deterioration and

19 Lox activity & Carotenoids level after artificial aging Expression of transgenic rice seeds after accelerated aging (storage) treatment at 45 C and 85 % RH for 14 days Analysis of rice seeds after artificial ageing treatment Estimation of carotenoids by HPLC Reduction of carotenoids level after storage

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21 Biosafety: Substantial Equivalence Study For biosafety assessment of GM crops, different countries follow their own protocol developed by their biosafety authority. Comparative analysis approach based on substantial equivalence concept (FDA 1992; OECD,1993) involves identification of differences between a GM product and its conventional counterpart and assessment of safety impact of such differences.

22 SAFETY PARAMETERS Gene / Protein Safety Food / Feed Safety Substantial Equivalency Gene(s) - Source(s) - Molecular characterization - Gene integrity Protein(s) - History of safe use - Toxicology/allergenicity - Specificity/mode of action Environmental safety - Non-target organisms - Soil degradation - Biodiversity - Pest resistance Crop characteristics - Morphology - Yield Food / feed composition - Proximate analysis - Key nutrients - Key anti-nutrients tmm

23 Case Study: High Iron Ferritin Rice Molecular & Morphology Analysis

24 Proximate analysis & Mineral content

25 Fatty acids, Vitamins, antinutrients

26 Amino acids

27 Hypothetical proteins: Determination of allergens Searching allergen from: Known Database from allergenonline version 15, NCBI BLASTP (non redundant protein database), FASTA3, 80mer amino acid (aa) sequence and 8mer aa exact match ( by Food Allergy Research & Resource Program (FARRP) [ 80mer and 8mer exact match depicts the cross reactivity with IgE antibody] Full FASTA sequence analysis: identity matches greater than 50% indicating possible crossreactivity (Aalberse, 2000). 80 aa alignments: identities greater than 35% indicating possible cross-reactivity (according to CODEX Alimentarius guidelines, 2003). Tissue SSP No Accession No of identified Protein Allergen Protein from FASTA3 (Full) Toatal aa sequence identity Identity E Score (< ) organism 80 mers over 35% identity No of 80-mers Identity No of 8- mers Exact 8 mers Identity Seed 7806 gi Mala S 12 allergen precursor % 0.76 Malassezia sympodialis 550 nil 623 nil 8605 gi nil nil 294 nil 8401 gi nil nil nil nil 8402 gi nil nil nil nil 4901 gi nil nil nil nil 4403 gi nil nil nil nil 0204 gi nil nil nil nil Shoot 1601 gi nil nil nil nil 2203 gi nil nil nil nil 4201 gi nil nil nil nil

28 Research highlights: The nutritional composition of grains of high iron transgenic rice (FR-19-7, the transgenic IR68144 line developed,vasconcelos et al,2003) is comparable to that of non-transgenic counterpart. The nutritional components of transgenic ferritin seeds are well within the accepted range of reported values. Based on the substantial equivalence concept of OECD, the analyzed transgenic seeds are safe for human consumption

29 GROUP MEMBERS We thank DBT, Govt. of India and ICAR for financial support