Progress and Prospects of Biofortification of Wheat for Higher Grain Iron and Zinc Contents and Their Enhanced Bioavailability

Transcription

1 Progress and Prospects of Biofortification of Wheat for Higher Grain Iron and Zinc Contents and Their Enhanced Bioavailability H. S. Dhaliwal Akal School of Biotechnology Eternal University Baru Sahib Himachal Pradesh To use any of the contents in this presentation please contact the author

2 Nearly half of the world population is affected from iron deficiency Welch and Graham, 2005.

3 Wide Spread Zinc Deficiency in Soils and Humans under Risk of its Deficiency (Alloway, 2004, In: Zinc in soils and Crop Nutrition. IZA Publications, Brussels)

4 Target To meet RDA of Zn or Fe for human beings, cereal grains should contain around mg kg -1 Zn and Fe Current level mg kg -1

5 STRATEGIES TO ALLEVIATE MICRONUTRIENT DEFICIENCY Supplementation Nutrients directly delivered by means of syrups or pills Dietary diversification Production, processing, marketing and consumption of a wide variety of foods. Fortification Utilize widely accessible, commonly consumed foods to deliver one or more micronutrients. Agronomic fortification Enrichment of nutrients through fertilizer application Biofortification Genetic enhancement of nutritional content of crop cultivars Bioavailability Enhanced bioavailability at the existing and biofortified levels

6 Wheat Biofortification Initiatives CGIAR s HarvestPlus Challenge Program to breed nutrient dense staple foods Synthetic hexaploid wheat from T. dicoccon and Aegilops taushii with high micronutrients were used in CIMMYT wheat breeding program. Developed agronomically superior wheat with 100% more zinc and 30% more iron than the modern cultivars. Zn intake was 72% higher from the biofortified wheat with 95% extraction and 0.5mg/d higher absorption than the control wheat. Department of Biotechnology, Govt. India Biofortification of Wheat for enhanced micronutrients using conventional and molecular breeding Phase I (2005) and Phase II (2011) PAU, Ludhiana using progenitor A and B genomes and related species IARI, New Delhi using progenitor D genome IIT Roorkee; Eternal University, Baru Sahib; GB Pant UA&T, Pantnagar using non-progenitor species with S, U and M genomes

7 Range and mean of grain iron and zinc content of wheat and durum cultivars and wild Triticum and Aegilops species S.No. Species Number of accessions Genome Iron mg/kg Zinc mg/kg Range Mean Range Mean 1 T. aestivum 13 ABD T. durum 2 AB T. boeoticum 19 A m T. dicoccoides 17 AB T.arraraticum 6 AG Ae longissima 5 S l ** Ae. kotschyi 14 US ** Ae. peregrina 10 US ** Ae. cylindrica 3 CD ** Ae. ventricosa 3 DN ** Ae. ovata 3 UM **

8 Germplasm Enhancement: Gene Pools of Bread Wheat Primary Wheat cultivars, Landraces, Primitive wheat, Diploid genome donors, Emmer wheat, Synthetic amphiploids Secondary Timopeevi wheats, Aegilops cylindrica, Ae. crassa, Ae. juvenalis and other with one genome in common with wheat Tertiary Aegilops species with non-progenitor C,U, S, M and N genomes, Secale cereale, Agropyron species, Hordeum species, Dasopyrum,

9 Biofortification through molecular breeding Screening of germplasm for Grain iron and zinc content Wide hybridization to introgress gene/qtls Recurrent back-crossing to get fertile derivatives Selection of high iron and zinc containing fertile derivatives Recombination and/or radiation induced transfer of critical chromatin with reduced linkage drag Cytological analysis for chromosome stability and pairing Molecular characterization: Using anchored SSR markers In-situ hybridization and chromosome identification

10 Micronutrient concentration (mg/kg) GRAIN IRON AND ZINC CONTENT OF SOME SELECTED Aegilops DONORS Micronutrient content of top wild relatives T. aestivum WL711 Ae. ventricosa Ae kotschyi 3573 Ae. kotschyi 3790 Ae. kotschyi 394 Ae. perigrina 3791 Ae. longissima 3770 Ae. kotschyi 393 Accessions Ae.ovata 3800 Ae. longissima 3507 Ae. kotschyi 387 Fe Ae. longissima 28 Ae. kotschyi 3774 Zn

11 Aegilops species used as donors for high grain iron and zinc content

12 Total chromosome number =21+14=35 Meiotic chromosome pairing in F1 hybrids of CS(PhI) X Ae. kotschyi F1 hybrid ABDUS 6 II + 23 I 3 II + 29 I a b CSPh I) AABBDD Ae. kotschyi UUSS 1 III + 2 II + 30 I c 1 II + 32 I 1 III + 3 II + 27 I e 2 II + 31 I d f (a) F 1 CS(Ph I )/ Ae. kotschyi 396 (b) CS(Ph I )/ Ae. kotschyi 3774 (c) F 1 CS (Ph I )/Ae. kotschyi 393 (d) F 1 WL711/ Ae. kotschyi 393 (e) F 1 WL711/ Ae. kotschyi 391 (f) F 1 WL711/ Ae. kotschyi 3790 Rawat et al., 2009

13 STERILE WHEAT/ Aegilops HYBRIDS

14 BACKCROSSES TO RECOVER WHEAT BACKGROUND WITH DESIRED VARIABILITY

15 Cytological stabilization of BC 1 F 4 and BC 2 F 3 of Ae. kotschyi 3790 derivatives a. BC 2 F chr, 20II+ 2I b.bc 1 F chr, 21II +1I a b c. BC 1 F chr, 21 II c d d. BC 2 F (x)-2 42 chr, 21 II e. BC 2 F (x)-9 43chr, 1III +19II+ 2I e f f (x) Chr, 21 II

16 Iron and zinc content(mg/kg) Grain iron and zinc content of selected introgressive derivatives of Ae. kotschyi Iron content (mg/kg) Zn content (mg/kg) Control WL711 Control CS (x) BC1F BC1F BC1F Control and derivative plants

17 SDS-PAGE of HMW Glutenins for identification of group 1 chromosomes of Aegilops spp in wheat- Aegilops derivatives

18 TRANSFERABILITY AND POLYMORPHISM OF WHEAT SSR MARKERS IN Ae. kotschyi Homoeologous genome Markers Tested Markers Transferable Markers Polymorphic Transferable (%) Polymorphic % A B D Total

19 Wheat group 2 and 7 SSR markers for identification of Aegilops chromosomes in the derivatives

20 Disomic subsitution line for Aegilops kotschyi chromosome 2S Disomic substitution line with 7U of Aegilops kotschyi 3790 Molecular markers also confirmed introgression of group2 chromosomes of Aegilops kotschyi 3790 in and group 7 in

21 Cytological stabilization of BC 2 F 3 Ae. kotschyi 396 derivatives a. BC 2 F chr, 21II+ 1I a b b. BC 2 F chr, 20II+3 I c. BC 2 F , 45 chr, 22II + 1I c d d d. BC 2 F chr, 21II e. BC 2 F , 40 chr, 19 II+ 2II e f f. BC 2 F chr, 20II+ 1 I

22 Micronutrient content (mg/kg) Grain iron and zinc content of some selected introgressive derivatives of Ae. kotschyi WL711 BC2F BC2F BC2F BC2F BC2F BC2F BC2F BC2F BC2F BC1F BC1F BC2F BC2F Iron Zinc

23 Wheat -Aegilops kotschyi 396 addition and substitution lines with high grain protein, iron and zinc

24 Cytological details of BC 1 F 3 Ae. longissima 3506 derivatives a.bc 1 F chr, 20 II+ 1I b. BC 1 F c a b d 46 chr, 22II+ 2 I c. BC 1 F chr, 21II+ 1I d. BC 1 F chr, 21II+ 1I e. BC 1 F chr, 21II+ 4I f. BC 1 F chr, 21II+ 2I e f

25 Wheat-Ae. longissima derivatives with high grain iron and zinc content Plant derivatives Introgressed chromosome Fe Conc. (mg/kg) %Increase over WL 711 Zn conc. (mg/kg) % Increase over WL 711 WL-711` a 26.4 a - HD a a - L c d 71.7 BC1F4 HD2687/l-3506//WL711(79) ,2, , , ,2, ,2, , , ,4,

26 Wheat-Ae. longissima disomic addition lines with one or two chromosomes

27 Available wheat-aegilops substitution and addition lines with high grain iron and zinc Species Acc. 2n Ch. Aegilops Chrom. Ae. kotschyi , 1U, 2S, 7S, 7U and comb. Ae. kotschyi S, 7U, 2+7 Ae. peregrina 1155, 3519, S, 7S, 7U Ae. longissima S, 2S, 5S, 7S and comb.

28 Biofortified wheat derivatives

29 Wheat -Aegilops addition and substitution lines

30 Recipient Cultivars Donor Species S. No. Recipient Cultivar Fe (ppm) 1. PBW WL UP S. No. Donor Species Fe (ppm) Zn (ppm) Zn (ppm) 1. Ae. peregrina Ae. kotschyi Ae. longissima ICPMS Analysis of plants harvested in S. No. Derivative Fe (ppm) Zn (ppm) Harvest Index Chromosome & 2S U S & 2S Derivatives developed U S S U & 7S S S & 7U S

31 Mapping of QTL for Fe and Zn in diploid wheat RILs Fe Zn

32 Chromosome(s) controlling grain iron and zinc content in Triticeae Triticeae species Chromosome (s) Reference T. monococcum 2A &7A Tiwari et al T. dicoccoides 2A & 7A Peleg et al T. dicoccoides Zinc QTL on 7A Shi et al. 2008; Genc et al T. diccocoides TtNAM-B2 on 2BS Uauy et al and Waters et al Ae. kotschyi 1, 2 & 7 Tiwari et al. 2010; Rawat et al, 2011 Ae. peregrina 4& 7 Neelam et al, 2011 Ae. longissima 1, 2 & 7 Neelam et al, 2011 (Inpress) Hordeum vulgare 2H & 5H Lonergan et al Agropyron intermedium L1, L4 and L7 Schlegel et al Haynaldia villosa V2 and V7 Schlegel et al Secale cereale 1R, 2R & 7R Cakmak et al. 1997, Schlegel et al Homoeologous groups 1, 2 and 7 chromosomes gave higher grain Zn and Fe concentrations

33 Compensating transfers without linkage drag Alien transfers, addition and substitution lines Induced homoeologus transfers Mono 5B ph1b Ae. speltoides Ph I Irradiation VIGS of Ph1 Molecular cytogenetics MAS and pyramiding

34 Chromosome number and pairing at metaphase 1 of pentaploid F1 hybrid Pavon mono 5B x Ae. kotschyi 3790 (AABDUS) + 5B - 5B

35 Induced homoeologous chromosome recombination using ph1b mutant Wheat cv. ph1b/ph1b x Wheat-Aegilops substitution line Ph1/ph1b + Subst. + Wheat homoeologue A, B or D ph1b/ph1b + Subst./wheat monosome X X Wheat cv. ph1b/ph1b Wheat cv. Ph1/Ph1 Ph1/ph1b+ Recombinant Chromosome +normal wheat homoeologue Self Ph1/Ph1 + Homozygous Recomb. Molecular markers, GISH, Nutrient analysis

36 Pollen irradiation induced compensating translocations Elite Wheat Cultivar Elite Wheat Cultivar x x Monosomic alien addition/substitution line GISH, SSRs, Micronutrient analysis Gamma radiations Ae A B D Selfing Homozygous compensating translocation

37 Seed and pollen Irradiation and recombination induced compensating translocations from 7U and 7S Seed Pollen ph1b

38 Genes and Genomics for Biofortification

39 Iron Uptake, Translocation and regulation in Higher Plants Kobayashi and Nishizawa, Annu. Rev. Plant Biol :131 52

40 µ g of Fe mobilised/g dry root wt/ 3 hr µg of Fe mobilised/g dry root wt/3 hr Phytosiderophore released among Aegilops and wheat cultivars under Fe deficient condition Fe deficient media Fe deficient th 11th 14t 17th th 11th 14th 17th Days after transfer Days after transfer Triticum.aestivum Ae.kotschyi Ae.peregrina Ae.longissima Ae.ventricosa Ae.geniculata Triticum.aestivum Aegilops species

41 Iron Transport and Deposition in Developing Grains of barley Borg et al. 2009, Plant Soil 325: 15-24

42 Laser capture microdissection transcriptome road map for zinc trafficking from the phloem to the final sequestering sites in the developing grain in barley Tauris et al J Experimental Botany, 60,: ,

43 Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition Fe concentration of T2 polished seeds. T2 seeds from the ear of the main tiller were harvested in a greenhouse and polished Masuda et al SCIENTIFIC REPORTS 2 : 543 DOI: /srep00543 Fe concentration in T3 polished seeds obtained from the paddy field

44 Selection of potential genes involved in Iron and Zinc uptake, translocation and regulation Potential genes were screened on the basis of available literature of system in different plants i.e. Arabidopsis, rice, barley, wheat etc.

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46 Inhibitors and enhancers if micronutrient bioavailability in human Inhibitors Enhancers Phytic acid Ascorbic, Citric, Malic acids Lignins, Cellulose, Hemicelluloses Inulin Tannins, Polyphenols Heavy metals Phytoferritin Vitamin A

47 Why reduce Phytic Acid? Chelates Fe +++, Zn ++, Ca ++ reducing the bioavailability of these micronutrients in monogastric animals causing hidden hunger Phosphorus has to be supplemented additionally in diets for swine, poultry, fish etc. Myo-inositol-1,2,3,4,5,6-hexakis phosphate Unutilized excreted phytate runs off to lakes and seas, causing eutrophication

48 Enhancing bioavailability of micronutrients Mechanical processing Soaking Fermentation Germination/Endogenous phytases Low phytate mutants Microbial phytases Genetic engineering for phytases Down regulation of MIPs Enhancing promoters Reducing other inhibitors

49 General Procedure for Simulated Digestion of Foods for Determination of In Vitro Fe Bioavailability Using Caco-2 cells Differentiation of Caco-2 Cells into Enterocytes Adjust ph to 2 Raise the ph to 6.5 Food or Meal Sample Lyophilized or dry samples rehydrated using deionized water and homogenized Gastric Phase Pepsin, HCl (Incubate at 37 o C, 1hr in shaking water bath or rocking shaker) Small Intestinal Phase Bile-Pancreatin, NaHCO 3, ph 7 in bicameral inserts in wells with the cell monolayers (Incubate at 37 o C, on rocking shaker in CO 2 incubator) After 2 hr Uptake Phase Insert ring with digest removed and further incubation for 22 hr in CO 2 incubator) Harvesting of Caco-2 cell monolayer Cell monolayers washed with PBS, harvested in PBS, scraped and sonicated at 4 o C 10 µl each of sonicated Caco-2 cell monolayer used for Ferritin Analysis by sandwich ELISA and Protein estimation using BCA kit

50 Phytic acid content and bioavailability of iron at different hours of germination of wheat seeds using the coupled in vitro digestion/caco-2 model S.No. Hours of Germination Phytic Acid (g/100g) Reduction in Phytic Acid (%) Fe Bioavailability (% of Control) ± ± ± ± ± ± ±

51 Spikes and seeds of wild type T. monococcum and EMS induced mutants MM225 and MM169 Genotype T. monococcum Lpa mm169 Lpa mm225 Content mg/g Phytic acid % change over wild type T. T. monococcum MM225 MM169 MM225 MM169

52 Percent of Control IRON BIOAVAILABILITY OF LOW PHYTIC ACID MUTANTS MM-169 & MM-225 RELATIVE TO THE CONTROL Triticum monococcum 200 b 150 a T. monococcum MM-169 MM-225

53 MTF1 MRE Driven Luciferase Reporter Gene Transactivation Assay Zn 2+ Zinc Transporter / ZIP-4 Zn 2+ MT MTF-1 MT Cytosol MT MREx4 TATA Luc MT Nucleus

54 Enhanced Zn Bioavailability in low phytic acid mutants of T. monococcum

55 Future prospects Precise transfer of genes/qtls of high grain iron and zinc content to continue through irradiation or induced homoeologous pairing and molecular cytogenetics Pyramiding of QTL for micronutrient uptake, translocation and deposition in grains through MAB for higher level of biofortification Further dissection of pathways for micronutrient homoeostasis and cloning of genes for genetic engineering Reduce micronutrient losses during processing through their uniform distribution in the grains Enhance bioavailability of micronutrients at the biofortified levels through phytic acid manipulation

56 Our Collaborators Indian Institute of Technology Roorkee Dr. R. Prasad, Dr. G. S. Randhawa, Dr. Partha Roy Students: Vijay Tiwari, Nidhi Rawat, Kumari Neelam, Shailender Verma, Satish Kumar, Rajani Shalunke Punjab Agricultural University, Ludhiana Dr. Kuldeep Singh, Dr. Parveen Chhuneja G. B. Pant University of Agriculture & Technology, Pant Nagar, Uttarakhand Dr. Sundip Malik and research Scholars Kansas State University, Manhattan, Kansas, USA Dr. Bikram S. Gill, Dr. Bernd Friebe, Dr. Nidhi Rawat National Agri-Biotech Institute (NABI), Mohali, Punjab Dr. Rakesh Tuli, Dr. Sudhir P. Singh, Dr. Joy K. Roy

57 Indian Institute of Technology Roorkee

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60 GISH analysis of mitotic chromosomes of wheat- Ae. peregrina derivatives (a) BC 2 F with single 7S P chromosome (green), a b (b) BC 2 F with single 4S P chromosome (green), c d (c) metaphase and (d) anaphase BC 2 F with single translocated 5U P chromosome (red), (e) metaphase and (f) anaphase, BC 2 F showing single 7U P chromosome (red). e f

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62 MORPHOLOGY AND CHROMOSOME PAIRING OF WHEAT-Ae. kotschyi AMPHIPLOIDS Chr- 64,1III+28II+5I Chr-68, 32 II + 4 I Chr-69, 31 II + 7 I Chr- 69, 32 II + 5 I Chr-64, 30II + 4I Chr- 67, 29 II + 8 I Rawat et al., Plan. Gent. Resour.

63 Black et al., 2008 Prevalence of stunting in children under 5 years Prevalence of stunting among children under 5 years old in India by state

64 Constitutive Overexpression of the OsNAS2 Gene for Effective Iron- and Zinc-Biofortification of Rice Endosperm WT Transgenic Fe Zn Jonson et al Plos One 6: e24476

65 Seed and pollen Irradiation and recombination induced compensating translocations from 7U and 7S Seed Pollen ph1b

66 Fe bioavailability of wheat cultivars, landraces and interspecific derivative BTC17 by the coupled in vitro digestion/caco-2 model

67 Large scale multiplication of wheat-aegilops derivatives for bioavailability studies Donor Aegilops species Parentage and pedigree of Wheat- Aegilops Aegilops derivatives chromosome Ae. kotschyi acc CS/3790//UP2338-2///WL711-2(63)-2-13 (Bulk ) 7U K -do- CS/3790//UP2338-2///WL711-2(77) (Bulk) 2S K -do- CS/3790//PDW274-2///PBW343-1(66)-1-89 (Bulk) 2S K. 7S K -do- CS/3790//UP2338-2(77) (Bulk) 2S K -do- CS/3790//UP2338-2(77) (Bulk) 2S K, 7S K Ae. kotschyi acc. 396 CS/396//PBW343-3///UP2425(49) (Bulk) 1S K, 2S K, 7S K -do- CS/396//PBW343-3///PBW373(48) (Bulk) 7S K Ae. peregrina acc CS/13772//PBW373-1///WL711(16) (Bulk) 7U P -do- CS/13772//PBW373-1///WL711(16) (Bulk) 7U P Ae. peregrina acc.1155 CS/1155/PBW373-1///WL711(1) (Bulk) 7S P Ae. peregrina acc.3519 CS/3519//WL711(17)-1///WL (Bulk) 7S P Ae. longissima acc HD2687/L-3506//WL711(79) (Bulk) 2S l, 7S l -do- HD2687/L-3506//WL711(79) (Bulk) 2S l -do- HD2687/L-3506//WL711(79) S l, 2S l

68 Designing of gene-specific primers Example: HvIDS2 gene Search the HvIDS2 protein in Uniprot (

69 Uniprot reference protein database

70 Sequence annotation feature of HvIDS2 protein

71 Protein sequence of HvIDS2 protein FASTA format of HvIDS2 protein

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76 Paste full length target gene coding sequence (cds) for finding genomic sequence contigs and intronic regions between cds (exons).

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80 On the basis of comparison between matched sequence contigs (wheat 5x cereal database) and full length target gene coding sequence (mrna), we had predicted introns location and size. Later we had designed the primers using the full length target gene coding sequence considering the open reading frame (ORF), location and size of introns using Primer 3.0 and BLAST at NCBI (

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82 Graphical view of 5 primers for first half of gene

83 Five primer sets were designed for half of the sequence.

84 Future Strategy

85 Kim et al Science 314:

86 Biofortification Initiatives CGIAR s HarvestPlus Challenge Program in association with CIMMYT, IRRI, CIP and ICRISAT for Fe, Zn and β- carotene Department of Biotechnology, Govt. India, Networks on wheat, maize, rice and sorghum for Fe and Zn and β-carotene in pigeon pea and groundnut Micronutrient Initiative of Canada, a NGO with support from CIDA for Vitamin and mineral supplementation and fortification

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88 GISH and FISH of wheat-ae kotschyi 3790 derivatives (a) Somatic chromosomes of showing the presence of one pair of 7Uk chromosome (red) after GISH, (b) Somatic chromosomes of showing the presence of a pair of 2Sk chromosome (green) after GISH (c-d) Somatic chromosomes of (c) showing the presence of a pair of 2Sk (green) and a single chromosome 7Uk (red) after GISH,(d) The same chromosomes of as in (c) after the sequential FISH using pas1 (red) and phvg38 (green).

89 Effects of equimolar concentration of Fe & Zn in the transactivation assay