MOLECULAR ANALYSIS OF WHEAT - Ae. kotschyi DERIVATIVES WITH HIGH GRAIN IRON AND ZINC CONTENT

Abstract

Over two billion people ofthe world suffer from micronutrient deficiency also known as 'hidden hunger'. Among the various approaches to overcome micronutrient deficiency, biofortification is the most sustainable, cheapest and long lasting solution. Combination of conventional and molecular breeding methods is the most desirable approach for biofortification ofwheat having diverse germplasm sources. Grains of 80 accessions ofnine species ofwild Triticum and Aegilops along with 15 semi-dwarf cultivars of wheat and durum grown over two years at Indian Institute of Technology, Roorkee, were analyzed for grain iron and zinc contents. The wheat and durum cultivars had very low content and little variability for both of these micronutrients. The related non-progenitor wild species with S, Uand Mgenomes showed upto 2-3 fold higher grain iron and zinc content. There were highly significant differences for iron and zinc contents among various cultivars and wild relatives over both the years with very high broad sense heritability. There was a significantly high positive correlation between flag leaf iron with grain iron content (r=0.82) and flag leaf zinc with grain zinc content (r=0.92) of the selected donors suggesting that the leaf analysis could be used for early selection and enrichment ofsegregants with high iron and zinc content for effective breeding for yield and other traits. Chinese Spring with Ph' gene from Aegilops speltoides was used to transfer useful variability from Aegilops to elite cultivars for inducing homoeologous chromosome pairing between Aegilops and wheat genomes. Amajority of the interspecific hybrids had higher leaf iron and zinc content than their wheat parents and equivalent or higher content than their Aegilops parents, strongly supporting a proof of the concept that the parental Aegilops donors possess superior genetic systems for efficient uptake and translocation ofthe micronutrients which could ultimately be utilized for wheat grain biofortification. Meiotic metaphase chromosome analysis of the Fi hybrids (ABDUS1) showed expected chromosome number of 35 and very little but variable homoeologous chromosome pairing. Partially fertile to sterile BCi derivatives with variable chromosomes of Aegilops species and nearly 75% of the expected wheat background had also higher leaf iron and zinc content confirming the transfer of required variability and ultimate expression of the efficient superior genetic systems of the donor parents for the micronutrient content in wheat grains. BC2Fi and BCiF2 progenies were cytologically and morphologically nearer to wheat cultivars. Selection among these progenies was done on the basis of grain iron and zinc content. Subsequently BC2F2 and BC1F3 progenies were analysed for grain micronutrient content. The recovery of fertile derivatives with seeds as bold as that of the wheat cultivars and micronutrient content as high as that of the wild donors gives unequivocal proofof the concept thatAegilops kotschyi possess efficient genetic system for uptake and translocation of the micronutrients which could be effectively used for biofortification of wheat cultivars. Thirteen derivatives were finally selected for detailed analyses using morphological markers, chromosome pairing, HMW- Glutenin subunit profile, GISH and anchored wheat SSRmarkers. Group 1, 2 and 7 chromosomes of Ae. kotschyi were found to be present in the selected derivatives carrying genes for high grain ironandzinc. Synthetic amphiploids between Triticum aestivum (AABBDD) landrace Chinese Spring (Ph1) and cultivar WL711 with different accessions of Aegilops kotschyi (UUS'S1) were developed through colchicine treatment of sterile Fi hybrids. The Fi hybrids and amphiploid plants were intermediate between the parents for plant morphology and spike characteristics. The amphiploids (AABBDDUUS S), however, had variable frequency of univalents at meiotic metaphase-I. The SDS-PAGE of 11 HMW glutenin subunits of amphiploids along with the parents showed the presence and expression of all the parental genomes in the amphiploids. The amphiploids with bolder seeds had higher grain and grain ash iron and zinc content than the wheat parents and comparable to those of their Ae. kotschyi parents suggesting that Ae. kotschyi possesses superior genetic system for the micronutrient uptake and translocation than the wheat cultivars. The variable chromosome number in PMCs of different tillers, spikelets and florets in some of the amphiploids suggests somatic chromosome elimination in the amphiploids. The amphiploids can be used for transfer of high iron and zinc content and development of alien addition and substitution lines in wheat. Lowering the content of anti-nutritional factor, phytic acid, in wheat may increase the bioavailable content of micronutrients iron and zinc. A set of 76 EMSinduced mutants of T. monococcum obtained from P.A.U. Ludhiana were screened for low phytic acid content. On the basis of initial screening two mutants having high inorganic phosphate content were selected as putative low phytic acid (Ipa) mutants. Phytic acid content in the two putative mutants viz., MM225 and MM169 was reduced by 57 %and 46 %respectively over the wild type. The decrease in phytic acid content ofthe mutants was paralleled by increase in total Fe, Zn and P. Available iron content increased by 57 % and 19 % in Ipa mutants MM225 and MM169, respectively over wild type T. monococcum. Scanning Electron Microscope-Energy Dispersive X-Ray (SEM-EDX) mapping of grains of wild type T. monococcum showed compactly arranged phytic acid granules whereas phosphorus was more loosely packed in the aleurone of Ipa mutant MM225. Higher Fe and Zn content in the endosperm of MM225 than that of T. monococcum wild type was also visible in SEM-EDX maps. Thus the novel T. monococcum Ipa mutants- MM225 and MM169 in had lower phytic acid, higher micronutrient content and increased bioavailability. Thorough understanding of increase in phosphate and micronutrients in Ipa mutants will beof tremendous help in biofortification with enhanced bioavailability. Effect ofgermination ofwheat grains on phytic acid content, mineral elements P, K, Mg, Ca, Fe and Zn was studied using SEM-EDX analysis along with other constituents. The minerals showed peripheral distribution within the wheat grain, being sequestered mainly in the aleurone layer ofthe grains. Phytic acid represented by the phosphorus rich granules ofthe aleurone layer ofgrains was reduced by 81 % after 120 hours of germination. The SEM-EDX profile of the minerals also showed their reduction in the aleurone layer with the progress of germination. SEM images revealed significant degradation of starch granules after 72 hours of germination. SDS-PAGE of the seed storage proteins indicated that the protein profile remained unaffected till 96 hours of germination. HMW glutenin proteins remained intact even after the fifth day of germination, whereas LMW glutenin proteins were preferentially degraded. Analysis ofprotein content on dry weight basis ofthe partially germinated seeds indicated a progressive increase over control. To reduce phytic acid content and enhance iron and zinc bioavailability to humans and monogastric animals, wheat seeds can be partially germinated upto 72 hours without any significant deterioration ofprocessing, nutritional characteristics and palatability. The precise transfer ofAe. kotschyi genes for high grain Fe and Zn content and their marker assisted pyramiding in elite wheat cultivars can nearly double the micronutrient content over the existing levels. The combination of biofortified wheat with low phytic acid mutants and improvised processing will be absolutely essential to enhance bioavailability of micronutrients to alleviate the hidden hunger.