Genetic engineering to enhance drought and salt tolerance of wheat (Triticum aestivum L.)

During their life cycle, plants are challenged by various unfavorable biotic and abiotic factors. All these constraints tend to prevent the plants from expressing their full genetic potential and yield capabilities. It has been estimated that almost 70 % of yield related losses are due to different abiotic stresses especially drought and surface salinity. Rapid land degradation and uncontrolled world population are serious threats to our food security which has almost reached its tipping point. To ensure the sustainable food supply, development of drought and salt stress tolerant crops has become imperative. Wheat crop is the staple food for 35 % of world's population including Pakistan and highly vulnerable to environmental changes like global warming, erratic rainfall and poor surface and subsurface irrigation water that cause land salinization. Genetic engineering approaches are offering a tangible alternative to conventional breeding techniques with its target specificity and swift outputs. Late Embryogenesis Abundance (LEA) protein gene HVA1 plays a major role in membrane protection, osmotic regulation, desiccation tolerance and cell membrane integrity during different abiotic stresses thus, a strong candidate for wheat genetic improvement. In current PhD research work, the main focus was to screen out wheat germplasm and wheat cultivars for high embryogenic and regeneration potential and to develop an optimized wheat tissue culture system for our locally adaptive but abiotic stress sensitive wheat varieties that will reduce our dependency on exotic but non-adaptive regenerable wheat lines like Bobwhite. Barley HVA1 gene, a member of Late Embryogenesis Abundance (LEA) group-3 protein was cloned under Arabidopsis thaliana rd29A stress inducible promoter and transformed in a commercial wheat cultivar Seher-2006 through Agrobacterium-mediated transformation with 1.84 % transformation efficiency. Transgenic plants were confirmed for successful gene integration through GUS histochemical assay, PCR, DNA hybridization, RT-PCR and basta leaf paint bioassay. T1 progeny of six selected transgenic plants showed better germination, root and shoot length at three NaCl treatments (0, 100 and 200 mM), three PEG-6000 treatments (0, 10 and 20 %) and four doses of basta herbicide (0, 2, 3 and 4 mg ml-1). At 200 mM NaCl, transgenic event ST6 showed 41 %, 42.6 % and 45.5 % higher germination rate, root and shoot length than the non-transgenic plant. Similarly at 20 % PEG-6000, same ST6 event showed 41.2 %, 67.7 % and 38.3 % higher germination rate, root and shoot length than non-transgenic plant. Whereas at 4 mg ml-1 basta, again ST6 was better performer with 96 %, 97.6 % and 95.3 % higher germination, root and shoot length than the non-transgenic plant. RT-PCR demonstrated higher transcript level of transgenes at both NaCl and PEG-6000 in comparison non-transgenic plants. Similarly, HVA1 transgenes exhibited improved membrane stability index (MSI), minimum electrolyte leakage (EL), better photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (C), water use efficiency (WUE), osmotic potential, water potential and turgor potential under both drought and salinity treatments. Much higher K+, K+/Na+ ratio and proline contents and relatively lower Na+ contents were observed in case of transgenic events ST1, ST5 and ST6 when compared to non-transgenic plant. Transgenes showed less yield penalty under reduced irrigation treatments and gave 13.14 % and 78 % higher grain yield than non-transgenic plant under severe drought and salinity treatments. Similarly, transgenes ST3, ST4, ST5 and ST6 performed much better than nontransgenic plant with respect to plant height, flag leaf area, number of tillers, root shoot weight, root shoot length and grain weight under both drought (4, 2 and 0 irrigations in field) and salinity stress (0.4, 10 and 20 dS m-1 in pots). Current study offers highly efficient transformation system for local high yielding but abiotic stress sensitive wheat varieties that got the potential to perform much better in open field. Transgenic events ST6, ST5 and ST4 proved to be highly salt and drought tolerant under different water deficit and salinity treatments both in lab and field based experiments and thus can be further utilized for varietal development for drought and salinity prone areas of sub-continent.