Development of transgenic wheat (Triticum aestivum L.) for drought tolerance

Abstract

Drought is a major limiting factor for plant growth and crop production. It is essential to understand and control transgene expression in plant transformation and biotechnology. The aim of this study was to develop drought tolerant crops especially wheat. Promoters play a pivotal role in controlling gene expression. Introns and sequences surrounding translation initiation codons are important cis-elements that modulate the expression of genes in drought stress. Using both GFP and GUS as reporter genes, expression pattern of rice SalT promoter was characterized in barley. Agrobacterium mediated method was used to transform SalT-GUS and SalT-GFP constructs in barley. Promoter activity was tested in embryo, callus, Leaf, shoots and root of barley. GFP and GUS under SalT promoter were observed in all plant parts. On the basis of these results, DREB1A gene was cloned under SalT promoter to express first in tobacco and then in wheat. In an attempt to develop drought tolerant tobacco, an expression cassette containing the Arabidopsis DREB1A cDNA under the Figwort Mosaic Virus promoter (FMV) and SalT promoter was transformed into tobacco via Agrobacterium mediated transformation. FMV is a strong and constitutive promoter while SalT is an inducible promoter that can be used for enhancing expression of AtDREB1A gene in tobacco. Putative transgenic T0 plants were confirmed by PCR and copy number was determined by Southern hybridization. RTPCR confirmed the expression of gene in transgenic plants. Selected single copy transgenic plants were further analyzed for drought stress tolerance at T1 generation. Seed germination results showed that transgenic tobacco seeds of both FMVDREB1A and SalT-DREB1A were able to germinate on 20 % PEG and 300 mM mannitol while wild type seeds failed to germinate. Different physiological tests demonstrated enhanced tolerance to drought stress in transgenic tobacco plants than their wild type counterparts. Transgenic tobacco plants of both constructs showed enhanced drought tolerance and produced more seeds than control plants when water was withheld for 10 days. The present investigation clearly showed that overexpression of the AtDREB1A gene under FMV and SalT promoters enhanced drought tolerance in transgenic tobacco and offers applications in developing drought tolerant crops. Based on the results in tobacco, three drought responsive constructs (SalT-DREB1A, rd29A-DREB1A and LEA-SUT2) were transformed in wheat. Wheat transformation was done through Agrobacterium mediated method. Putative transgenes were confirmed by PCR and Southern hybridization. One to four copies of insertions were found in Southern hybridization analysis. Single copy plants were selected for further analysis. RT-PCR showed the expression of transgene while no expression was observed in wild type. Osmotic stress (by mannitol) showed higher seed germination in transgenic wheat plants of all constructs than wild type. Transgenic progenies obtained significantly more number of tillers, increased flag leaf area and 1000 grain weight than wild type. Similarly physiological results indicated that most of the transgenic plants of all constructs gained higher relative water content, photosynthetic rate, stomatal conductance, transpiration efficiency than wild type plants. The present research clearly showed that over-expression of the AtDREB1A gene under rd29A and SalT promoters and HvSUT2 gene under LEA promoter enhanced drought tolerance in transgenic wheat. This study offers applications to grow transgenic wheat in drought prone environment for food security.