PLANT CELL BIOTECHNOLOGY AND MOLECULAR BIOLOGY

Aims: In-planta genetic transformation has been carried out to overcome problems associated with host/tissue specificity and to increase the Agrobacterium infection in the target plant tissue, besides overcoming labour-intensive and tedious tissue culture which can also result in somaclonal variation.

Place and Duration of Study: Department of Agricultural Biotechnology, College of Agriculture, Bhubaneswar during 2014 to 2015.

Methodology: Apical meristem of the differentiated embryo of the germinating seedlings of green gram cultivar- Sujatais infected with Agrobacterium tumefaciens carrying the genes for a transcription activator (Dehydration Responsive Element Binding factor-DREB1A) with a stress inducible promoter. T₁transformants were identified by PCR analysis with gene- specific primer, positive control and negative control.

Results: Transgenic green gram plants have been produced by a tissue-culture independent Agrobacterium tumefaciens–mediated transformation procedure carrying the genes for transcription activator (DREB1A) with a stress inducible promoter (rD29A). Since the transgene is integrated into the cells of already differentiated tissues, the T₀ plants were chimeric and stable integration could be seen only in the T₁generation.There were 80 plants (T₀) in 16 pots at 5plants/pot. One pot carrying 5 such plants (T1) showed PCR amplification with gene specific primer and were selected for continuing into the next generations. Molecular characterization of the T₁ generation with a transformation frequency of 1.25-6.25% suggested the feasibility of the method to generate transgenic plants in greengram.

Conclusion: The in planta protocol developed in the present study would be beneficial to transfer the economically and nutritionally important genes into different varieties of green gram and other pulse crops recalcitrant to tissue culture, and the transgenic research in such crops can be easily harnessed.