An efficient high throughput plant regeration protocol for production of transgenic plants tolerant to salt in Finger millet (Eleusine coracana (L.) Gaertn.)
*Dept. of Crop Physiology, University of Agricultural sciences, College of Agriculture Dharwad-580005, Karnataka, India, E mail: email@example.com
**Department of Agronomy, UAS, GKVK, Bangalore-560065, Karnataka, India.
***O/o Assistant Director of Agriculture, Bagepally (Tq), Chikkaballapur (Dist) -561207, Karnataka, India.
*****Department of Crop Physiology, UAS, GKVK, GKVK, Bangalore-560065, Karnataka, India.
Finger millet (Eleusine coracana (L.) Gaertn.) is the primary food source for millions of people in tropical dry land regions of the world. Development of efficient and genotype-independent tissue regeneration system is an essential prerequisite for successful production of transgenic plants. In this direction we established efficient reproducible protocols for in vitro plant regeneration and genetic transformation in finger millet using PDH45 as a candidate gene to develop transgenic Finger millet for salinity tolerance by Agrobacterium mediated gene transfer method (in vitro method) by using actively dividing embryogenic calli which obtained from Finger millet seeds. Here the seed calli was co cultivated with Agrobacterium Plasmid carrying binary vector pCAMBIA construct containing PDH45 candidate gene, nptII gene as bacterial selection marker, hptII gene as plant selectable marker, and GUS reporter gene driven by CaMV 35S promoter. The co cultivated callus was regenerated in half strength MS media with 0.5 mg.L−1BA, 3.0 mg.L−1 2, 4-D, and hygromycin antibiotic supplemented with acetosyringone (100 100 g.mL−1), a potent inducer of virulence genes. Successful transformation at callus stage was initially confirmed by GUS histochemical assay. And by PCR amplification genomic DNA of putative transformed calli showed positive for hptII primers. And the results by RT-PCR showed that the level of transcripts overexpression in transformed calli was relatively higher than nontransformed control calli. Putative regenerated transgenic were confirmed by PCR amplifying the genomic DNA of putative transformed plants.
Agrobacterium, callus, Finger millet, PDH45, regeneration.