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Online ISSN : 2349-8080 Issues : 12 per year Publisher : Excellent Publishers Email : editorinchiefijcrbp@gmail.com |
2International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi-110 067, India
3Jawaharlal Nehru Technological University, Kukatpally, Hyderabad-500 085, Telangana, India
Coleoptilar nodal explants, which are independent of season, were used to develop high efficiency transformation method in maize. Conditions which affect Agrobacterium-mediated genetic transformation have been standardized by using these explants on hygromycin selection regime. Hyper virulent Agrobacterium strain EHA105 containing gateway vector pMDC99 with Ascorbate - Glutathione pathway coding genes harboring with hygromycin phosphotransferase (hpt II) and bar as plant selectable marker genes was used. The survival frequency of calli at three different stages viz., at the end of I sub-culture on normal medium and II selection as well as on regeneration media supplemented with hygromycin were taken into consideration for the assessment of optimal conditions after checking with PCR. Of the different parameters used with different conditions, one day pre-conditioning of explants, 0.8 optical density of bacterial culture in plain MS liquid infection medium having 5.8 pH along with 200 µM acetosyrengone, 15 minutes infection time for the explants after treating with 0.1% Macerozyme, application of vacuum infiltration for 10 minutes, three days of co-cultivation period were found to be optimal for getting high frequency of transformation through Agrobacterium-mediated genetic transformation in maize. Transformation frequency of 3.25% was obtained on average with these optimal conditions, for which the explants were co-cultivated with these conditions and inoculated on callus induction medium having hygromycin at 10 mg l-1 for selection. The selected calli were transferred onto regeneration medium supplemented with BAP and Kinetin, at a concentration of 1 mg l-1 each for plantlet development. Putative transformants were screened by using hpt II gene specific primers and confirmed after performing southern analysis.
