An efficient and universal Agrobacterium-mediated transformation protocol in rice
2012
Puhan, Puspasree | Vipparla, Abhilash | Vemireddy, Lakshminarayana R. | Anuradha, G. | Siddiq, E. A.
For development of transgenic varieties of interest in rice, we have developed a simple, efficient and universal Agrobacterium mediated transformation protocol. Mature seeds of two indica (IR64 and Jaya), one each from japonica (AC41039) and aromatic (Basmati370) varieties were used as explants in the present study. Agrobacterium tumefaciens strain EHA 105 carrying Ti plasmid (pBI121) with the selectable marker npt II along with the reporter gene uidA encoding β-glucuronidase (GUS) was successfully integrated with rice genome without use of acetosyringone. Sterile distilled water washing in place of cefotaxime in the elimination process has been used to control excess growth of Agrobacterium. All the material after transformation germinated in 2 to 3 days of co-cultivation on MS basal medium. Germinated seeds transferred to the selection medium i.e. plain MS medium with 50 mg/l of kanamycin, produced two to three primary tillers within 2 weeks. Mesocotyls from 2 week old in vitro grown plants were taken and cultured in the multiplication medium (MS supplemented with 0.5 mg/l BA and 50 mg/l Kanamycin) where within 6–8 days they produced 3–4 secondary tillers. All the five to six tiller shoots so produced in the process developed roots on plain MS and grew well when transferred to pots containing autoclaved soil and vermicompost in the proportion of 4:1. Transient expression of GUS was observed in all the tissues of recipient plants. Integration of the transgene was confirmed by employing southern blotting and real-time PCR technique. The transformation protocol developed can be efficiently used across the two major subspecies/ecotypes of the Asian rice cultivar Oryza sativa.
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