Agrobacterium-mediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp indica cv. ADT 43)

作者：Karthikeyan Alagarsamy; Pandian Shunmugiah Karutha; Ramesh Manikandan^*

来源：Plant Science, 2011, 181(3): 258-268.

DOI：10.1016/j.plantsci.2011.05.011

• Abiotic factors
• Acetosyringone
• Coconut water
• GUS (beta-glucuronidase)
• HPT (hygromycin phosphotransferase)
• Polyvinyl pyrrolidone (PVP)

A simple and efficient protocol for the Agrobacterium-mediated transformation of an agronomically useful abiotic sensitive popular indica rice cv. ADT 43 has been developed. Initiation of calli were best achieved from the leaf bases of 4 days old rice seedlings on LS medium supplemented with 2.5 mg/L 2,4-D and 1.0 mg/L thiamine-HCl. Rice calli immersed in Agrobacterium suspension (strain EHA 105, OD600=0.8) were co-cultured on LS30-AsPC medium for 2 days at 25 +/- 2 degrees C in the dark. Based on GUS expression analysis, 10 min co-cultivation time with 100 mu M acetosyringone was found optimum for the delivery of gus gene. Calli were proved to be very sensitive to Agrobacterium infection and we found that the level of necrotic response can be minimized after co-cultivation with 30% LS, 10 g/L PVP, 10% coconut water and 250 mg/L timentin which improved the final transformation efficiency to 9.33%. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgene in the progeny (T-1) of these plants. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants (T-0 and T-1).

• 出版日期2011-9