Gene delivery using microinjection of Agrobacterium to embryonic shoot Apical meristem of elite Indica rice cultivars

Abstract

A method of gene transfer to elite indica rice cultivars (BPT5204, IW-Ponni and CR1009) by microinjecting the embryonic shoot apical meristem (ESAM) of seeds with Agrobacterium is described. The ESAM microinjected with Agrobacterium cells containing binary plasmid was incubated in co-cultivation media at 28°C for 3 days in the dark and subsequently transferred to selection medium with appropriate supplements and antibiotic. The frequency of gene transfer under various concentrations of Agrobacterium and acetosyringone used were analyzed by staining for the presence of the marker enzyme β -glucuronidase (GUS) carried as part of the binary plasmid and growth in the presence of antibiotic hygromycin. Results of this investigation show that the maximum efficiency of gene transfer took place when Agrobacterium was used at an OD600 of 0.3 and grown in the presence of 20 mg l^-1 acetosyringone.