A simple and efficient agrobacterium tumefaciens-mediated plant transformation of brassica rapa ssp. Pekinensis

Abstract

Abstract The present study aims to investigate the numerous factors influencing Agrobacterium tumefaciensmediated genetic transformation of Chinese cabbage (Brassica rapa ssp. pekinensis). Factors affecting transformation efficiency, such as age of explants, Agrobacterium concentration, and effect of acetosyringone, precultivation, infection and co-cultivation time of Agrobacterium were examined. The pre-cultured hypocotyls from young seedlings prior to exposure to Agrobacterium showed higher shoot regeneration. The plant transformation with the modest A. tumefaciens concentrations (0.8 OD) and the 3 days co-cultivation periods increased transformation efficiency. Plant growth hormones [1- naphthyl acetic acid (NAA) and 6-benzyl amino purine (BAP)] were essential for callus and shoot formation. Root formation was effective in half strength MS medium without supplementation of root-inducing hormones. To maintain selection pressure, plant subculture was carried out every 2 weeks with selective antibiotics. The putative transgenic plants were acclimatized in the greenhouse. Polymerase chain reaction was performed to confirm the integration of T-DNA into the genome of transgenic plants. A transformation efficiency of 15% was obtained. This protocol allows effective transformation and indirect regeneration of Brassica rapa.