An efficient Agrobacterium tumefaciens-mediated transformation of apical meristem in radish (Raphanus sativus L.) using a needle perforation

Abstract

Radish (Raphanus sativus) is a rich source of glucosinolates (GSLs) and their hydrolytic products such as isothiocyanates (ITCs). GSLs and ITCs enhance plant defense responses to biotic and abiotic stresses and are health promoting effect in human. The branched-chain aminotransferase 4 (BCAT4) gene encode an enzyme catalyzing the deamination of methionine in the first step in the chain elongation of aliphatic GSL biosynthesis. Previously, plant transformation in radish has been successfully performed using several methods such as floral dipping, vacuum infiltration and sonic infiltration, protoplast transformation and microspore culture. However, the recalcitrant of regeneration in radish affects the transformation efficiency remain relatively low. Therefore, there is still a need to improve the transformation methods for radish. In this study, we used a simple method for the efficient transformation of radish using Agrobacterium tumefaciens strain GV3101 and tested it with the radish BCAT4 (RsBCAT4) transgene. The PCR, RT-qPCR, Southern blot, GFP fluorescence, and HPLC analyses were used to confirm the transgene integration. Positive correlations between the expression of RsBCAT4 and downstream genes (i.e., CYP79F1, CYP83A1, and GRS1) were also observed in selected T-2 transgenic lines. RsBCAT4 transgenic lines exhibited significantly increased levels of aliphatic GSLs compared to the levels in wild type plants, particularly glucoraphasatin. This needle perforation technique is simple in plant transformation method significantly enhancing transformation efficiency in radish, which could be utilized for molecular breeding of radish to improve its traits. Key message An efficient protocol for stable transformation of apical meristems of radish (Raphanus sativa L.) seedlings using a needle perforation and Agrobacterium tumefaciens incubation method. We developed a needle perforation and Agrobacterium tumefaciens incubation method for in planta transformation