Factors influencing the efficiency of Agrobacterium tumefaciens-mediated transformation and regeneration in Brussels sprouts

Abstract

An efficient method for Agrobacterium tumefaciens-mediated genetic transformation of Brussels sprouts was developed. We tested two strains, LBA4404 and EHA105 both with the binary vector p35SGUSINT containing the NPT11 gene for kanamycin resistance. Explants were derived from in vitro-grown seedlings of two commercial cultivars, Winter Pick and Troika. Transformation and regeneration efficiency were based on transient GUS expression and the ability of the explants to grow on media supplemented with kanamycin. Factors influencing GUS expression and shoot regeneration included the plant genotype, the A. tumefaciens strain, and the explant type. Winter Pick had significantly (P ≤ 0.05) higher GUS expression, up to 44.3% in leaf sections compared to 25.6% in Troika, and significantly (P ≤ 0.05) higher shoot regeneration. Agrobacterium strain LBA4404 was easier to control in culture and it achieved significantly (P ≤ 0.05) higher infection rate, 46.1 %, than EHA105 (22.0%), ultimately achieving higher shoot regeneration. The length and temperature of co-cultivation did not significantly affect GUS expression, but co-cultivation for 2 d at either 21°C or 24°C resulted in more and healthier shoots as opposed to the vitreous shoots obtained from 3 d and 5 d co-cultivation periods. Compared to hypocotyl and petiole explants, leaf sections were the best, with highest GUS expression (44.8%) and moderate shoot regeneration of 45.8% on MS medium with 2.0 mg L-1 BAP, 5.0 m L-1 AgNO3 and 50 mg L-1 kanamycin. This optimised protocol can be used to introduce the thaumatin gene for sweetness to mask the bitter taste in Brussels sprouts.