Successful Agrobacterium-mediated transformations of Chinese cabbage have been limited owing to the plant’s recalcitrant nature, genomic background and explant necrosis upon infection, which hinders the transfer of T-DNA region into the Chinese cabbage. Consequently, in the current experiment, a stable Agrobacterium tumefaciens-mediated transformation method for Chinese cabbage cv. Kenshin established by employing important anti-oxidants in the co-cultivation and subsequent regeneration media. Four-day-old in vitro derived cotyledon explants were infected with A. tumefaciens strain GV3101 harboring the vector pCAMIBA1303. Cotyledon explants exposed to an Agrobacterium suspension (OD600 of approximately 0.6) for 10 min and then incubated for 3 days co-cultivation in Murashige and Skoog medium containing an L-cysteine + AgNO3 combination exhibited the highest β-glucuronidase (GUS) expression (94%) and explant regeneration efficiency (76%). After 3 days, the cotyledon explants were subjected to three selection cycles with gradually increasing hygromycin B concentrations (10 to 12 mg/L). The incorporation and expression of hptII in T0 transformed plants were verified by polymerase chain reaction and Southern blot analyses. These transgenic plants (T0) were fertile and morphologically normal. Using the present protocol, a successful transformation efficiency of 14% was achieved, and this protocol can be applied for genome editing and functional studies to improve Chinese cabbage traits.

An easy and efficient in vitro regeneration protocol was established for Chinese cabbage, cv. Kenshin (Brassica rapa ssp. pekinensis), using hypocotyl and cotyledon explants. The individual and combined effects of different plant growth regulators were examined in detail at various stages of regeneration through the direct regeneration method. Maximum shoot induction (3.41 shoots/ hypocotyl explant) occurred on Murashige and Skoog medium containing benzyladenine (4 mg•L -1 ), naphthalene acetic acid (1 mg•L -1 ), and silver nitrate (4 mg•L -1 ). These in vitro-regenerated shoots elongated on the same medium and produced abundant roots on half-strength MS medium. Rooted plantlets were successfully hardened and acclimatized with a survival rate of 86%. This optimized method used for cv. Kenshin was studied on two more lines, CNU_11635 and rapid-cycling RCBr cv.R-o-18.Among them, cv. Kenshin was found to be the best responding cultivar with a higher shoot production response of 79%. This method can be applied to regenerate plants after gene transfer and genome editing.