Abstract
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.
Highlights
Chinese cabbage (Brassica rapa ssp. pekinensis; Brassicaceae) is an important leafy vegetable crop that is cultivated worldwide
The best hygromycin B concentration was determined for the selection of transgenic shoots by in vitro culturing cotyledon explants on shoot-induction medium supplemented with several hygromycin B concentrations (0, 2, 4, 6, 8, 10 mg/L) along with optimum concentrations of benzyl adenine (BA) at 5 mg/L, 1naphthaleneacetic acid (NAA) at 0.5 mg/L and silver nitrate (AgNO3) at 4 mg/L
The cotyledon explants were extremely susceptible to different hygromycin B concentrations, and the shoot induction capacity dramatically decreased in cotyledon explants as the hygromycin B concentration increased (Figure 1A)
Summary
Chinese cabbage (Brassica rapa ssp. pekinensis; Brassicaceae) is an important leafy vegetable crop that is cultivated worldwide. Pekinensis; Brassicaceae) is an important leafy vegetable crop that is cultivated worldwide. It has abundant healthful properties and is rich in phytochemicals, such as phenolics, carotenoids, flavonoids, glucosinolates, and anthocyanins. Increased Transformation Efficiency in Chinese Cabbage and other Brassicas in South Korea during 2018–2019 was 2.57 million tons, making South Korea the fifth largest producer in the world.. During 2016–2017, South Korea imported 13.7 thousand tons of cabbage and related vegetables, having a US $5.2 million import value (see text footnote 2). The demand for Chinese cabbage is increasing annually, but the present annual production does not meet the demand (see text footnote 2). Increasing the cultivation land for Chinese cabbage is one solution, but it does not increase the production rate. Conventional plant breeding methods have significantly improved Chinese cabbage, certain limitations, such as a complex genome, susceptibility to different stresses and long selection regimes, have limited its development (Velasco et al, 2017)
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