An improved method for Agrobacterium-mediated genetic transformation from cotyledon explants of Brassica juncea

作者：Bhuiyan Mohammed Shafi Ullah^*; Min Sung Ran; Jeong Won Joong; Sultana Sayeda; Choi Kwan Sam; Lim Yong Pyo; Song Won Yong; Lee Youngsook; Liu Jang R

来源：Plant Biotechnology, 2011, 28(1): 17-23.

DOI：10.5511/plantbiotechnology.10.0921a

• Cotyledon explants
• co-cultivation
• genetic transformation
• hygromycin
• Indian mustard

An efficient Agrobacterium-mediated genetic transformation method was established for Brassica juncea by investigating several factors responsible for successful gene transfer. Four-day-old cotyledon explants from in vitro grown seedlings were co-cultivated with Agrobacterium strain GV3101 harboring the binary vector EnPCAMBIA1302-YCF1, which contained the hygromycin phosphotransferase (HPT) gene as a selectable marker and the yeast cadmium factor 1 (YCF1) gene. Two days co-cultivation period on shoot induction medium (MS medium supplemented with 0.1 mg l(-1) alpha-naphthaleneacetic acid, 1.0 mg l(-1) 6-benzyladenine, and 2.0 mg l(-1) silver nitrate) containing 20 mg l(-1) acetosyringone and five days delaying exposure of explants to selective agent enhanced transformation efficiency significantly. A three-step selection strategy was developed to select hygromycin resistant shoots. Hygromycin-resistant shoots were subsequently rooted on root induction medium. Rooted plantlets were transferred to pot-soil, hardened, and grown in a greenhouse until maturity. Using the optimized transformation procedure, transformation efficiency reached at 16.2% in this study. Southern blot analysis was performed to confirm that transgenes (HPT and YCF1) were stably integrated into the plant genome. All transgenic plants showed single-copy of transgene integration in the host genome. Segregation analysis of T(1) progeny showed that the transgenes were stably integrated and transmitted to the progeny in a Mendelian fashion.

• 出版日期2011