Journal
PLANT BIOTECHNOLOGY
Volume 28, Issue 1, Pages 17-23Publisher
JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY
DOI: 10.5511/plantbiotechnology.10.0921a
Keywords
Cotyledon explants; co-cultivation; genetic transformation; hygromycin; Indian mustard
Funding
- Korea Research Foundation [KRF-2007-211]
- Korea Ministry of Education, Science and Technology
- Marine Extreme Genome Research Center
- Korean Ministry of Land, Transport and Maritime Affairs
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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.
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