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

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Title
An improved method for Agrobacterium-mediated genetic transformation from cotyledon explants of Brassica juncea
Author(s)
M S U Bhuiyan; Sung Ran MinWon Joong Jeong; S Sultana; K S Choi; Y P Lim; W Y Song; Y Lee; Jang Ryol Liu
Bibliographic Citation
Plant Biotechnology, vol. 28, no. 1, pp. 17-23
Publication Year
2011
Abstract
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 0.1mgl -1 α- naphthaleneacetic acid, 1.0mgl 1 6-benzyladenine, and 2.0mgl -1 silver nitrate) containing 20mgl -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 T1 progeny showed that the transgenes were stably integrated and transmitted to the progeny in a Mendelian fashion.
Keyword
Co-cultivationCotyledon explantsGenetic transformationHygromycinIndian mustard
ISSN
1342-4580
Publisher
Japanese Soc Plant Cell & Molecular Biology
DOI
http://dx.doi.org/10.5511/plantbiotechnology.10.0921a
Type
Article
Appears in Collections:
Division of Research on National Challenges > Plant Systems Engineering Research > 1. Journal Articles
Synthetic Biology and Bioengineering Research Institute > Cell Factory Research Center > 1. Journal Articles
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