<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for large-scale producion of transgenic chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp.<Emphasis Type="Italic">pekinensis</Emphasis>) plants for insertional mutagenesis |
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Authors: | Mi-Kyung?Lee Hyoung-Seok?Kim Jung-Sun?Kim Sung-Hoon?Kim Email author" target="_blank">Young-Doo?ParkEmail author |
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Institution: | (1) Graduate School of Biotechnology and Department of Horticultural Biotechnology, KyungHee University, 447-501 Yongln-Si, Korea;(2) Brassica Genomics Team, National Institute of Agricultural Biotechnology, Rural Development Administration, 441-701 Suwon, Korea;(3) Graduate School of East-West Medical Science, KyungHee University, 447-501 Yongln-Si, Korea |
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Abstract: | In order to better utilize insertional mutagenesis and functional genomics in Chinese cabbage, we have developed an improved
transformation system that more efficiently produces a large number of transgenic plants. Hypocotyl explants were inoculated
withAgrobacterium tumefaciens LBA4404. This strain harbors tagging vector pRCV2, which contains a hygromycin-resistance gene, an ampicillin resistance
gene, and a bacterial replication origin within the T-DNA. Transformation efficiency was highest when the explants were first
co-cultivated for 3 d in a medium supplemented with 5 mg L-1 acetosyringone, then transferred to a 0.8% agar selection medium containing 10 mg L-1 hygro-mycin. In addition, maintaining a low pH in the co-cultivation medium was critical to enhancing transformation frequency.
A total of 3369 transgenic plants were obtained, with efficiencies ranging from 2.89% to 5.00%. Southern blot analysis and
T, progeny tests from 120 transgenic plants confirmed that the transgenes were stably inherited to the next generation. We
also conducted plasmid rescue and inverse PCR with some transformants, based on their phenotype, to demonstrate the applicability
of T-DNA tagging in Chinese cabbage. The tagged sequences were then analyzed. |
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Keywords: | acetosyringone Agrobacterium tumefaciens genetic transformation T-DNA tagging |
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