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A new
time-saving transformation system for Brassica napus
Fanming Kong1,
Juan Li1, Xiaoli Tan1*, Lili Zhang1,
Zhiyan Zhang1, Cunkou Qi2 and Xiaoke
Ma1
1Institute
of Life Sciences, Jiangsu University, 212013, P.R. China.
2Institute
of Industrial Crop, Jiangsu Academy of Agricultural
Sciences, Nanjing, 200014, P. R. China.
*Corresponding author. E-mail:
xltan@ujs.edu.cn.
Fax: 86-511-88791923.
Accepted 9 April, 2009
Abbreviations:
AS, acetosyringone; 6-BA, 6-benzyladenine;
CaMV35S, cauliflower mosaic virus 35S; CTAB,
cetyl trimethyl ammonium bromide; GFP, green
fluorescent protein; NAA, naphthalene acetic acid;
NOS, nopaline synthase; OD, optical density. |
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By optimizing different parameters for infection, an
efficient Agrobacterium-mediated transformation system for
Brassica napus was developed. A series of
combinations of optical densities,
infection durations, concentrations of acetosyringone and
silwet L77 were evaluated.
Maximum transformation efficiency reached 18.93% when
cotyledonary nodes were infected for 10 min with an
Agrobacterium suspension of 0.8 OD and 200 µM
acetosyingone and 0.02% silwet L77 in the culture
medium. In addition, the concentrations of
6-benzyladenine and naphthalene acetic acid were also
optimized. Meanwhile, maximum transformation efficiency for
green plantlet was 94.38% when cotyledonary nodes were
cultivated in the medium containing 4 mg/L
6-benzyladenine and 0.04 mg/L
naphthalene acetic after co-cultivation. Transformation was
confirmed by polymerase chain reaction analysis, southern
blotting analysis and green fluorescent protein assay.
Key words:
Agrobacterium tumefactiens,
cotyledonary node, 6-benzyladenine, naphthalene acetic acid,
transformation, Silwet L 77. |
