An efficient regeneration system and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Chinese upland rice cultivar Handao297 |
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Authors: | Weina Zhao Shusong Zheng Hong-Qing Ling |
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Institution: | (1) The State Key Laboratory of Plant Cell and Chromosome Engineering, National Centre for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101, China;(2) Graduate University of the Chinese Academy of Sciences, Yuquan Road, Shijingshan District, Beijing, 100049, China; |
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Abstract: | A highly efficient tissue culture system and Agrobacterium-mediated transformation protocol for Chinese upland rice cultivar Handao297 has been established with mature embryos as explants.
Up to 81.2% of mature embryos were induced to regenerate good-quality calli on NB medium (a medium combining N6 macronutrient
components and B5 micronutrient and organic components) containing 3 mg/l 2,4-dichlorophenoxyacetic acid in 10 days. More
than 80% of the calli were morphogenic within 1 week and regenerated green plantlets within 1 month on Murashige and Skoog
medium supplemented with 0.5 mg/l 6-benzyladenine, 0.5 mg/l kinetin, 1 mg/l zeatin, 0.5 mg/l thidizazuron (TDZ), 0.5 mg/l
naphthaleneacetic acid, 0.15 mg/l indoleacetic acid, and 0.15 mg/l indolebutyric acid. This tissue culture system was suitable
for Agrobacterium-mediated transformation of upland rice Handao297. Furthermore, some important factors affecting transformation frequency
were investigated with Agrobacterium strain AGL1 containing the plasmid pCAMBIA1381. The addition of 30 mg/l hygromycin B followed by 60 mg/l hygromycin B to
the selection induction medium facilitated the revival of calli from selection and reduced false positive calli. Hygromycin
B at 10 mg/l was most effective in suppressing non-transgenic callus growth in the differentiation medium. The addition of
TDZ to the differentiation medium promoted the morphogenesis of calli and facilitated the generation of adventitious shoots
by five to tenfold in comparison to medium without TDZ. |
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