Abstract
Rapeseed (Brassica napus) is an important oil crop that supplies a considerable amount of global vegetable oil production. Genetic transformation system is important to gene functional analysis and molecular breeding. Here, an efficient Agrobacterium-mediated transformation protocol using hypocotyl of rapeseed as explants is described. To develop this protocol, we compared several essential factors that would affect the transformation efficiency, such as Agrobacterium strains, selection marker genes, and genotypes of rapeseed. Comparison of different Agrobacterium strains showed that the GV3101 had higher transformation efficiency than that of C58C1 and EHA105. HPTII, NPTII, and RePAT were used as selection marker genes in tissue culture. The results showed that the transformation efficiency was 3.7–4.8%, 2.2–22.5%, and 1.6–5.9% when the hypocotyl of Westar was infected by GV3101 and screened under hygromycin, kanamycin, and basta, respectively. The transformation efficiency of Westar was the highest and ZS11 was the lowest when five different genotypes of rapeseed (Westar, ZS9, ZS11, GY284, and WH3417) were infected by GV3101. Using this protocol, it will take 8–10 weeks to obtain transgenic plants. This protocol has been used to study gene function in several genotypes of rapeseed in our laboratory. These results indicate that it is efficient to obtain transgenic plant of rapeseed using this protocol.
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Acknowledgments
We are grateful to Prof. Qijun Chen at China Agricultural University for kindly providing pKSE401 and pCBC-DT1T2 vectors for constructing CRISPR/Cas9 system.
Funding
This study was supported by the National Program of Transgenic Variety Development of China (2018ZX08020-001), the National Natural Science Foundation of China (31701458), the Fundamental Research Funds for the Central Universities (2662018QD064), and the China Postdoctoral Science Foundation (2015M580651, 2016T90704).
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SP.L., C.D., and L.G. designed the research. YQ. L, L.L, ZL.D., SL. L., X.T., SJ. L., B.Y., W.Y., and J.W. performed the experiments. SP.L., and C.D. analyzed the data. C.D. and SP.L. wrote the manuscript. L.G. revised the manuscript. All authors read and approved the manuscript.
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Dai, C., Li, Y., Li, L. et al. An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus. Mol Breeding 40, 96 (2020). https://doi.org/10.1007/s11032-020-01174-0
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DOI: https://doi.org/10.1007/s11032-020-01174-0