Establishment and optimization of hairy root genetic transformation system in Cyclocarya paliurus

Gao Yilin, Gao Kaidi, Wang Yiting, Fang Shengzuo, Shang Xulan

Journal of Nanjing Forestry University (Natural Sciences Edition） ›› 0

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南京林业大学学报（自然科学版）
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Journal of Nanjing Forestry University (Natural Sciences Edition） ›› 0 DOI: 10.12302/j.issn.1000-2006.202606004

Establishment and optimization of hairy root genetic transformation system in Cyclocarya paliurus

  • Gao Yilin, Gao Kaidi, Wang Yiting, Fang Shengzuo, Shang Xulan*
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Abstract

【Objective】 This study aims to establish and optimize the hairy root genetic transformation system of Cyclocarya paliurus mediated by Agrobacterium rhizogenes.It will lay a solid technical foundation for gene functional verification, secondary metabolic regulation and germplasm innovation of C. paliurus. 【Method】 Sterile seedlings of Wufeng diploid C. paliurus were used as explants for genetic transformation mediated by Agrobacterium rhizogenes harboring the 35S::eGFP plasmid. Orthogonal tests and progressive optimization experiments were performed to screen the optimal transformation conditions, and the effects of genotype on the hairy root transformation efficiency of C.paliurus were further analyzed. 【Result】 Agrobacterium rhizogenes can successfully infect root-cut seedlings and cotyledons of C. paliurus and induce the formation of positive hairy roots. When root-cut seedlings were used as explants, immersion inoculation was performed with MSU440 bacterial suspension at an OD600 of 0.4 for 15 minutes. Hairy roots were obtained after 20 days of culture, with a hairy root induction rate of 80.00%, genetic transformation rate of 70.00%, hairy root positive rate of 48.60%, and an average root number of 4.67. For cotyledon explants, inoculation was carried out for 20 minutes under a negative pressure of -0.04 MPa using MSU440 suspension with an OD600 of 0.8. Hairy roots emerged after 11 days of culture. The hairy root induction rate, genetic transformation rate and positive rate reached 86.67%, 76.67% and 53.00% respectively, and the average root number was 10.16. In addition, significant differences were found in the hairy root formation capacity between sterile materials of Wufeng tetraploid and Muchuan tetraploid C. paliurus. Their positive transformation rates were considerably lower than that of Wufeng diploid materials. These results demonstrate that genotype exerts a remarkable effect on the hairy root transformation efficiency of C. paliurus. 【Conclusion】 For the first time, this study successfully constructed a simple and efficient hairy root genetic transformation system for C. paliurus, which provides crucial technical support for subsequent research on gene function and molecular-assisted breeding of C. paliurus.

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Cyclocarya paliurus / Agrobacterium rhizogenes / hairy root / genetic transformation

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Gao Yilin, Gao Kaidi, Wang Yiting, Fang Shengzuo, Shang Xulan. Establishment and optimization of hairy root genetic transformation system in Cyclocarya paliurus[J]. Journal of Nanjing Forestry University (Natural Sciences Edition）. 0 https://doi.org/10.12302/j.issn.1000-2006.202606004

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