Tissue culture regeneration and genetic transformation of Coptis chinensis / 中草药

ABSTRACT

Objective: To establish the system of tissue culture regeneration and gene transformation for Coptis chinensis. Methods: The cotyledon and hypocotyl of C. chinensis were used as explants, and the effects of different basic media with different plant growth regulators on in vitro tissue culture regeneration were compared; The embryogenic calli of C. chinensis were used as recipients for genetic transformation by particle bombardment method. Results: The induction rates of cotyledon and hypocotyl were 86.31% and 54.34%, respectively; The 6, 7-V medium was more suitable for callus induction and proliferation rate of C. chinensis than the MS medium. The optimal hormone combination for callus induction was 0.5 mg/L 2, 4-D + 0.5 mg/L KT in MS medium; Either the appropriate concentration of cytokinin alone or the combination of both cytokinin and auxin could induce the continuous proliferation of calli; However, only the hormone combination 0.5 mg/L KT + 0.5 mg/L IAA and 1.0 mg/L 6-BA + 0.5 mg/L NAA could generate the somatic embryos. 6, 7-V medium in absence of hormone could maintain the continuous growth of the embryogenic calli. The somatic embryos could germinate and grow up to plantlets on 6, 7-V medium containing 1 mg/L GA3 + 0.5 mg/L IBA. The embryogenic calli were transformed by particle bombardment method and screened under 3 mg/L Basta, then the activity of β-glucuronidase was detected. PCR analysis showed that the bar gene was successfully transferred to the regenerated plants. Conclusion: The tissue culture regeneration and genetic transformation system of C. chinensis is established, which lays the foundation for its genetic improvement.