关键词: 84K杨, 发根农杆菌, 毛状根, 金钗石斛, 遗传转化

Abstract:

Objective: In order to address the technical challenge of transformation of hairy roots in medicinal plants, and based on the development potential of heterologous transformation technology, the fast-growing 84K poplar was selected to construct an efficient genetic transformation system of hairy roots in this study. Method: In this study, the tissue-cultured seedlings of 84K poplar were used as materials to construct an efficient hairy root genetic transformation system by comparing the strain types, explant types, bacterial liquid concentrations, and infection times. Furthermore, the downstream gene LOC110095726 (LOC2) of the terpenoid alkaloid synthesis pathway in Dendrobium nobile was cloned, and the effect of heterologous transformation was evaluated. Result: The best inducing strain was C58C1, and the induction rate of hairy roots reached 100% after 21 days. The average number of induced roots was 5.06 ± 2.36, the average root length was （12.83 ± 5.75） mm, and the transformation rate was 46.67% ± 11.55%. The best explant for induction was the leaf, and the induction rate of hairy roots reached 93.33% ± 11.55% after 14 days, with an average number of induced roots of 3.89 ± 2.53 and an average root length of （8.36 ± 4.24） mm. The optimal infection concentration was 0.8 （OD₆₀₀） value, and, the induction rate of hairy roots reached 100% after 21 days, with the average number of induced roots of 6.03 ± 2.10, and the average root length of （17.77 ± 9.23） mm. The optimal infection time was 15 minutes, and the induction rate of hairy roots reached 100% after 21 days, with an average number of induced roots of 6.03 ± 2.10 and an average root length of （17.76 ± 9.23） mm. LOC2 gene of D. nobile was successfully cloned into an overexpression vector and transferred into Agrobacterium rhizogenes C58C1. Positive hairy roots of LOC2 were obtained in 84K poplar. Conclusion: The optimal transformation system for the hairy roots of 84K poplar is to infect the leaves with C58C1 bacterial solution at a bacterial solution concentration (OD₆₀₀) of 0.8 for 15 minutes. This set of methods, with the advantages of simplicity, rapidity, high stability, etc., can be applied in the functional verification of active ingredients of metabolites in medicinal plants and the creation of plants with high active ingredients.This system is not only applicable to the functional verification of the active ingredients of medicinal plants with similar metabolic pathways, but also provides a reliable technical basis for the large-scale production of medicinal active ingredients.

Key words: 84K poplar, Agrobacterium rhizogenes, hairy root, Dendrobium nobile, genetic transformation