小豆子叶节遗传转化体系建立

Establishment of Genetic Transformation System using Cotyledon Nodes in Adzuki Bean

以小豆品种‘京农6号’子叶节为外植体进行离体再生,旨在建立‘京农6号’子叶节再生遗传转化体系。本研究对农杆菌菌株EHA105侵染子叶节的时间、诱导愈伤生成的适宜6-BA浓度、诱导不定芽的激素浓度及配比及诱导不定根生长的IBA浓度等影响子叶节再生遗传转化体系的因素,结果发现农杆菌EHA105侵染的最佳时间为50 min,GUS阳性率为67.5%;诱导愈伤的适宜6-BA浓度为2.0 mg/L,诱导率为91.4%;诱导不定芽的最适激素是5.0 mg/L ZT,出芽率为62.0%;而0.1 mg/L IBA诱导不定根的效果最好,生根率可达100.0%。本研究建立了小豆‘京农6号’子叶节外植体的遗传转化体系,获得了再生植株,为小豆的CRISPR/Cas9基因编辑技术在小豆功能基因组学和育种中的应用提供了技术参考。

To establish the genetic transformation system,the cotyledon nodes of adzuki bean variety'Jingnong6'that had been sequenced for reference genome were used as explants in vitro.The infection time of Agrobacterium strain EHA105 on cotyledon node,optimal 6-BA concentration of callus induction,different hormone ratio of inducing adventitious buds,and optimal IBA concentration of adventitious root induction were analyzed.The results showed that the optimal infection time of Agrobacterium EHA105 was 50 min with a 67.5%positive rate of GUS.The optimal concentration of callus induction was 2.0 mg/L 6-BA with the callus induction frequency of 91.4%.The optimum hormone for inducing adventitious buds was 5.0 mg/L ZT,and the budding frequency was 62.0%.The adventitious roots induced by 0.1 mg/L IBA had the best effect,and the rooting percentage was 100.0%.The genetic transformation system of cotyledon node explants using'Jingnong6'in adzuki bean was established,and regeneration plants was obtained.This research provided support for the application of CRISPR/Cas9 gene editing technology in functional genome and improvement of adzuki bean.