农杆菌侵染条件对柱花草遗传转化效率的影响

在前期建立的柱花草高效组织再生体系基础上,进一步以柱花草热研5号的下胚轴为受体材料,以 GUS基因为报告基因,研究了农杆菌介导的柱花草遗传转化的几个影响因素,包括根癌农杆菌菌液浓度、浸染时间和共培养时间等对农杆菌介导的柱花草遗传转化效率的影响。结果表明,以柱花草下胚轴为外植体,在农杆菌菌液浓度(OD₆₀₀)为0.4~0.6,农杆菌浸染时间为15 min和共培养3 d的条件下,愈伤的转化效率为72%。愈伤组织进一步经过分化、生根和炼苗等过程后,对转化植株进行GUS染色和PCR检测,结果表明外源基因已成功整合到柱花草基因组中。本研究结果对柱花草遗传转化和关键基因功能研究具有重要的参考价值。

Factors affecting genetic transformation efficiency for stylo (Stylosanthes guianensis) with Agrobacterium tumefaciens

The objective of this study was to investigate effects of Agrobacterium concentration,infection time and co-culture duration on the genetic transformation efficiency for stylo (Stylosanthes guianensis )using a high-efficiency regeneration system that had been developed previously.Hypocotyls from stylo seedlings were used as plant material and theβ-glucuronidase (GUS)gene was used as the reporter gene.The callus transfor-mation efficiency was evaluated by GUS staining.The callus transformation efficiency reached 72% under the following conditions:Agrobacterium at a concentration of OD600 =0.4-0.6,with 15 mins infection and 3 days of co-cultivation.The exogenous gene had been successfully introduced into the stylo genome as indicated by GUS staining and PCR detection after callus selection,shoot differentiation and rooting.The results indicate that this A.tu-mefaciens-mediated transformation system is suitable for genetic improvement and functional gene analysis of stylo.