根癌农杆菌介导的金发草遗传转化条件的优化

金发草(Pogonatherum paniceum)是一种多年生岩生草本植物,在生态恢复和景观建设中起着重要的作用。利用根癌农杆菌介导转化金发草胚性愈伤组织,通过GUS(β-葡萄糖苷酸酶)瞬时表达率研究菌液浓度、浸染时间、乙酰丁香酮(AS)浓度、葡萄糖浓度、共培养时间等因素对金发草转化的影响,并利用确定的最佳条件将GUS基因转入金发草,获得稳定表达转化植株。结果表明:菌液浓度(OD₆₀₀)为0.6,浸染时间为10min,添加20mg/L的乙酰丁香酮(AS)和10g/L的葡萄糖,共培养时间5d为最佳条件,GUS瞬时表达率最高。经过抗性筛选后最终获得阳性转基因植株频率为57%。再生植株经GUS染色和PCR检测证明,GUS基因已成功整合到金发草基因组中。此转化体系的建立为金发草的遗传改良及相关功能基因的研究奠定了基础。

Optimization of Genetic Transformation Conditions in Pogonatherum paniceum Mediated by Agrobactrium tumefaciens

A perennial herbaceous plant, Pogonatherum paniceum, plays an important role inecological restoration and landscape construction. To establish highly efficient transformation system, factors which influenced transformation including Agrobacterium concentration, infection time, concentration of acetosyringone and glucose, and co-culture time were studied with the mature embryo callus of Pogonatherum paniceum based on the variance of GUS (β-glucuronidase) trsnsient expression rate. The results showed that the transformation efficiency was improved with Agrobacterium concentration of OD₆₀₀=0.6, incubation for 10 min, co-culture time for 5d. Acetosyringone with the concentration of 20mg/L and 10g/L glucose could significantly enhance the transformation rate. Utilizing this system, more than 57% kanamycin resistance selection efficiency was obtained. GUS gene has successfully integrated into the plant genome by GUS and PCR detection. The establishment of this transformation system lays a foundation for genetic modified and study of functional genes of Pogonatherum paniceum.