3种农杆菌对茶树发状根诱导的影响

以茶树‘福云6号’和‘铁观音’成熟种子下胚轴、未成熟种子下胚轴和愈伤组织为材料,以发状根诱导率为指标,探究菌液浓度、农杆菌菌株、外植体类型和预培养时间对发状根诱导的影响。结果表明:(1)菌液浓度OD600在0.4~1.2范围内,‘福云6号’成熟种子下胚轴发状根诱导率先升高后降低,ATCC15834在OD600为0.6时发状根诱导率最高为23.96%,A4和K599在OD600为0.8时,发状根诱导率最高,分别为10.51%和13.11%。(2)3种发根农杆菌致根能力不同,ATCC15834侵染力最强,致根能力大小依次为ATCC15834>K599>A4。(3)发状根诱导率与外植体有关,茶树成熟种子下胚轴可被诱导产生发状根,诱导率‘福云6号’大于‘铁观音’,未成熟种子下胚轴和愈伤组织难以产生发状根。(4)不经预培养和预培养时间为1~2 d的愈伤组织易褐变,无法产生发状根;预培养3 d的愈伤组织经侵染可产生发状根,诱导率为1.85%(‘福云6号’)和0.59%(‘铁观音’)。(5)PCR琼脂糖凝胶电泳检测和GUS组织化学染色证实GUS基因已被整合进‘福云6号’和‘铁观音’愈伤组织和成熟种子下胚轴发状根基因组中并表达。研究发现,发根农杆菌ATCC15834在OD600为0.6时,对茶树‘福云6号’成熟种子下胚轴发状根诱导率最高。该研究结果对改进发根农杆菌介导的茶树遗传转化系统提供理论依据,对茶树高效遗传转化体系的优化和新型受体的开发具有重要意义。

Induction of Hairy Roots of Tea Plant by Three Kinds of Agrobacterium rhizogenes

The hypocotyls of mature seeds,hypocotyls of immature seeds and callus of tea‘Fuyun 6’and‘Tieguanyin’were used as materials,and the hairy root induction rate was used as an indicator to explore the bacterial concentration,Agrobacterium rhizogenes strain,explant type and pre-culture time on the induction of hairy roots.The results showed that:(1)the induction rate of hairy roots increased first and then decreased when the bacterial concentration OD600 was in the range of 0.4 to 1.2.At OD600=0.6,ATCC15834 had the highest hairy root induction rate of 23.96%,while at OD600=0.8,A4 and K599 had the highest hairy root induction rates of 10.51%and 13.11%,respectively.(2)All three kinds of A.rhizogenes can induce hairy roots in tea plants,but their rooting abilities are different,of which ATCC15834 has the strongest rooting ability,for ATCC15834,K599 and A4,the ability of hairy root induction on tea plant was declining in turn.(3)The induction rate of hairy roots is related to the explants.The hypocotyls of mature seeds of tea plants can be induced to produce hairy roots,and the induction rate is‘Fuyun 6’>‘Tieguanyin’,while the hypocotyls of immature seeds and callus are difficult to produce hairy roots.(4)Callus without pre-culture and the pre-culture time of 1-2 days is easy to brown and cannot produce hairy roots,while callus pre-cultured for 3 days can produce hairy roots after infection,with an induction rate of 1.85%(‘Fuyun 6’)and 0.59%(‘Tieguanyin’).(5)Genomic integration and transgene expression of hairy roots of mature cotyledons of‘Fuyun 6’and‘Tieguanyin’were confirmed by polymerase chain reaction(PCR)detection and beta-glucuronidase(GUS)assays.The above results showed that ATCC15834 had the highest induction rate of hairy roots with hypocotyls of mature seeds of‘Fuyun 6’when OD600 was 0.6.These findings provide a theoretical basis for improving the tea tree genetic transformation system mediated by Agrobacterium rhizogenes,and is of great significance to the optimization of the high efficiency genetic transformation system and the development of new receptors in tea plants.