影响虎杖毛状根高频诱导的因素探讨

Factors Affecting High-frequency Induction of Hair-like Roots of Reynoutria Japonica Houtt.

利用发根农杆菌ATCC 15834对虎杖(R eynoutria jap on ica Hou tt.)进行遗传转化,探讨影响其毛状根高频诱导的因素.结果表明:成熟叶片是转化效率较高的外植体,感染10 d左右可形成毛状根,平均诱导率为55.66%,诱根密度为4.30.经PCR检测,证实农杆菌R i质粒中的rolB及tm s基因全部转入毛状根中.改良YEB培养液活化菌种后对出根作用不显著;叶片未经预培养而直接进行感染,其出根效果要明显好于经过M S0或利用TDZ预培养处理(P<0.01),毛状根平均诱导率可达到81.84%,诱根密度为7.04;稀释菌液浸泡、超声振荡或微波辐射处理均可促使叶片提前出根,其中以超声处理30 s效果最显著(P<0.01);感染叶片上表皮向上放置进行共培养有利于毛状根形成.在优化各种转化条件后,虎杖毛状根平均诱导率可提高到97.1%,诱根密度达到11.3.

Using Agrobacterium rhizogene ATCC15834,the study transformed Reynoutria japonica to probe the factors affecting the induction of its hair-like roots. The results revealed that the mature leaf blades were the explants with high transformation frequency,which could forms hair-like roots about ten days after the inoculation with their induction rate averaging 55.66% and their root density standing at 4.30. It was proved by PCR test that the genes rolB and tins in Ri plasmid of Agrobacterium rhizogene were completely transformed into the hair-like roots. The inoculum activation by improved YEB culture solution did not significantly affected hair like root occurrence ;the leaf blades directly inoculated without being pre-cultured performed in rooting significantly better than those pre cultured with MS0 or TDZ and showed a hair- like root induction rate as high as 81.84% and an induced-root density of 7.04;soaking with diluted inoculure solution,supersonic oscillation or microwave radiation could make the leaf blades form roots early and that 30 second supersonic oscillation showed the most significant root inducing effect （P〈0.01）;The culture of the inoculated leaf blades with their epicuticles facing upwards was conducive to hair-like root formarion. With the different transformation factors optimized,the induction rate of the hair-like roots of Reynoutria japonica could be raised to 97.1% and its hair-like root density could reach 11.3.