转基因杜仲再生体系的优化

Optimization of Regeneration System of Transgenic Eucommia Ulmoides Oliver

为了提高杜仲不定芽的获得率及生根率,选用B5、MS和WPM 3种培养基,以杜仲下胚轴为外植体,利用农杆菌介导pGM 626-Act1-EuDIR3和pSH 737-35S-EuDIR1遗传转化杜仲,并将浸染后的各外植体分别接种于3种不同培养基上,观察记录各培养基上外植体的生长情况,通过GUS染色检测和PCR验证统计得到阳性芽数。结果表明:转化质粒在B5、MS和WPM培养基上的平均出芽率分别为(14.79±6.20)%、(7.68±2.37)%和(4.05±0.64)%,相较于MS和WPM培养基,B5培养基上的愈伤组织生长旺盛,色泽鲜艳呈绿色,质地紧密脆嫩,平均出芽率显著高于另外2种培养基,但是WPM培养基的生根率显著高于另外2种培养基,WPM和B5培养基的生根率分别为53.85%和3.57%,在MS培养基上的阳性芽并未生根。因此,在杜仲遗传转化过程中,B5培养基更适合诱导不定芽,WPM培养基利于不定芽生根。本研究优化了转基因杜仲再生体系,为杜仲基因功能研究提供技术支持。

In order to increase the yield and rooting rate of adventitious buds of Eucommia ulmoides Oliver,three different basal media were used to culture genetically transformed explants.Using the hypocotyl of Eucommia ulmoides as the explant, agrobacterium-mediated pGm 626-Act1-EuDir3 and pSH 737-35 S-EuDIR1 were used to genetically transform Eucommia ulmoides. The explants were inoculated in three different culture mediums to observe and record the growth of the explants, and numbers of successfully transformed buds were obtained by GUS detection and PCR verification. Results indicated that the average germination rates of the transforming plasmids on B5, MS and WPM media were 14.79±6.20%, 7.68±2.37% and 4.05±0.64%, respectively. Compared with MS and WPM media, callus on B5 medium grew vigorously with bright green color and tight crisp texture, whose germination rate was significantly higher than the other two media, but the rooting rate of WPM medium was significantly higher than the other two kinds of culture medium, the rooting rate of WPM and B5 medium were 53.85% and 3.57% respectively, positive buds on MS medium did not take root. Therefore, in the process of genetic transformation of Eucommia ulmoides, B5 medium was more suitable for inducing adventitious buds during the genetic transformation of Eucommia ulmoides Oliver, while WPM medium was beneficial to rootage of adventitious buds. This study optimizes regeneration system of transgenic Eucommia ulmoides Oliver and provides technical support for gene function research of Eucommia ulmoides.