一种根癌农杆菌介导的耐盐椒样薄荷含芽茎段转化系统

An Efficient Transformation of Salinity-resistant Peppermint Stem Mediated with Agrobacterium tumefaciens

椒样薄荷的转基因技术是提高精油产量和品质的重要手段,为解决耐盐椒样薄荷叶片不定芽诱导过程中易褐化,难分化,不适合作为转基因外植体的问题,利用含芽的椒样薄荷茎段作为外植体建立了一种根癌农杆菌介导的外源基因转化体系。利用正交试验对转化所用的根癌农杆菌菌液浓度、转化时间和共培养时间进行优化,结果显示:3个因素对转化效果的影响作用表现为转化时间(B)>菌液浓度(A)>共培养时间(C),筛选出的最优转化条件为菌液浓度为OD_(600)=0.8,转化时间为15 min,共培养时间为3 d。对不同转化液组分进行优化筛选,结果获得最适的转化液组分为:1/5MS+0.5 g/L MES(2-(N-吗啡啉)乙磺酸)+1%葡萄糖+2%蔗糖,PH5.4。利用不同植物表达载体进行转化,并通过基因组PCR和GUS染色进行转化植株的阳性检测,结果表明该转化体系适用于多种植物表达载体的转化,容易获得再生植株,为耐盐椒样薄荷的基因转化提供新的方法与体系。

Transgenic technology is an important means to improve the essential yield and quality of peppermint（Mentha piperitaL.）oil. An Agrobacterium tumefaciens-mediated transformation using bud peppermint stem was established,in order to solve the problemsin adventitious bud induction of salinity-resistant peppermint leave,such as easy browning and difficult differentiation. The orthogonal testwas carried out to screen the optimal concentration of A. tumefaciens,transformation time and co-culture time. The results showed that theeffect of the three factors on transformation displayed as the transformation time（B） bacterial concentration（A） co-culture time（C）.The bacterial concentration OD_（600） = 0.8,the transformation time 15 min and the co-culture time 3 d proved to be the optimal transformationconditions. The optimal composition of transformants was screened to be 1/5MS ＋ 0.5 g/L MES（2-（N-morpholino）ethanesulfonic acid）＋1% glucose ＋ 2% sucrose and PH 5.4. The transformation was conducted using different plant expression vectors,and the positive plants weredetected by genomic PCR and GUS staining. And the results revealed that this transformation system was suitable for the transformation of manyplant expression vectors,and the regenerated plants were easily obtained. This provides a new method and system for gene transformation ofsalinity-resistant peppermint.