Krasnyanski S, May R A, Loskutov A, Ball T M, Sink K C
NRES, University of Illinois, IL 61801, Urbana, USA, US.
Theor Appl Genet. 1999 Aug;99(3-4):676-82. doi: 10.1007/s001220051284.
Agrobacterium-mediated and direct gene transfer into protoplasts using PEG were both successfully used to produce stable, transformed peppermint plants (Mentha×piperita L. cultivar Black Mitcham) with the limonene synthase gene. Stem internode explants found to possess a high level of organogenesis through adventitious shoot formation were subjected to Agrobacterium tumefaciens disarmed strain GV3101 (pMP90). Following the development of an efficient protoplast-to-plant cycle from stem-isolated protoplasts, they were used in direct gene transformations. In both cases the binary vector pGA643 carrying the nptII/GUS genes, both driven by the CaMV35S promoter, was used in preliminary plant-transformation studies. Later, GUS was replaced with the limonene synthase gene. Kanamycin was used as a selective agent in all transformation experiments to obtain both transformed protoplast-derived calli as well as putative transgenic shoots regenerated from internode explants. Both types of transformation resulted in transgenic plants which were detected using PCR and confirmed by Southern-blot hybridizations. Southern analysis revealed that the method of Agrobacterium-mediated transformation is superior to the direct DNA uptake into protoplasts with regard to the stability of the insert during the transformation event. Single transgenic plants were grown to 10% flowering in a greenhouse and the plants derived both by the Agrobacterium and the protoplast-derived methods were generally observed to have essential oil profiles characterized by a high-menthone, low-menthol, high-menthofuran and -pulegone content in comparison to a typical mid-west peppermint. Limonene varied only slightly, around 1.2%, in transgenic plants produced by both methods.
农杆菌介导法和使用聚乙二醇（PEG）将基因直接导入原生质体的方法都成功用于培育携带柠檬烯合酶基因的稳定转化薄荷植株（薄荷×辣薄荷L.品种黑米奇姆）。通过不定芽形成发现具有高水平器官发生能力的茎节间外植体用根癌农杆菌无致病力菌株GV3101（pMP90）处理。从茎分离的原生质体建立了高效的原生质体到植株周期后，将其用于直接基因转化。在这两种情况下，携带由花椰菜花叶病毒35S启动子驱动的nptII/GUS基因的双元载体pGA643用于初步的植物转化研究。后来，GUS被柠檬烯合酶基因取代。在所有转化实验中，卡那霉素用作选择剂，以获得转化的原生质体来源的愈伤组织以及从节间外植体再生的推定转基因芽。两种转化类型都产生了转基因植株，通过聚合酶链反应（PCR）检测并通过Southern杂交确认。Southern分析表明，就转化事件中插入片段的稳定性而言，农杆菌介导的转化方法优于将DNA直接导入原生质体的方法。单个转基因植株在温室中生长至10%开花，与典型的中西部薄荷相比，通过农杆菌法和原生质体衍生法获得的植株通常观察到具有以高薄荷酮、低薄荷醇、高薄荷呋喃和胡薄荷酮含量为特征的精油谱。两种方法产生的转基因植株中柠檬烯含量仅略有变化，约为1.2%。
