A high throughput Agrobacterium tumefaciens-mediated transformation method for functional genomics of perennial ryegrass (Lolium perenne L.)
A high throughput Agrobacterium tumefaciens-mediated transformation method for functional genomics of perennial ryegrass (Lolium perenne L.)
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DOI：
10.1007/s00299-005-0099-9
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发表时间：
2006-07-01
影响因子：
6.2
通讯作者：
Puthigae, Sathish
中科院分区：
文献类型：
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作者：
Bajaj, Shivendra;Ran, Yidong;Puthigae, Sathish
以多年生黑麦草为材料，建立了一种高效的农杆菌遗传转化体系。选择胚性愈伤组织系并保持为离体植株。将来自营养分蘖分生组织区的胚性愈伤组织与携带质粒pCAMBIA 1305.1的根癌农杆菌菌株EHA 101在乙酰丁香酮存在下共培养3-4天。愈伤组织分别在94.8和151.6 μ M潮霉素选择下生长两个周期，每个周期2周，然后转移到含有47.4 μ M潮霉素的再生培养基上。再生的植物生根并成功转移到土壤中。DNA凝胶印迹分析证实了再生植株的转基因性质，GUS组织化学分析和/或逆转录PCR证实了基因表达。在开发转化程序后，我们使用携带修饰的含有目的基因的二元质粒pMH的农杆菌菌株EHA 101。在过去的2年中，我们已经生产了1,000多株植物，这些植物具有编码来自多年生黑麦草的不同感兴趣基因的构建体。
A robust and high throughput Agrobacterium genetic transformation procedure has been developed for perennial ryegrass (Lolium perenne L.). Embryogenic callus lines were selected and maintained as plants in vitro. Embryogenic calli derived from meristematic regions of the vegetative tillers were co-cultivated with Agrobacterium tumefaciens strain EHA101 carrying the plasmid pCAMBIA 1305.1 in the presence of acetosyringone for 3-4 days. The calli were grown under 94.8 and 151.6 mu M hygromycin selection, respectively for two cycles of 2-weeks each, followed by transfer to regeneration medium with 47.4 mu M hygromycin. Regenerated plants were rooted and successfully transferred to soil. The transgenic nature of the regenerated plants was confirmed by DNA gel blot analysis and gene expression demonstrated by GUS histochemical assay and/or reverse transcription PCR. After development of the transformation procedure, we used Agrobacterium strain EHA101 carrying a modified binary plasmid pMH bearing genes of interest. In the past 2 years, we have produced more than 1,000 plants with constructs encoding different genes of interest from perennial ryegrass.