Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620024, Tamil Nadu, India.
Plant Cell Rep. 2013 Oct;32(10):1557-74. doi: 10.1007/s00299-013-1467-5. Epub 2013 Jun 8.
An efficient, reproducible and genotype-independent in planta transformation has been standardized for sugarcane using seed as explant. Transgenic sugarcane production through Agrobacterium infection followed by in vitro regeneration is a time-consuming process and highly genotype dependent. To obtain more number of transformed sugarcane plants in a relatively short duration, sugarcane seeds were infected with Agrobacterium tumefaciens EHA 105 harboring pCAMBIA 1304-bar and transformed plants were successfully established without undergoing in vitro regeneration. Various factors affecting sugarcane seed transformation were optimized, including pre-culture duration, acetosyringone concentration, surfactants, co-cultivation, sonication and vacuum infiltration duration. The transformed sugarcane plants were selected against BASTA(®) and screened by GUS and GFP visual assay, PCR and Southern hybridization. Among the different combinations and concentrations tested, when 12-h pre-cultured seeds were sonicated for 10 min and 3 min vacuum infiltered in 100 µM acetosyringone and 0.1 % Silwett L-77 containing Agrobacterium suspension and co-cultivated for 72-h showed highest transformation efficiency. The amenability of the standardized protocol was tested on five genotypes. It was found that all the tested genotypes responded favorably, though CoC671 proved to be the best responding cultivar with 45.4 % transformation efficiency. The developed protocol is cost-effective, efficient and genotype independent without involvement of any tissue culture procedure and can generate a relatively large number of transgenic plants in approximately 2 months.
已针对甘蔗，通过种子外植体，标准化了一种高效、可重现且与基因型无关的体内转化方法。利用根癌农杆菌感染和体外再生进行转基因甘蔗生产是一个耗时且高度依赖基因型的过程。为了在相对较短的时间内获得更多数量的转化甘蔗植株，使用携带 pCAMBIA 1304-bar 的根癌农杆菌 EHA 105 感染甘蔗种子，并成功地在不进行体外再生的情况下建立了转化植株。优化了影响甘蔗种子转化的各种因素，包括预培养时间、乙酰丁香酮浓度、表面活性剂、共培养、超声处理和真空渗透时间。利用 BASTA(®)对转化的甘蔗植株进行选择，并通过 GUS 和 GFP 可视检测、PCR 和 Southern 杂交进行筛选。在所测试的不同组合和浓度中，当 12 小时预培养的种子在 100 µM 乙酰丁香酮和 0.1% Silwett L-77 中的根癌农杆菌悬浮液中超声处理 10 分钟和真空渗透 3 分钟，并在共培养 72 小时时，显示出最高的转化效率。该标准化方案的适用性已在五个基因型上进行了测试。结果发现，所有测试的基因型都反应良好，尽管 CoC671 被证明是反应最好的品种，转化率为 45.4%。所开发的方案具有成本效益、高效且与基因型无关，无需涉及任何组织培养程序，大约在 2 个月内可产生相对大量的转基因植株。
