Karthik Sivabalan, Pavan Gadamchetty, Sathish Selvam, Siva Ramamoorthy, Kumar Periyasamy Suresh, Manickavasagam Markandan
1Department of Biotechnology, Bharathidasan University, Tiruchirappalli, 620024 Tamil Nadu India.
2School of Bio Sciences and Technology, VIT, Vellore, 632014 Tamil Nadu India.
3 Biotech. 2018 Apr;8(4):202. doi: 10.1007/s13205-018-1231-1. Epub 2018 Mar 29.
infection and regeneration of the putatively transformed plant from the explant remains arduous for some crop species like peanut. Henceforth, a competent and reproducible in planta genetic transformation protocol is established for peanut cv. CO7 by standardizing various factors such as pre-culture duration, acetosyringone concentration, duration of co-cultivation, sonication and vacuum infiltration. In the present investigation, strain EHA105 harboring the binary vector pCAMBIA1301- was used for transformation. The two-stage selection was carried out using 4 and 250 mg l BASTA to completely eliminate the chimeric and non-transformed plants. The transgene integration into plant genome was evaluated by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot hybridization. Among the various combinations and concentrations analyzed, highest transformation efficiency was obtained when the 2-day pre-cultured explants were subjected to sonication for 6 min and vacuum infiltrated for 3 min in suspension, and co-cultivated on MS medium supplemented with 150 µM acetosyringone for 3 days. The fidelity of the standardized in planta transformation method was assessed in five peanut cultivars and all the cultivars responded positively with a transformation efficiency ranging from minimum 31.3% (with cv. CO6) to maximum 38.6% (with cv. TMV7). The in planta transformation method optimized in this study could be beneficial to develop superior peanut cultivars with desirable genetic traits.
对于花生等一些作物品种而言,从外植体诱导出假定转化植株并使其感染和再生仍然很困难。因此,通过对预培养时间、乙酰丁香酮浓度、共培养时间、超声处理和真空渗透等各种因素进行标准化,为花生品种CO7建立了一种高效且可重复的体内遗传转化方案。在本研究中,使用携带双元载体pCAMBIA1301-的EHA105菌株进行转化。采用4和250 mg l的BASTA进行两阶段筛选,以完全消除嵌合体和未转化的植株。通过GUS组织化学分析、聚合酶链反应(PCR)和Southern杂交来评估转基因整合到植物基因组中的情况。在分析的各种组合和浓度中,当将预培养2天的外植体在悬浮液中进行6分钟的超声处理和3分钟的真空渗透,并在添加150 µM乙酰丁香酮的MS培养基上共培养3天时,获得了最高的转化效率。在五个花生品种中评估了标准化体内转化方法的准确性,所有品种均有积极响应,转化效率范围从最低的31.3%(CO6品种)到最高的38.6%(TMV7品种)。本研究中优化的体内转化方法可能有助于培育具有理想遗传性状的优良花生品种。