Dabauza Mercedes, Velasco Leonardo
Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), Murcia, Spain.
Methods Mol Biol. 2012;847:227-35. doi: 10.1007/978-1-61779-558-9_19.
Genetic engineering of grapevine is a powerful tool to study gene function as well as to introduce new traits into existing Vitis cultivars without altering their essential characters and identity. Agrobacterium-mediated transformation is one of the most efficient methods for gene transfer, but the efficiency of the procedure depends on several parameters such as the grapevine genotype, the selection strategy, the Agrobacterium strain, and concentration used to infect as well as the culture method among others. This chapter describes highly efficient genetic transformation protocols for seedless table grapevine cultivars Sugraone and Crimson Seedless by co-culturing embryogenic calli with Agrobacterium tumefaciens. The procedures are specific for each cultivar by adjusting the kanamycin concentration used to select transformed cells (20 mg/L and 50 mg/L kanamycin for Crimson Seedless and Sugraone, respectively) and the low Agrobacterium density used to infect the embryogenic calli (0.06 OD(600) being more effective for the transformation of Crimson Seedless and 0.2 OD(600) for Sugraone). Other factors that affect the transformation efficiency are the initial amount of embryogenic calli used to co-culture with Agrobacterium and the culture method of calli.
葡萄基因工程是研究基因功能以及将新性状引入现有葡萄品种而不改变其基本特征和特性的有力工具。农杆菌介导的转化是最有效的基因转移方法之一，但该过程的效率取决于几个参数，如葡萄基因型、选择策略、农杆菌菌株、用于感染的浓度以及培养方法等。本章描述了通过将胚性愈伤组织与根癌农杆菌共培养，对无核鲜食葡萄品种苏格拉诺（Sugraone）和深红无核（Crimson Seedless）进行高效遗传转化的方案。通过调整用于选择转化细胞的卡那霉素浓度（深红无核为20 mg/L卡那霉素，苏格拉诺为50 mg/L卡那霉素）以及用于感染胚性愈伤组织的低农杆菌密度（600 nm波长下吸光度为0.06 OD对深红无核的转化更有效，苏格拉诺为0.2 OD），这些方案针对每个品种都是特定的。影响转化效率的其他因素包括用于与农杆菌共培养的胚性愈伤组织的初始量以及愈伤组织的培养方法。
