Cruz-Hernández A, Litz R E, Lim M Gomez
CINVESTAV-IPN Unidad Irapuato, Apartado Postal # 629, Irapuato, Guanajuato, Mexico, , , , , , MX.
Tropical Research and Education Center, University of Florida, 18905 SW 280 St, Homestead, FL 33031, USA, , , , , , US.
Plant Cell Rep. 1998 Apr;17(6-7):497-503. doi: 10.1007/s002990050431.
Embryogenic avocado cultures were genetically transformed with the uidA (GUS) and nptII genes, and transformed somatic embryos were recovered from these cultures. Embryogenic avocado cultures derived from zygotic embryos of `Thomas' and consisting of proembryonic masses were gently separated and co-cultivated with disarmed, acetosyringone-activated Agrobacterium tumefaciens strain A208, which contained the cointegrative vector pTiT37-ASE::pMON9749 (9749 ASE). Kanamycin-resistant embryogenic suspension cultures were selected in two steps: (1) initial selection in maintenance medium, consisting of MS basal medium, supplemented with 0.1 mg l picloram and 50 mg l kanamycin sulfate for 2-4 months and (2) subsequent selection in maintenance medium with 100 mg/ml kanamycin sulfate for 2 months in order to eliminate chimeras. Somatic embryo maturation was initiated by subculture onto semisolid maturation medium (without picloram) followed by transfer to maturation medium with 100 mg l kanamycin sulfate. Genetic transformation of embryogenic cultures and somatic embryos was confirmed by the X-gluc reaction, and integration of nptII and uidA into the avocado genome was confirmed by PCR and Southern hybridization, respectively.
将携带uidA（GUS）和nptII基因的载体对胚性鳄梨培养物进行遗传转化，并从这些培养物中获得转化的体细胞胚。从‘托马斯’合子胚衍生而来的、由原胚团组成的胚性鳄梨培养物被轻轻分离，并与携带共整合载体pTiT37-ASE::pMON9749（9749 ASE）且经过减毒处理、乙酰丁香酮激活的根癌农杆菌菌株A208共培养。通过两步筛选获得抗卡那霉素的胚性悬浮培养物：（1）在维持培养基中进行初次筛选，该培养基由添加了0.1 mg/l毒莠定和50 mg/l硫酸卡那霉素的MS基本培养基组成，筛选2 - 4个月；（2）随后在添加100 mg/ml硫酸卡那霉素的维持培养基中筛选2个月，以消除嵌合体。体细胞胚的成熟通过转接至半固体成熟培养基（不含毒莠定）开始，随后转移至添加100 mg/l硫酸卡那霉素的成熟培养基中。通过X - 葡糖反应确认胚性培养物和体细胞胚的遗传转化，分别通过PCR和Southern杂交确认nptII和uidA整合到鳄梨基因组中。
