【摘要】 用五种不同的根癌土壤杆菌转化咖啡栽培品种小粒种的成熟胚培养物,筛选出对咖啡侵染力较强的菌株GV3111(pTiB6S3)和A281(pTiB0542)。在无激素的MS培养基(MSD)上得到淡黄色、松脆、生长迅速的瘤性愈伤组织,继代培养4—6周后,产生大量的畸胎芽。转化体经Opine检测,分别含有特异的章鱼碱或农杆碱。GV3111转化体的DNA分子杂交结果表明,在咖啡的基因组内有T-DNA的插入。用带有GUS基因和NPT Ⅱ基因的双元载体系统pBI121/GV3111和pBI121/A281转化咖啡子叶,转化后第三天可以检测到GUS基因的瞬时表达。在含卡那霉素(Kanamyvin,Km)的MS选择培养基上,得到抗性愈伤组织,这些愈伤组织仍能检测到GUS基因的表达。更多还原

【Abstract】 We have transformed embryo-derived culture of coifee cultivars (Coffee arabica L.) using five different Agrobacterium tumefaciens. Two kinds of strain GV3111 (pTiB6S3) and A281 (pTiBo542) have been screened out in regard to their high infection capacity. Lightyellow, fragil and fast grow of tumorigenic callus was formed on the hormone-free MS medium. A mass of teratoma-like shoots developed from the tumorigenic callus after subculture 4—6 weeks on hormone-free medium. Opine detection showed all teratoma-like shoots presented specific octopine or agropine. Southern hybridization of DNA from transgenic tissue of strain GV3111 confirmed the presence of DNA fragments containing TDNA sequence in the coffee genome. Coffee cotyledons was transformed using binary vector system of PBI121/GV3111 and PBI121/A281, which containing GUS gene and NPT Ⅱ gene. Three days after transformation, the transient expression of GUS gene was detected. The kanamycin-resistant callus have been obtained on the medium of MS+ 100mg/l km. and the expression of GUS gene was also detected.更多还原