Parveez Ghulam Kadir Ahmad, Bahariah Bohari
Advanced Biotechnology and Breeding Centre, MPOB, Kajang, Selangor, Malaysia.
Methods Mol Biol. 2012;847:163-75. doi: 10.1007/978-1-61779-558-9_14.
The effectiveness of mannose (using phosphomannose isomerase [pmi] gene) as a positive selection agent to preferably allow the growth of transformed oil palm embryogenic calli was successfully evaluated. Using the above selection agent in combination with the previously optimized physical and biological parameters and the best constitutive promoter, oil palm embryogenic calli were transformed with pmi gene for producing transgenic plants. Bombarded embryogenic calli were exposed to embryogenic calli medium containing 30:0 g/L mannose to sucrose 3 weeks postbombardment. Selectively, proliferating embryogenic calli started to emerge around 6 months on the above selection medium. The proliferated embryogenic calli were individually isolated once they reached a specific size and regenerated to produce complete plantlets. The complete regenerated plantlets were evaluated for the presence of transgenes by PCR and Southern analyses.
成功评估了甘露糖(使用磷酸甘露糖异构酶[pmi]基因)作为阳性选择剂以优先促进转化油棕胚性愈伤组织生长的有效性。将上述选择剂与先前优化的物理和生物学参数以及最佳组成型启动子相结合,用pmi基因转化油棕胚性愈伤组织以产生转基因植株。轰击后的胚性愈伤组织在轰击后3周暴露于含有30.0 g/L甘露糖与蔗糖的胚性愈伤组织培养基中。选择性地,增殖的胚性愈伤组织在上述选择培养基上约6个月开始出现。增殖的胚性愈伤组织一旦达到特定大小就被单独分离,并再生以产生完整的植株。通过PCR和Southern分析评估完整再生植株中转基因的存在情况。