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基因枪介导的转基因油棕生产

Biolistic mediated production of transgenic oil palm.

作者信息

Kadir Ahmad Parveez Ghulam

机构信息

Advanced Biotechnology and Breeding Centre, Biological Research Division, Malaysian Palm Oil Board, Kuala Lumpur, Malaysia.

出版信息

Methods Mol Biol. 2008;477:301-20. doi: 10.1007/978-1-60327-517-0_23.

Abstract

Physical and biological parameters affecting DNA delivery into oil palm embryogenic calli using the biolistic device are optimized. Five different promoters are also evaluated to identify the most suitable promoter for use in oil palm transformation. Finally, the effectiveness of kanamycin, geneticin (G418), neomycin, hygromycin, and herbicide Basta as selection agents to inhibit growth of oil palm embryogenic calli is evaluated. Combination of optimized parameters, best promoter and selection agent is later used to transform oil palm embryogenic calli for producing transgenic oil palm plants. Bombarded embryogenic calli are exposed to 50 mg/l of Basta after 3 weeks. Basta-resistant embryogenic calli started to emerge five to six months in medium containing Basta. The Basta-resistant embryogenic calli are proliferated until they reach a specific size, and the Basta-resistant calli are later individually isolated and regenerated to produce complete plantlets. The complete regenerated plantlets are evaluated for the presence of transgenes by PCR, Southern and thin layer chromatography analyses.

摘要

优化了使用生物射弹装置将DNA导入油棕胚性愈伤组织的物理和生物学参数。还评估了五种不同的启动子,以确定最适合用于油棕转化的启动子。最后,评估了卡那霉素、遗传霉素(G418)、新霉素、潮霉素和除草剂Basta作为选择剂抑制油棕胚性愈伤组织生长的有效性。随后,将优化参数、最佳启动子和选择剂的组合用于转化油棕胚性愈伤组织,以生产转基因油棕植株。轰击后的胚性愈伤组织在3周后暴露于50mg/l的Basta中。在含有Basta的培养基中,抗Basta的胚性愈伤组织在五到六个月后开始出现。抗Basta的胚性愈伤组织增殖直至达到特定大小,随后将抗Basta的愈伤组织单独分离并再生以产生完整的植株。通过PCR、Southern和薄层色谱分析评估完整再生植株中转基因的存在情况。

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