摘要
本研究以柱型苹果"鲁加6号"试管苗叶片外植体为转化材料,以卡那霉素抗性基因为选择标记,建立了根癌农杆菌介导的柱型苹果"鲁加6号"苹果的遗传转化体系。研究结果表明:在MS+6-BA4.0mg/L+NAA0.05mg/L培养基上,叶片不定芽分化阶段Km的选择压力为20mg/L,不定芽生根阶段Km的选择压力为15mg/L,脱菌培养阶段头孢霉素的适宜浓度为300mg/L。在农杆菌介导的遗传转化过程中,外植体经过36h预培养,侵染10min,共培养48h时,有利于提高遗传转化效率,获得的抗性愈伤组织和抗性芽数均最高。获得的Km抗性植株经PCR检测,扩增出特异条带,初步证明目的基因已整合到苹果基因组DNA中。
In this research,we established an Agrobacterium tumfaciens-mediated genetic transformation system of columnar apples named Lujia No.6,which in vitro cultured leaves were used as the explants for genetic transformation and the Kanamycin resistant gene as selective marker.The optimizing transformation system would be based on the culture media of MS＋6-BA 4.0 mg/L＋NAA 0.05 mg/L with the 20 mg/L Km selective pressure for differentiating the infinitive leaf shoots and 15 mg/L best for inducing their roots.The optimal concentration of Cef was 300 mg/L during the sterile culture statge of Agrobacterium tumefaciens.Further results indicated that efficiency of genetic transformation,such as the percentages of resistant callus and resistant buds,were increased with the procedures of the 36 h pre-culture,10 min infection and 48 h co-cultivation.In this experiment we obtained some regenerating columnar apple plantlets with Kanamycin resistance.PCR validation might imply that the target gene was integrated into the genomes of tested apples based on the specific band amplified by PCR.
出处
《分子植物育种》
CAS
CSCD
2009年第6期1130-1136,共7页
Molecular Plant Breeding
基金
国家现代苹果产业技术体系项目(620836)
国家十一五科技支撑计划项目(2008BAD92B01)
山东省良种产业化工程项目共同资助
关键词
柱型苹果
根癌农杆菌
遗传转化体系
Columnar apple Agrobacterium tumfaciens Genetic transformation
