纯合四倍体马铃薯遗传转化体系优化及转基因块茎的褐化鉴定

以农杆菌介导的反义POT32基因对纯合四倍体马铃薯不同外植体类型进行遗传转化,发现叶片预培养2d,侵染10min,共培养3d所得的抗性愈伤组织频率最高,污染频率最低。茎段预培养2～3d,侵染8min,共培养2d能够获得较高的抗性愈伤组织频率和较低的茎外植体污染率。试管微型薯在农杆菌工程菌侵染10min时获得高达52.8%的抗性愈伤组织频率。另外,1mg/LNAA 5mg/LGA3 2mg/LZT的激素配比能够有效促进不定芽分化并提高转化频率,其不定芽及转化频率分别为12.5%和8.3%。对块茎褐化的检测结果表明,转基因块茎的褐变强度和PPO活性明显低于对照(未转基因品种),且PPO活性与褐变强度及褐变指数呈正相关(r1=0.8244**,r3=0.7524*)。损伤后的转基因块茎的褐化出现时间及褐化指数明显迟于及低于对照。

Optimization Transformation System of Homozygous Tetroploid Potato and Identification Browning Degree of Transgenic Tuber

In this article, some factors of transformation were investigated. The Agrobacterium tumefaciens with anti-sense POT32 gene was used to infect different kinds of explants of homozygous tetroploid potato. The result showed that the highest resistant calli and lowest pollution frequency were obtained when leaf explants were precultivated for 2d, infected in 10min and cocultured for 3d. The same result was also obtained when stem explants were precultivated for 2 to 3 days, infected in 8min , cocultivated for 2d. Microtuber was best material for transformation, the resistant calli got up to 52.8% after infecting in 10min. In addition, the medium with 1mg/L NAA 5mg/L GA3 2mg/L zeatin could enhance the frequency of plant regeneration and transformation. It was up to 12.5% and 8.3% respectively. Otherwise, the browning degree and PPO activity was significant lower than that of control for most of transgenic tubers. The correlation was 0.8244 between browning intensity and PPO activity,and 0.7524 between browning intensity and PPO activity. The time to appear browning was later and browning index was lower than that of control after transgenic tuber were wounded.