反义AcInv基因转化马铃薯方法的研究

马铃薯反义AcInv基因,采用农杆菌介导法,选用生产上推广的9个普通四倍体栽培种,对影响转化的各个因素进行了优化研究。结果表明:在愈伤组织诱导过程中,卡那霉素(Km)的适宜浓度为100 mg/L;生根过程中为50 mg/L。外植体的预培养为:茎段2 d,叶盘3 d效果较好;农杆菌浓度当OD600值为0.5时对茎段和叶盘的转化效果均较佳;茎段和叶盘侵染时间分别达8 min和10 min时转化率较高;外植体与农杆菌共培养时间分别为2 d和3 d时,茎段和叶盘的转化率较高;各个品种的外植体在卡那霉素抗性培养基上均能形成愈伤组织,但只有1号(“大西洋”)最后从愈伤组织上分化成苗,形成正常植株,植株再生率为11.2 %,PCR扩增检测表明大部分转基因植株为阳性。

Methods of transformation of AcInv gene into potato plants mediated by Agrobacterium tumefaciens

AcInv antisense gene was transformed by the Agrobacterium tumefaciens, LBA 4404 in which the plasmid pBIRA was as the vector for the gene expression. The results showed that: The selected callus induction media for the two kinds of explants was: MS+NAA 0.5 mg/L + 6BA 2.25 mg/L+GA310 mg/L, regeneration media was: MS + 6BA 2.25 mg/L + GA3 10 mg/L, the selected media for rooting was:MS+NAA 0.1 mg/L. The selective pressure during the callus induction and regeneration was Kanamycin 100 mg/L, thes selected pressure during regeneration was Kanamycin 50 mg/L. In the optimum regeneration system, the optimum bacterium concentration in value of OD600 was 0.5, it had a best promoting effect on stem segments and the leaf of discs. the optimum time of pre-culture, infection and co-culture for leaf discs were 3 days, 12 mins and 3 days respectively; to stem segments, they were 2 days, 8 mins and 2 days. Nine cultivars were chosen for transformation. In the transformation of potato using AcInv Antisense gene, transgenic plants were obtained from the callus derived from the stem segments of "Atlantic" cultivar. Using PCR methods assay showed that the regenerated plants had postive reaction, thus it could be proved that they are transformed plants, the primary detection expressed that AcInv antisense gene was transformed into potato genome.