石斛兰转ACS反义基因抗性原球茎及转化植株的筛选与鉴定

该试验就石斛兰转化ACS(1-氨基环丙烷-1-羧酸合成酶)反义基因的不同筛选方法和筛选处理对抗性原球茎筛选的影响,以及石斛兰转基因植株的再生与鉴定进行研究.结果表明:(1)石斛兰原球茎经带有gus报告基因和ACS反义基因的农杆菌LBA4404侵染共培养5d后除菌,采用逐渐提高选择压浓度的延迟筛选,并在低选择压浓度下切割而高选择压浓度下不切割的处理方式为抗性原球茎的最佳筛选途径,抗性原球茎获得率可达14.97％.(2)抗性原球茎繁殖时应逐渐降低选择压浓度,且在低选择压浓度下进行切割处理,繁殖倍数达到1.15倍,且原球茎生长势好.(3)抗性原球茎在1/2 MS+0.5 mg/L 6-BA培养基中的分化率达到73.85％；107株无根小苗培养于1/2 MS+1.0 mg/L NAA+50.0 mg/L Km(卡那霉素)+100.0 mg/L Cef(头孢霉素)培养基中进行生根培养,共获得了13株具有卡那霉素抗性的转化植株,转化效率达到12.15％.(4)转化植株经报告基因产物GUS组织化学检测和gus的PCR检测,证实带ACS反义基因的T-DNA已整合进石斛兰基因组中,且转基因植株在形态上与未转基因植株无明显差别,3株转基因植株移栽2个月后均已成活.

Selection and Identification of Resistant PLBs and Transgenic Plants Mediated by Agrobacterium tumefaciens with Antisense ACS Gene in Dendrobium spp.

This experiment was studied on the effect of different selection methods and selection treatments on resistant protocorm-like bodies (PLBs),the selection and examination of transgenic plant regeneration in Dendrobium spp.The results showed that:(1)The method of retarding selection with the gradual enhancement of kanamycin (Km) concentration and selecting treatments with cutting under low Km after sterilizating PLBs mediated by Agrobacterium tumefaciens strain LBA4404 with antisense ACS gene and gus gene was optimal.The rate of transgenic resistant PLBs was up to 14.97%.(2)The multiplication of resistant PLBs was cultured on medium with gradual reduction of Km concentration and cutting under low Km concentration.The rate was 1.15 and the growth of PLBs was the best.(3)The differential rate of resistant PLBs cultured on the 1/2 MS medium supplemented with 0.5 mg/L 6-BA was 73.85%,13 resistant plantlets rooted on the 1/2 MS medium supplemented with 1.0 mg/L NAA and 50.0 mg/L Km and 100.0 mg/L Cefotaxime (Cef),the transgenic rate was 12.15%.(4)GUS histochemical assay and PCR assays proved that the T-DNA containing antisense ACS gene was integrated into the genome of these resistant plantlets.There was no morphological difference between transgenic plantlets and no-transgenic plantlets.Three transgenic plantlets transferred after 2 months were survival.