番茄组培苗的不同阶段对抗生素和PPT的抗性筛选试验

Screening Test of Resistance to Antibiotics and PPTat Different Stages of Tomato Tissue Culture

以番茄高效再生体系为基础,在番茄子叶和下胚轴的出愈、芽分化生长以及生根的各阶段培养基中,分别添加不同质量浓度的卡那霉素、新霉素和除草剂PPT三种选择性试剂,以确定番茄遗传转化所需的最佳筛选质量浓度.试验表明:T79和T151两个自交系对卡那霉素、新霉素、除草剂的抗性差异不大;卡那霉素与PPT在组培的各个阶段均对外植体有强烈的抑制作用,而新霉素对它们的抑制作用则不明显;在番茄组培的不同阶段、不同外植体对卡那霉素和PPT的反应存在较大差异,以下胚轴出愈对卡那霉素反应最敏感,子叶出愈和生根阶段对其反应迟钝;下胚轴出愈和芽分化对PPT的反应较敏感,子叶出愈对其反应较迟钝.因此建议在番茄遗传转化中,卡那霉素对子叶出愈和生根阶段的筛选质量浓度为100mg·L-1,对下胚轴出愈为50mg·L-1,对芽分化及其生长为70mg·L-1;而除草剂PPT对子叶出愈阶段的筛选质量浓度为1.0mg·L-1,对下胚轴出愈为0.5mg·L-1,对芽分化和生根阶段为0.75mg·L-1.

Based on the high efficienct regeneration system of tomato, 3 kinds of screening reagents-kanamycin(Kan), neomycin(Neo), and herbicide PPT were respectively added into each medium of tomato tissue culture through different massconcentrations during the stages of the callus formation of tomato's cotyledon and hypocotyl, the differentiation and growth of tomato's bud, and the rootgrowth of tomato plantlets, so that the optimum screening massconcentrations needed by genetic transformation of tomato could be determined. The experiment showed that there was not distinct difference in resistance to above reagents between the two selfbred lines-T79 and T151; at each stage of tomato tissue culture, both kanamycin and PPT had a very stronger inhibition on explants, while neomycin had no obvious inhibition on them; on the other hand, during the different stages, there existed obvious difference in different explants' reaction to kanamycin and PPT, and particularly the callus formation of hypocotyl was the most susceptible to Kan, though the callus formation of cotyledon and the rootgrowth of plantlets were unsusceptible to it; in addition, the callus formation of hypocotyl and the differentiation of buds were susceptible to PPT, though the callus formation of cotyledon was unsusceptible to it. Hence, it was suggested that for genetic transformation of tomato, Kan 100 mg·L-1 should be selected as ideal screening massconcentration at the stage of hypocotyl callus inducing and plantlets' rootgrowth, Kan 50 mg·L-1 at the stage of hypocotyl callus inducing; and Kan 70 mg·L-1 at the stage of bud differentiation. However, for PPT, 1.0 mg·L-1 should be taken as ideal screening massconcentration at the stage of cotyledon callus inducing, 0.5 mg·L-1 at the stage of hypocotyl callus inducing; and 0.75 mg·L-1 at the stage of bud differentiation and plantlets' rootgrowth.