Preliminary study on the isolation and culture of protoplast from Apocynum venetum callus | 罗布麻愈伤组织原生质体分离培养

Chinese. ［目的］ 探究罗布麻愈伤组织原生质体的分离培养及其影响因素。［方法］ 以罗布麻无菌苗子叶与下胚轴诱导的愈伤组织为材料，研究MS培养基附加不同激素组合对原生质体分离培养的影响以及愈伤组织继代培养、不同浓度纤维素酶与离析酶组合、DPD和MS等4种培养基对原生质体产量的影响。［结果］激素组合中以6-BA 0.5 mg/L + NAA 0.5 mg/L组合获得最高愈伤组织诱导率，达100%；用固体培养基进行继代培养的愈伤组织的原生质体产量高于液体培养基，尤其以从继代1次愈伤组织收集的原生质体最为适宜；纤维素酶0.30%+离析酶0.30%组合获得的原生质体产量最高，且细胞碎片较少；DPD培养基的原生质体较其他培养基的分裂得早，MS培养基的培养效果最差。［结论］ 该研究为罗布麻遗传转化、突变体筛选及体细胞融合育种提供了技术途径。

English. [Objective] The study aimed to explore the protoplast isolation and culture of callus in Apocynum venetum and the influence factors. [Method] With callus induced from the cotyledon and hypocotyls in aseptic seedlings of A. venetum as material，the effects of MS medium supplemented with different hormone combinations on the protoplast isolation and culture and the effects of the callus subculture，different concn. of cellulase enzymes combined with the maceration enzyme，4 kinds of culture mediums such as DPD and MS on the protoplast yield were researched. [Result] Hormone combination with 6-BA 0.5 mg/L + NAA 0.5 mg/L combination received the highest callus induction rate，up to 100%；The protoplast yield of the callus cultured on solid medium for subculture was higher than that of the callus cultured on the liquid medium and specially the protoplasts collected from the callus subcultured for once were most suitable. The protoplast yield obtained with the combination of cellulase enzyme 0.30% + maceration enzyme 0.30% was the highest，with the less segments of cells. The division of the protoplast cultured on DPD medium was earlier than that on other mediums and the culture effect of MS medium was the worst. [Conclusion] This study provided a technicall approach for the genetic transformation，mutant screening and somatic fusion breeding of A. venetum.