甜叶菊离体保存技术研究

The Studies on the Conservation Technology in vitro of Stevia rebaudiana Bertoni

为探究适合甜叶菊种质资源中长期保存的方法,以甜叶菊无菌苗为材料,研究蔗糖、多效唑、培养温度等3个因素对甜叶菊无菌苗离体保存的影响,并对保存后材料生根继代培养,进而运用生化和分子标记技术鉴定保存材料的遗传稳定性。研究结果表明,在MS+1.0 mg/L 6-BA+0.2 mg/L NAA+琼脂5.0 g/L基础保存培养基和1 000 Lx、12 h/d的培养条件下,30 g/L蔗糖、2.0 mg/L多效唑以及培养温度为10℃~15℃时,能有效抑制甜叶菊无菌苗生长、延长其保存时间,保存12个月试管苗存活率为70.4%~78.5%;离体生根后再生苗生长旺盛,形态正常,其过氧化物酶酶谱和SRAP分子标记扩增图谱与对照苗无明显差异,遗传性稳定。本研究结果为延缓甜叶菊种苗生长、种质资源离体保存及遗传稳定性提供了理论依据和技术参考。

In order to explore the method of medium and long term conservation ofStevia rebaudiana germplasm resources, the aseptic seedlings of Stevia rebaudiana were used as experimental materials in this study to explore the effects of sucrose, PP333 and culture temperature on the conservation of Stevia rebaudiana in vitro. The genetic stability of the preserved materials was evaluated by biochemical and molecular marker techniques after rooting and subculture. The results showed that the seedlings growth were effectively inhibited and their conservation time was prolonged by the medium MS＋6-BA 1.0 mg/L＋NAA 0.2 mg/L＋agar 5.0 g/L supplemented with 30 g/L sucrose and 2.0 mg/L PP333 under temperature 10℃ ~15℃ in the condition of 1 000 Lx illumination intensity and 12 h/d photopcriod. The survival rate of aseptic seedlings which were conserved for 12 months was 70.4%-78.5%. The regenerational seedlings grew well and the morphology of regenerational seedlings were normal after conservation seedlings subculture. The peroxidase isoenzyme patterns and SRAP-PCR amplification profiles had no significant differences with the regenerational seedlings of the control （non-preserved）. In addition, the heredity was stable. This study could provide theoretical basis and technical reference for postponing the growth of conservation seedlings, in vitro conservation of germplasm resources, and genetic stability of Stevia rebaudiana.