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目的:以甜叶菊组培苗为材料,研究培养温度、甘露醇及多效唑等三个因素对种质保存时再生苗生长的影响,并检测离体保存过程中甲基化水平与模式的变化。方法:离体保存试验采用正交设计法,培养温度设定5、10、15℃3个温度,甘露醇浓度设定0、20、40 g/L 3个浓度,多效唑浓度设定0、0.4、0.8 mg/L 3个浓度;甲基化水平和模式的变化采用甲基化敏感扩增多态性技术(MSAP)进行检测。结果:T4处理(10℃条件下添加20 g/L的甘露醇)能有效地减缓甜叶菊种质的生长速度,并保持较高的存活率;与对照相比,保存6个月后各处理再生苗的甲基化水平存在不同程度的降低,且T4处理的甲基化模式以超甲基化为主。结论:甜叶菊离体保存中的最佳培养体系为:MS+30 g/L蔗糖+5 g/L琼脂粉+20 g/L甘露醇的培养基中10℃低温处理,该处理条件下组培苗的表观遗传变化为甲基化水平的降低和以超甲基化模式为主。
OBJECTIVE: To study the effect of culture temperature, mannitol and paclobutrazol on the growth of regenerated seedlings when the germplasm was preserved, and to detect the change of the methylation level and pattern during the in vitro preservation process. Methods: The orthogonal design method was used in vitro. The temperature was set at 5, 10 and 15 ℃. The concentration of mannitol was set at 0, 20 and 40 g / L. The concentration of paclobutrazol was set at 0 and 0.4 , 0.8 mg / L, respectively. The changes of methylation level and pattern were detected by methylation sensitive amplification polymorphism (MSAP). Results: T4 treatment (20 g / L mannitol at 10 ℃) could effectively reduce the growth rate of Stevia germplasm and maintain a higher survival rate; compared with the control, after 6 months of storage, The methylation level of regenerated seedlings decreased to some extent, and the methylation pattern of T4 treatment was mainly hypermethylation. Conclusion: The best culture system of Stevia rebaudiana in vitro is: 10 ℃ low temperature treatment with MS + 30 g / L sucrose + 5 g / L agar powder + 20 g / L mannitol, The epigenetic changes of plantlets were the decrease of methylation level and the hypermethylation pattern.