留兰香茎尖超低温保存及遗传变异分析

为了长久保存留兰香种质资源,以留兰香(Menthae spicatae L.)茎尖为材料,对其玻璃化超低温保存条件进行研究,并运用扩增片段长度多态性(AFLP)和甲基敏感扩增多态性(MSAP)对玻璃化超低温保存再生材料(处理组)的遗传与表观遗传稳定性进行分析。结果表明:留兰香试管苗于4℃低温锻炼28d,于添加2mol/L甘油的MS培养基中预培养4d,0℃下于PVS2中脱水50min,液氮保存1h或者更长时间,成活率最高,可达60%左右,再生植株分化正常;超低温处理后再生材料(处理组)与正常继代材料(对照组)之间未发现有DNA片段的差异;与对照相比,处理组的材料均发生了甲基化状态与水平的变化,全基因组DNA胞嘧啶甲基化水平降低,另外,甲基化与去甲基化位点比率分别为8.93%和12.3%,说明超低温处理能够引起DNA甲基化的变化。由此推测,DNA甲基化可能是植物适应超低温保存的机制之一。

Cryopreservation of Mentha spicata L.Germplasm and Analysis of Genetic Variation

In order to conservation of spearmint（Mentha spicata L.） for a long time,the shoot-tips of spearmint were treated by vitification method of cryopreservation.The technologies of methyl-sensitive amplified polymorphism（MSAP） and amplified fragment length polymorphism（AFLP） were used to analyze the genetic and epigenetic stability of regenerated plants.The results demonstrated that the shoot tips were cryoacclimatized at a low temperature（4℃） for 28 days,pre-cultured in the MS medium with addition of 2 mol/L glycerol after 4 days,dehydrated in PVS₂ at 0 ℃ for 50 min,and then liquid nitrogen preserved for 1 hours or longer,the survival rate was highest up to 60%.No variation band was found between the treatment and the control.The DNA methylation level was reduced in recycled materials treated by cryopreservation.Methylation and demethylation of DNA were 8.93%and 12.3%,respectively.It suggests that DNA methylation might be one of the mechanisms used by plants to combat cryopreservation.