Cryopreservation Of Mentha Spicata L. Germplasm And Analysis Of Genetic Variation

Cryopreservation is considered to be the most effective way to long-term preservation of plant germplasm resources. In this paper, we used the spearmint (Mentha spicata L.) tips as the materials to exploratory research the vitrification cryopreservation procedure. And studied the effects of cryopreservation on physiology , genome stability and DNA methylation in Mentha Spicata. The main results as follows:1. The procedure of spearmint shoot tips vitrification cryopreservation was exploratory studied. The results showed that: the shoot tips were cryoacclimatized at a low temperature (4℃) for 3-4 weeks; pre-cultured in the MS medium with adding 2 mol.L^-1 glycerol after 3 days; treated with liquid pretreatment medium for 20mins at room temperature and dehydrated in PVS2 at 0℃for 50mins, the survival rate was highest up to 60%. Regenerations differentiated normally.2. The content of malondialdehyde (MDA) of the spearmint treated by Cryopreservation increased significantly compared with controls, the activity of peroxidase (POD) and catalase (CAT) aslo significantly enhanced. The physiological indexes illustrated ultra-low temperature treatment affected the normal physiological and biochemical reactions of spearmint, resulted in MDA accumulation, activity of some antioxidant enzymes changing.3. The technique of amplified fragment length polymorphism (AFLP) was used to analyze the genome DNA of the treatments and the controls, no variation of bands was found between the treatment and the control, which indicated that there were no changes in the genomic DNA sequences of the materials during cryopreservation.4. The methyl-sensitive amplified polymorphism (MSAP) markers techlologe was used to analysis the variation of DNA methylation level of samples between the treatment and the control. The results indicated that there were changes in the DNA methylation states compared with controls.The recycled materials treated by cryopreservation , have occurred in different degrees of methylation changes.the demethylation phenomenon occurred in the sites. Methylation and demethylation of DNA were 8.93%and12.3%.