Cryopreservation Of Arabidopsis Thaliana Seedlingsand Analysis Of Genetic Variation

Cryopreservation is one of the long-term conservation ways of plant germplasm. Vitrification as a simple method has been successfully used for cryopreservation of many materials.This paper studies furtherly the vitrification procedure of Arabidopsis thaliana seedlings. And genetic integrity after cryopreservation has been evaluated by developmental phenotypes and DNA molecular marker techniques. The main results are as follows:1. The procedures of vitrification for Arabidopsis thaliana seedlings were modified, and high rates of survival had been evaluated. 1～2 days seedlings were pretreated in a solution containing 2 mol/L Gly and 0.4 mol/L sucrose at room tempreture. After 20 minutes, the solution was replaced by PVS2 (MS containing 30%w/v Gly +15%w/vEG+15% w/v DMSO +0.4mol/L sucorse ) and kept for about 50 minutes at 0℃, then the cryovials with the seedlings were immersed into liquit nitrogen directly. After been thawed rapidly in a water bath at 40℃, the seedlings were washed with another solution (MS liquid medium + 1.2 mol/L sucrose) for 30 minutes, and the solution was replaced once 10 min. The seedlings were then cultured on MS medium and maintained under the exactly same condition as that for seeds germination. The survival rates were evaluated after 5 days of thawing. Seedlings were transferred into solid and placed in the same growth chamber after growing on MS about 10 days, Regular management was kept until seeds were harvested.2. The development of the samples with treatment and the controls were observed. All seedlings with treatment (include the seedlings cryostoraged in LN and those without cryostorage) grew very little but the controls grew faster than those after thawing 2 days. Almost all seedlings treated without cryostorage in LN (pre-treated, cryopretected with PVS2, unloaded and then recultured) were survived and started to grow up within five days, but the seedlings after cryostorage in LN and thawing in a water bath decreased its survival ability little, and the average survival rate was 93.8%. Twenty days after re-culture, all the plants with treatments (involving freezing-thrawing or not) did not show morphological alterations as compared with the untreated and unfrozen controls, but in the first, seedlings with treatment grew more slowly than the controls. And all the plants flowered and had seeds normally at last.3. The genome DNA of treatments and controls were analyzed by amplified fragment lenth polymorphism (AFLP) technique. The DNA were digested by EcoRⅠand MseⅠ, and 549 bands were scored using 7 pairs of primers combination. No variation of bands was found during the three samples, which suggested that no nucleotide sequence of the seedlings had altered during cryopreservation.4. Methylation-sensitive amplification polymorphism (MSAP) was used to analyze the DNA methylation status of the samples and their descendants. HapⅡ/ MspⅠand EcoRⅠdigested the DNA. In all the 662 bands, 64 bands were found changerable in the samples, and there were 48 bands could maintain the same between two descendants among them. Compared to the controls and the samples treated without in LN and 40℃water bath, both de novo methylation and demethylation were observed in the samples after cryopreservation, but demethylation were the main trend.