| Cryopreservation by vitrification has been thought one of the most practical and effective technique for plant germplasm in vitro conservation.The method has some fortes at saving space,without repeated subculture,avoiding pollution,low equipment request and simple,convenient operating procedure.On the basis of previous established cryopreservation technique system of Siraitia grosvenorii by vitrification,this paper using single factors and orthogonal experiments to study the cryopreservation of S.grosvenorii by droplet-vitrification for exploring the most suitable procedure.The changes of structure and methylation of DNA of their subcultured plants and regenerated plants after cryopreservation with AFLP and MSAP analysis were studied to evaluate their genetic stability.The establishment of cryopreservation technique system by droplet-vitrification could supply the morphous and molecular basis on S.grosvenorii cryopreservation and its germplasm resource protection and exploitation.The main results were as follows:1.Effects of different preculture times on survival and regeneration rates were compared.Results showed that the suitable preculture time was 1 day,survival and regeneration rates of Qingguan were up to 67%and 21%,respectively.2.Results of different concentration of sucrose in preculture mediums on cryopreservation showed that the effects of medium supplemented with 0.7mol/L sucrose were the best,its survival and regeneration rates of Qingguan were 68%and 21%,respectively.3.The suitable loading time with 60%plant vitrification solution 2(PVS2)was screened.Results showed that the best loading time was 30 min,the survival and regeneration rates of Qingguan could reach 66%and 27%,respectively.4.Different PVS2 treatment time had notable effects on survival and regeneration rates,100min was the best PVS2 treatment time,the survival and regeneration rates of Qingguan were 67%and 31%,respectively.5.Using 3 factors(preculture,loading and dehydration)and 3 levels orthogonal experiments,a suitable combination of Qingguan for cryopreservation was screened which was that shoot-tips precultured for 1d on MS liquid medium supplemented with 0.7mol/L sucrose,loaded with 60%PVS2 for 30 min at 25?,and dehydrated with 100%PVS2 for 90min at 0?.On which,the survival and regeneration rates were 76%and 62%,respectively.Applying this method to Lvxing,Keji 1 hao and Zhengdong cryopreservation,their survival and regeneration rates were 62%and 34%,53%and 19%,41%and 14%,respectively,which indicated that there were obviously difference among varieties.6.DNA structures of 4 subcultured varieties and 3 regenerated varieties after cryopreservation were analyzed by Amplified fragment length polymorphism(AFLP).The results showed that variation bands were produced in 4 subcultured varieties,their average percentage of mutant bands was 4.94%.The variation of regenerated varieties after cryopreservation was little higher than that of subcultured varieties,their average percentage of mutant bands was up to 6.65%,which indicated that DNA structures were changed during subculture and cryopreservation.The degree of DNA structural variation during subculture and cryopreservation was different among different varieties.7.The technology of methyl-sensitive amplified polymorphism(MSAP)was used to analyze the methylation states of 4 subcultured varieties and 3 regenerated varieties after cryopreservation.The results demonstrated that total methylated ratio of DNA was decreased by subculture and cryopreservation treatment,total methylated ratio of regenerated varieties was higher than subcultured varieties,and total methylated ratio of Qingguan was the lowest among all varieties.Three types of methylation variation were produced in both subcultured varieties and regenerated varieties.Demethylation was the main trend of methylation changes.The variation rates of materials after cryopreservation were higher than those of subculture.The highest and lowest variation rate were produced in Keji 1 hao,7.60%,and Qingguan 4.81%,respectively. |
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