| As a simple method, vitrification has been successfully used for cryopreservationof many materials, so it is one of the long-term conservation ways of plant germplasm.Potato (Solanum tuberosum L.) is the third largest stample crop in the whole worldafter rice and wheat. Avilability of germplasm resources is a basic requirement forbreeding of potato. Now there have been a numerous reports on cryopreservation ofpotato in the world, and many countries have been set up cryopreservation gene banks,such as IPK in Germany, CIP in Peru and NAC in Republic of Korea. However,information on cryopreservation of China’s potato has been quite liminted. Therefore,the objective of the present study was to establish the vitrification procedures forpotato germplasm pool.In this study, Qingshu9, Atlantic, Zihuabai and E107were used to study thevitrification of cryopreserved potato shoot tips.In addition, studied the effects ofcryopreservation on genetic stability and DNA methylation in Potato.The main resultsas follows:1.The main factors affecting the pos-thaw regeneration of shoot tips wereanalyzed by orthogonal test, and the preserved protocol was optimized. In vitro shoottips (2-3mm in length with two leaf primordia) from30or40-day-old stock cultures,were pre-cultured for4hours, then loaded with a mixture of2mol/L glycerol plus0.4mol/L sucrose for40min at room temperature, the shoot tips were sufficientlydehydrated in PVS2for35min at0℃. And then plunged directly into liquid nitrogenand conserved (conserved at least24h). After rapidly thawed in1.2mol/L sucrosesolution for90s at room temperature, the shoot tips were incubated25min with it,and then the shoot tips were transferred to the solid culture medium in dark for10d,and another10d in dim light, finally transfered them to normal light for recovery andregeneration.2.With optimized parameters, we saved four potato cultivars genomes, Theshoot tips’ survival rate of Qingshu9, Atlantic, Zihuabai and E107was86.93%,78.03%,71.57%and65.82%, and the regrowth rate reached77.05%,48.02%,49.45%and40.95%, respectively. The survival rate and regrowth rate varied with thegenotypes, as well as significant difference between different genotypes.3.In vitro cultured potatoes were used to study on genetic stability after cryopreservation in morphology and molecular biology, the results as follows:Ⅰ. There are no significance difference about leaf figure, leaf color, plant type,flower color, potato type and potato color between the treated plantlet and its control;Ⅱ. The technique of simple sequence repeat (SSR) was used to analyze theDNA of the treatments and the controls. Results indicate30primers were screend,from which10primers produced reproducible and well resolved bands. Each primerwas able to produce6bands, and obtained314bands in total. Compared with controls,no variation of bands was found. So there were no changes in the genomic DNAsequences of the materials after cryopreservation.4.Variation patterns of DNA methylation of potato genotypes between thetreatment and the control were assessed by using the technique of methylation-sensitive amplified polymorphism (MSAP). MSAP analysis on the plant methylationindicated that average493fragments each genotype were amplified using16pairs ofselective primers. Compared to the control, DNA methylation events were detected inthe percentages from8.4%to14.3%in the CCGG sequence of potato genomes.Demethylation and de novo methylation were produced after cryopreservation, anddemethylation was the main trend of methylation changes. |
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