| The life activity of plant materials is minimized near to the zero in liquid nitrogen, so cryopreservation is a safe and cost-effective method for long-term preservation of plant materials at an ultra-low temperature in liquid nitrogen. Vitrification is a main cryopreservation method because of its simple operating and simplified procedure. In this study, main steps of vitrification cryopreservation consisting of sub-culture, cold hardening, preculture, frozen storage, thawing and recovery-medium were experimented using the apple shoot tips as the experimental materials. A suitable cryopretectant (PVS3) was selected and a method of the vitrification cryopreservation procedure of apple shoots tips was established at a high survival rate. In addition, the survival shoots were examined by morphological, biochemical and molecular analysis, which confirmed their genetic stability. So vitrification cryopreservation was a practicable method for apple germplasm storage. The main results were as follows:1 The shoots were cultured on MS base medium with a high sucrose concentration (l.Smol.L"1) and ABA (0.5 mg.L"1) for different sub-culture time and then cold-hardened for 0-6 weeks under 0-7. A higher survival rate was gained under 3 months sub-culture and 4-5 treatment for at least 4 weeks.2 The survival rate was influenced by the composition and concentration of sugar in preculture medium. Prescriptions of O.Tmol.L"1 sucrose and 0.5mol. L"1 sucrose + 0.2mol. L"1 mannitol were suitable. When NH was excluded from the precluture medium and 5% DMSO was added, the survival rate would be enhanced at some degree. A highest survival rate was attained in 2 days' preculture with the lowest water content, highest soluble sugar content andhighest cell viability.3 Five compound cryopretectants were designed and the effects were investigated. Of all the compound cryopretectants, PVS3, containing 40%(W/V) glycerol, 45%(W/V) sucrose, 10%(W/V) ethylene glycol and 10%(WAO dimethyl sulfoxide (DMSO) was most effective. Higher survival rate was gained by dehydration in PVS3 for 90 min directly or step by step in diluted PVS3.4 The survival rate did not change when stored in liquid nitrogen during 1-90 days. Different thawing methods had much effect on survival rate. By washing the PVS3 with 1.2 mol L"1 sucrose after thawing the survival shoot tips would grow normally after a 20-30 days lag stage in recovery medium containing 0.6mg - I/'BA, 0.1 mg L"'NAA, and 0.5 mg . L"'GA3. Excluding NH% in recovery medium could shorten the lag stage for 4-5d. Cryopreservation tolerance was different in different apple genotypes. The survival rate of the cultivar "world one'' was the highest of all the six cultivars experimented.5 The shoot tips cryopreserved by vitrification could develop normally and no difference was found in morphology. SDS-PAGE technique was used to analyze the soluble proteins and PAGE to POD isozymc. No differences were found in soluble proteins and POD isozyme map between the control and survival shoots. RAPD analysis also showed no difference at DNA level. All the results suggested genetic stability of the survival shoots on morphological, biochemical and molecular level after Cryopreservation and vitrification Cryopreservation was a practicable method to store apple germplasm.
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