马铃薯(Solanum tuberosum L.)茎尖的超低温保存及其遗传变异的初步观察

Cryopreservation of Potato (Solanum tuberosum L.) Shoot-Tips and Preliminary Observations of Its Genetic Variation

研究马铃薯茎尖超低温保存技术的结果表明,4℃低温下锻炼6d,在添加二甲基亚砜(DMSO)和乙酰胺的培养基中预培养5d,60%PVS2于室温下装载30min,0℃下PVS2脱水40min时,茎尖成活率最高(71.6%),再生植株生长分化正常。进一步对再生植株进行AFLP分析,6对引物组合共扩增出385条带,超低温保存前后的材料之间未见到明显差的异带,但用MSAP技术分析超低温保存前后植株甲基化的结果显示:超低温保存后的材料均有不同程度的甲基化。在扩增的624条带中,处理与否之间完全一致的带型为584条;有变化的带型为40条,处理2(茎尖经过完整的超低温保存过程,区别于处理1,增加了冷冻、解冻和洗涤后恢复培养)有13个位点的甲基化增加,21个位点去甲基化。

Abstract： A technology of potato shoot-tip-cryopreservation was studied in this paper. The highest survival rate of shoot-tips could reach 71.6% after trained at 4 ~C low temperature for 6 days, pre-cultured on the medium with DMSO and acetamide for 5 days, loaded with 60% PVS2 for 30 rain at room temperature, and dehydrated with PVS2 for 40 min at 0 ~C. The regeneration plants differentiated normally. 385 bands were amplified from the regeneration plants using 6 pairs of primers combination for AFLP analysis. Before and after cryopreservation the samples showed no obvious variation bands. It suggested that the plants maintained a good genetic stability at DNA level. But MSAP analysis on the plant methylation indicated that after cryopreservation the materials had different methylation changes. Among 624 bands, the control and the treated samples had 584 bands of identical type and 40 bands of changerable type. Treatment 2 （shoot-tips culture after the complete cryopreservation process, is different from Treatment 1 with an increase of freezing, thawing and washing recovery training） had 13 de novo methylation sites and 21 demethylation sites.