超低温对降香种子遗传变异和生理特性的影响

Effect of Cryopreservation on Genetic Variation and Physiological Characteristics of Dalbergia odorifera Seeds

采用单因素随机区组试验方案探究降香种子玻璃化超低温保存最适条件,结合显微切片观察和生理生化特性检测对冷冻后细胞形态和生理生化指标变化进行分析,同时运用形态观察法和相关序列扩增多态性(SRAP)分子标记法对超低温保存后的降香植株遗传稳定性进行检测。结果表明:降香种子玻璃化超低温保存最佳条件为:室温(25℃)装载25min,0℃玻璃化脱水30min,40℃水浴解冻5min。对比检测不同冷冻时间的降香种子超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、α-淀粉酶、脱氢酶活性、丙二醛(MDA)含量以及电导率等生理生化指标,无明显差异。显微观察超低温保存前后的降香种胚内部结构,未见明显变化。超低温保存后的降香种子发芽时,出现短暂的生长停滞期,恢复培养后,其表观形态指标与对照植株无明显差异。SRAP技术分析了超低温冷冻前后的降香再生植株,其SRAP带型基本一致,未见明显差异带型。综上表明,超低温保存降香种子是长期稳定、安全可靠的。

In this study, we used a single factor random block test to optimize cryopreservation procedure of Dalbergia odorifera T. Chen seed by vitrification. The embryo cells of Dalbergia before and after frozen were inspected with microscope by paraffin section method, and the change of physiological and biochemical indexes of seeds before and after frozen were analyzed. In addition, the genetic stability of regenerated plantlets after cryopreservation was tested with sequence—related amplified polymorphism（SRAP） molecular marker method. The results showed that the optimal conditions for Dalbergia odorifera seeds vitrification were 25 min loading condition at room temperature, 30 min dehydration condition at 0 ℃ and 5 min thawing condition at 40 ℃. The was no significant difference in seed physiological and biochemical indexes of different freezing time, and when inspected by microscope before and after cryopreservation, the internal structure of the embryo had no obvious variations. These results suggest that the time of cryopreservation had no significant effect on germplasm preservation of Dalbergia odorifera. When sprouted the seed of Dalbergia odoriferaafter freeze in liquid nitrogen, there had a short period of stagnation, but the morphological indexes did not vary greatly with those of the control after renewal cultivation. No nucleotide sequence had been altered during cryopreservation, which was indicted by no obvious differences in the banding patterns of DNA fragments between survival material and the control material of the Genomic DNA analyzed by SRAP after cryopreservation. In conclusion, the ultarlow-tem-perature preservation of Dalbergia odorifera seeds was stable, safe and reliable.