低温预处理对百子莲体细胞胚胎诱导的影响及生理机制研究

Effects and Physiological Mechanisms of Cold Pre-treatment on Somatic Embryo Induction in Agapanthus praecox ssp.orientalis

为研究低温预处理对百子莲体胚诱导的影响,并初步揭示其生理调节机制,通过4℃低温预处理0、2、4、6 d,优化百子莲体胚诱导体系,并利用酶联免疫吸附法(ELISA)测定了体胚诱导期激素代谢、糖代谢以及氧化胁迫相关生理指标。结果表明,与未低温处理(对照)相比,4℃预处理2 d可使体胚数量显著增加63.81%(P<0.05),处理4、6 d分别降低17.43%、75.78%。低温预处理显著调节体胚诱导期生理代谢,对内源IAA、GA含量和体胚诱导数量具有相似的调节规律,均在2 d的处理中达到较高水平,而细胞分裂素(CTK)含量随低温预处理时间逐渐降低,且具有梯度效应,IAA、CTK含量与体胚数量的相关系数分别为0.880、0.705。4℃处理2 d,体胚诱导材料中的总糖、淀粉、麦芽糖含量分别显著增加31.44%、51.91%、36.50%(P<0.05),其中淀粉、麦芽糖含量与体胚诱导数量的相关系数分别为0.786、0.742。低温预处理可使活性氧(ROS)活性降低,过氧化氢酶(CAT)活性整体升高,4℃处理2 d,体胚诱导材料内H2O2含量、过氧化物酶(POD)活性比对照分别显著降低25.35%、18.41%(P<0.05),与体胚数量的相关系数分别为-0.886、-0.993。低温预处理模式下,内源IAA、GA、总糖、淀粉、麦芽糖含量积累利于百子莲体胚诱导,而H2O2含量、POD活性增强对体胚诱导不利。

This study aims to evaluate the effects of cold pre-treatment on somatic embryo induction efficiency,and reveal the physiological mechanisms preliminarily in Agapanthus praecox ssp.orientalis.Cold pre-treatment at 4℃for 0 d,2 d,4 d and 6 d was conducted to optimize the somatic embryo induction system of A.praecox.Furthermore,the physiological indicators including hormone metabolism,sugar metabolism and oxidative stress were investigated by using the method of enzyme linked immunosorbent assay(ELISA).The results showed that the number of somatic embryos significantly increased by 63.81%with 4℃treatment for 2 d compared with control(P<0.05).However,the number of somatic embryos decreased by 17.43%and 75.78%under 4 d and 6 d treatment compared with control,respectively.Cold pre-treatment significantly regulated physiological metabolism during the somatic embryo induction stage.Cold pre-treatment had similar regulatory effects on endogenous IAA,GA contents and somatic embryo number,all of which reached a high level with 2 d treatment,while CTK content gradually decreased with the increase of treatment time.The correlation coefficient between active IAA content and somatic embryo quantity was 0.880,while the correlation coefficient between CTK content and somatic embryo number was 0.705.The contents of total sugar,starch and maltose in somatic embryo increased significantly by 31.44%,51.91%and 36.50%with 2 d treatment,respectively(P<0.05).The correlation coefficient between starch content and somatic embryo number was 0.786,and the correlation coefficient between maltose content and somatic embryo number was 0.742.Cold pre-treatment reduced the ROS level,and increased the activity of CAT.The content of H2O2 and activity of POD were significantly decreased by 25.35%and 18.41%compared with control(P<0.05)with 2 d treatment,and the correlation coefficients with the number of somatic embryos were-0.886 and-0.993,respectively.Collectively,the results suggested that the accumulation of endogenous IAA,GA,total sugar,starch and maltose was beneficial to somatic embryo induction with cold pre-treatment.In contrast,the enhancement of H2O2 content and POD activity was unfavorable for somatic embryogenesis.