PVP处理对黑皮油松外植体酚类物质形成及酶活性的影响

doi:10.11707/j.1001-7488.20211016

摘要/Abstract

摘要：

目的: 探讨黑皮油松组织培养中聚乙烯吡咯烷酮（PVP）处理对外植体中相关酶活性、酚酸类物质含量的影响，以寻找酶促褐变的主要底物，为黑皮油松组织培养褐变这一瓶颈问题的解决提供理论依据和技术支撑。方法: 以黑皮油松休眠叶芽为外植体，研究不同浓度PVP处理对其休眠芽褐化与萌发的影响，筛选适宜的PVP处理浓度，测定PVP最佳处理浓度下外植体中相关酶活性的变化，并利用高效液相色谱仪对酚酸进行定性和定量分析。结果: 添加300~1 500 mg·L^-1 PVP可以有效抑制黑皮油松外植体褐变，促进其休眠芽的萌发。PVP处理第8天开始抑制外植体褐化，第24天褐化率达到峰值，随后趋于稳定，并以添加1 500 mg·L^-1 PVP抑制外植体褐化的效果最佳，褐化率仅为25.56%，休眠芽萌发率也最高（37.78%）。与对照相比，PVP处理下多酚氧化酶（PPO）与苯丙氨酸解氨酶（PAL）的活性在处理过程中均处于较低的水平，但过氧化物酶（POD）活性变化规律不明显。从PVP处理的第4天开始，PPO活性显著下降；从第8天开始，PAL活性明显被抑制。在褐变加重的第8~12天，PVP处理下绿原酸含量下降幅度显著小于对照。与对照相比，PVP处理下，在0~12天内阿魏酸含量变化较小，但在16~32天时阿魏酸含量显著高于对照组。香豆酸含量在处理全程均维持在较低的水平。结论: PVP处理可有效抑制黑皮油松休眠芽的褐化并促进其萌发，并以1 500 mg·L^-1的PVP处理效果最佳；PVP处理抑制PPO、PAL活性上升而使外植体褐变较轻；同时PVP处理可以保护绿原酸与阿魏酸不被氧化进而抑制组织褐变。

关键词: 黑皮油松, 休眠芽, 褐化, PVP, 酶活性, 酚类物质

Abstract:

Objective: To investigate the effects of polyvinyl pyrrolidone (PVP) treatment on the activities of related enzymes and phenolic acid content in explants during tissue culture of Pinus tabulaeformis var. mukdensis, in order to find the main substrates for enzymatic browning, and to provide theoretical basis and technical support for solving the bottleneck problem of browning during tissue culture of P. tabulaeformis var. mukdensis. Method: Using dormant buds of P. tabulaeformis var. mukdensis as explants, the effects of PVP treatments with different concentrations on the browning and germination of explants were detected. The optimal PVP concentration was determined, the changes of activities of the relevant enzymes in the explants under the optimal treatment concentration of PVP were detected, and the qualitative and quantitative analyses of the phenolic acid were conducted by using high performance liquid chromatography. Result: Addition of 300-1 500 mg·L^-1 PVP effectively inhibited the browning of explants and promoted the germination of the dormant buds of P. tabulaeformis var. mukdensis. Inhibition of the browning of explants was started on the 8th day of PVP treatment, the browning rate peaked on the 24th day, and then tended to stabilize. The most effective concentration of PVP for inhibiting the browning of P. tabulaeformis var. mukdensis explants was 1 500 mg·L^-1 under which the browning rate was only 25.56%, and the dormant bud germination rate was also the highest (37.78%). Analysis of changes in enzyme activities and phenolic acid content showed that polyphenol oxidase(PPO) and phenylalanine ammonia-lyase(PAL) activities were at a relatively low level during the whole treatment process under PVP treatments compared with the control, whereas the change of peroxidase (POD) activity was not significant. PPO activity under PVP treatments was significantly lower than that of the control starting from day 4, but PAL activity was obviously inhibited since day 8. The decrease of chlorogenic acid content under PVP treatments during days 8-12 when browning was aggravated was significantly lower than that of the control; the ferulic acid content under PVP treatments during days 0-12 did not change much, but was significantly higher than that of the control during days 16-32. The content of coumaric acid was maintained at a low level throughout the treatment. Conclusion: PVP effectively inhibits the browning of P. tabulaeformis var. mukdensis explants and promotes germination of the dormant buds. And the addition of 1500 mg·L^-1 PVP obtained the best effects. PVP treatment prevented browning of P. tabulaeformis var. mukdensis explants by reducing the activity of PPO and PAL. PVP treatment protects chlorogenic acid and ferulic acid from being oxidized so as to inhibit tissue browning.

Key words: Pinus tabulaeformis var. mukdensis, dormant buds, browning, PVP, enzyme activity, phenolic substances

中图分类号:

S718.43

梁艳,赵雪莹,白雪,刘德强,张妍,潘朋. PVP处理对黑皮油松外植体酚类物质形成及酶活性的影响[J]. 林业科学, 2021, 57(10): 166-174.

Yan Liang,Xueying Zhao,Xue Bai,Deqiang Liu,Yan Zhang,Peng Pan. Effects of PVP Treatments on Phenolic Contents and Enzyme Activities in Explants of Pinus tabulaeformis var. mukdensis[J]. Scientia Silvae Sinicae, 2021, 57(10): 166-174.

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处理 Treatment	PVP浓度 Concentration/(mg·L^-1)	褐化率 Browning rate(%)	褐化程度 Browning degree	萌发率 Germination rate(%)

CK	0	76.67±1.93 a	+ + +	12.22±1.11 d
P1	100	66.67±1.93 b	+ + +	13.32±1.93 d
P2	300	52.22±1.11 c	+ +	24.44±1.11 c
P3	500	41.11±1.11 d	+ +	23.33±1.93 c
P4	800	50.00±1.92 c	+ +	25.56±1.11 c
P5	1 000	37.78±1.11 d	+	32.22±1.11 b
P6	1 500	25.56±1.11 e	+	37.78±1.11 a
P7	2 000	65.66±1.11 b	+ + +	16.67±1.93 d

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