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