Dynamic changes in polyamines, endogenous hormones and oxidase activities during rooting of in vitro plantlets of Pyrus betulifolia Bunge

Abstract: 【Objective】Pyrus betulifolia Bunge is widely used as a pear rootstock in China. Propagation with seeds has large genetic variation and poor consistency. Difficulty in rooting limitsits propagation by traditional vegetative propagation methods such as layering and cutting. With the increasingly perfecting of in vitro propagation technology, virus-free seedlings can be produced by micropropagation method. Root induction is one of the important processes in in vitro propagation. The transplanting survival rate and seedling quality are affected by rooting efficiency. There are many internal and external factors affecting the rooting process, including endogenous hormones, polyamines, related oxidases and exogenous substances such as ethylene and salicylic acid. The study aimed at revealing the roles of endogenous hormones, polyamines and related oxidases in root induction of tissue-cultured seedlings.【Methods】Subcultured plantlets of Pyrus betulifolia Bunge were cultured on rooting medium with1/2 MS, 7.5 g · L-1 agar, 25.0 g · L-1 sucrose and different ratios of hormones. A total of 24 treatments using five different concentrations (0, 0.2, 0.5, 1 and 2 mg · L-1) of IAA and IBA were assessed in terms of rooting percentages at 40 days after transfer to the media. Each treatment consisted of 6 bottles each comprising 5 subcultured plantlets. Uniform subcultured plantlets were selected to be cultured on the optimized rooting medium. Approximately 500 plantlets were used to monitor the dynamic changes in polyamines, endogenous hormones and related enzyme activities during root regeneration. All of the cultures were incubated at 25 ℃ under a 16 h light/8 h dark cycle and 2 000-3 000 lx light intensity. The basal stem cuttings were taken 0, 3, 7, 10 and 15 days after transfer to media. Polyamines, endogenous hormones and related enzyme activities were determined.【Results】The treatment of 2.0 mg · L-1 IAA +0.5 mg · L-1 IBA increased rooting rate and the percentage of plantlets with ≥3 roots compared with the other treatments. The rooting rate was 86.7% and the percentage of plantlets with ≥3 roots was53.3%. The combination of IAA and IBA was better than single hormone. Callus was observed between the 7 th and 10 th day, growing fast from the 10 th to 15 th day. The roots generated 20 days after transfer to media. The contents of three polyamines declined after 3 days in the root induction medium. The content of spermine (Spm) and spermidine (Spd) fluctuated during root induction period. The content of putrescine declined in the first three days and rose between the 3 th day and the 15 th day. The contents of IAA and IBA decreased firstly and then increased, and were the lowest at the 7 th day. The content of GA was the lowest at the 3 rd day and increased rapidly from the 3 th to the 10 th day, but then the increasing speed slowed down. The variation in ZR content followed a pattern of "decrease→increase→decrease", lowest at the 7 th day. The ratio of IAA/ABA was relatively stable during the first seven days, and increased from the 7 th day to the 15 th day. The ratio of IAA/ZR decreased slightly within the first 3 days and then increased from the 3 th day to the 15 th day. The activity of IAAO remained stable from the 7 th day to the 10 th day, and increased rapidly from the 10 th to the 15 th day, indicating that decomposition of IAA was accelerated during this period. There was a significant negative correlation between Spm and ZR contents p < 0.01, r=-0.898) . Spd content had significant positive correlations (p < 0.01) with the contents of IAA and ABA, the ratios of IAA/ABA and IAA/ZR and the activity of IAAO. Put content was positively correlated with GA (p < 0.01, r=0.646) , IAA and ABA (p < 0.05) . IAAO activity was significantly positively correlated with PPO activity (r=0.843) , Spd content (r=0.643) and IAA/ZR ratio (r=0.662) . PPO enzyme activity was significantly positively correlated with IAA and ABA contents and the ratio of IAA/ZR (p < 0.01) . The content of IAA had moderate correlations with the contents of Spd, GA and ABA, PPO activity and the ratios of IAA/ZR and IAA/ABA. The content of ABA had moderate correlations with the contents of IAA, Spd and GA and the ratios of IAA/ZR and IAA/ABA. The results showed that Spd, IAA and ABA played important regulation roles in rooting process.【Conclusion】The suitable rooting medium for Pyrus betulifolia Bunge was 1/2 MS + 2.0 mg· L-1 IAA +0.5 mg · L-1 IBA + 7.5 g · L-1 agar + 25.0 g · L-1 sucrose. The increase in IAA/ABA and IAA/ZR ratios induced adventitious roots formation. Spd might be closely related to the adventitious root formation. In the late stage of root induction, the increase in GA content had no significant effect on the adventitious root formation.