A Study On Anti-browning During Tissue Culture Of Musella Lasiocarpa

Musella lasiocarpa which belongs to a monotypic genus, is endemic to southwest of China, including Yunnan and Sichuan province. It’s a precious wild flowers germplasm resource as famous for dignified plant type and beautiful inflorescence. Callus necrosis during in vitro propagation is a large obstacle in breeding and large scale production of M. lasiocarpa. In this paper, effects of sterilizing way of explants, the ratio of plant growth regulators and 4 kinds of browning inhibitors on callus browning were studied with the callus induced from its explants of immature male flowers. The difference between M. lasiocarpa var. lasiocarpa and M. lasiocarpa var. rubribracteata during tissue culture was studied. In addition, the dynamic changes of phenols content and PPO activity were analyzed during tissue culture of the two varieties. The phenolic content and PPO activity in different parts of different varieties of M. lasiocarpa were quantificationally analysed. The major conclusions were as follows:(1)The contamination rate of unsterilized explants was 0, and its browning rate and browning index were significantly lower than explants after disinfection; 6-BA played a major role in callus induction of M. lasiocarpa, which was conducive to callus induction and significantly reduced browning index with its concentration of 2 mg·L-1 or 3 mg·L-1; Adding VC on the surface and VC mixture of citric acid failed to suppress browning. However, Ginger and ferulic acid gave satisfactory result to reduced browning. The ferulic acid with its best concentration of 48.5 mg·L-1 could significantly reduce the callus browning and increased the rate of differentiation to17.9%, 15% higher than control.(2)The browning index was on the rise during tissue culture of M. lasiocarpa. It was 3.1% and 8.3% respectively in M. lasiocarpa var. lasiocarpa and its variety after 30 days culture, and which rose to 71.7% and 89.3% after 180 days. In the same period, the browning index of M. lasiocarpa var. lasiocarpa was significantly lower than its variety. The relative growth weight of callus were 487.3% and 244.0% in M. lasiocarpa var. lasiocarpa and its variety respectively after 30 days culture, and was declining all through the subculture, reaching to 33.3% and 10.7% respectively after 180 days culture. M. lasiocarpa var. rubribracteata was harder to get tissue culture seedling successfully than M. lasiocarpa var. lasiocarpa because its callus necrosis during tissue culture was more serious.(3)Polyphenol oxidase activity of callus was increase dramatically at the beginning of culture, which of M. lasiocarpa var. lasiocarpa was significantly higher than its variety during this period. The specific activity of PPO were 3948.1 and 2037.2(0.01 A·g-1·min-1)respectively after 14 days culture. The specific activity of PPO of M. lasiocarpa var. lasiocarpa began to decrease after 60 days, and remained at 1700~1900(0.01 A·g-1·min-1)after 120 days. However, the same circumstance did not occur in the variety that the specific activity of PPO maintained at 2200~2550(0.01 A·g-1·min-1). The total phenolic content of M. lasiocarpa var. rubribracteata was significantly higher than its variety during callus induction and differentiation stages. The total phenolic content of M. lasiocarpa var. rubribracteata increased during the later stage of culture, however, M. lasiocarpa var. lasiocarpa remained relatively stable. The PPO activity and the phenolic content of M. lasiocarpa var. rubribracteata were both higher on the differentiation stage of callus. Meanwhile, the browning index was consistent with the change of the PPO activity and the phenolic content which indicating that severe browning of M. lasiocarpa var. rubribracteata might associated with its high PPO activity and high total phenolic content.(4)The total phenolic content of stamen was the highest among different parts of M. lasiocarpa, 21 mg·L-1. The total phenolic content of leaf and leaf veins of M. lasiocarpa var. rubribracteata were higher than M. lasiocarpa var. lasiocarpa, which were 2.0 times and 3.2 times of latter respectively. The PPO activity of leaf, veins and female ovule were higher, but the other parts such as bracts, ovary had very low or no PPO activity. The total phenolic content and PPO activity were reduced when the callus differentiated into bud, the total phenolic content of the bud was higher in the variety, on the contrary, the PPO activity of the bud lower than M. lasiocarpa var. lasiocarpa. In the process of buds developed into leaves, the total phenolic content increased, from 0.56 mg·L-1 to 4.67 mg·L-1 and 2.34 mg·L-1 to 9.43 mg·L-1 respectively in M. lasiocarpa var. lasiocarpa and its variety, Neverthless the difference of PPO activity was not significant between buds and leaves.(5) Unsterilized immature male flowers of M. lasiocarpa were used as explant in this study. The results showed that callus were cultured on MS medium supplemented with 2 mg·L-1、3 mg·L-1 6-BA, 3 mg·L-1 2,4-D and 48.5 mg·L-1 ferulic acid could significantly reduced browning. The study could optimize tissue culture system of M. lasiocarpa.