| Tree peony has a long history of cultivation in China.It belongs to rare woody flowers.It not only has good ornamental value and medicinal functions,but also has high development and utilization value in oil use.Paeonia ostii‘Feng Dan'is a cultivar of the wild peony Yangshan peony and is a major cultivar of oil peony in China.Many scholars have tried and done some results in the tissue culture of'Fengdan'peony.However,during the experiment,it was found that the browning of tissue culture materials was a major obstacle to the establishment of the'Fengdan'peony rapid propagation system.At the same time,the mechanism of anti-browning agent in the browning process of peony callus is not clear enough.Therefore,investigating the mechanism of inhibition of browning by silver nitrate as the anti-browning agent in the process of tissue culture of'Fengdan'peony can provide theoretical support for the analysis of the underlying causes of the'Feng Dan'peony browning and the development of new anti-browning agents.In this experiment,the proliferating seedlings obtained from tissue culture of'Fengdan'peony were cultured in a medium containing 0-5.0 mg·L-1 silver nitrate,and the materials were collected in three periods to measure the callus.Changes in total phenolic content,monophenols,total flavonoids,and related enzyme activities,and selected transcription factors and key enzymes that have important biological functions in the synthesis pathway of polyphenols for real-time quantitative detection from molecules The effect of different concentrations of silver nitrate on callus browning inhibition was examined.The main findings are as follows:?1?After comparing the growth of explants,it was found that adding silver nitrate to the medium can significantly reduce browning of the callus,of which 2.0 mg·L-1 had the best comprehensive effect and normal growth of the tissue culture seedlings.influences.?2?It was found by HPLC that the monophenols that affected the browning process of the tissue culture seedlings by silver nitrate were chlorogenic acid,rutin,coumaric acid,p-coumaric acid,epicatechin,and Hydroquine,which has the highest rutin content,has the largest increase in phenols compared to several other monomeric phenols.?3?Under the influence of silver nitrate,the total phenol content,total flavonoid content and browning grade of'Fengdan'peony callus showed extremely significant correlation?P<0.01?,and the activity of peroxidase?POD?and degree of browning were present.Significant negative correlation?P<0.05?,polyphenol oxidase?PPO?,catalase?CAT?activity,malondialdehyde?MDA?content and browning degree showed a significant positive correlation?P<0.05?,superoxide There was no significant correlation between the activity of the dismutase?SOD?and the browning grade.?4?The fluorescence real-time quantitative assay of silver nitrate-treated callus revealed that the transcription factors MYB2,WD40-1,WD40-2 and the structural gene phenylalanine ammonia-lyase?PAL?in the Polyphenol synthesis pathway,Flavanone reductase?DFR?,Chalcone synthase?CHS?,Chalcone isomerase?CHI?,3-dihydroflavonol hydroxylase?F3H?,flavonol 3'-hydroxylation There was a significant correlation between the expression level of enzyme?F3'H?and browning grade and total phenolic content?p<0.05?. |
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