Optimization Of Regeneration Conditions And Regulation Of Metabolite Saponin B4 In Pulsatilla Chinensis

Pulsatilla chinensis?Bge.?Rege is a perennal herb in the Ranunculaceae family commonly used in traditional Chinese medicine.It is cold,tasted bitter and has the effects of clearing heat detoxification,cooling blood,stopping diarrhea and so on^[1-2].Modern pharmacological studies have shown that Pulsatilla has the efficacy on anti-inflammatory,tumor-resistant,immune-enhancing and curing Alzheimer^[3-5].Through the optimization of different medium types and induction of callus,this paper explores the effects of different plant hormone on callus proliferation;physiological biochemistry characteristics in callus proliferation;different types of hormones on adventitious bud induction and the optimization of the adventitious bud proliferation by using the response surface method.Adding different concentrations of exogenous elicitors to the culture medium,to explore the influence on the content of callus saponin B4,it provides a reference for the further use of mass-produce the medicinal ingredients in Pulsatilla.The results of this paper are as follows:1.The effect of different sterilization time on seed germination was studied through single-factor test design using full seed of Pulsatilla chinensis?Bge.?Rege;the results showed that 0.1%liter mercury sterilization 4min was the best sterilization time,and the sterile seedlings were successfully obtained.Three different types of materials?leaves,stems,and petioles?were taken to investigate the effects of different media types?MS,1/2MS,and B5?on callus induction.The effect of different concentration ratios of 6-BA,T Z and NAA on the formation of callus was studied by orthosis test design,and the results showed that the leaves of sterile seedlings had the best induction effect on MS culture.By using orthopaedic test design,the callus treated with 6-BA,T Z and NAA at different concentrations can grow to varying degrees and all have significant differences,the results are shown that the medium suitable for induction of Pulsatilla callus is MS+6-BA 0.1mg/L+T Z 0.3mg/L+NAA0.3 mg/L,induction rate 100%,6.36g,large amount,yellow-green and loose.2.Using MS+6-BA0.1mg/L+T Z0.3 mg/L+NAA0.1 mg/L as the basic medium for inducing Pulsatilla callus,healthy callus as the culture material,the effects of different hormone concentration ratios of VC,T Z and NAA on the proliferation of callus were studied by orthopaedic test design.The results show that the influence of 3 factors on the proliferation of callus is T Z>NAA>VC,through range analysis.The results show that T Z and NAA have significant differences efficacy on callus proliferation,and VC has no significant difference in the proliferation of callus.Therefore,combining all factors,the best hormone combination is MS+T Z1.2mg/L+NAA0.1mg/L+VC2.0 mg/L.3.?1?By comparing the physiological and biochemical changes of Pulsatilla callus in the process of proliferation,it was found that the CAT?PO?M A gradually increasing during the first 10-40 days;CAT?PO?M A and SO increased sharply to the maximum in 40-50 days.aftet 50d began to decrease;and the activity decreased to the lowest after 70d.It is indicated that the metabolic ability of the callus is enhanced and then decreased,and 50d is the optimal growth state.?2?By comparing the physiological and biochemical characteristics of different callus,it was found that the results of determination of CAT,SO,PO and M A in embryogenic and non-embryonic,browned callus tissues showed that:the content of CAT in yellow-green embryogenic callus was significantly higher than that of non-embryogenic callus;the content of SO was lower than that of non-embryogenic callus,but none reached a significant difference;the content of PO was under yellow-green embryogenic callus significantly higher than non-embryonic callus,yellow soft type content is lower than non-embryonic,and all have significant differences.In summary,through the study of the related physiological and biochemical changes in different types of callus and proliferation,it has laid a scientific foundation for the establishment of the tissue culture and rapid propagation system of Pulsatilla.4.The optimal medium for the differentiation of Pulsatilla adventitious buds by single factor experiment is MS+NAA0.1mg/L+0.3mg/L hydrolyzed milk protein+ZT0.2mg/L.The response surface method is used to optimize the adventitious buds of Pulsatilla propagation conditions,the most suitable medium for the selection of adventitious bud growth was Huabao 3g/L+6-BA0.75mg/L+NAA0.1mg/L+ZT0.25mg/L.5.Taking healthy green embryogenic callus as material,studied the effects of different concentrations of me JA and SA on the growth of callus and the content of active ingredients in Pulsatilla saponin B4,the results showed that the effects of different concentrations of Me JA and SA on the growth of healing tissue and the content of Pulsatilla saponin B4 had significant differences.With the increasing of Me JA and SA,the content of Pulsatilla saponin B4 increases and then decreases,reaching the maximum when the SA concentration is 600 mg/L,which is 8.33 times that of the control,Me JA reaches the maximum when the concentration is 150ug/L,which is 32.45 times of the reference substance.