Ttissue Culture Of Millettia Reticuiata Benth. And The Change Of Endogenous Hormones

Millettia reticuiata Benth.is a Spatholobus suberectus of fabaceae.As a traditional Chinese medicine,it includes the dry stem of Spatholobus suberectus Dunn which can enrich blood and promote blood,regulate painful menstruation,relax tendon and activation collaterals,and so on.Modern pharmacology have demonstrated that Millettia reticuiata Benth.have the functions of improving the blood system,anti-tumor,anti-virus,antioxidant,two-way adjusting tyrosinase and relieving the cardiovascular.Also on the immune regulation,protecting liver,anti-inflammatory and analgesic,sedative and hypnotic have good curative effects.In recent years,as the increasing demand for medicine,the genuine Millettia reticuiata Benth.of wild resources is sharply down and become in short supply state,coupled with slow growth and less artificial cultivation of Millettia reticuiata Benth.,all of this will affect our country's medical needs.Using the plant tissue culture technology to quickly reproduce regeneration plant will play an important role in the protection of germplasm resources and expanding the scale of artificial cultivation,and also providing a powerful guarantee to meet the market demand for medicinal Millettia reticuiata Benth.In this study,the medicinal Millettia reticuiata Benth.of artificial cultivation was used as experimental materials for callus induction,proliferation and differentiation of adventitious bud,and many factors were screened in the process of plant regeneration.In addition,the content of four kinds of endogenous hormones IAA,ABA,ZT and GA3 was estimated and analyzed in the process of callus induction and differentiation.Besides,the effects of internal and external hormones on callus differentiation of adventitious bud were also discussed preliminarily.All above lead to the excellent germplasm resources preservation and rapid propagation of cultivated Millettia reticuiata Benth.and providing the reference for subsequent cell and molecular breeding.The main research results were as follows:1.The best disinfection methods are obtained for explants of Millettia reticuiata Benth..The best treatment schedule for tender leaf was:75%alcohol for 20 s and 0.1%mercuric chloride treatment 5 min,under this condition of the tender leaf survival rate reached 91.7%;The best treatment schedule for immature stem was:75%alcohol for 10 s and 0.2%mercuric chloride treatment 5 min,under this condition of the immature stem survival rate was 88.3%;The best treatment schedule for initial roots was:75%alcohol for 20 s and 0.1%mercuric chloride treatment 5 min,under this condition of the immature stem survival rate was 95%.2.Optimal explant and medium were screened for callus induction.Optimal explant was tender leaf and the best medium was MS+6-BA 0.9 mg/L + NAA 0.5 mg/L+KT 0.2mg/L+ Sucrose 30g/L+ Agar 8g/L,induction rate reached 88.3%.3.The optimal culture medium for callus proliferation was MS+6-BA 0.9 mg/L+NAA 0.5 mg/L+KT 0.1mg/L+ Sucrose 30g/L+ Agar 8g/L,callus proliferation coefficient was 4.59.4.Selected the optimal culture medium for callus differentiation of adventitious bud was MS+6-BA 2.5 mg/L + NAA 0.4 mg/L+GA3 1.0mg/L+ Sucrose 30g/L+ Agar 8g/L,the rate of adventitious bud differentiation was 88.33%;The optimal medium for tissue culture plantlet was 1/2MS+NAA 0.1mg/L+IBA 0.5mg/L+ Sucrose 30g/L+Agar 8g/L,the rooting rate 81.67%.5.In the process of callus induction,the content of IAA always kept a higher level and reached maximum when the callus growth was the most exuberant;the content of ABA,GA3 and ZT has remained at low levels;IAA/ABA on callus induction and growth has an important regulatory role.6.During the course of callus differentiation,the content of endogenous hormones changed regularly:IAA(high-low-high),reached the highest in differentiated into bud and higher than that of callus induction period,between 1.15-1.87?g/g;ZT and ABA(low-high-low);GA3(low-high);IAA/ABA(high-low-high),played an important role in promoting bud differentiation and growth;GA3/ABA and ZT/ABA(high-low-high),had cooperation effects of successfully promoted the adventitious bud differentiation.7.Taking the callus of Millettia reticuiata Benth.cultured in different differential medium for 28d as materials,the endogenous hormone IAA and ABA levels were determined.We found that as differentiation rates of callus treated by different exogenous hormones increased,the corresponding endogenous IAA levels dropped,while the ABA and ABA/IAA increased gradually.Consequently,we speculated that there was a close relationship between the ratio of callus endogenous ABA/IAA and callus differentiation ability.