| Tripterygium wilfordii Hook.F. is our country’s important toxic natural resources of traditionalchinese medicine and Its main active ingredients include second terpenes and alkaloids. Because of its lowcontent of plant medicinal ingredients, and it is difficult for industrial production,So this experiment baseson the research status of plants at home and abroad and the summary of their previous research work,through the optimization of tissue culture formula of Tripterygium wilfordii Hook.F. and physiological andbiochemical study on tissue culture seedlings of various growth stages to provide a certain reference valuefor exogenous hormones and nutrition of reasonable growth substances added,and also providing technicalsupport for the realization of the tissue culture plants of Tripterygium wilfordii Hook.F.At the sametime,through adding the different concentration and types of elicitors and precursor substances in tissueculture rooting phase by tripterygium wilfordii,and through the analysis and research on the lactone alcoholcontent,finally finding out the different concentration and types of elicitors and precursor substances whichcan effectively improve the lactone alcohol content, to provide new way for increasing tripterygiumwilfordii’s lactone content and also solve the problems of its low content and long production period.Main results of this study reflect as follows:（1） In the process of tissue culture shoot induction of tripterygium wilfordii, Ms+6-BA1.5mg L-1+IBA0.1mg L-1（Hy7） induces most out of the total number of buds and seeding growing of best,the useof hormones on the effect of bud induction by tripterygium wilfordii’s primary relationship is6-BA> NAA> IBA.At the same time, by analyzing the results of the external plant hormones content in the plant,wefind that when buds induction in cultured21d has been completed, and enters the vegetative growth phase.（2） In buds subculture proliferation stage,the treat of Ms+6-BA1.0mg L-1+NAA0.1mg L-1+KT0.5mg L-1（Hz9） obtains the largest number of buds and average monthly multiplication coefficient is26.7which is the best culture medium formula. And the primary and secondary relationshipon of all of theabove factors on buds proliferation effect is6-BA＞NAA＞KT.By the experimental study on the externalplant hormones in the plant,we find that lower ratio of cytokinin and Auxin is good for buds proliferationand growth, as6-BA added concentration increases, endogenous IAA as rose. This experiment also provesagain that buds induction cultured for21d has been completed, and enters the vegetative growth phase. （3） In seedling culture stage, this test mainly uses in different concentrations of peptone and add VB1to make the buds growing robust. In the trial, the best training and formulation of both for the shoots growwell and buds to gain further proliferation is Ms+6-BA0.5mg L-1+VB10.5mg L-1+蛋白胨0.5g L-1（即LD6）.And the primary and secondary relationshipon of effect on seedling cultivation of tripterygium wilfordii is6-BA＞蛋白胨＞VB1. By the experimental study on the external plant hormones in the plant,we find that inseedling culture stage the endogenous hormones of IAA and GA3play a key role and it needs high contentof growth hormone as well as a certain content of gibberellin.（4） In the rooting culture one, the best training and formulation is1/2Ms+NAA2.0mg L-1+KT0.1mg L-1whichcan make up to100%rooting rate. Three factors above the primary and secondary relationshipon of effect on rooting rateis基本培养基＞NAA＞KT; By the experimental study on the external plant hormones in the plant,we findthat the growth hormone plays an important role in the rooting culture, high ratio of auxin and cytokinin isgood for rooting.Through observing and analyzing the root proliferation situation in the process ofrooting,we find that the increase in the number of adventitious root is when culture time increased.In the rooting culture two that is ABT on the effect of rooting cultivation experiment by tripterygiumwilfordii, we can draw out that three kinds of ABT used can induct root in varying degrees. But the effectof ABT1is better than the same concentration of ABT3and GGR6. The best mix of media on different typesof ABT on the rooting culture of tripterygium wilfordii are the AT1-3:1/2Ms+ABT11.5mg L-1+蔗糖30g L-1+琼脂8g L-1+Ac1g L-1;AT3-2:1/2Ms+ABT31.0mg L-1+蔗糖30g L-1+琼脂8g L-1+Ac1g L-1;GR6-2:1/2Ms+GGR61.0mg L-1+蔗糖30g L-1+琼脂8g L-1+Ac1g L-1. At the time of determination and analysis of endogenous hormone,we find thatin rooting culture stage, compared with the addition of auxin IBA,the addition of three kinds of ABT abovereduces the endogenous hormone contents of IAA.So we can think that add ABT to some extent inhibits thesynthesis and the content of IAA, And also between the different ABT,the effect difference,so when we use it weshould accord to actual situation to choose the appropriate category and concentration.（5） In adventitious root proliferation, according to root growth and proliferation status, weget the bestmedium formula is1/2Ms+IBA1.5mg L-1+ABT11.0mg L-1+KT0.5mg L-1.On the endogenous hormonesdetermination of the root,we find that In the course of adventitious root proliferation requires high levels ofIAA and ZT, and high levels of GA3is good for roots growth and thickening.（6） In the process of refine seeding transplant,after closing bottle hardening-off4d and openingbottle hardening-off3d,transplanting it to the1:1mixture of vermiculite and sterile peat soil and irrigatingit.After these,every other week when the fungicide（500times1:1mixture of carbendazim and DuPont Benlate） and shudaduo（500times） sprays.one months later,we find that survival rate of tripterygiumwilfordii can reach to84.5%.（7） On determination of soluble sugar content about the tissue culture of tripterygium wilfordii in allphases,we find that the changes of soluble sugar content in the callus induction of bud stage is differentfrom other training phases.In other phases,soluble sugar content has two peaks appear when cultured for7days and21days.In the rooting culture one,the second peak is located in the culture of28d because ofhigh concentrations of phytohormones NAA added.（8） The experiment about the influence on the changes of soluble sugar content for the different carbon sourceadded on tripterygium wilfordii in rooting stage,we find that soluble sugar content in addition to P2treatment, theremaining processings of the double-peak,most appeardd when cultured14days and28days（except Z3treatment）.From the test results we can draw that any concentration of glucose added and sucrose concentration of10g L-1and20g L-1added to some extent inhibit the growth of root induction.the best sucroses of the sugarconcentration and the type is30g L-1, followed by the20g L-1, finally10g L-1,the using of glucose has the worsteffect even no effect.（9） In the experiment on the influence of the basic media culture factor added in different concentration ontripterygium wilfordii induction of adventitious roots and pigment content,we find that no matter how much theculture factor added which has no significant effect on rooting rate of tripterygium wilfordii. But for pigmentcontent,the using concentration of40g L-1sucrose and0.5g L-1toner （Ac） can achieve the best results.We alsodraw from this experiment that the best way for rooting culture is after vaccination for the dark culture about1weeks, then transfer it to light training which the roots can get more volume and grow well.（10） In the experiment on the influence of the precursor substances added to the tissue culture by tripterygiumwilfordii’s adventitious root induction and pigment content,we can find that the best precursor materialtypes,concentration and cultivation condition respectively:citric acid40mg L-1（HNnm2）,dark culture;Sodium pyruvate1.0mg L-1（HNbt2）,dark culture;2.0mg L-1sodium acetate （HNys3）,dark culture;0.5mg L-1of phenylalanine （BA1）,lighttraining;Tyrosine0.5mg L-1（HLUA1）,dark culture;Lysine1.0mg L-1（LAA2）,light training.However the bestconcentration of precursor material and training condition for tripterygium wilfordii root induction are:Citric acid80mg L-1（Nnm4）,light training;Sodium pyruvate3.0mg L-1（Nbt4）,light training;1.0mg L-1sodium acetate （HNys2）,darkculture;phenylalanine1.0mg L-1（BA2）,light training;tyrosine1.0mg L-1（HLUA2）,lysine0.5mg L-1（HLAA1）,darkculture. （11） In the experiment on the influence of the elicitor added to the tissue culture by tripterygium wilfordii’sadventitious root induction and pigment content,we draw out that the best elicitor concentration and cultureconditions to improve pigment content were as follows:Salicylic acid10mg L-1（Nsy4）,light training;Lauren richalcohol0.5ml L-1（HNfn2）,dark culture;Yeast extract from1.0g L-1（Njm2）,light training.However the bestconcentration of the elicitor and training condition for tripterygium wilfordii root induction are:Salicylic acid1.0mg L-1（Nsy2）,light training;Yeast extract from1.5g L-1（HNjm3）,dark culture;And the lauren rich alcohol whether thelight or the dark,all of its concentrations play a role of inhibition effect of tripterygium tripterygium wilfordiiadventitious roots’ pigment synthesis.（12） In the experiment on the influence of the elicitor added to the tripterygium wilfordii proliferation ofadventitious root and pigment content, we conclude that when yeast extract and peptone, two kinds of elicitorsadded and cultured in the dark can make the adventitious root proliferation coefficient is improved and promot itsgrowth.And the best concentration and culture conditions for improving the pigment content are as follows:Yeastextract1.0g L-1（HBjm2）,dark culture;peptone0.5g L-1（HBdb1）,dark culture.From the test results of the remainingtwo kinds of elicitors on the beef extract and alcohol,we can find that low concentration of beef extract added canmake the adventitious root grow well,regardless of light or dark,but the high concentration inhibit thegrowth.While lauren rich alcohol added irrespective of the concentration and the culture condition, inhibits thegrowth of adventitious roots.In the test,the best elicitor concentration and culture condition which can improvepigment content are2.0g L-1extract of beef （HBnr2）, dark culture;Lauren rich alcohol0.1ml L-1（HBfn1）, darkculture. |
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