Optimize The Inducement And The Culture Conditions Of Hairy Root Of Gynostemm Apentaphyllum

Gynostemma pentaphyllum belongs to Cucurbitaceae Gynostemma pentaphyllum,which is a perennial herbaceous vine.Gynostemma pentaphyllum mostly contain gypenosides,flavonoids, polysaccharides,8 kinds of essential amino acids and various trace elements to human.Gynostemma pentaphyllum has a wide range of pharmacological function,which can effectively protect the heart, brain,blood vessels and liver,can regulate the immunity and anti-mutagenic and so on.At the same time,Gynostemma pentaphyllum not only used for anti-aging,anti-fatigue,anti-radiation and the elimination of free radicals,but also improved nervous system function,anti-ulcer,inhibiting gallstone formation and regulating endocrine activities.There is large demand for the drug in market.In the study about medicinal plants,it has become a hot,which by means of induction and culture of hairy roots for culturing new breeds,extracting the active ingredients of medicinal herbs. In nay study,I have set up Gynostemma pentaphyllum hairy root induction and culture system,which offer foundation for the study of the late Gynostemma pentaphyllum.Single factor experiments for several influential factors were carried out to find the best condition for inducement of Gynostemma pentaphyllum hairy roots.The influential factors contain Agrobacterium type,explant type, concentration of bacteria and infection time,pre-culture(co-culture) time,different antibiotic medium on the hairy root induction.At the same time,we have analyzed the type of basic medium and the medium of carbon sources on hairy root growth.This experiment by the following major conclusions:1.Comparison to A4 and R1601,according to induction rate,we can educe the most suitable Agrobacterium strains are A4.By filtrating the induction rate about leaves,petioles,stems as explant, according to the result,young and more mature leaves as explant were more suitable experimental material.2.Bacteria concentration have a lot of impact on Gynostemma pentaphyllum hairy root induction.On the basis of experiment result,the most appropriate OD600 of A4 suspension was about 0.8,the induction rate was 75%.3.The number of days pre-culture,co-culture days,infecting time were carried out to find most suitable Gynostemma pentaphyllum hairy roots induced conditions.As a results,pre-culture is unnecessary for Gynostemma pentaphyllum,induction rate was 88.4 percent,the most appropriate co-culture time was 2-3 days,the most appropriate infecting time was about 10min.4.AS has significant influence to plant hairy roots induction.However,different plants are different to the most appropriate concentration of AS.The experimental results show that along with the increase in the concentration of AS,the hairy root induction rate was first gradually increased and then decreased trend.AS concentration was 100umol/L,the induction rate is highest,about 79.7%.AS concentration was 400umol/L,it have more higher than not AS in the rate of hairy root induction,but the leaf explants greater harm,easily browning,so that the hairy root induction rate was relative lower.5.The inducing rate in MS+500mg/L(cef) medium culture,was not a patch on in MS+300mg/L(Cab) culture for Gynostemma pentaphyllum receptor leaves.Earliest rooting time is about seven days in the cab medium,but in the cef medium was about 11 days,it was later than 4 days at the root Cab cultivated.After three weeks,in hairy root induction rate is as high as about 85%in Cab medium,while only 51.72%in cef medium.Hence MS+300 mg/L cab medium are more suitable medium for hairy roots.6.PCR analysis for rolC confirmed that the T-DNA fragment of Ri plasmid of A.rhizogene was integrated into genome of hairy roots of Gynostemma pentaphyllum.7.It was obtained that 1/2 MS culture medium was the most favorable one for growth of hairy roots of Gynostemma pentaphyllum,among MS,1/2MS,B5 and1/2 B5 basic culture medium,in which the average growth fold of fresh weight was 9.71.8.Changes in the types of sugar on the basis of at 1/2MS medium,The experimental results obtained,sucrose as sugars was best of the hairy root growth,fresh weight of hairy roots can be increased 10.62 times.