Agrobacterium Rhizogenes-mediated Genetic Transformation System Of Ardisia Crenata Sims And Determination Of Triterpenoid Saponin In Hairy Roots

Ardisia crenata Sims as a typical ornamental and medicinal plant,either from ornamental and medicinal value,or from the use of ecological and economic value,has highly potential of exploitation and utilization.In this paper,we studied the aseptic rapid propagation of Ardisia crenata Sims,hairy roots inducing conditions optimization and testing,and content determination of triterpenoid saponin in it.The main results were as follows:1,Through sterile cultivation of the four kinds of explants,we obtained the following conclusions:(1)Selecting of healthy stems with buds,basal sprouting,2-month ages seedlings and seeds as four kinds of explants to sterile culture,basal sprouting and seeds were the best explants material,having lower pollution rate and degree of browning.Continuously sterilized twice with 0.1%HgCl2,basal sprouting and 2-month ages seedlings optimal disinfection time were(3+3)min,and the seeds and stems with buds optimal sterilization time were(4+4)min.(2)MS medium was optimal growth medium for the four kinds of explants of further buds induction and buds multiplication hormone selecting.For the four kinds of explants,the best combination of buds induction medium was MS + TDZ 0.1 mg/L + NAA 0.1 mg/L,and the best combination of buds multiplication medium is MS + 6-BA 1.0mg/L + NAA 0.1 mg/L + TDZ 0.05mg/L.2,We selected A4,ATCC15834,LBA9402,R1601 four kinds of Agrobacterium rhizogenes strains to infect the aseptic explants leaves(approximate 1×cm2)and young stems(about 1cm long)to induce hairy roots.Agrobacterium rhizogenes resistance were selected and growth curves were plotted,also pre-culture time,co-culture time,infection methods were selected,as well as the hairy roots induction rate of different A.rhizogenes and different explants were compared.We used A.rhizogenes to infect A.crenata Sims and abtain hairy roots firstly,and established genetic transformation system preliminarily.The results were as follows:(1)Through A.rhizogenes resistance selecting,A4,ATCC15834 optimally growed on YMB + Rif 50mg/L medium,and LBA9402,R1601 optimally growed on YMB + Amp 50mg/L medium.In the activation processes of strains,LBA9402,ATCC15834,A4,R1601 were cultured 8?10h,11?13h,12?14h,12.5?14h in liquid medium separately could reach OD600 of 0.5?0.8.This time was the logarithmic growth phase of the strains and also was the best time to infect.(2)In hairy roots induction processes,1/2MS medium was the most suitable induction medium.Pre-culture 2d on 1/2MS medium and co-culture 2d on 1/2MS + AS 100mg/L medium could increase the induction rate.Puting leaves and bacteria together in a thermostatic shaker and setting conditions of 28?.180r/min darkness co-oscillation 8?15 min was the best inducting method.We took 1/2MS + Cef as sterilization medium and replaced it every 7d with Cef concentration decreasing(500mg/L,400mg/L,300mg/L,200mg/L,100mg/L,Omg/L)gradually.Aseptic leaves were the best hairy roots induction explants.Four kinds of A.rhizogenes strains were able to induce hairy roots of A.crenata Sims of aseptic leaves,but A4,ATCC15834 were the most effective strains,and their abilities to induce hairy roots were ATCC15834>A4>LBA9402>R1601.(3)Through hairy roots intuitive morphological observation,recording and PCR molecular detection,we found that ATCC 15834 and A4 T-DNA of Ri plasmid had been transferred and integrated into the leaves nucleus genome successfully.3,We used RP-HPLC method for triterpenoid saponins content testing of hairy roots which were mediated by A.rhizogenes ATCC15834 on leaves.The results showed that the triterpenoid saponins content of hairy roots was 8.21%,but CK(tissue culture root)was 0.18%and(sterile leaves)was host cell(sterile leaves)was 0.79%.This results were in accordance with the rules that the secondary metabolites content in hairy roots were much higher than it.