Study On The Tissue Culture Of Artemisia Annua L. And Artemisinin Content In The Culture Substance

The stem fragmens,leaves,apical buds and flowerets of Artemisia annua L. were used as explants in this study in order to establish the effective tissue culture system of A. annua . In addition the factors influencing artemisinin biosynthesis in A.annua shoot cluster cultures were also investigated in this paper so as to select the opimum cultural conditions and the most suitable condition had been determined to provide the scientific basis for screening A.annua plant with high artemisinin content.The results are shown as follows:(1) The best sterilizing effect on stem segments is 7 minutes with 0.1% HgCl2 in which condition the living rate can reach 66.3%.While as for the leaves and flowerets,it is 5 minutes and the living rate can reach 71.8% and 76.8% separately.(2) The favorite culture medium for callus induction from leaves is MS+2, 4-D 2.0 mg.L-1, induction rate can reach as high as 92%, but the most suitable culture medium for callus induction from flowerets is MS+6-BA 4.0 mg.L-1+NAA 0.05mg.L-1, induction rate has also achieved 46.4%.(3) If we take the stem segments as explants,the culture medium MS+6-BA 0.3 mg.L-1+IBA 1.0 mg.L-1+KT 2.0 mg.L-1 was more fitable to induce shoot clusters and the induction rate is 55.6%.But it is difficult to differentiated shoot clusters from the callus induced from leaves and flowerets.The best culture medium is MS+6-BA 1.0 mg.L-1,and the differentiating rate is only 30.4%.(4) The optimum medium for multiplicating of shoot cluster is MS+6-BA 0.5 mg.L-1+NAA0.05 mg.L-1, under this culture condition, the multiplication time is 4.7.(5) During initial and multiplication culture, shoot clusters frequently appear vitrification. The MS culture medium attaching the 50g.L-1 sucrose or the 9g.L-1 agar can effectivly prevent shoot cluster from vitrification.(6) The culture medium suitable to root for shoot is MS+ NAA 0.05 mg.L-1 or MS+IBA0.3 mg.L-1 in which condions the rooting rate can reach 87.6% and 82.5% separately. (7)The optimum cultural conditions of growth and artemisinin accumulation of shoot cluster in A. annua were identified as follows : sucrose concentration,30 g.L-1 ; nitrate resource concentration,45mM;inorganic phosphate concentration,200mg.L-1;NH4+-N/NO3--N ratio ,1:3;gibberellic acid concentration,3.0mg.L-1;Under the above conditions , artemisinin pruduction can reach 15.8 mg.L-1 after 25 days of culture.