| Camptotheca acuminata Decne. was a peculiar anticancer plant native in South China. It could produce CPT (Camptothecin), a monoterpenoid indole alkaloid, which was known for its remarkable inhibitory activity against tumour cells. It was the only one inhibitor found up until now to inhibit Topo I (Topoisomerase I), and was praised as one of the 3 anticancer drugs of mankind first-selected in 21st century. The effects of various basic culture mediums, explant types and sampling seasons on the percentage of callus induction was discussed in this paper, and studied on the changes of CPT content in callus and leaves by HPLC, the results were as follows:1.The content of CPT was determined by HPLC on a ODS C18 column with a mobile phase consisted of acetonitrile-water (30:70) at a flow rate of 1.0 ml/min , by the detective wavelength 254 run, and the detective temperature 25 ℃. Under these chromatographic conditions, retention time of CPT was about 6.3 minutes, and the shape of chromatographic peak of CPT was very well.2.Among these three extraction technology, Soxhlet extraction technology was the best one by which the content of CPT was remarkably higher than that of Supersonic extraction technology and NaOH solution extraction technology.3.With the induction cultuer medium of MS+NAA1.0 mg/L+IBA1.0 mg/L+KT0.1 mg/L, choosing the tender leaf or stem as the explant, the percentage of callus induction could reach even more than 90.0 %. The callus have good quality and growing rapidly. But only those callus which inducted by stem could induce bud. With the cultuer medium of MS+NAA0.05 mg/L+6-BA0.25 mg/L+KT0.5 mg/L, the coefficient of bud split could reach up to 4.2.4.Compared with 1/2MS , MS , B5 and White as basic culture medium, choosing the tender leaf as explant to induction callus with SH as basic culture medium, the content of CPT in callus was the highest,witch could reach 0.0382 ‰.5.With the culture medium of SH+NAA1.0 mg/L+IBA1.0 mg/L+KTO.l mg/L ,choosing the tender leaf as explant to induction callus, the best sampling season was in July. During this time, the percentage of callus induction could reach 82.2 %, the content of CPT in callus could reach 0.0412 %o, and the polluting rate of explant was 18.5 %.6.The best concentration of NAA and IBA to influence the content of CPT in callus induction by tender leaf was 3.0 mg/L, 10 d later under the tratment with NAA and IBA, the content of CPT in callus respectively got 25.7 % and 11.4 % higher than CK (contrast).7.Both survival rate of callus and content of CPT in callus got down rapidly after being dealt with 10 d by various concentration of colchicines. But another result showed that the content of CPT in callus was higher than CK (contrast) after being dealt with 10 d by various concentration of 8-hydroxy quinoline, the best concentration of 8-hydroxy quinoline was 20 mg/L.Under this concentration, the content of CPT in callus got 51.4 % higher than CK (contrast) and the survival rate of callus could reach 90 %.8.There were two peaks of CPT content both in tender leaf and older leaf around the year,in June and September. The content of CPT in June was the highest around the year. In this month, the content of CPT in tender leaf could reach 1.941 %o, and the content of CPT in older leaf could reach 1.325 %o correspondingly. In all months, the content of CPT in tender leaf was extremely remarkable higher than that in older leaf.9.1n April, the age of leaf within 5 d has the highest content of CPT, it could reach above 1.114 %o. In July, the age of leaf within 3 d has the highest content of CPT, it could reach above 1.960 %o. In October, the age of leaf within 7 d has the highest content of CPT, it could reach above 0.950 %o. At the same ages, the content of CPT in leaf in June was extremely remarkable higher than in April and in October.The result indicated that choosing the tender leaf in July as explant, and using SH as basic culture medium with 3.0 mg/L NAA or dealing the callus with 8-hydroxy quinoline by the concentration of 20 mg/L were able to remarkably increase the content of CPT in callus. It is observed from its growth and development seasons.The best collecting season of leaf was in June. At this time the leaves grow rapidly and the content of CPT come to the highest around the year. |
