Study On Tissue Culture And Cryopreservation Of Camptotheca Acuminate

Camptotheca acuminata is the secondary protection plant, meanwhile the anticancer activity substances-camptothecin and its derivatives in vivo is also a research hotspot in the medical field today. In order to cultivate and utilize the resources of Camptotheca acuminate in large-scale, realize the value of Camptotheca acuminata resources and achieve the preservation and development of Camptotheca acuminata germplasm resources in the use of resources, this study respectively explored the optimum conditions of adventitious buds regeneration by using stems or shoot tips and callus induction by using stems or leaves in vitro, and carried out the shoot tips’ and callus’ cryopreservation of Camptotheca acuminata. The main results are as follows:1. Camptotheca acuminata fruits are disinfected by using0.1%HgCl2. After the seeds preculture in MS medium for15days and then treat3minutes by using10%NaCIO, they germinate well, and also seedlings grow well.2. Using shoot tip and stem segments as explants to induce adventitious bud differentiation, the best medium is MS medium supplemented with6-BA1.0mg/L, NAA0.1mg/L,3%sucrose and0.8%agar.3. When inducing callus by leaves and stems, the better medium are MS medium with2,4-D2.0mg/L and6-BA0.2mg/L or MS medium with2,4-D2.0mg/L,6-BA2.0mg/L and NAA2.0mg/L. The former calli induced are soft and white, which are suitable for cell suspension; the latter calli induced are also loose but present yellow-green, they are suitable for cell suspension and organ regeneration.4.Establishing the primary method for cryopreservation of Camptotheca acuminata. The main procedures are as follows:10mm stem tips cut from3months growth materials are precultured on MS semi-solid medium with5%mannitol and5%DMSO under the condition of4℃for6days; then detaching2mm stem apex and pretreating them with60%PVS2for30minutes at0℃, then dehydrating with100%PVS2for30minutes at0℃.After replacing fresh100%PVS2solution, the stem apexes are immediately plunged into liquid nitrogen; the materials are taken off from liquid nitrogen after24hours, and then thawed rapidly in the water bath at40℃for2minutes, the stem apexes are washed by MS liquid medium with1.5mol/L sucrose for2times, each time is10minutes; after drying excess liquid, the stem apexes are transferred to MS regeneration medium supplemented with6-BA1.0mg/L and NAA0.1mg/L, and cultured in darkness for10days at25℃and then transferred to normal condition in lightness.Counting the survival rate after15d. The survival rate and regeneration rate of shoot tips can respectively reach73.33%and43.33%under the best combination conditions.