Research On The Germplasm Preservation And Genetic Variation Of Taxus Cell

The taxus was instability during the long time subculture which serious restrict the industrialized process of large-scale culture of taxus. Till now, the mechanism of the instability was not very clear althought there were many researches focused on this. So, it was very important to establish a stably germplasm preservation approach. In this research, the possibility of slow-growth conservation and crypreservation for the taxus cell were conducted. The results as follows:1) Established a series of taxus cell lines of anti-precursor by add phenylalanine, sodium benzoate and sodium acetate in the medium. After six months's selection, the output of taxol was enhanced to 1.35 times.2) The variation of taxus cell lines during the slow-growth conservation was carried out, the results showed:(1) There was no significant difference in the cell morphology and taxol production between the cell lines bebore and after preservation, but the taxol production decreased during the subculture after recovery, and the taxol content decreased faster when conserved a long time in the low temperature;(2) Reversed-phase high performance liquid chromatography(HPLC) were used to detect the DNA methylation level during the conservation and the subculture after recovery, the results showed that DNA methylation level was increased during the subculture after recovery which accompanied a decrease in the taxol content. Maybe there was relationship between the DNA methylation and the taxol content;3) The possibility of the three crypreservation method such as Vitrification, Pregrowth-Desiccation, Desiccation- Vitrification for the taxus cell preservation was conducted, and constructed the preservation methods both for the Taxus Chinese and the Taxus media cv. Hicksii.(1) For the method of Vitrification, the factors of preculture method, the cryoprotective agent and the dehydration time were more important for cryopreservation of taxus cell; but the recovery of the reserved cell was very difficult, and it began to growth after 45 days, the growth rate was very low; (2) The Taxus Chinese was seemed to easy to conservation than Taxus media cv.Hicksii, it can easy to recovery from preservation both by Pregrowth-Desiccation and Desiccation- Vitrification; and the Taxus media cv.Hicksii was succeded by Desiccation-Vitrification; further research showed that the critic water content of Taxus media cv.Hicksii was higher than the Taxus Chinese, so it wouldpass the freezing point only with the help of cryoprotectant.