| Panax ginseng C. A. Meyer is a precious Chinese medicinal plant that belongs to Araliaceae family. Although China is rich in Panax genus, many genetic resources were lost due to the imcomplete collection and preservation system, which brought many difficulties for Panax breeding and technical innovations. Cryopresevation offers an efficient method for the long-term preservation of germplasm resources. In this research, buds and callus of ginseng were used as material to studuy the factors that influence the effect of cryopreservation and genetic variation characteristics of regeneration material. The main results were as followed:1. In this experiment, vitrification cryopreservation was used to store adventitious buds, a suitable procedure was established as follows: Choosing tissue culture plants had been subcultured for 45-60 days, dissected adventitious buds of 2-3mm were pretreatment with MS culture medium containing 0.5mol/L Suc for 2 days after 14-28 days of cold-hardening. Following, adventitious buds were loaded in a mixture of 2mol/L Gly and 0.4mol/L Suc for 30 min at room temperature, then under the condition of PSV2 dehydration at ice-water bath for 90-120 min. After dehydration, adventitious buds were directly plunged into liquid nitrogen. Stored in liquid nitrogen for 30 days, and then plunged into 38-40℃ water bath for rapidly thaw, the adventitious buds were washed three times with 1.2mol/L Suc of MS culture medium. Finally, recovery culture in MS medium with 4mg/L 6-BA+5mg/L GA3+0.25mg/L NAA, the highest survival rate was up to 72.39%.2. In this experiment, ladder-style freezing was adopt to store Panax ginseng callus, factors affecting cryopreservation were analyzed, results showed pretreatment had effect on both cell activity and water content, cell activity came to the highest value while water content was 30% after 10 days of pretreatment; the optimum cryoprotective agent was 10% DMSO+0.5mol/L Suc+10% Gly. Recovery growth was characterized by new callus growing out of the surface of initial callus, it entered into fast growth stage after a period of time called stagnation stage.3. In this experiment, genetic variation characteristics of experimental material after cryopreservation were analyzed using morphology observation, SSR molecular marker technique and two-dimensional gel electrophoresis, results were as followed: in morphology, there was no difference in growth state; in molecular level, there was no differential band;in the level of protein expression, according to differentially expressed proteins identified by MALDI-TOF-TOF,several proteins showed changed expression with different extent.Inclusion, in this research, cryopreservation system was established through discussing the factors effect cryopreservation, and then the genetic variation characteristics were explored. These results can provide method and material basis for the storage and innovation of Panax ginseng germplasm resources. |
