Establishment Of Embryogenic Cell Suspension System And Cryopreservation Of Japanese Lawngrass (Zoysia Japonica Steud.)

Zoysia japonica steud.'Zenith'and native germplasm collected from Shandong and Liaoning were used for establishment of cell suspension lines, somatic embryo inducement, and cryopreservation. The objective of this study was to establish high efficiency regeneration system and conserve the system safely even after long-term of preservation, provide theoretical and practical basis for cell project and gene project of zoysiagrass. Conclusions showed as follows:(1)In vitro cell suspension cultures of zoysia japonica were established and optimized. Light yellow, grainy and incompact calli were selected and cultured in liquid MS medium with 2 mg/L 2, 4-D. During early stage, the rate between condition medium and fresh medium was 1:3. With the increasing of subculture times, time spent to establish a cell suspension line was shortened.(2)Under micro observation, at early stage, cells spread out from calli were unregular, filled with big vacuole; with the increasing of subculture times, more and more cells were elliptic, and filled with grainy inclusions; finally almost all the cells were elliptic and full of inclusions.(3)Growth curve of suspension cells were divided into four phases: lag, exponential, stationary, and senescent phases. 5～7 ml(PCV)/100 ml density, pH 5.5～6.2, and 5 days'subculture period were appropriate for cell suspension culture of zoysiagrass.(4)In vitro somatic embryo inducement and plant regeneration system of zoysiagrass were established. In vitro somatic embryo could be induced either by solid MS medium or liquid MS medium, but somatic embryo induced from liquid medium did not have the capacity to grow into mature plants. Somatic embryo induced from solid medium regenerated into plants after subcultured for two months.Two embryogenic cell suspension cultures Clone A, Clone B and one non-embryogenic cell culture Clone J of Zenith established in this study were used as materials. Vitrification,rapid prefreezing and desiccation protocols were studied to establish cell suspension culture preservation system. Conclusions were as follows:(1)Cell survival rate of embryogenic suspension lines were higher than non-embryogenic cell suspension lines after cryopreservation. Preculture and desiccation following preculture also could increase cell survival rate of suspension culture lines of zoysiagrass. Transition dehydration reduced cell survival rate of zoysiagrass cell suspension culture lines. Thaw at 37～40 oC water bath was the best way for cell suspension culture of zoysiagrass.(2)Differences of cell survival rate by TTC assay between different growth phases were significant after vitrification preservation. Cell survival rate by TTC assay of later exponential and early stationary phases were 56% and 74% respectively. Survival rate of cells in early exponential and senescent phases were 12% and 11%. But there were no significant differences between cell survival rates of different phases by rapid prefreezing preservation.(3)Cell survival rate by TTC assay was increasing with the time extending of precuture and reached the peak value after 7 days. During vitrification protocol, sucrose medium was more efficient in improving cell survival rate at early stage(1～5d), then with the time extending, sorbitol medium reached up and finally there were no significant differences between them after 7 days'preculture. All in all, as preculture medium, sucrose medium was more efficient than sorbitol medium followed by vitrification preservation. To rapid prefreezing, 0.8 mol/L sorbitol medium was much more efficient in improving cell survival rate of zoysiagrass.(4)20 mins was the appropriate time in room temperature for dehydration by cryoprotective agent(PVS2), however, 40～60mins was needed in ice and water bath. Also dehydrated cells in ice and water bath could increase survival rate of cell suspension lines. Somehow, cells treated with preculture also can shorten the dehydration time. The proper time for zoysiagrass dehydration was 20～40mins.(5)Influences of different cryopreservation protocols and preculture mediums on cells regrowth rate were significant. High osmoticum preculture medium could improve cell regrowth rates of cell suspension culture of zoysiagrass. Cells survived and regrowing after thawed could be divided into two types: one kind was grainy as calli prior cryopreservation and the other kind was soft and unregular. Those soft and unregular calli died or grow into grainy calli with the time of subculture.