| Plant genetic resources are one of the most important substance bases for human to survive and develop, and to keep their diversity is the important guarantee for sustainable development. However, planting in open field, as a traditional method for conservation of persimmon germplasm, has emerged so many faults such as diseases and insect pests, effects from natural climate,space limitation,lack of managerial cost,and other reasons.Genetic variation may be caused in sub-culture process when the plant resources was cultured in vitro.Recent years developed from tissue culture technique,LN cryopreservation was proved to be an ideal way to conserve plant resources due to its advantages,such as conserving stability,high efficience,less expense and so on.Cryopreservation of plant germplasm by encapsulation -vitrification technique has been appreciated extensively to date because its simple operating and simplified procedure.Many successful studies on the cryopreservation of germplasm by encapsulation–vitrification technique have been reported so far.Significant potential have been showed for applying this method to cryopreservation of germplasm.Cassava(Manihot esculenta Crantz) is the most important energy produced root tuber crop in southeast,tropic Africa and Central America.Shoot-tips micropropagation,recovery micropropagation,cryopreservation by encapsulation- vitrification,genetic stability of three cassava cultivars:"huanan wu hao","huanan liu hao","huanan ba hao"were studies in this experiment to investigate primary influencing factors.The main results are as follow:1.Medium with different hormone levels of TDZ for cassava somatic embryogenesis was optimized and selected.The shoot-tips were cultured preferably on the medium containing 0.05mg/LCuSO4 and 10μmol/LTDZ.2.Shoot tips of cassava in vitro plantlets were successfully cryopreserved using the encapsulation-vitrification technique. Excised shoot tips were encapsulated into alginated-gel beads. Then alginate-coated shoot tips were loaded with a mixture of 2 mol.L-1 glycerol and 0.4 mol.L-1 sucrose for 30 to 40 min at 25 oC and precultured on sucrose enriched (0.5 mol.L-1) medium for 2 d. After preculture, encapsulated shoot tips were dehydrated with a highly concentrated vitrification solution(PVS2 solution)for 4 h at 0℃, and then plunged directly into liquid nitrogen. After rapid warming in water for about 90 sec at 40℃, the shoot tips were rinsed with the modified MS medium containing 1.2 mol.L-1 sucrose for 20 min, and then cultured on the modified medium (MS+0.02mg.L-1 NAA) in dark prior to exposure to the light. The best survival rate of encapsulation vitrified shoot tips amounted to nearly 70% and no abnormality was observed. Thus the protocol using shoot tips appeared to be promising as a routine method for the long-term conservation of Cassava germplasm.3.A selected medium consists of MS+0.02mg/LNAA that suitable to recovery of cassava shoot-tips after cryopreservation.4.The survival rate was influenced by the composition and concentration of sugar in preculture medium.A highest survival rate was attained in 2 days'preculture with the lowest water content,highest soluble sugar content and higest cell viability.5.The MDA content after loading and dehydrating the beads was not changed, while it increased dramatically after storing the beads in LN for 24h.6. In vitro cultured cassavas were used to study on genetic stability after cryopreservation by SRAP mark.Results showed that DNA polymorphism was not different by SRAP. So we could conclude that genetic stability of cassava was not altered after cryopreservation.
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