Study On Establishment Of Tissue Culture Regeneration System And Conservation In Vitro Of Cymbidium Hybridum Walu 'Idol'

In order to estsablish the technological procedure of conservation in vitro and provide technical support for standardized production, The tissue culture and the conservation in vitro of Cymbidium hybridum Walu'Idol were studied in the paper.The main results are as follows:(1)The tissue culture system are established by different methods using shoot tip Cymbidium hybridum Walu'Idol .①The way to induce protocorm by shoot tip:shoot tips are treated with 70% alcohol disinfects for 30s, and immerged in 0.1% HgCl2 for 10min.Then the shoot tips are stripped to the last leaf and immerged in 0.05% HgCl2 for 2 min.MS+6-BA1.5mg/L+NAA0.1mg/L is the optimum medium for the induction of protocorm.MS+6-BA2.0mg/L+NAA0.1mg/L is the optimum medium for protocorm multiplication and 1/2MS+6-BA2.0mg/L+NAA0.5mg/L is the optimum medium for protocorm differrentiation . The optimum medium for rooting is 1/2MS+NAA0.1mg/L+AC0.5g/L . The transplanting medium of vermiculite and peat with the proportion was 1:2,which can save production cost.②The way to induce clump shoot by shoot tip:sterilization method is the same as above.MS+6-BA1.5mg/L+NAA0.1mg/L is the optimum medium for the induction of clump shoot from shoot tip;young seedlings can root in initial medium.They are transplanted into the culture medium of the mixture of vermiculite and peat with the proportion 1:2 after 80d.The survival rate of young seedlings is 100%.(2)The technological procedure of restricting growth conservation in vitro of protocorm and plantlets of Cymbidium hybridum Walu'Idol'are established.①The optimum method of protocorm restricting growth conservation:the protocorms of 30d-subculture can be preserved for 3 months at 10℃.The survival rate is 53.3% after renewing culture.The average differentiation multiple is 2.5.②The optimum method of plantlets restricting growth conservation:the plantlets of 30d-subculture could be preserved for 12 months at 10℃.Under 10℃, the plantlets are not frostbited and grow well after recovery growth.Further experiments is in progress.The plantlets have shorter conservation period by the medium with different chemical elements or different concertration of osmotic reagents or inhibitors.The plantlets grow fast and have the possibility of somaclonal variation.Therefore,cryopreservation method is beneficial to keep the genetic stability of plant germplasm resources,which can be applied in large-scale practice.(3)The cryopreservation procedure by protocorm is preliminarily established.The protocorms of 30d-subculture are pre-cultured for 5 days in the MS culture medium supplemented with 60g/L sorbitol at 25±1℃,and treated in Loading solution for 40min.Then it is treated in PVS2 vitrification solution at 0℃for 50min.The protocorms are directly plunged into liquid nitrogen.Following that, rapid thawing in a water bath at 40℃for 60s, washing in Unloading solution for two times, each time washing for 10min.Then they are transferred into the regeneration medium of MS+6-BA2.0mg/L+NAA0.1mg/L in dark for two weeks and prior to exposure to the light.protocorms are regenerated after cryopreservati- on,the survival rate is 10%, The plantlets can normally proliferate,root and transplantation as the control.Shoot tips can not be regenerated after cryopreservation though there was survival by TTC.That is probably closely related to seedling age,subculture time,growth condition which needed further study.