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Effect of different pretreatment on cryopreservation injury of native Cymbidium sinense rhizome tips
|摘要:||本實驗以台灣原生之報歲蘭（Cymbidium sinense （Andr.）Willd.
This study used native Cymbidium sinense as materials for investigation. The main purpose was to investigate the cryopreservation protocols of seeds and rhizome tips, and understand the degrees of damage on cell membrane after dehydration, cryoprotectant pretreatment or preculture in different sucrose concentrations of semi-solid culture medium. In the meanwhile, ultrastructural changes in rhizome tips were used to show the damage of cryopreservation procedure. Cymbidium sinense seeds were transferred to cryogenic vial, and immersed in liquid nitrogen for cryopreservation. The seeds still can germinate after cryopreservation for two years. There was no survival after the rhizome tips were pretreated with dehydration, cryoprotectant, encapsulation-dehydration or different sucrose concentrations of semi-solid culture medium, and then cryopreserved in liquid nitrogen. Furthermore, lipid peroxidation, ion leakage, and ultrastructural change were detected to judge the degree of damage on rhizome tips through cryopreservation procedure. The MDA production and ion leakage were all increased after treated with dehydration, cryoprotectant or different sucrose concentrations of semi-solid culture medium only without cryopreservation. The TEM ultrastructural examination revealed that the cell was plasmolyzed, cytoplasm concentrated and cell wall deformed, but mitochondria were still seen after dehydration for 60 mins. The cell was also plasmolyzed after cryoprotectant treatment but many vesicles appeared. Furthermore, cytosol was accumulated at the periphery of plasma membrane, and part of membrane systems was broken after dehydration and PVS2 pretreatment followed by cryopreservation. This result showed that membrane systems were damaged in accordance with the high lipid peroxidation and ion leakage detected after cryopreservation. The most serious injury of three pretreatments mentioned above was cryoprotectant pretreatment. Thus, the cryopreservation of rhizome tips of Cymbidium sinense should avoid using cryoprotectant composed with PVS2 ingredient. The result of this study suggests that pretreatment with sucrose semi-solid culture medium and dehydration with silica gel within 60 mins can be the basic procedure for cryopreservation of rhizome tips. To enhance the survival after freeze-thawing may need to combine additional treatment in the procedure.
|Appears in Collections:||生命科學系所|
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