| Summary: | 碩士 === 國立中興大學 === 植物學系 === 86 === 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.
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