Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20658
標題: 不同前處理對原生報歲蘭超低溫冷凍保存傷害之探討
Effect of different pretreatment on cryopreservation injury of native Cymbidium sinense rhizome tips
作者: 羅智明
Luo, Jyh-Ming
關鍵字: 報歲蘭;超低溫冷凍保存;微細構造;離子滲漏;根莖莖頂;MDA生成量;Cymbidium sinense;cryopreservation;ultrastructure;ion leakage;MDA production;rhizome tips
出版社: 植物學系
摘要: 
本實驗以台灣原生之報歲蘭(Cymbidium sinense (Andr.)Willd.
)為材料,主要目的在研究種子及根莖莖頂超低溫冷凍保存之方法,並探
討乾燥劑脫水、保護劑與不同濃度蔗糖培養基前處理對報歲蘭根莖莖頂細
胞膜系傷害之影響。同時配合穿透式電子顯微鏡觀察細胞微細構造之變化
,以瞭解冷凍保存之處理過程對報歲蘭根莖莖頂影響之情形。 蘭花種
子移入冷凍管中,直接置入液態氮桶中進行超低溫冷凍保存,經液態氮冷
凍保存2年後仍能存活發芽。根莖莖頂先以乾燥劑脫水、保護劑、裹膠脫
水(encapsulation-dehydration)及不同濃度蔗糖培養基等前處理後,
進行超低溫冷凍保存結果均未存活,因此進一步測定乾燥劑脫水、保護劑
與不同濃度蔗糖培養基前處理後,膜脂質過氧化程度、離子滲漏及微細構
造之變化,以作為判斷冷凍保存處理過程之傷害依據。根莖莖頂未經液態
氮冷凍保存,僅以乾燥劑脫水、保護劑與不同濃度蔗糖培養基處理,莖頂
MDA生成量、離子滲漏程度皆已上升。穿透式電子顯微鏡觀察,發現經乾
燥劑脫水60分鐘後造成細胞質離、細胞質濃厚,但仍可見到粒線體,而細
胞壁開始變形;保護劑處理則造成細胞質離及產生小囊泡。以乾燥劑脫水
或PVS2冷凍保護劑處理根莖莖頂,再經冷凍保存後細胞膜附近皆有物質累
積,部份膜系產生破洞,顯示膜系構造已經受傷,此結果亦與冷凍保存後
高膜脂質過氧化及離子滲漏之結果相符合。 上述三種前處理結果以冷凍
保護劑之傷害最嚴重,故進行報歲蘭根莖莖頂冷凍保存應避免使用PVS2組
成之冷凍保護劑。由各項前處理傷害程度之結果獲知,以蔗糖培養基前處
理配合乾燥劑脫水60分鐘為限,可作為冷凍保存前處理模式,若能再進一
步探討相配合之其他處理或可獲致有效之冷凍保存技術。

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.
URI: http://hdl.handle.net/11455/20658
Appears in Collections:生命科學系所

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