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標題: 臺灣原生藥用植物石薺薴和六角草玻璃化法超低溫冷凍保存流程之探討
Study of Protocol on the Cryopreservation of Taiwan Native Medicinal Herbs Mosla scabra (Thunb.) Wu & Li and Laggera alata (D. Don) Sch. Bip. ex Oliver by Vitrification.
作者: 林經剴
Lin, Jing-Kai
關鍵字: cryopreservation
出版社: 生命科學系所
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摘要: 本研究以兩種臺灣原生藥用植物 - 石薺薴 [Mosla scabra (Thunb.) Wu & Li] 及六角草 [Laggera alata (D. Don) Sch. Bip. ex Oliver] 組織培養苗為材料,探討超低溫冷凍保存前處理流程,並以水分生理及細胞形態變化為輔,深入了解各流程對芽體之影響。 研究結果顯示,石薺薴以含0.3 M蔗糖MS基礎鹽類培養基預培養7天後,切取莖頂組織,處理化學性冷凍保護劑LS (loading solution) 60 min,及PVS2 (plant vitrification solution 2) 120 min,冷凍保存後,可得最佳存活率80.5 ± 6.8 %。六角草以含 0.4 M蔗糖 MS基礎鹽類培養基預培養21天後,切取莖頂組織,再經LS處理120 min、PVS2處理180 min,冷凍保存後可得最佳存活率 77.7 ± 4.0 %。 石薺薴植株經0.3 M培養基預培養7天後,水分生理及醣類累積結果與未經預培養處理組比較,細胞含水量由88.0 %下降至79.4 %,滲透潛勢由 -0.81 MPa 下降至 -1.79 MPa,水分潛勢也由 -0.92 MPa 降至 -1.77 MPa,經預培養後可溶性醣由13.82 mg g-1增加為35.86 mg g-1,可溶性蛋白質累積由4.08 mg g-1 提升至5.73 mg g-1,藉此誘導植株之冷凍或脫水忍受力,增進冷凍保存之存活率。 石薺薴植株經預培養後,頂端分生組織原生質濃縮、核質比提升,並有原生質分離現象。石薺薴芽體經液態氮處理後,頂端分生組織及葉原體之核質比提高及輕微質壁分離,原生質膜與細胞壁分離間隙為168.42 nm,頂端分生組織下方4 – 5層細胞,質壁分離情形較為嚴重,原生質膜與細胞壁分離間隙為795.83 nm,為頂端分生組織的4.73倍,可能對細胞產生不可回復之傷害。推測於冷凍保存後存活細胞,應為原生質較濃稠的細胞,如頂端分生組織及葉原體,經成長後成為新植株。 本研究得知石薺薴及六角草冷凍保存之較佳前處理條件,及各處理對於石薺薴芽體水分生理及微細構造之變化,期望能供為種原超低溫冷凍保存之參考。
This study examined the pretreatment protocols for cryopreservation of in-vitro grown apical buds of two Taiwan native medicinal herbs Mosla scabra (Thunb.) Wu & Li and Laggera alata (D. Don) Sch. Bip. ex Oliver. In the meanwhile, the apical buds were used to examine the changes in water status and ultrastructure in order to correlate with the effects of cryopreservation procedure. The plantets of M. scabra were precultured for 7 days on hormone-free solidified Muashige and Skoog medium (MS medium) supplemented with 0.3 M sucrose. The excised shoot tips were treated with loading solution (LS) for 60 min and dehydrated with a highly concentrated plant vitrification solution 2 (PVS2) for 120 min. Following this protocol, we achieved the best survival rate of 80.45 6.75 %. The plantets of L. alata were precultured for 21 days on MS medium supplemented with 0.4 M sucrose. The excised shoot tips were treated with LS for 120 min and PVS2 for 180 min. Following this protocol, we achieved the best survival rate of 77.71 4.03 %. The physiological analyses were performed using shoot tips of M. scabra which have been precultured on hormone-free MS medium and supplemented with 0.3 M sucrose for 7 days. The relative water content reduced from 88.02 % to 79.38 %, and the amounts of soluble sugar and soluble protein increased from 13.82 mg g-1 to 35.86 mg g-1, and 4.08 mg g-1 to 5.73 mg g-1, respectively. The osmotic potential and water potential reduced from -0.81 to -1.79 MPa, and -0.92 to -1.77 MPa, respectively. This results showed the preculture procedures greatly enhanced the freezing or dehydration tolerance and improved the survival rate of cryopreservation shoot tips. The apical meristem of M. scabra shoot tip during preculture showed dense cytoplasm and slight plasmolysis. TEM ultrasctuctural examination of shoot tips after cryopreservation revealed the cells had high nucleo-cytoplasm ratio and slight plasmolyzed at first few layers of cells in apical meristem. The estimated interval between plasma membrane and cell wall was 168.42 nm. The 4 to 5 layers of cells below apical meristem had high degree of plasmolysis and the interval between plasma membrane and cell wall was 795.83 nm. This result showed the survived cells after cryopreservation were mainly localized in apical meristem or leaf primordia and subsequently regenerated into plantlets. This study provided an optimal pretreatment protocol for cryopreservation and the results showed greatly improved water relations and ultrastructural changes in of M. scabra and L. alata. This study was one step forward to develop long-term cryopreservation technology.
其他識別: U0005-0408200917161400
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