Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22529
標題: 番木瓜玻璃質化法超低溫冷凍保存流程之探討
Investigation of Protocol on the Cryopreservation of Papaya (Carica papaya L.) Treated by Vitrification
作者: 羅文陽
Ro, Wen-Yang
關鍵字: papaya;番木瓜;cryoperservation;vitrification;PVS2;超低溫冷凍保存;玻璃質化法;抗凍保護劑
出版社: 生命科學系所
引用: 王源龍。2004。番木瓜莖頂之液態氮超低溫保存方法及傷害之探討。國立中興大學生命科學研究所博士論文。 王德男。1990。本省木瓜優良品種簡介。台灣省農業試驗所技術服務。1: 17-21。 王德男。1991a。木瓜。臺灣果樹彩色圖說。pp 191-196。 王德男。1991b。台灣木瓜栽培之回顧與展望。台灣果樹之生產及研究發展研討會專刊。pp 357-371。 王德男。1991c。番木瓜。台灣農家要覽 農作篇(二)。pp 129-136。 王德男,翁瑞亨,黃基倬,徐秀鳳。1999。木瓜新品種-台農六號(朱玉)之育成。中華農業研究。48: 65-74。 朱麗淑。1997。不同前處理對番木瓜莖頂超低溫保存存活率之影響。國立中興大學植物學研究所碩士論文。 許圳塗。1995。作物種質體外保存系統。作物種原保育技術研習會專刊。台灣省農業試驗所特刊。55: 69-80。 劉壹介。1997。缺水及鹽處理對冰花生理代謝之影響。國立中興大學植物學研究所碩士論文。 蔡淑華。1975。染色與染料。植物組織切片技術綱要。pp 47-58。 Ashmore SE, Azimi M, Drew RA (2001) Cryopreservation trials in Carica papaya. Acta Horti. 560: 117-120 Azimi M, O''Brien C, Ashmore SE, Drew RA (2005) Cryopreservation of papaya germplasm. Acta Horti. 692: 43-50 Bryant G, Koster KL, Wolfe J (2001) Membrane behavior in seeds and other systems at low water content: the various effects of solutes. Seed Sci. Res. 11: 17-25 Chu C, Dai Z, Maurice SB, Edward GE (1990) Induction of crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallium by abscisic acid. Plant Physiol. 93: 1253-1260 Crowe JH, Crowe LM, F. CJ, Wistrom CA (1987) Stabilization of dry phospholipid bilayers and proteins by sugars. Biochem. J. 242: 1-10 Dhindsa RS (1991) Drought stress, enzymes of glutathione metabolism, oxidation injury, and protein synthesis in Tortula ruralis. Plant Physiol. 95: 648-651 Dussert S, Mauro MC, Engelmann F (1992) Cryopreservation of grape embryogenic cell suspensions: influence of post-thaw culture conditions and application to different strains. Cryo Lett. 13: 15-22 Engelmann F (1991) In vitro conservation of tropical plant germplasm - a review. Euphytica 57: 227-243 Helliot B, Swennen R, Poumay Y, Frison E, Lepoivre P, Panis B (2003) Ultrastructural changes associated with cryopreservation of banana (Musa spp.) highly proliferating meristems. Plant Cell Rep. 21: 690-698 Hirai D, Sakai A (2003) Simplified cryopreservation of sweet potato [ Ipomoea batatas (L.) Lam.] by optimizing conditions for osmoprotection. Plant Cell Rep. 21: 961-966 Houssa P, Bernier G, Kinet JM (1991) Qualitative and quantitative analysis of carbohydrates in leaf exudate of the short-day plant, Xanthium strumarium L. during floral transition. J. Plant Physiol. 138: 24-28 Ishikawa M, Suzuki M, Nakamura T, Kishimoto T, Robertson AJ, Gusta LV (2006) Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses. Ann. Bot. 97: 453-459 Ishikawa M, Tandon P, Suzuki M, Yamaguishi-Ciampi A (1996) Cryopreservation of bromegrass ( Bromus inermis Leyss) suspension cultured cells using slow prefreezing and vitrification procedures. Plant Sci. 120: 81-88 Jensen WA (1962a) Carbohydrates and Cell Wall Constituents. In Botanical Histochemistry, pp 198-199 Jensen WA (1962b) Histological Procedures. In Botanical Histochemistry, pp 55-99 Jitsuyama Y, Suzuki T, Harada T, Fujikawa S (1997) Ultrastructural study on mechanism of increased freezing tolerance due to extracellular glucose in cabbage leaf cells. Cryo Lett. 18: 33-44 Kendall EJ, Kartha KK, Qureshi JA, Chermak P (1993) Cryopreservation of immature spring wheat zygotic embryos using abscisic acid pretreatment. Plant Cell Rep. 12: 89-94 Kohmura H, Ikeda Y, Sakai A (1994) Cryopreservation of apical meristems of Japanese shallot (Allium wakegi A.) by vitrification and subsequent high plant regeneration. Cryo Lett. 15: 289-298 Koster KL, Lei YP, Anderson M, Martin S, Bryant G (2000) Effects of vitrified and nonvitrified sugars on phosphatidylcholine fluid-to-gel phase transitions. Biophysical J. 78: 1932-1946 Lambardi M, Fabbri A, Caccavale A (2000) Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips. Plant Cell Rep. 19: 213-218 Mari S, Engelmann F, Chabrillange N, Huet C, Michaux-Ferriere N (1995) Histocytological study of apices of coffee (Coffea racemosa and C. sessiliflora) in vitro plantlets during their cryopreservation using the encapsulation-dehydration technique. Cryo Lett. 16: 289-298 Matsumoto T, Mochida K, Itamura H, Sakai A (2001) Cryopreservation of persimmon (Diospyros kaki Thunb.) by vitrification of dormant shoot tips. Plant Cell Rep. 20: 398-402 Matsumoto T, Sakai A (2003) Cryopreservation of axillary shoot tips of in vitro-grown grape (Vitis) by a two-step vitrification protocol. Euphytica 131: 299-304 Matsumoto T, Sakai A, Nako Y (1998) A novel preculturing for enhancing the survival of in vitro-grown meristems of wasabi (Wasabia japonica) cooled to -196 ℃ by vitrification. Cryo Lett. 19: 27-36 Matsumoto T, Sakai A, Yamada K (1994) Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration. Plant Cell Rep. 13: 442-446 Nishizawa S, Sakai A, Amano Y, Matsuzawa T (1993) Cryopreservation of asparagus (Asparagus officinalis L.) embryonic suspension cells and subsequent plant regeneration by vitrification. Plant Sci. 91: 67-73 Panis B, Lambardi M (2005) Status of cryopreservation technologies in plants(crops and forest trees). In The role of biotechnology, pp 43-54 Panis B, Piette B, Swennen R (2005) Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Sci. 168: 45-55 Pennycooke JC, Towill LE (2000) Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification. Plant Cell Rep. 19: 733-737 Pihakaski-Maunsbach K, Tamminen I, Pietiainen M, Griffith M (2003) Antifreeze proteins are secreted by winter rye cells in suspension culture. Physiol. Plant. 118: 390-398 Sakai A (1985) Cryopreservation of shoot tips of fruit trees and herbaceous plants. In Cryopreservation of Plant Cells and Organs, pp 135-158 Sakai A (2004) Plant Cryopreservation. In Life in the frozen state, pp 329-345 Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. Var. brasiliensis Tanaka) by vitrification. Plant Cell Rep. 9: 30-33 Steponkus PL, Langis R, Fujikawa S (1992) Cryopreservation of plant tissues by vitrification. In Advances in Low-Temperature Biology, Vol 1, pp 1-61 Stressmann M, Kitao S, Griffith M, Moresoli C, Bravo LA, Marangoni AG (2004) Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye. Plant Physiol. 135: 364-376 Suzuki M, Ishikawa M, Okuda H, Noda K, Kishimoto T, Nakamura T, Ogiwara I, Shimura I, Akihama T (2006) Physiological changes in gentian axillary buds during two-step preculturing with sucrose that conferred high levels of tolerance to desiccation and cryopreservation. Ann. Bot. 97: 1073-1081 Suzuki T, Kaneko M, Harada T (1997) Increase in freezing resistance of excised shoot tips of Asparagus officinalis L. by preculture on sugar-rich media. Cryobiology 34: 264-275 Takagi H, Thinh N, Islam O, Senboku T, Sakai A (1997) Cryopreservation of in vitro-grown shoot tips of taro(Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of vitrification procedure. Plant Cell Rep. 16: 594-599 Tao D, Li PH, Carter V (1983) Role of cell wall in freezing tolerance of cultured potato cells and their protoplasts. Plant Physiol. 58: 527-532 Tarcznski MC, Byrne DN, Millerr WB (1992) High performance liquid chromatography analysis of carbohydrates of cotton-phloem sap and honeydew produced by Bemisia babaci feeding on cotton. Plant Physiol. 98: 753-756 Touchell DH, Chiang VL, Tsai C-J (2002) Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification. Plant Cell Rep. 21: 118-124 Turner SR, Senaratna T, Touchell DH, Bunn E, Dixon KW, Tan B (2001) Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation. Plant Sci. 48: 489-497 Uemura M, Steponkus PL (2003) Modification of the intracellular sugar content alters the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves. Plant Cell Environ. 26: 1083-1096 Uragami A, Sakai A, Nagai M, Takahash T (1989) Survival of cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification. Plant Cell Rep. 8: 418-421 Uragami A, Sakai A, Nagai N (1993) Cryopreservation of asparagus (Asparagus officinalis L.) cultured in vitro. Japan Agri. Res. Quart. 27: 112-115 Vandenbussche B, Weyens G, De Proft MP (2000) Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique. Plant Cell Rep. 19: 1064-1068 Verleysen H, Fernandes P, Sanchez Pinto I, Van Bockstaele E, Debergh P (2005) Cryopreservation of Robinia pseudoacacia. Plant Cell Tiss. Org. Cult. 81: 193-202 Wang Q, Laamanen J, Uosukainen M, Valkonen JP (2005) Cryopreservation of in vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation-vitrification and encapsulation-dehydration. Plant Cell Rep. 24: 280-288 Wang QC, Mawassi M, Sahar N, Li P, Violeta C-T, Gafny R, Sela I, Tanne E, Perl A (2004) Cryopreservation of grapevine (Vitis spp.) embryogenic cell suspensions by encapsulation–vitrification. Plant Cell Tiss. Org. Cult. 77: 267-275 Wowk B, Leitl E, Rasch CM, Mesbah-karimi N, Harris SB, Fahy GM (2000) Vitrification enhancement by synthetic ice blocking agents. Cryobiology 40: 228-236 Yamada T, Sakai A, Matsumura T, Higuchi S (1991) Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Sci. 78: 81-87 Yie ST, Liaw SI (1977) Plant regeneration from shoot tips and callus of papaya. In Vitro 13: 564-568 Yoshida S, Forno DA, Cock JH, Gomez KA (1976) Determination of sugar and starch in plant tissue. In Laboratory manual of physiological studies for rice, pp 46-49 Zhu GY, Geuns JMC, Dussert S, Swennen R, Panis B (2006) Change in sugar, sterol and fatty acid composition in banana meristems caused by sucrose-induced acclimation and its effects on cryopreservation. Physiol. Plant. 128: 80-94
摘要: 
本研究以台農二號的番木瓜(Carica papaya L.)組織培養苗為材料,成功地進行玻璃質化法超低溫冷凍保存。將株齡6~8週大的番木瓜以0.09 M蔗糖預培養3天,誘導產生脫水及冷凍的耐受性,切取莖頂組織後,分別處理LS (loading solution) 60分鐘和PVS2 (plant vitrification solution 2) 60分鐘(0℃),接著置入液態氮中保存。保存的莖頂在回溫後,約二到四星期即可觀察到明顯的生長,二個月後即長成完整植株,存活率可高達96%。本流程也成功地在其他六個品種的番木瓜上得到穩定的存活率,大約介於85~96%,有效地達成種原長期保存的目的。
實驗中進行生理分析和切片觀察,探討玻璃質化法流程中預培養對冷凍保存存活率之影響。結果發現番木瓜隨著預培養基中蔗糖濃度增加,組織內會累積可溶糖,造成相對含水量、水分潛勢及滲透潛勢的下降。在低濃度(0.09 M)的蔗糖預培養下,冷凍保存的存活率最佳,蔗糖濃度太高(0.5~0.6 M)反而會導致嚴重的滲透逆境,冷凍保存存活率因此降低。顯示番木瓜不適合高糖預培養,提供適當濃度的蔗糖預培養加上前處理條件的配合,才是成功進行番木瓜玻璃質化法超低溫冷凍保存的關鍵。

Shoot tips of in vitro-grown plantlets of papaya (Carica papaya L. cv. Tai-nung No.2) from seven cultivars were successfully used for cryopreservation by vitrification. To induce the osmotolerance and tolerance to subsequent freezing, 6 to 8-week-old papaya plantlets were precultured in 0.09M sucrose for 3 days. After preculture, excised shoot tips were pretreated with LS (loading solution) for 60 min and dehydrated with PVS2 (plant vitrification solution 2) for 60 min at 0℃ and then were directly immersed into liquid nitrogen. Cryopreserved shoot tips after being warmed resumed growth between 2 and 4 weeks and developed plantlets after 2 months. The recovery percentages of shoot tips were about 96%. This study has successfully developed a simple and effective protocol for the cryopreservation of papaya shoot tips from other six cultivars and the recovery percentages were steady between 85~96%.
To investigate effects of the preculture protocol by vitrification on the survival of papaya, we analyzed the physiological responses and observed histological sections. Soluble sugar accumulated in papaya shoot tips with increase of the sucrose concentration, resulting in decrease of relative water content, water potential and osmotic potential. The best survival was obtained when papaya was precultured in a low sucrose concentration (0.09M). Preculturing with high sucrose concentrations (0.5~0.6M) could cause excessive osmotic stress, resulting in decrease of the survival of cryopreservation. The result indicated preculture in a high sucrose concentration was not suitable for papaya. It suggested preculture with a suitable sucrose concentration and pretreated durations were achieved through investigating two key steps of the vitrification protocol.
URI: http://hdl.handle.net/11455/22529
其他識別: U0005-1508200717492700
Appears in Collections:生命科學系所

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.