Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22836
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
冷凍保存
ultrastructure
微細構造
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冷凍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。 朱麗淑。1997。不同前處裡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。 李坤弘。2008。臺灣藥用植物食茱萸與牛樟玻璃化法超低溫冷凍保存前處理流程之探討。國立中興大學生命科學研究所 碩士論文。 林維熙。2008。臺灣原生藥用植物-高氏柴胡玻璃質化法超低溫冷凍保存流程之探討。國立中興大學生命科學研究所 碩士論文。 柯勇。2002。植物生理。藝軒出版社。pp.667-679。 武艳琪、李娜、王明伟。2006。我国六棱菊属植物化学成分研究进展。中国中葯杂志 31(3) 181-184。 許圳塗。1995。作物種質體外保存系統。臺灣省農業試驗所編印。農業試驗所特刊第55號 pp. 69-80。 陳嘉吉、陳世賢。1995。植物種源保育方向及政策。作物種源保育技術研習會專刊,溫英杰編。臺中,臺灣省農業試驗所。pp.150-156。 陳岱陽。2008。臺灣四種中草藥-地筍、臺灣黃岑、石香薷、生毛將軍玻璃質化法超低溫冷凍保存處裡流程之探討。中興大學生命科學研究所 碩士論文。 郭明科。2008。臺灣四種藥用植物增殖系統之建立。國立中興大學生命科學院在職專班 碩士論文。 莊明珈。2005。六種唇形花科植物精油於改善青春痘之應用。嘉南藥理科技大學化妝品科技研究所 碩士論文。 楊遠波、劉和義、彭鏡毅、施炳霖、呂勝由。2001。臺灣維管束植物簡誌第肆卷。行政院農委會 pp.141、267。 楊忠祐。2002。臺農69號品種甘藷之超低溫冷凍保存研究。國立中興大學植物研究所 碩士論文。 廖松淵、陳清義。1992。大豆滲透調節與耐旱性之關係。中華農學會報158: 19-28。 劉新裕、林俊義、張成國。2002。藥用植物專輯。行政院農委會農業試驗所編印。農業試驗所特刊第98號 pp.1-12。 劉淑芬。2004。臺農十六號鳳梨玻璃化法超低溫冷凍保存前處裡流程之探討。國立中興大學生命科學系 碩士論文。 蔡淑華。1975。植物組織切片技術綱要。臺北,茂昌圖書有限公司。pp. 47-58。 蔡新聲、陳忠川、李鎮宇、郭昭麟、羅淑芳。2003。臺灣珍稀藥用植物。科學發展364:42-49。 羅智明。1998。不同前處理對原生報歲蘭超低溫冷凍保存傷害之探討。國立中興大學植物學研究所 碩士論文 羅文陽。2007。番木瓜玻璃質化法超低溫冷凍保存流程之探討。國立中興大學生命科學系 碩士論文。 鐘錠全。1997。青草世界彩色圖鑑。臺北,協聯印書館有限公司。pp.466-467。 Ahuja, S., Mandal, B., Dixit, S. and Srivatava, P. S. 2002. Molecular, phenotypic and biosynthetic stability in Dioscorea floribunda plants derived from cryopreserved shoot tips. Plant Sci. 163:971-977. Bartolomé, A. P., Rup, rez P. and Fúster, C. 1995. Pineapple fruit: morphological characteristics, chemical composition and sensory analysis of Red Spanish and Smooth Cayenne cultivars. Food Chem. 53:75-79 Benson, E. E. 1999. Conservation, In:Benson, E. E. (Eds.), Plant Conservation Biotechnology, Taylor and Francis, London, 1999 pp. 83-95. Bowman, J. L. and Eshed, Y. 2000. Formation and maintenance of the shoot apical meristem. Trends Plant Sci. 163:971-977. Bradford, M. 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248-254 Bowman, J. L. and Eshed, Y. 2000. Formation and maintenance of the shoot apical meristem. 5 (3): 110-115. Chandel, K. P. S., Chaudhurt, R., Radhamani, J. and Malik, S. K. 1995. Desiccation and freezing sensitivity in recalcitrant seeds of tea, cocoa and Jackfruit. Ann. Bot. 76:443-450. Chen, J., Henny, R. J., Devanand, P. S. and Chao, C. T. 2006. AFLP analysis of nephthytis (Syngonium podophyllum Schott) selected from somaclonal variants. Plant Cell Rep. 24:743-749. Chu, C., Dai, Z., Maurice, S.B. and Edward, G.E. 1990. Induction of crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallium by abscisic acid. Plant Physiol. 93:1253-1260 Crowe, J. H., Crowe, L. M., Carpenter, J. F. and Wistriom, C. A. 1987. Stabilization of dry phospholipids bilayers and proteins by sugars. Biochem. J. 242:1-10. Dhindsa, R.S. 1991. Drought stress, enzymes of glutathione metabolism, oxidation injury, and protein synthesis in Tortula ruralis. Plant Physiol. 95:648-651. Dussert, S., Chabrillang, N., Engelmann, F., Anthony, F. and Hamon, S. 1997. Cryopreservation of coffee (Coffea arabica L.) seed: importance of the precooling temperature. CryoLett. 18:269-276. Ellis, R. E., Hong, T. and Roberts, E. H. 1990. An intermeditate category of seed storage behaviour?. J. Exp. Bot. 41:1167-1174. Engelmann, F. 1991 In vitro conservation of tropical plant germplasm – a review. Euphytica 57:227-243. Engelmann, F. 1997 In vitro conservation methods. In: Callow J. A., Ford-Lloyd B. V., Newbury H. J. (Eds.), Biotechnology and Plant Genetic Resources. CAB International, Oxford, pp.119-161. Engelmann, F. 2004. Plant cryopreservation: progress and prospects. In Vitro Cell Dev. Biol. Plat 40:427-433. Fahy, G. M., Lilley, T.H., Linsdell, H., Douglas, M. S. and Meryman, H. T. 1990. Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms. Cryobiol. 27:247–268. Fang, D., Jin, S. Hong, N., Zhong, T., Cao, Q., Yi, G. and Wang, G. 2008. Vitrificcation-cryopreservation, an efficient method for eliminating Candidatus Liberobacter asiaticus, the citrus Huanglongbing pathogen, from in vitro adult shoot tip. Plant Cell Rep. 27:241-250. Frankel, O. and Soule, M. 1981. Conservation and Evolution. Cambridge Univ. Press, Cambridge. Franks, F., Hatlet, R. H. M. and Mathias, S. 1991. Materials science and the production of shelf-stable biologicals. Pharm. Technol. Int. 3: 24-34. Franks, J. R. 1999. In situ conservation of plant genetic resources for food and agriculture: a UK perspective. Land Use Policy 16:81-91. Frankham, R. and Briscoe, D.A. 2002. Introduction to Conservation Genetics. New York, Cambridge. Gnanapragasam, S. and Vasil, I. K. 1992. Ultrastructural changes in suspension culture cells of Panicum maximum during cryopreservation. Plant Cell Rep. 11:169-174. Handa, S., Bressan, R. A., Handa, A. K., Carpita, N. C. and Hasegawa, P. M. 1983. Solute contributing to osmotic adjustment in cultured plant cells adapted to water stress. Plant Physiol 73:834-843. Helliot, B. 1998. Croissance et Stabilite genetique des Vitroplants de Prunier Ferlenain Plumina (R) apres cryoconservation des meristemes. PhD thesis, Universite de Bordeaux 1, France. Helliot, B., Swennen, R., Poumay, Y., Frison, E., Lepoivre, P. and Panis, B. 2003. Ultrastructural changes associated with cryopreservation of banana (Musa spp.)highly proliferating meristems. Plant Cell Rep. 21:690-698. Hirai, D. and Sakai, A. (2003) Simplified cryopreservation of sweet potato (Ipomoea batatas (L.) Lam.) by optimizing conditions for osmoprotection. Plant Cell Rep. 21:961-966. Hitmi, A., Barthomeuf, C. and Sallanon, H. 1999a. Cryopreservation of Chrysanthemum cinerariaefolium shoot tips. Effects of pretreatment conditions and retention of biosynthetic capacity. CryoLett. 20:109-120. Hitmi, A., Coudret, A., Barthomeuf, C. and Sallanon, H. 1999b. The role of sucrose in freezing tolerance in Chrysanhemum Cineraiaeflium L. cell cultures. CryoLett 20:45-54. Hoekstra, F. A., Golovina, E. A. and Buitink, J. 2001. Mechanism of plant desiccation tolerance. Trends Plant Sci. 6:431-438. Hsieh, T. H. and Huang T.C. 1999. Revision of Mosla (Lamiaceae) in Taiwan. Taiwania 44: 72-81. Ishikawa, K., Harata, K., Mii, M., Sakai, A., Yoshimatsu, K. and Shimomura, K. 1997. Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Rep. 16:754-757. Ishikawa. M, Suzuki. M., Nakamura, T., Kishimoto, T., Robertson, A. J. and Gusta, L. V. 2006. Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses. Ann. Bot. 97:453-456. Jensen W. A. 1962. Histological Procedures. In: Botanical Histochemistry, W.H. Freeman and Co, San Francisco pp 55-99. Jitsuyama, Y., Suzuki, T., Harada, T. and Fujikawa, S. 1997. Ultrastructural study on mechanism of increased freezing tolerance due to extracellular glucose in cabbage leaf cell. CryoLett. 18:22-44. Jitsuyama, Y., Suzuki, T., Harada, T. and Fujikawa, S. 2002. Sucrose incubation increases freezing tolerance of asparagus (Asparagus officinalis L.) embryogenic cell suspensions. CryoLett. 23:103-112. Kartha, K. K. 1985. Meristem culture and germplasm preservation. In:Cryopreservation of plant cell and organs. Kartha, K. K. (Eds.), Boca Raton, Florida. CRC Press. pp.115-134. Kartha, K. K. and Engelmann, F. 1994. Cryopreservation and germplasm storage. In: Vasil, I. K., Thorpe, T. A. (Eds.), Plant Cell and Tissue Culture. Dordrecht: Kluwer pp.195-230. Kim, H. H., Kim, J. B., Baek, H. J., Cho, E. G., Chae, Y. A. and Engelmann, F. 2004. Evolution of DMSO concentration in garlic shoot tips during a vitrification procedure. CryoLett. 25:91-100. Kurkela, S. and Franck, M. 1990. Cloning and characterization of a cold- and ABA-inducivle Arabidopsis gene. Plant Mol. Biol. 15:137-144. Lambardi, M., Fabbri, A. and Caccavale, A. 2000. Cryopreservation of white poplar (Populus alba L.) by virification of in vitro-grown shoot tip. Plant Cell Rep. 19:213-218. Liu, Y., Wang, X. and Liu, L. 2004. Analysis of genetic variation in surviving apple shoot following cryopreservation by vitrification. Plant Sci. 166:677-685. Malcolm, L. and Hunter, J. 2002. Fundamentals of Conservation Biology. Backwell. Massachusetts, U.S.A. pp.53-67. Matsumoto, T., Sakai, A. and Nako, Y. 1998. A novel preculturing for enhancing the survival of in vitro grown meristems of wasabi (Wasabia japonica) cooled to -196 ℃ by vitrification. CryoLett. 19:27-36. Matsumoto, T., SaKai, A. and Yamada, K. 1994. Cryopreservation of in virto-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration. Plant Cell Rep. 13:442-446. Matsumoto, T. and Sakai, A. 2003. Cryopreservation of axillary shoot tip of in vitro-grown grape (Vitis) by a two-step vitrification protocol. Euphytica. 131:299-304. Maxted, N., Ford-Lloyd, B. V. and Hawkes, J. G. 1997. Complementary conservation strategies. In: Maxted, N., Ford-Lloyd, B. V. and Hawkes, J. G. (Eds.) Plant Genetic Conservation. Chapman & Hall, London, pp. 15-39. Mazur, P. 1984. Freezing of living cell: mechanisms and applications. Am. J. Physiol. Cell Physiol. 247: C125 - C142. Meryman, H. T., Williams, R. J. and Douglas, M. S. J. 1997. Freezing injury from solution effects and its prevention by natural or artificial cryoprotection. Cryobiol. 12:287-302. Moges, A. D., Shible, R. A. and Karam, N. S. 2004. Cryopreservation of Afircan violet (Saintpaulia Ionantha Wendl.) shoot tips in vitro cell. Dev. Biol. Plant 40:389-395. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15(3): 473-497. Niino, T. and Sakai, A. 1992. Cryopreservation of alginate-coated in vitro grown shoot tips of apple, pear and mulberry. Plant Sci. 87:199-206. Niino, T., Tanaka, D., Tantely, R. R., Fukui, K. and Shirata, K. 2007. Cryopreservation of basal stem buds of in vitro-grown mat rush (Juncus Spp.) by vitirification. CryoLett. 28:197-206. Panis, B. and Lambardi, M. 2005. Status of Cryopreservation Technologies in Plants (Crops and Forest trees). In: The Role of Biotechnology, Villa Gualino, Turin, Italy pp 43-54. Panis, B., Piette, B. and Swennen, R. 2005. Droplet vitrification of apical meristems : a cryopreservation protocol applicable to all Musaceae. Plant Sci. 168:45-55. Panis, B., Tottk, N., Van, N. K., Withersb, L. A. and Swennen, R. 1996 Cryopreservation of banana (Musa spp.) meristem cultures after preculture on sucrose. Plant Sci. 121:95-106. Pennycooke, J. C. and Towill, L. E. 2000. Cryopreservation of shoot tips from in vitro plants of sweet potato (Ipomoea batatas L. Lam.) by vitrification. Plant Cell Rep. 19:733-737. Phila, R., Bianchini, J. P., Ramanoelina, A. R. P., Rasoarahona, J. R. E., Faure, R. and Cambon, A. 1998. Eudesmane sesquiterpenes form Laggera alata. Phytochemistry 46:1085-1088. Plessis, P., Leddet, C., Collas, A. and Dereuddre, J. 1993. Cryopreservation of Vitis vinifera L. cv. chardonnay shoot tip by encapsulation-dehydration: effects of pretreatment , cooling and post culture conditions. CryoLett. 14:309-320. Reed, B. M. and Chang, T. 1997. Medium- and long- term storage of in vitro cultures of temperate fruit and nut crops. In: Razdan, M.K., Cocking, E. C. (Eds.), Conservation of Plant Genetic Resources in vitro, vol. 1. General Aspects. Enfield: Science Publishers. pp.67-105. Reed, B. M. Kovalchuk, I., Kushnarenko, S., Meier-Dinkel, A., Schoenweiss, K., Pluta, S., Straczynska, K. and Benson, E. E. 2004. Evaluation of critical points in technology transfer cryopreservation protocols to international plant conservation laboratories. CryoLett. 25:341-352. Reed, B. M., Schumacher, L., Dumet, D. and Benson, E. E. 2005. Evaluation of a modified encapsulation-dehydration procedure incorporating sucrose pretreatments for the cryopreservation of ribes germplasm in vitro cell. Dev. Biol. Plant 41:41-436 Sakai, A., Kobayashi, S. and Oiyama, I. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep. 9:30-33. Sadraei, H., Ghannadi, A. and Malekshahi, K. 2003. Relaxant effect of essential oil of Melissa officinalis and citral on rat ileum contractions. Fitoterapia 74:445-452. Sarer, E. and Kökdil, G. 1991. Constituents of the essential oil from Melissa officinalis. Planta Med. 57:89-90. Sarkar, D. and Nail, P. S. 1998. Cryopreservation of shoot tips of tetraploid Solanum tuberosum L. clones by vitrification. Ann. Bot. 82:455-462. Seijo, G. 2000. Effects of preculture with sucrose and ABA on cell suspensions water status and its relation with vitrification resistance. Revista Brasileira de Fisiologia Vegeta. 12:35-50. Shibli, R. A., Haagenson, D. M., Cunningham, S. M., Berg, W. K. and Volence, J. J. 2001. Cryopreservation of alfalfa (Medicago sativa L.) cell by encapsulation-dehydration . Plant Cell Rep. 20:445-450. Slavik, B. 1974. Water in cells and tissues. In : B, Slavik.(Eds.), Methods of Studying Plant Water Relations. Academid Publishing House of the Czevhoslovak Academy of Science Prague, New York .pp.1-20 Smith, M. and Croft, S. 1991. Embedding and thin section preparation. In: Harris, I. R. (Eds.), Electron Microscopy in Biology. IRL press, Oxford, New York, Tokyo. pp.17-37. Steponkus, P. L. 1992.Advances in Low Temperature Biology. London, UK, JAI Press, pp 807-834. Suzuki, M., Akihama, T. and Ishikawa, M. 2005. Cryopreservation of encapsulated gentian axillary buds following 2 step-preculture with sucrose anddesiccation. Plant Cell 83:115-121. Suzuki, T., Kaneko, M. and Harda, T. 1997. Increase in freezing resistance of excised shoot tip of Asparagus officinalis L. by preculture on sugar-rich medium. Cryobiol. 34:264-275. Swan, T. W., Hare, D., Gill, R. A. and Lynch, P. T. 1999. Influence of preculture conditions on the post-thaw recovery of suspension cultures of Jerusalem artichoke (Helianthus tuberosus L.). CryoLett. 20:25-36. Takagi, H., Thinh, N. T., Islam, O. M., Senboku, T. and Sakai, A. 1997. Cryopreservation of in vitro-grown shoot tip of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure. Plant Cell Rep. 16:594-599. Thierry, C., Florin, B. and Petiard, V. 1999. Changes in protein metabolism during the acquisition of tolerance to cryopreservation of somatic embyros. Plant Physiol. Biochem. 37:145-154. Touchell, D. H., Chiang, V. L. and Tsai, C. J. 2002. Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification. Plant Cell Rep. 21:118–124. Towill, L. E. 1991. In vitro methods for conservation of plant genetic resources .In: Dodds, J. H. (Eds.), Cryopreservation. Chapman & Hall, London. pp.41-70. Towill, L. E. and Jarret, R. L. 1992. Cryopreservation of sweet potato (Ipomoea batatas L. Lam.) shoot tip by vitrification. Plant Cell Rep. 11:175-178. Turner, S. R., Senaratna, T., Bunn, E., Tan, B., Dixon, K. W. And Touchell, D. H. 2001a. Cryopreservation of shoot tip from six endangered Australian species using a modified vitrification protocol. Ann. Bot. 87:371-378. Turner, S. R., Senaratna. T., Touchell. D., Bunn, E., Dixon, K. and Tan, B. 2001b. Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation. Plant Sci. 160:489-497. Tsai, S. F., Yen, S. D., Chen, C. F. and Liaw, S. I. 2009. High-efficiency vitrification protocols for Cryopreservation of in vitro grown shoot tips of transgenic papaya lines. Plant Cell Tiss Organ Cult 98:157-164. Uragami, A., Sakai, A. and Nagai, M. 1990. Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro. Plant Cell Rep. 9:328-331. Uragami, A., Sakai, A. and Nagai, M. 1993. Cryopreservation of asparagus (Asparagus officinalis L.) cultured in vitro. Japna Agri. Res. Quart. 27:112-115. Urbanová, M., Košuth, J. and Čellárová, E. 2006. Genetic and biochemical analysis of Hypericum perforatum L. plants regenerated after cryopreservation. Plant Cell Rep. 25:104-147. Vandenbussche, B., Weyens, G. and De Proft, M. P. 1999. Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by vitrification technique. Plant Cell Rep. 19:1046-1068. Volk, G. H. and Walters, C. 2006. Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryoprotection. Cryobiol. 52:29-34. Volk, G. H., Harris, J. L. and Rotindo, K. E. 2006. Survival of mint shoot tips after exposure to cryoprotectant solution components. Cryobiol. 52:305-308. Wang, Q. C., Batuman, Ö., Li, P., Bar-Joseph, M. and Gafny, R. 2002. Cryopreservation of in vitro-grown shoot tips of ''Troyer'' citrange [Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-dehydration. Plant Cell Rep. 20:901-906. Wang, Y. L., Fan, M. J. and Liaw, S. I. 2005. Cryopreservation of in vitro-grown shoot tips of papaya (Carica papaya L.) by vitrification. Bot. Bull. Acad. Sin. 46:29-34. Yoshida, S., Forno, J.H., Cook, H. and Gornez, K.A. 1976. Determination of chlorophyll in plant tissue. Laboratory manual of physiological studies for rice. IRRI 26:43-49. Zheng, Q., Xu, Z., Sun, X., Yao, W., Sun, H. Cheng, H. K. and Zhao, Y. 2003. Eduesmane and megsatigmane glucosides from Laggera alata . Phytochemistry 63:835-839.
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.
URI: http://hdl.handle.net/11455/22836
其他識別: U0005-0408200917161400
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0408200917161400
Appears in Collections:生命科學系所

文件中的檔案:

取得全文請前往華藝線上圖書館

Show full item record
 
TAIR Related Article
 
Citations:


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