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標題: 大葉佛來明豆頂芽及芥藍花粉超低溫冷凍保存流程之探討
Investigation on Protocols of Cryopreservation in Apical Buds of Flemingia macrophylla (Willd.) Kuntze ex Prain and Pollen of Brassica oleracea var. alboglabra
作者: 招彥明
Chao, Yen-Ming
關鍵字: 大葉佛來明豆
Flemingia macrophylla (Willd.) Kuntze ex Prain
Brassica oleracea var. alboglabra
apical buds
出版社: 生命科學系所
引用: 宋芬玫、沈競辰、林淡櫻、施小玲、許佳玲、謝素芬。2010。蔬果.野菜圖鑑。晨星出版社。pp. 88-89。 何偉真、林德勳、謝昌衛、張世良、黃雯雯、陳信泰。2011。台灣野生藥用植物圖鑑(三)。行政院農業委員會林務局。pp. 86-87。 周廷光。1987。蔬菜。淑馨出版社。pp. 30。 陳嘉吉、陳世賢。1995。植物種原保育政策及方向。作物種原保育技術研習會專刊。pp. 155-161。 王欽麗、盧龍鬥、吳小琴、陳祖鏗、林金星。2002。花粉的保存及其生活力測定。植物學通報 19: 365-373。 林維熙。1998。臺灣原生藥用植物-高氏柴胡玻璃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 邱智聖。2011。青花菜和臺灣蝴蝶蘭花粉乾燥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。 陳世賢。1997。植物種原保育政策及方向。蔬菜種原保育技術訓練專刊。64: 99-108。 陳榮才。2002。中藥材一條根的成分分離.。嘉南藥理科技大學醫藥化學系 專題研究計畫。 莊蟬伊。2003。蔗糖前處理對山藥種間超低溫冷凍保存及生理之影響。國立中興大學生命科學系 碩士論文。 陳玟君。2006。台農13號鳳梨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系 碩士論文。 郭宏遠、宋妤。2007。花粉保存與利用。植物種苗 9: 48-58。 陳威臣、夏奇妮。2004。植物表皮蠟質與組織培養苗馴化之關係。技術服務 58: 32-35。 黃敬涵。2011。臺灣原生植物牛樟玻璃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。 詹金鳳。2010。臺灣原生藥用植物艾納香玻璃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。 蔡叔芬。2004。臺農25、臺農64與臺農66品種甘藷玻璃化法超低溫冷凍保存前處理流程之探討。中興大學生命科學系 碩士論文。 戴元熏。2012。花椰菜和青花菜花粉乾燥法超低溫冷凍保存流程之探討。中興大學生命科學系 碩士論文 Benson, E.E. (2008). Cryopreservation theory. In Plant Cryopreservation: A Practical Guide, B. Reed, ed. (New York: Springer), pp. 15-32. Engelmann, F., and Engels, J. (2002). Technologies and strategies for ex situ conservation. In Managing plant genetic diversity, J. Engels, ed.( Rome: IPGRI), pp. 89-103. Reed, B. (2008). Cryopreservation—Practical Considerations. In Plant Cryopreservation: A Practical Guide, B. Reed, ed. (New York: Springer), pp. 3-13. Sakai, A. (2000). Development of cryopreservation techniques. In Cryopreservation of tropical plant germplasm: current research progress and applications, F. Engelmann, and H. Takagi, eds. (Tsukuba: JIRCAS; Rome: IPGRI), pp. 1-7 Sakai, A., Matsumoto, T., Hirai, D., and Charoensub, R. (2002). Survival of tropical apices cooled to -196℃by vitrification. In Plant Cold Hardiness, P.H. Li, and E.T. Palva, eds. (New York: Kluwer Academic/Plenum Publishers), pp. 109-119. Salisbury, F.B., and Ross, C.W. (1992). Plant Physiology and Plant Cells. In Plant Physiol, F.B.Salisbury, and C.W. Ross, eds. (Belmont, CA: Wadsworth, Inc.), pp. 3-26. Takagi, H. (2000). Recent developments in cryopreservation of shoot apices of tropical species. In Cryopreservation of tropical plant germplasm: current research progress and applications, F. Engelmann, and H. Takagi, eds. (Tsukuba: JIRCAS; Rome: IPGRI), pp. 178-193. Towill, L.E., and Walters, C. (2000). Cryopreservation of pollen. In Cryopreservation of tropical plant germplasm: current research progress and applications, F. Engelmann, and H. Takagi, eds. (Tsukuba: JIRCAS; Rome: IPGRI), pp. 115–129. Withers, L.A., and Engelmann, F. (1998). In vitro conservation of plant genetic resources. In Biotechnology in agriculture, A. Altman, ed. (New York: Marcel Dekker, Inc.), pp. 57-88. Yoshida, S., Forno, J.H., Cook, H., and Gornez, K.A. (1976). Determination of chlorophyll in plant tissue. In Laboratory manual of physiological studies for ice. (Int. Rice. Res. Inst.), pp. 43-45. Antony, J.J.J., Keng, C.L., Rathinam, X., Marimuthu, S., and Subramaniam, S. (2011). Effect of preculture and PVS2 incubation conditions followed by histological analysis in the cryopreserved PLBs of ''Dendrobium'' Bobby Messina orchid. Aust. J. Crop Sci. 5, 1557. Barnabas, I.J., Dean, J.R., and Owen, S. (1994). Supercritical fluid extraction of analytes from environmental samples. A review. Analyst 119, 2381-2394. Berjak, P., Farrant, J., Mycock, D., and Pammenter, N. (1989). Homoiohydrous (recalcitrant) seeds: the enigma of their desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer. Planta 186, 249-261. Brewbaker, J.L., and Kwack, B.H. (1963). The essential role of calcium ion in pollen germination and pollen tube growth. Am. J. Bot. 50, 859-865. Crisp, P., and Grout, B. (1984). Storage of broccoli pollen in liquid nitrogen. Euphytica 33, 819-823. Chaves M. M., Maroco J. P., Pereira J. S. (2003). Understanding plant responses to drought — from genes to the whole plant. Funct. Plant Biol. 30, 239-264. Dumet, D., Engelmann, F., Chabrillange, N., and Duval, Y. (1993). Cryopreservation of oil palm (Elaeis guineensis Jacq.) somatic embryos involving a desiccation step. Plant Cell Rep. 12, 352-355. Engelmann, F. (2004). Plant cryopreservation: Progress and prospects. In Vitro Cell. Dev. Biol.: Plant 40, 427-433. Fabre, J., and Dereuddre, J. (1990). Encapsulation–dehydration: a new approach to cryopreservation of Solanum shoot tips. Cryoletters 11, 413-426. Gill, P.K., Sharma, A.D., Singh, P., and Bhullar, S.S. (2002). Osmotic stressinduced changes in germination, growth and soluble sugar content of Sorghum bicolor (L.) Moench seeds. Bulg. J. Plant Physiol. 28, 12-25. Hamilton, M.B. (1994). Ex situ conservation of wild plant species: time to reassess the genetic assumptions and implications of seed banks. Cons. Biol. 8, 39-49. Hirsh, A.G., Williams, R.J., and Meryman, H.T. (1985). A novel method of natural cryoprotection : intracellular glass formation in deeply frozen populus. Plant Physiol. 79, 41-56. Jitsuyama, Y., Suzuki, T., Harada, T., and Fujikawa, S. (1997). Ultrastructural study on mechanism of increased freezing tolerance due toextracelluar glucose in cabbage leaf cells. Cryoletters 18, 33-44. Ko, Y. J., Lu, T. C., Kitanaka, S., Liu, C.Y., Wu, J. B., Kuo, C. L., Cheng, H. Y., Lin, Y. C., and Peng, W. H. (2010). Analgesic and anti-inflammatory activities of the aqueous extracts from three flemingia species. Am. J. Chin. Med. 38, 625. Kaczmarczyk, A., Shvachko, N., Lupysheva, Y., Hajirezaei, M. R., and Keller, E.R.J. (2008). Influence of alternating temperature preculture on cryopreservation results for potato shoot tips. Plant Cell Rep. 27, 1551-1558. Leprince, O., Harren, F. J.M., Buitink J., Alberda, M., and Hoekstra, F. A. (2000). Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles. Plant Physiol. 122, 597-608. Lora, J., Oteyza, M., Fuentetaja, P., and Hormaza, J.I. (2006). Low temperature storage and in vitro germination of cherimoya (Annona cherimola Mill.) pollen. Sci. Hortic. 108, 91-94. Lloyd, G., and McCown, B. (1980). Commercially feasible micropropagation of mountain laurel Kalmia latifolia, by use of shoot tip culture. Intl. Plant Prop. Soc. Proc. 30, 421-427. Mix‐Wagner, G., Conner, A., and Cross, R. (2000). Survival and recovery of asparagus shoot tips after cryopreservation using the “droplet” method. N. Z. J. Crop Hortic. Sci. 28, 283-287. Myers, J.A., and Kitajima, K. (2007). Carbohydrate storage enhances seedling shade and stress tolerance in a neotropical forest. J. Ecol. 95, 383-395. Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473-497. Marassi, M., Scocchi, A., and Gonzalez, A. (2006). Plant regeneration from rice anthers cryopreserved by an encapsulation/dehydration technique. In Vitro Cell. Dev. Biol.: Plant 42, 31-36. Maxted, N., Tan, A., Amri, A., and Valkoun, J. (2001). In Situ Conservation. Plant Genet. Resour. 39, 292. Mbogning, J.B.D., Youmbi, E., and Nkongmeneck, B.A. (2007). Morphological and in vitro germination studies of pollen grains in kola tree (Cola sp.). Akdeniz Un. Ziraat Fak. Dergisi 20, 311-318. 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. Nishizawa, S., Sakai, A., Amano, Y., and Matsuzawa, T. (1993). Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Sci. 91, 67-73. Niino, T., Sakai, A., Yakuwa, H., and Nojiri, K. (1992). Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification. Plant Cell, Tissue Organ Cult. 28, 261-266. Pacini, E., Guarnieri, M., and Nepi, M. (2006). Pollen carbohydrates and water content during development, presentation, and dispersal: a short review. Protoplasma 228, 73-77. Panis, B., Totte, N., Van Nimmen, K., Withers, L.A., and Swennen, R. (1996). Cryopreservation of banana (Musa spp.) meristem cultures after preculture on sucrose. Plant Sci. 121, 95-106. Reed, B.M., and Chang, Y. (1997). Medium-and long-term storage of in vitro cultures of temperate fruit and nut crops. Conserv. Plant Genet. Resour. In Vitro 1, 67-105. Rosell, P., Herrero, M., and Galan Sauco, V. (1999). Pollen germination of cherimoya (Annona cherimola Mill.): In vivo characterization and optimization of in vitro germination. Sci. Hortic. 81, 251-265. Rao, G., Jain, A., and Shivanna, K. (1992). Effects of high temperature stress on Brassica pollen: viability, germination and ability to set fruits and seeds. Ann. Bot. 69, 193-198. Reinhoud, P.J., Schrijnemakers, E.W., van Iren, F., and Kijne, J.W. (1995). Vitrification and a heat-shock treatment improve cryopreservation of tobacco cell suspensions compared to two-step freezing. Plant Cell, Tissue Organ Cult. 42, 261-267. Siddiqi, E.H., and Ashraf, M. (2008). Can leaf water relation parameters be used as selection criteria for salt tolerance in safflower (Carthamus tinctorius L.). Pak. J. Bot. 40, 221-228. Sarasan, V., Cripps, R., Ramsay, M., Atherton, C., McMichen, M., Prendergast, G., and Rowntree, J. (2006). Conservation In vitro of threatened plants progress in the past decade. In Vitro Cell. Dev. Biol.: Plant 42, 206-214. Sakai, A., and Engelmann, F. (2007). Vitrification, encapsulation-vitrification and droplet-vitrification: a review. CryoLetters 28, 151-172. Syiem, D., and Khup, P. (2007). Evaluation of Flemingia macrophylla L., a traditionally used plant of the north eastern region of India for hypoglycemic and anti-hyperglycemic effect on mice. Pharmacologyonline 2, 355-366. Sato, S., Katoh, N., Iwai, S., and Hagimori, M. (1998). Establishment of reliable methods of in vitro pollen germination and pollen preservation of Brassica rapa (syn. B. campestris). Euphytica 103, 29-33. 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. Sheoran, I., Pedersen, E., Ross, A.S., and Sawhney, V. (2009). Dynamics of protein expression during pollen germination in canola (Brassica napus). Planta 230, 779-793. Shinozaki, K., and Yamaguchi-Shinozaki K. (2000). Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways, Curr. Opin. Plant Biol. 3, 217-223. Thomas, T.D. (2008). The role of activated charcoal in plant tissue culture. Biotechnol. Adv. 26, 618-631. Thierry, C., Florin, B., and Petiard, V. (1999). Changes in protein metabolism during the acquisition of tolerance to cryopreservation of carrot somatic embryos. Plant Physiol. Biochem. 37, 145-154. Tyagi, R.K., and Hymowitz, T. (2003). Pollen from Glycine species survive cryogenic exposure. CryoLetters 24, 119-124. Tannoury, M., Ralambosoa, J., Kaminski, M., and Dereuddre, J. (1991). Cryopreservation by vitrification of coated shoot tips of carnation (Dianthus caryophyllus L.) cultured in vitro. Comptes Rendus de I’Acad. des Sci. Paris Ser III 313, 633-638. Turner, S., Senaratna, T., Touchell, D., Bunn, E., Dixon, K., and Tan, B. (2001). Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation. Plant Sci. 160, 489-497. Uragami, A., Sakai, A., and Nagai, M. (1993). Cryopreservation of asparagus (Asparagus officinalis L.) cultured in vitro. JARQ 27, 112-115. Vendrame, W.A., Carvalho, V.S., Dias, J.M., and Maguire, I. (2008). Pollination of Dendrobium hybrids using cryopreserved pollen. HortScience 43, 264-267. Volk, G.M., Harris, J.L., and Rotindo, K.E. (2006). Survival of mint shoot tips after exposure to cryoprotectant solution components. Cryobiology 52, 305-308. Wang, Y. L., Fan, M. J., and Liaw, S. I. (2005a). Cryopreservation of in vitro-grown shoot tips of papaya (Carica papaya L.) by vitrification. Bot. Bull. Acad. Sin. 46, 29-34. Wang, Y., Gao, C., Liang, Y., Wang, C., Yang, C., and Liu, G. (2010). A novel bZIP gene from Tamarix hispida mediates physiological responses to salt stress in tobacco plants. J. Plant Physiol. 167, 222-230. Williams, R.J., and Leopold, A.C. (1989). The glassy state in corn embryos. Plant Physiol. 89, 977-981. Wang, Q., Laamanen, J., Uosukainen, M., and Valkonen, J.T. (2005b). Cryopreservation of in vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation–vitrification and encapsulation–dehydration. Plant Cell Rep. 24, 280-288.
摘要: 培植體特性不一,適合的冷凍保存方法與流程也不盡相同,本試驗以大葉佛來明豆頂芽(Flemingia macrophylla (Willd.) Kuntze ex Prain)和芥藍(Brassica oleracea var. alboglabra)花粉當作研究材料,並檢測各項生理分析結果,以期建立這兩種培植體的超低溫冷凍保存流程。 大葉佛來明豆組織培養苗以玻璃質化法保存,將株齡40-60天的植株移植至含0.3M蔗糖的MS培養基預培養3天,頂芽在室溫下以LS處理120min,再於0℃下以PVS2處理150min後快速放入液態氮中進行長期保存。回溫時以1.2M蔗糖溶液處理,並放在回復培養基上培養一個月,最高平均存活率可達47.8%。經生理分析後,發現高濃度蔗糖處理會促使頂芽內部的可溶糖和可溶蛋白累積,LS和PVS2避免細胞產生冰晶,因此頂芽對冷凍逆境的耐力增加。 芥藍花粉則用脫水乾燥法保存,將花藥置於開放小盒,再放入有矽膠乾燥劑的玻璃罐中脫水,經液態氮保存一週後回溫,並塗抹在含23%蔗糖的B&K培養基上,12hr後檢測花粉的萌發率。得知黑芥藍的花粉最佳脫水乾燥時間是80-100min,含水率為31.8-51.0%時會有最佳冷凍保存後的花粉萌發率;而翠津的花粉最佳脫水乾燥時間亦是80-100min,含水率為46.8-54.2%時會有最佳冷凍保存後的花粉萌發率,兩個品種授粉後都會有良好的結莢率或結籽數,表示經液態氮保存花粉仍能保持活性與受精能力。證明此處理流程適合芥藍花粉超低溫冷凍保存。 無論是玻璃質化法或脫水乾燥法,都是為了減少細胞內冰晶形成和降低滲透潛勢,讓培植體達到適合放入液態氮保存的狀態,以維持細胞活性與保留完整基因,藉以達到保存種原之目的。
The appropriate cryopreservation methods and protocols for different explants will not be the same because of their unique features. The apical buds of Flemingia macrophylla (Willd.) Kuntze ex Prain and the pollen of Brassica oleracea var. alboglabra are the experimental materials for this study. According to the investigation of many physiology analyses, the expection is setting up protocols of cryopreservation of these two explants. Apical buds of F. macrophylla were cryopreservated by vitrification. A first, the 40-60 days old micropropatagation plantlets were transplanted to 0.3M sucrose medium for 3 days. Excised buds were loaded to LS 120 min at room temperature, dehydrated to PVS2 150 min at 0℃and then immediately put into liqid nitrogen (LN) for a long-term storage. After the rewarm treatment, the buds were transferred to recovery medium for one month. The best survival rate could reach 47.8%. physiology analyses indicated that plant precultured in high sucrose medium would induce accumulation of soluble sugar and soluble protein in cells of apical buds. LS and PVS2 could also help cells avoid ice formation. Therefore, Apical buds can endure frozen stress. Pollen of B. oleracea var. alboglabra was cryopreservated by desiccation. Anthers were put in open box and then dehydrated in glass jar by silica desiccant. After preservated in LN for one week and rewarm treatment, pollen was smeared on B&K medium with 23% sucrose. Pollen germination was counted after 12 hours. It tuned out that the best desiccation duration of ‘Black leaf kai-lan’ was 80-100min. There would be the highest germination of cryopreservated pollen when water content was 31.8-51.0% . The best desiccation duration of ‘Chui-Chun’ was also 80-100min. There would be the highest germination of cryopreservated pollen when water content was 46.8-54.2% . Both of two varieties had pretty good podding and seedling after pollination. It means that pollen still can maintain the vitality and fertilizing capacity. This procedure is surely suitable for cryopreservation of pollen of B. oleracea var. alboglabra. Either vitrification or desiccation, they are used to protect cells from ice formation and decrease osmotic potential of cells. So treated explants can be proper for cryopreservation. These procedures can eventually achieve germplasm conservation by keeping cell vitality and integral genome.
其他識別: U0005-1008201305553500
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