臺灣博碩士論文加值系統

本試驗利用小型感測元件搭配資料記錄器，長時間且連續記錄組織培養瓶內CO2濃度日韻律，以判別蝴蝶蘭實生瓶苗之固碳模式轉變過程，同時量測苗株葉片PEPC的活性及蛋白質濃度以驗證瓶內CO2濃度日韻律與固碳模式間的關係，建立一方便可靠的組培瓶內CO2量測系統。
偵測結果顯示，蝴蝶蘭繼代初期苗固碳模式屬於C3型，其瓶內CO2濃度日韻律變化類似菊花，至繼代中後期於光期和暗期中瓶內皆有CO2濃度下降現象，同時呈現C3和CAM特性，並隨著株齡的成熟，CAM特性的表現更明顯。在苗株展現CAM特性時，瓶內CO2濃度日韻律變化可分為四個時期：phase I為整個暗期，在短暫的「光呼吸延續」現象後，以暗呼吸為主，容器內CO2濃度持續升高，PEPC開始作用固定CO2；phase II為光期來臨時CO2吸收時期，夜間殘存的PEPC活性持續作用，瓶內CO2濃度快速下降；phase III為去羧化作用時期，夜間儲存於液胞中的蘋果酸還原為CO2，供Calvin 循環所需，組培容器內CO2濃度再度上升；phase IV為C3型光合作用時期，夜間儲存的蘋果酸耗盡，苗株恢復對CO2的吸收，於光期中以Rubisco進行羧化作用，此時期結束前3-4小時，瓶內CO2降低至不利光合作用進行之濃度 (350 ppm以下)。

苗株葉片測量結果顯示，PEPC活性及蛋白質濃度與株齡和光週期時程有關；繼代後期苗活性與蛋白質濃度高於初期苗，同一株齡中暗期高於光期。其變化與瓶內CO2濃度日韻律相吻合，具體呈現苗株固碳模式由C3型轉為CAM型。

This study was to develop a reliable CO2 measuring and recording system, which can be applied in tissue culture environment for long-term, real-time monitoring and recording of CO2 circadian rhythm of Phalaenopsis seedlings. Carbon dioxidide sensors and a data logger were employed to continuously monitor the change of CO2 concentrations in vitro and tests of enzyme, PEPC, activities and protein concentration in leaves were conducted.
Concerning the CO2 circadian rhythm inside culture vessel, results of the study showed that subculture seedlings of chrysanthemum and Phalaenopsis of the first growth stage presented the same characteristic as C3 plant. Depending on species, seedlings presented C3 or both of C3 and CAM characteristics progressively in the middle growth stage. Then, seedlings presented a more remarkable CAM characteristic of CO2 circadian rhythm in the last growth stage for both species.
When seedlings presenting CAM characteristics, the CO2 circadian rhythm inside culture vessel can be divided into four phases: phase I, at the onset of dark period, a rapid increase of CO2 concentration was caused by the postillumination burst and followed by the start of CO2 fixation with PEPC. Phase II, at the beginning of lighting period, the remaining PEPC activity in the dark period carried on carboxylation and caused a rapid increase of CO2. Phase III, decarboxylation stared and the malate that stored in the vacuole in the dark period converted to CO2 for Calvin cycle. Consequently, CO2 concentration increased with time. Phase IV, seedlings restarted the process of C3 carboxylation. Rubisco is the primary enzyme to fix CO2 during this period. Moreover, it was found that the CO2 concentration in vitro decreased to a level much lower than the atmosphere’s (350 ppm).
The PEPC activity and protein concentration were related to the growth stage and the status of photoperiod. The activity and protein concentration of the seedlings in the last growth stage were higher than those of the first and middle growth stages. They were also higher in the dark period than in the light period. These results were in agreement with the CO2 circadian rhythm of seedlings in vitro and showed the CO2 fixation pattern was transferring from C3 to CAM over three growth stages.

目錄
中文摘要 I
Abstract III
誌謝 V
目錄 VI
圖目錄 IX
表目錄 XI
壹、前言 1
貳、文獻回顧 3
一、台灣蝴蝶蘭產業與瓶苗生產概況 3
二、光合作用：碳的固定 5
(一) 固碳的模式 5
(二) 模式間的轉換 6
(三) 景天酸型植物的分類 7
三、組培苗的生理行為 9
(一) 組培瓶內環境的影響 9
(二) 碳源對培殖體生長的影響 11
四、蝴蝶蘭固碳模式之界定 13
(一) 一般方式 13
(二) 氣體交換方法 14
(三) 磷烯醇丙酮酸羧化酶 (PEPC) 活性方法 16
參、材料與方法 18
一、組培瓶內二氧化碳濃度的量測 18
(一) 植物材料 18
(二) 培養基與組培室環境條件 20
(三) 二氧化碳感測元件與資料記錄器 20
二、磷烯醇丙酮酸羧化酶 (PEPC) 活性分析 22
(一) 酵素萃取 22
(二) 蛋白質定量與酵素活性分析 22
1. 蛋白質定量分析 (Bradford test) 22
2. 酵素活性分析 23
(三) 統計分析方法 23
肆、結果與討論 24
一、組培苗瓶內二氧化碳日韻律 24
(一) 菊花瓶苗 24
(二) 蝴蝶蘭瓶苗 25
1. 蝴蝶蘭苗品種一“Phal. new cinderella × Dpps. i-hsin parikia” 25
2. 蝴蝶蘭苗品種二“Phal. taida pinlong × join diamond” 28
二、磷烯醇丙酮酸羧化酶 (PEPC) 活性之探討 31
伍、討論 36
一、組培苗瓶內二氧化碳日韻律與固碳模式轉變的關係 36
二、磷烯醇丙酮酸羧化酶 (PEPC) 與瓶內CO2日韻律的關係 41
三、光週期對瓶內CO2濃度的影響 43
陸、結論與建議 46
參考文獻 47
附錄 57
作者簡介 63

參考文獻
王振武。1996。光與細胞分裂素對Mesembryanthemum crystallinum L. 景天酸代謝之調節。國立台灣大學農藝學系碩士論文。
朱建鏞、王美陽。1996。光和二氧化碳對玫瑰組織培養培殖體生長的影響。農林學報。45(1)：23-32。
吳安娜。1994。蔗糖、光度和添加二氧化碳對玫瑰花枝梢培殖體生長之影響。國立中興大學園藝學系碩士論文。
吳昭慧、朱鈞。1997。光度對Mesembryanthemum crystallinum L. 景天酸代謝之調節。中華農藝。7：127-137。
李哖、郭瑋君。2002。台灣蝴蝶蘭和大白花雜交種之光合作用。蝴蝶蘭繁殖、生育特性、產期調節及產後品質。財團法人台灣區花卉發展協會。台北。pp: 278-192。
邱雯卉。2002。環境因子、乙烯與培養基添加物對蝴蝶蘭瓶苗品質的影響。國立中興大學園藝學系碩士論文。
侯鳳舞。2004。優質外銷蝴蝶蘭生產體系之建構（上）。農業試驗所技術服務。60：12-15。
郝岫音。2005。分進合擊的藍海策略～台灣蝴蝶蘭外銷策略(下)。台灣花卉園藝月刊。218：42。
涂美智、李哖。1988。培養基成份對蝴蝶蘭種子發芽和幼苗生長。蝴蝶蘭生產改進研討會專輯。台東改良場。pp. 57-66。
涂美智、李哖。2002。培養基成份對蝴蝶蘭種子發芽和幼苗生長。蝴蝶蘭繁殖、生育特性、產期調節及產後品質。財團法人台灣區花卉發展協會。台北。pp. 39-48。
陳加忠、林瑞松、陳駿季。1998。植物組織培養瓶內微氣候之量測。農林學報。47(4)：77-91。
陳加忠、林裕彥、林瑞松。1999。植物組織培養瓶換氣率之影響因子。農林學報。48(3)：17-30。
陳怡靜、李哖。2002。台灣蝴蝶蘭瓶苗內二氧化碳與乙烯濃度及有機酸含量之日韻律變化。中國園藝。48：157-166。
楊于萱。2003。培養基成份、IBA及香蕉成熟度對朵麗蝶蘭瓶苗生長的影響。國立中興大學園藝學系碩士論文。
廖玉琬、徐善德、林美華、謝永祥、吳弘達、鐘仁彬。2002。光合作用：碳的代謝。廖玉琬、徐善德。植物生理學。啟英文化。台灣。pp. 231-262。
廖成康。1991。離層酸對Mesembryanthemum crystallinum L.景天酸代謝之促進。國立台灣大學農藝學系博士論文。
廖麗蘭。1998。發育階段、培養基、光線及容器對火鶴花、彩色海竽與數種觀賞植物組織培養苗瓶內CO2和乙烯濃度之影響。國立台灣大學園藝學系碩士論文。
蔡媦婷。2004。通氣性和液體培養基對蝴蝶蘭瓶苗生長之影響。國立中興大學園藝學系博士論文。
蔡瑜卿。2006。蘭花產業經營效率改善座談會記實。台灣花卉園藝月刊。222：54。
羅聖賢。2002。台灣蝴蝶蘭之種源鑑識與光合特性之探討。國立中興大學生命科學系博士論文。
饒瑞佶。2000。發光二極體應用於蝴蝶蘭、彩色海芋與馬鈴薯組織培養的研究。國立台灣大學生物產業機電工程研究所博士論文。
Arigita, L., A. Gonzalez and R. S. Tames. 2002. Influence of CO2 and sucrose on photosynthesis and transpiration of Actinidia deliciosa explants in vitro. Plant Physiol. 115: 166-173.
Borland, A. M. 1996. A model for the partitioning of photosynthetically fixed carbon during the C3-CAM transition in Sedum telephium. New Phytol. 134: 433-444.
Borland, A. M. and H. Griffiths. 1992. Properties of phosphoenolpyruvte carboxylase and carbohydrate accumulation in the C3-CAM intermediate Sedum telephium L. grown under different light and watering regimes. J. Exp. Bot. 43: 353-361.
Borland, A. M. and T. Taybi. 2004. Synchronization of metabolic processes in plants with Crassulacean acid metabolism. J. Exp. Bot. 55: 1255-1265.
Borland, A. M., J. Hartwell, G. I. Jenkins, M. B. Wilkins and H. G. Nimmo. 1999. Metabolite control overrides circadian regulation of phosphoenolpyruvte carboxylasekinase and CO2 fixation in Crassulacean acid metabolism. Plant Physiol. 121: 889-896.
Bradford, M. 1976. A rapid and sensitive method for the quantition of microgram quantities of protein utilizing the principle of dye-binding. Anal. Biochem. 72: 248-254.
Brain, K. E. L., H. Fuchigami, S. Kwartkowski and C. S. Clark. 1981. Leaf anatomy and water stress of aseptically cultured Pixy plum growth under different environments. Hortscience 16: 173-175.
Bula, R. J., R. C. Morrow, T. W. Tibbitts and D. J. Barta. 1991. Light-emitting diodes as a radiation source for plants. HortScience 26: 203-205.
Capellades, M., R. Lemeur and P. Debergh. 1991. Effects of sucrose on starch accumulation and rate of photosynthesis in Rosa culture in vitro. Plant Cell Tiss. Org. Cult. 25: 21-26.
Cheng, S. E., B. D. Moore and J. R. Seemann. 1998. Effect of short- and long-term elevated CO2 on the express of Rubisco genes and carbohydrate accumulation in leaves of Arabidopsis Thaliana L. Heynh. Plant Physiol. 116: 715-723.
Chu, C., Z. Dai, M. S. B. Ku and G. E. Edwards. 1990. Induction of Crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum L. by abscisic acid. Plant Physiol. 93: 1253-1260.
Cushman, J. C. 2001. Crassulacean acid metabolism. A plastic photosynthetic adaptation to arid environments. Plant Physiol. 127: 1439-1448.
Cushman, J. C. and A. M. Borland. 2002. Induction of crassulacean acid metabolism by water limitation. Plant Cell and Environ. 25: 295-310.
Decker, P. 1955. A rapid, postillumination deceleration od respirationin green leaves. Am. Soc. Plant Physio. 30: 82-84.
Desjardins, Y., C. Hdider and J. Riek. 1994. Carbon nutrition in vitro – regulation and manipulation of carbon assimilation in micropropagated systems. In: Christie, J. A., T. Kozai, and M. A.L. Smith (ed.). Automation and environmental control in plant tissue culture. pp. 441-471. Kluwer Academic Publishers. Netherlands.
Dimasi-Theriou, K. and A. M. Bosabalidis. 1997. Effects of light, magnesium amd sucrose on leaf anatomy, photosynthesis, starch and total sugar accumulation in kiwifruit cultured in vitro. Plant Cell Tiss. Org. Cult. 47: 127-134.
Dodd, A. N., A. M. Borland, R. P. Haslam, H. Griffiths and K. Maxwell. 2002. Crassulacean acid metabolism: plastic, fantastic. J. Exp. Bot. 53: 569-580.
Drennan, F. M. and P. S. Nobel. 2000. Responses of CAM species to increasing atmospheric CO2 concentrations. Plant Cell and Environ. 23: 767-781.
Drew, A. P., K. L. Kavanagh and C. A. Maynard. 1988. Comparative growth of micropropagation Prunus serotina plantlets and seedlings in different containers. In: Valentine, F. A. (ed.). Progress and prospects in forest and crop biotechnology. pp. 99-111. Springer-Verlag. Berlin.
Endo, M. and Isao Ikusima. 1989. Diurnal rhythm and characteristics of photosynthesis respiration in the leaf and roof of a Phalaenopsis plant. Plant Cell Physiol. 30(1): 43-47.
Fujiwara, K. and T. Kozai. 1994. Physical microenvironment and its effects. In Christie, J. A., T. Kozai, and M. A.L. Smith (ed.). Automation and environmental control in plant tissue culture. pp. 319-333. Kluwer Academic Publishers. Netherlands.
Fujiwara, K., S. Kira, T. Kozai and I. Watanbe. 1987. Measurement of carbon dioxides gas concentration on closed vessels cultured plantlet and estimates of net photosynthetic rate of plantlets. J. Agr. Met. 43(1): 21-30.
Gouk, S. S., J. He and C. S. Hew. 1999. Changes in photosynthetic capability and carbohydrate production in epiphytic CAM orchid plantlet exposed to super-elevated CO2. Environ. Exp. Bot. 41: 219-230.
Gouk, S. S., J. W. H. Yong and C. S. Hew. 1997. Effects of super-elevated CO2 on the growth and carboxylating enzymes in an epiphytic CAM orchid plantlet. J. Plant Physiol. 151: 129-136.
Griesbach, R. J. 1983. The use of indoleacetylamino acids in the in vitro propagation of Phalaenopsis orchid. Sci. Hortic. 19: 363-366.
Gupta, S. K., M. S. B. Ku, J-H Lin, D. Zhang and G. E. Edwards. 1994. Light/dark modulation of Phosphoenolpyruvte carboxylase in C3 nad C4 species. Photosynthesis Research 42: 133-143.
Hahn, E. J. and K. Y. Paek. 2001. High photosynthetic photon flux and high CO2 concentration under increased number of air exchanges promote growth and photosynthesis of four kinds of orchid plantlets in virto. In virto Cellular and Developmental Biology-Plant 37: 678-682.
Hanscom, Z. and I. P. Ting. 1978. Response of succulents to water stress. Plant Physiol. 61: 327-330.
Haslam, R., A. Borland, K. Maxwell and H. Griffiths. 2003. Physiological responses of the CAM epiphyte Tillandsia usueoides L. (Bromeliaceae) to variations in light and water supply. J. Plant Physiol. 160: 627-634.
Hdider, C. and Y. Desjardins. 1994. Effects of sucrose on photosynthesis and phosphoenolpyruvate carboxylase activity of in vitro cultured strawberry plantlets. Plant Cell Tiss. Org. Cult. 36: 27-33.
Hew, C. S. and J. W. H. Yong. 1994. Growth and photosynthesis of Oncidium “Goldiana”. J. Hort. Sci. 69: 809-819.
Hew, C. S. and J. W. H. Yong. 1997. Photosynthesis. In: physiology of tropical orchids in relation to the industry. pp. 37-87. World Scientific. Singapore.
Holtum, J. A. M. 2002. Crassulacean acid metabolism: plasticity in expression, complexity of control. Funct. Plant Biol. 29: 657-661.
Hopkins, W. G. 1999. Photorespiration and the photosynthetic carbon oxidation cycle. Introduction to palnt physiology. pp. 197-200. John Wiley and Sons, Inc. New York.
Jeong, B. R., C. S. Yang and J. C. Park. 1996 Growth of Gerbora hybrida in vitro as effected by CO2 concentration and air exchange rate of the vessel. Acta Hort. 440: 510-514.
Kanechi, M., M. Ochi, M. Abe, N. Inagaki and S. Maekawa. 1998. The effects of carbon dioxide enrichment, natural environment ventilation, and light intensity on growth, photosynthesis, and transpiration of cauliflower plantlets cultured in vitro photoautotrophically and photomixotrophically. J. Amer. Soc. Hort. Sci. 123: 176-181.
Kozai, T. 1989. Autotrophic (sugar-free) micropropagation for a significant reduce of production costs. Chronica Hort. 29: 19-20.
Kozai, T. 1991. Acclimatization of micropropagated plants. In: Bajaj, Y. P. S. (ed.). Biotecnology in agriculture and forestry 17. High-tech and micropropagation I. pp. 127-130. Springer-Verlag. Berlin.
Langford, P. J. and H. Wainwright. 1987. Effects of sucrose concentration on the photosynthesis ability of rose shoots in vitro. Ann. Bot. 60: 633-640.
Lüttge U. 2000. Light-stress and crassulacean acid metabolism. Phyton 40: 65-82.
Lüttge U. 2004. Ecophysiology of Crassulacean acid metabolism (CAM). Ann. Botany 93: 629-652.
Malda, G., R. A. Backhaus and C. Martin. 1999. Alterations in growth and crassulacean acid metabolism (CAM) activity of in vitro cultured cactus. Plant Cell Tiss. Org. Cult. 58: 1-9.
Markovska, Y. K. and D. S. Dimitrov. 2001. The effect of leaf age on gas exchange and malate accumulation in C3-CAM plant Marrubium frivaldszkyanum (Lamiaceae). Photosynthetica 39: 191-195.
Martin, C. E. 1996. Putative cause and consequences of recycling CO2 via crassulacean acid metabolism. In: Winter, K. and J. A. C. Smith(ed.). Crassulacean acid metabolism: biochemistry, ecophysiology and evaluation. 114: 192-203. Springer-Verlag. Berlin.
McWilliams, E. I. 1970. Comparative rates of dark CO2 uptake and acidification in the Bromeliaceae, Orchidaceae and Euphorbiaceae. Bot. Gaz. 131: 285-290.
Minocha, S. C. and P. Mehra. 1974. Nutritional and morphogenetic investigations on callus culture of Neomammillaria prolifera Miller (Cactaceae). Am. J. Bot. 61: 168-173.
Murashige, T. 1977. Manipulation of organ initiation in the plant tissue culture. Bot. Bull. Academic Sinica. 18: 1-24.
Neales, T. F. and C. S. Hew. 1975. Two types of carbon fixation in tropical orchids. Planta 123: 303-306.
Nhut, D. T., T. Takamura, H. Watanabe, K. Okamoto and M. Tanaka. 2003. Responses of strawberry plantlets cultured in vitro under superbright red and blue light-emitting diodes (LEDs). Plant Cell Tiss. Org. Cult. 73:43-52.
Osmond, C. B. 1978. Crassulacean acid metabolism: a curiosity in context. Ann. Rev. Plant Physiol. 29: 379-414.
Osmond, C. B. and O. Björkman. 1972. Simultaneous measurement of oxygen effects on net photosynthesis and glycolate metabolism in C3 and C4 species of Atriplex. Carnegia institution of Washington Yearbook. 71: 141-148.
Ota, K. K. M. and Y. Yamamoto. 1991. Effects of leaf age, inflorescence, temperature, light intensity and moisture conditions on CAM photosynthesis in Phalaenopsis. J. Japan. Soc. Hort. Sci. 60(1): 125-132.
Sayed, O. H. 2001. Crassulacean acid metabolism 1975-2000, a check list. Photosynthetica 39: 339-352.
Suto, K. 1993. Effects of light on the CO2 metabolism in Phalaenopsis. J. Japan. Soc. Hort. Sci. 62: 380-382.
Sutter, E. and W. Langhans. 1982. Formation of epicuticular wax and its effect on water loss in cabbage plants regeneratied from shoot tip culture. Can. J. Bot. 60: 2869-2902.
Taiz, L. and E. Zeiger. 2002. Photosynthesis: Carbon reactions. In Sinauer, A. D. (ed.). Plant physiol. pp. 145-168. Sinauer Associates, Inc. USA.
Tanaka, M., D. C. H. Yap, C. K. Y. Ng and C. S. Hew. 1999. The physiology of Cymbidium plantlets cultured in vitro under conditions of high carbon dioxide low photosynthetic photon flux density. J. Hort. Sci. Biotech. 74: 632-638.
Taybi, T., J. C. Cushman and A . M. Borland. 2002. Environmental, hormonal and circadian regulation of crassulacean acid metabolism. Funct. Plant Biol. 29: 669-678.
Ting, I. PHAL., A. Patel, S. Kaur, J. Hann and L. Walling. 1996. Ontogenetic development of Crassulacean acid metabolism as modified by water stress in Peperomia. In: Winter, K. and J. A. C. Smith (ed.). Crassulacean acid metabolism: biochemistry, ecophysiology and evaluation. 114: 192-203. Springer-Verlag. Berlin.
Tolbert, N. E. 1980. Photorespiration. In: Davis, D. D. (ed.) The biochemistry of plants. 2: 487–523. Academic Press. London.
Vizkot, B. and Z. Jara. 1984. Clonal propagation of cacti through axillary buds in vitro. HortScience 59: 449-452.
Wardle, K., E. B. Dobbs and K. C. Short. 1983. In vitro acclimatization of aseptically cultured plantlets to humidity. J. Am. Soc. Hortic. Sci. 108: 386-389.
Ziv, M., G. Meir and A. H. Halevy. 1983. Factors influencing the production of hardened glaucous carnation plantlest in vitro. Plant Cell Tiss. Org. Cult. 2: 55-65.
農委會。2006a。蝴蝶蘭苗株歷年出口數量圖。2006年5月8日。取自：http://agrapp.coa.gov.tw:7001/TS2/ts/tsccko9003.jsp
農委會。2006b。單一農產品交易趨勢圖。2006年5月8日。取自：http://agrapp.coa.gov.tw:7001/TS2/ts/ts199tb.jsp#graph