| 摘要註: |
Part 1如何縮短培養時間與減少人工成本是維持台灣蝴蝶蘭國際競爭力的重要因素。本論文擬利用實驗室先前所開發的可程控生物反應器，藉由調控培養基區的淹灌頻率、栽培區的通氣量、以及更換培養液的頻率，找尋大量增生蝴蝶蘭(Phalaenopsis Sogo Yukidian ‘V3’)不定芽(adventitious buds) biomass及芽數的最佳培養條件。此外，本論文不僅比較不同培養條件對幼苗抗氧化相關的分析，以了解幼苗遭受逆境的程度；並且選定上述初步的最佳條件，分析幼苗在生長過程，反應器內的微環境的變化，以做為未來改善培養條件的依據。實驗結果發現，在每分鐘通氣4 ml、每2小時淹灌一分鐘、每週更換培養液的條件下培養6週，不定芽的biomass與芽數增生效率最佳，分別是傳統玻璃瓶培養的2.1與1.6倍。由反應器內每週CO2濃度日韻律變化及營養成份，包括NH4+、K+、malate、nitrate、phosphate的消耗率皆是第四週後明顯增加，上述結果暗示不定芽培養的前3週是生長潛伏期，第4週後進入生長的對數期。當每週更換或無更換培養液，皆會造成幼苗內H2O2含量的增加，但是分別表現較高的superoxide dismutase (EC1.15.1.1)及 ascorbate peroxidase (EC 1.11.1.11)活性，明顯說明不同培養系統會誘導幼苗表現不同的抗氧化機制。當每兩週更換培養液，則幼苗內H2O2含量較低，暗示此條件培養的幼苗遭受逆境程度最低。Part 2蝴蝶蘭在玻璃瓶內的小苗是屬於C3或CAM型式皆有研究者提及。為了釐清何種環境因子會影響蝴蝶蘭吸收CO2的型式，本實驗設計傳統玻璃瓶﹙對照組﹚、每分鐘通氣2 ml﹙通氣組﹚及不外加2%蔗糖﹙缺糖組﹚三種處理，除了追蹤不同培養時間瓶內CO2濃度的日韻律變化外，亦分析具有2~3片葉子的蝴蝶蘭幼苗(Phalaenopsis Sogo Yukidian ‘V3’)在不同條件下培養8週後，碳水化合物與蘋果酸含量及相關酵素–phosphoenolpyruvate carboxylase﹙EC 4.1.1.31, PEPCase﹚、ribulose-1,5-bisphosphate carboxylase/oxygenase﹙EC 4.1.1.39, rubisco﹚、NAD+- malic enzyme﹙EC 1.1.1.39, NAD+- ME﹚活性的日韻律變化。雖然由於樣品數過少，無法精確取得日韻律的樣品，導致澱粉與蘋果酸含量的變化及三種酵素–PEPCase、rubisco及NAD+- ME活性之間的相關性不高；然而瓶內CO2濃度的日韻律變化則三種處理呈現明顯差異。對照組及通氣組皆呈現白天低晚上高的C3模式，而且對照組的CO2濃度可達20000 ppm遠高於通氣組。在缺蔗糖的培養基，雖然在培養4週前仍是C3模式，但是於第8週則呈現CAM模式。培養基的碳水化合物含量分析亦確認培養4週後，培養基內已無蔗糖存在。因此，蝴蝶蘭幼苗可能會受到培養基碳源─尤其蔗糖而改變光合作用模式。另外，通氣與缺糖皆會誘導瓶苗生合成甘油，由於甘油是一種可親和性溶質﹙compatible solute﹚，因此合理推論此兩種條件會對蘭苗造成逆境。 Part 1How to reduce the culture time and labor cost both are the important factors for keeping the international competitiveness of Taiwan’s Phalaenopsis. A programmable bioreactor developed by our lab was adopted in this thesis to search the optimum culture condition for the mass propagation of the biomass and bud of Phalaenopsis Soga Yukidian ‘V3’ adventitious buds via controlling the immersion frequency of medium-holding compartment, the airflow rate of growth compartment, and the frequency of changing medium. In addition, not only the anti-oxidation related properties of seedlings cultured under different conditions were compared, thus could understand the seedlings in response to the stress degree; but also the microenvironmental variation of bioreactor during seedlings growth for the preliminary optimum condition was monitored, thus could act as the basis on the improving culture condition. The results clearly revealed that the best growth condition of adventitious buds in bioreactor until now was 4 ml per minute airflow rate, 1 min immersion every 2h, and simultaneously replacing medium every week, the biomass and bud was 2.1- and 1.6- fold, respectively, as compared to the traditional glass vessel after 6 weeks cultured. Under this condition, the diurnal change of CO2 concentration inside the bioreactor and the consumption of nutrients, including NH4+, K+, malate, nitrate, phosphate, all were significantly increased after 4 weeks cultured. Given these results, one might speculate that a lag phase growth of adventitious buds appeared to be during the first 3 weeks, then, they entered into a log phase after 4 weeks. When the medium without or with changing every week, the H2O2 content of the seedlings was significantly increased, but expressed ascorbate peroxidase (EC1.11.1.11) and superoxide dismutase (EC1.15.1.1) activity, respectively. This discrepancy may be explained by the different antioxidative mechanisms of seedlings under various culture conditions. In contrast, the lowest H2O2 content was exhibited in the seedlings when their medium was replaced every 2 weeks strongly implies this culture condition for seedlings in response to a minor stress.Part 2The photosynthetic pathway of Phalaenopsis plantlets in a glass vessel has been determined as either CAM or C3 mode by many studies. In order to elucidate what kind of environmental factors affect the type of fixing CO2 of Phalaenopsis Sogo Yukidian ‘V3’ plantlets with 2 to 3 leaves, three kinds of internal vessel conditions were designed in this thesis: a traditional glass vessel culture (control treatment); 2 ml airflow rate per minute (airflow treatment); and finally, 2% (w/v) sucrose in an agar medium (deficient carbon treatment). The CO2 concentration rhythm inside the vessel was continuously monitored, meanwhile, the diurnal changes in the carbohydrate and malate content, together with the activities of phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPCase), ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco), and NAD+-malic enzyme (EC 1.1.1.39, NAD+-ME) were analyzed when the plantlets were cultured under different conditions for 8 weeks . Due to the available sampling plantlets was scarce, it was difficult to get the accurately diurnal samples, rending no close correlation among the changes in the levels of starch and malate, and the activities of PEPCase, rubisco, and NAD+-ME. However, the diurnal changes in the CO2 concentration inside vessels for the various treatments were significantly different. In the control and airflow treatments, the CO2 concentration exhibited a C3 mode, namely, a decrease during the daytime and an increase during the nighttime, and it reached to 20000 ppm in the control treatment, which was considerably higher than that of the airflow treatment. In the deficient carbon treatment, even though the CO2 concentration rhythm also appeared to be a C3 mode during the first four weeks, the pattern changed into a CAM mode at the 8th week. The sugar analysis in the agar medium also showed that no sucrose appeared after 4 weeks culture. Hence, the photosynthetic mode of Phalaenopsis plantlets could be affected by the carbon source especially sucrose. In addition, glycerol was accumulated in the plantlets cultured in the airflow and deficient carbon treatments. Because glycerol is one of the compatible solutes, it is reasonably speculated that the plantlets exhibit a stress response under these conditions. |