乙烯抑制劑對報歲蘭根莖抽梢之影響及臺灣原生豆蘭屬相關種之無菌播種

Abstract

本論文分為兩部分，第一部分探討乙烯抑制劑對報歲蘭根莖抽梢之影響，而第二部分研究臺灣原生豆蘭屬相關種之無菌播種。 於根莖繁殖培養基中施用不同濃度之aminooxyacetic acid (AOA)、AgNO3、1-methylcyclopropene (1-MCP)，以抑制乙烯生合成或作用，間接探討乙烯對報歲蘭根莖器官分化的影響。另於報歲蘭根莖培養時期燻蒸乙烯，以瞭解乙烯對於報歲蘭器官生長分化之直接影響。 報歲蘭‘瑞寶’ × ‘光華蝶’根莖培養於添加AgNO3之液體根莖繁殖培養基中皆有芽體及根部分化，而培養於未添加AgNO3之根莖繁殖培養基中有最多之根莖分化數目。兩次重複試驗中，液體培養基添加50 μM AgNO3之處理組誘導較多芽體，培植體於培養10天後觀察到鞘葉 (sheath leaf) 的形成，代表此時頂端分生組織已趨向芽體分化。於培養基添加AgNO3 處理者之瓶內乙烯濃度均較未添加AgNO3者高。 報歲蘭根莖培養於添加AOA以及未添加乙烯相關藥劑之固體根莖繁殖培養基無芽體和根部形成，但培養於添加100 μM AgNO3者則有芽體和根系分化之情形，故乙烯作用抑制劑較乙烯生合成抑制劑能促進報歲蘭分化芽體。 另於不同培養期間對液體培養基添加AgNO3，探討不同長度之新生報歲蘭根莖其芽體分化受影響之情形。結果不同時期添加50 μM AgNO3者其培植體均有芽體發生，培養於未添加AgNO3培養基者無芽體分化。 於培養時期燻蒸1及10 μL•L-1 1-MCP無法有效誘導報歲蘭根莖分化芽體，而培養基添加100 μM AgNO3之處理有芽體分化，但培養基添加100 μM AgNO3且燻蒸1 μL•L-1乙烯之處理並未有芽體形成。故乙烯會抑制報歲蘭根莖分化芽體。

造成蘭科種子不易發芽之因素，包括胚發育不完全、缺乏胚乳、發芽抑制物質之累積及種皮不滲水性等。本論文第二部分針對可能影響豆蘭屬 (Bulbophyllum) 植物種子發芽之因素設計試驗，研究培養方式、種子前處理、果莢成熟度及不同濃度fluridone (ABA生合成抑制劑) 處理對豆蘭屬種子無菌發芽之影響且調查不同種豆蘭成熟種子的發芽率。 紋星蘭 (Bulb. affine) 與烏來捲瓣蘭 (Bulb. macraei) 種子無菌播種發芽率皆以1/2 MS固體培養基較液體培養者高。固體培養者其原球體發育較液體培養者快，且液體培養者之原球體於培養後期均有玻璃質化現象。 使用次氯酸鈉、氫氧化鈉與氯化氫三種化學藥劑及超音波震盪於無菌播種前處理小豆蘭種子一段時間，以未經前處理者發芽率最高 (59.3%)，各前處理組均無法有效提高發芽率，且小豆蘭 (Bulb. aureolabellum) 種子發芽率隨超音波震盪或浸泡1%次氯酸鈉處理時間增加而下降。 取自然授粉之果莢進行無菌播種，果莢開裂當天播種者之發芽率最低 (27.9%)，而果莢開裂前85天播種者之發芽率最高 (50.4%)。另取人工授粉後120、150、180及270天之果莢進行無菌播種，其中以人工授粉後270天者有最高的發芽率 (72.6%)，其果莢之成熟度相當於開裂前90天。 於授粉後130天對黃萼捲瓣蘭果莢處理不同濃度之fluridone不會影響採收果莢之大小及鮮重，處理50及100 μM fluridone皆可有效提高種子發芽率，其中以50 μM fluridone處理者之發芽率最高，為33.4%，而未施用者僅15.6%。 六種臺灣原生豆蘭屬植物皆有一部分種子發芽但種間的發芽階段有些許差異，而發芽率由高往低排序分別為狹萼捲瓣蘭、阿里山豆蘭、白毛捲瓣蘭、臺灣捲瓣蘭、小豆蘭，最低者為烏來捲瓣蘭。

The thesis is consistent of two chapters. The first chapter describes the effects of ethylene inhibitors on shooting of Cymbidium sinense rhizomes. The second chapter elucidates asymbiotic germination of Bulbophyllum related species native in Taiwan. Aminooxyacetic acid (AOA), silver nitrate (AgNO3), and 1-methylcyclopropene (1-MCP) were applied to indirectly clarify the ethylene effect on shoot growth and differentiation of Cym. sinense. Additionally, ethylene was supplied during culture to investigate its direct effect on organ differentiation of Cym. sinense. Cymbidium sinense ‘Rui-Bao’ × Cym. sinense ‘Guang-Hua-Die’ rhizomes cultured in liquid rhizome propagating medium supplied with AgNO3 all developed shoots and roots. Rhizomes cultured in AgNO3-free medium (control) had more newly formed rhizomes. Liquid medium supplied with 50 μM AgNO3 had more differentiated shoots in both replicated experiments. Sheath leaf formation was observed at 10 days after culture, indicating the differentiation of shoot from rhizome. Ethylene concentrations in cultural vessels were higher in AgNO3-supplied treatments than in AgNO3-free controls. Cymbidium sinense rhizomes cultured in solid rhizome propagating medium supplied with AOA or ethylene-inhibitor-free control did not form shoots and roots, but did differentiate some in medium supplied with 100 μM AgNO3. Thereby, ethylene perception inhibitors rather than ethylene synthesis inhibitors were more effective for Cymbidium shoot inducing. AgNO3 was added to cultural medium at different times after subculture to test the effect of rhizome lengths on subsequent organ differentiation. When 50 μM AgNO3 were supplied in liquid rhizome propagating medium at different times after subculture, all rhizomes formed shoots compared with that no shoot formation was found in AgNO3-free controls. More shoot formation was recorded when 50 μM AgNO3 was supplied at two weeks after subculture. Shoot differentiation of Cym. sinense. rhizomes were not induced by the fumigation of 1-MCP at 1 and 10 μL•L-1, but were induced by supplement of 100 μM AgNO3 in medium. However, combination of 1 μL•L-1 ethylene and 100 μM AgNO3 treatments did not induced the shoot differentiation of Cym. sinense rhizomes. These results indicated that ethylene inhibited shoot differentiation of Cym. sinense rhizomes.

Factors contributing to low germination of Orchidaceous plants include incompletely developed embryos, lack of endosperm, accumulation of germination-inhibiting metabolites, and seed coat impermeability. The second chapter focuses to overcome the possible difficulties in Bulbophyllum seed asymbiotic germination, so that cultural medium types, seed pretreatments, capsule maturities, fluridone (an ABA synthetic inhibitor) treatments were tested. Also, germination rates of mature seeds in different species were determind. Seed germination of Bulb. affine and Bulb. macraei were higher in solid 1/2 MS medium than in liquid medium. Protocorm growth were also faster in solid medium; protocorms cultured in liquid medium turned vitrificated consequently. Seeds of Bulb. aureolabellum were pretreated with sodium hyperchloride (NaOCl), sodium hydroxide (NaOH), hydrogen chloride (HCl), and ultrasonic vibration for various durations. Highest germination was recorded in control (59.3%), and treatments tested in this study did not improve germination. Moreover, germination decreased with increasing NaOCl concentration and ultrasonic vibration duration. The lowest germination percentage (27.9%) was observed when sowing seeds on the day of Bulb. retusiusculum capsule spliting, and the highest (50.4%) was obtained by sowing seeds 85 days before capsule spliting. In another experiment, Bulb. retusiusculum capsules were harvested at 120, 150, 180, and 270 days after artificial pollination (DAP) for in vitro germination. Fluridone application at 130 DAP did not affect the sizes and fresh weights of capsules at harvest; capsules treated with 50 and 100 μM fluridone had higher subsequent germination rates. The highest germination percentage was recorded in capsules treated with 50 μM fluridone, which was 33.4%, compared with that of 15.6% in controls. All six species of Bulbophyllum studied had some seeds germinated, but rate in protocorm development varied. The germination percentage from high to low were Bulb. drymoglossum, Bulb. pectinatum, Bulb. albociliatum, Bulb. taiwanense, Bulb. aureolabellum, and Bulb. macraei.