香莢蘭果莢發育、微體繁殖及調製之研究

香莢蘭是臺灣新興潛力作物，2020年國內栽培面積近20公頃，且國產的香草莢也陸續上市。因應我國的氣候條件及溫網室設施栽培環境，其生理特性、栽培管理技術、遺傳研究乃至品種改良之研究尚付闕如，本論文探討香莢蘭的花粉管、胚及果莢生長情況、建立無菌播種、側芽與多倍體誘導技術，並進行調製流程試驗之研究，結果摘錄如下： 1. 果莢及種子發育研究顯示：(1) 香莢蘭果莢在授粉成功後迅速生長大約在35 DAP (days after pollination) 果莢長度達最長，在49 DAP果莢直徑達最粗。(2) 在45 DAP種子成球狀胚，外種皮的最外層已發育增厚但未木質化，60 DAP外種皮最外層的細胞壁開始木質化並最終形成厚細胞壁，這些特徵與60 DAP種子發芽率的顯著下降符合。 2. 花粉發芽及花粉管生長試驗顯示：(1) 香莢蘭花粉體外發芽以不含蔗糖的BK培養基最佳，25℃ - 30℃為最適培養溫度。(2) 香莢蘭開花期間，花粉發芽率在午夜12點時最高，但在9點以前差異不大，而中午12點以後花粉發芽率就明顯下降。(3) 高溫會影響花粉發芽率，35℃ 處理1 h即有影響，除造成花粉管異常，也會降低生長速度，但只要回到25℃室溫下可恢復生長，高溫處理時間越久異常越嚴重，也越難恢復；40℃ 處理1 h就對花粉管造成嚴重傷害，並且難以恢復。(4) 花粉管在授粉後第一天已陸續萌發，5 - 7 DAP時生長至蕊柱基部，14 DAP已經進入子房，並28 DAP抵達子房的基部。大約在21 DAP始觀察有花粉管進入胚珠並完成受精，受精的胚珠在子房內發育成種子並非同步進行，蕊柱和子房連接處附近的受精胚珠比子房遠端附近的種子發育得更早。 3. 繁殖技術研究顯示：(1) 成熟種子在4%次氯酸鈉溶液中浸泡75到90 min可顯著增加發芽率；(2) 在45 DAP收集的未成熟種子具有最高的發芽率，而後即快速降低。(3) 培植體於含1 mg mL-1 BA的培養基中45天培養誘導側芽效果最佳，每個培植體可產生5.2個芽，經繼代於不含生長調節劑的培養基2個月進行馴化、生根後可出瓶種植。(4) 在多倍誘導體試驗中以0.625 mM - 1.2 mM的秋水仙素之液態培養基處理3天可成功誘導多倍體，成活植株以流式細胞儀及根尖染色體鏡檢確認染色體倍體數，獲得四倍體植株2株。多倍體植株可直接進行育種流程評估，或做為香莢蘭種內及種間雜交之親本。 4. 調製流程試驗顯示：(1) 臺灣種植之香草莢在授粉後24週已開始累積葡萄糖苷香草醛，第34週後累積速度趨緩，42週累積最高，考量其他綜合因素，以38週前後採收，調製可以得到較高品質之香草莢。(2) 在調製試驗以65℃殺菁3 min即可滿足大部分果莢殺菁處理需求，但果莢如在栽培時遮陰較為嚴重或施用氮肥較高，可以稍微降低殺菁溫度或維持時間，避免對於果莢外層組織造成過度的破壞。(3) 發酵及快速乾燥溫度皆以40℃進行，可維持香草莢內香草醛含量。(4) 果莢調製的前5天香草醛含量的增加速度最快，5-7天逐漸趨緩，8-10天達到高峰，再增加時間也無法提升香草醛含量，甚至讓香草醛揮發而降低整體含量。整體來說，國內現行的栽培技術生產之香草莢，在調製後和國外優質香草莢的品質水平相近。

關鍵字

香莢蘭；微體繁殖；胚；花粉管；多倍體；香草莢調製；香草醛；葡萄糖苷香草醛

並列摘要

Vanilla is an emerging cash crop in Taiwan, and its domestic cultivation area reached 20 hectares in 2020. Vanilla comes from the cured vanilla beans, which are the pods of the orchid plant Vanilla planifolia Andrews. In response to domestic requirements of vanilla production, related research on physiology, cultivation, genetics, and breeding is still awaiting exploration. This study investigated the pod/seed development, pollen tube growth, the optimal protocols for asymbiotic germination, the induction of lateral shoots and polyploidy, and the vanilla curing process. Results are summarized as below. 1. The pod and seed development study showed: (1) After successful hand-pollination, ovaries began to enlarge and elongate rapidly. The pod length increased steadily and reached the maximum size at 35 DAP (days after pollination), while the diameter of the pod reached the maximum size at 49 DAP. (2) The 45-DAP immature seeds have developed globular embryos and the thickened non-lignified cell wall at the outermost layer of the outer seed coat. After 60 DAP, the cell wall of the outermost layer of the outer seed coat became lignified and finally compressed into a thick envelope. These features match the significant decreases of immature seed germination percentage after 60 DAP. 2. The pollen germination and pollen tube development study indicated: (1) BK medium without sucrose is the best for in vitro pollen germination, and 25℃- 30℃ is the optimum temperature. (2) During vanilla flowers opened, the pollen activity was highest at midnight and had no significant reduction before 9 am, but decreased significantly after 12 pm. (3) High temperature significantly affects pollen activity: treatment at 35°C for 1 hour would have harmful effects, including abnormal pollen tubes and stunt growth of pollen tube, but the growth of pollen tube can be restored at 25°C. The longer the high-temperature treatment, the worse the harmful effects; treatment at 40°C for 1 hour causes severe damage to the pollen tube and unable to recover. (4) For the in vivo germination trials, the pollen tubes germinated one day after pollination, grows to the base of styles at 5 to 7 DAP, entered the ovary at 14 DAP, arrived in the middle of the ovary at 21 DAP, and finally reached the base of the ovary at 28 DAP. Pollen tubes enter ovules to fertilize at approximately 21 DAP. The development of fertilized ovules into seeds in an ovary is progressive: seeds near the joint between the style and ovary developed earlier than seeds near the distal end of the ovary. This study describes the flowering habit of vanilla and the observation of pollen tube growth after pollination, which can be used as a reference for field management and likely helps improve the success rate of pod setting. 3. The micropropagation study revealed showed: (1) mature seeds soaking in 4 % sodium hypochlorite solution for 75 - 90 min would increase germination significantly. (2) immature seeds at 45 days after pollination (DAP) had the highest germination percentage. (3) Plantlets in the medium containing 1 mg mL-1 BA for 45 d had the best effect to induce lateral shoots. Each node could produce 5.2 buds. These buds can produce roots in the basal medium without growth regulators and move out after two months subculture. (4) Treating liquid medium with 0.625 mM - 1.2 mM colchicine for three days can successfully induce polyploid. Two independent tetraploid plants were obtained. 4. The study of vanilla curing illustrated that: (1) the major component β‐glucovanillin started accumulating at 24 WAP. The cumulative efficiency of glucovanillin slowed down after 34 DAP and reached the highest cumulative content at 42 DAP. The cured beans which harvested at 38 DAP had the highest vanillin content. (2) The ideal killing condition was at 65℃ for 3 min while reducing the temperature or killing duration in order to prevent over shaded beans from heat damage. (3) Vanilla beans can keep vanillin content when sweated and dried at 40℃. (4)Vanillin content advanced quickly during the first 5 days after killing and slowed down from the 5th to 7th day. Vanillin content reached the highest level from the 8th to 10th day during the curing process and could not increase any more at the same condition. Overall, the condition established in this study can help promote curing process and produce beans which had similar vanillin content to abroad.