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研究生:周玉儒
研究生(外文):Chou, Yu-Ju
論文名稱:筆筒樹孢子體無菌生產系統之建立
論文名稱(外文):Establishment of in vitro Sporophyte Production System in Cyathea lepifera
指導教授:謝清祥謝清祥引用關係
指導教授(外文):Hsieh, Ching-Hsiang
口試委員:張正方中宜
口試委員(外文):Chang, ChenFang, Jong-Yi
口試日期:2017-07-31
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:農園生產系所
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:110
中文關鍵詞:筆筒樹孢子萌芽配子體孢子體綠球體
外文關鍵詞:Cyathea lepiferaspore germinationgametophytesporophytegreen globular body
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筆筒樹(Cyathea lepifera)孢子萌芽試驗處理使用1/2 MS巨量元素與0.1 g/L的活性碳,再添加不同濃度的GA3(0、3、6 mg/L)及蔗糖(0、5%)(Sucrose)處理之組合進行測試。孢子播種1周後,於添加3 mg/L與6 mg/L GA3的培養基中即有孢子萌芽,平均分別為1.5個與0.5個。孢子萌芽數量在播種後第7周達到高峰且配子體覆蓋面積及覆蓋率急速增加,添加3 mg/L GA3(含蔗糖5%)的處理最多,平均有266個;未添加GA3(含蔗糖0 %)的處理最少,平均有113個。筆筒樹孢子應以GA3 3 mg/L(含蔗糖5%)的1/2 MS培養基進行播種及培養,可得最大的孢子萌芽數與配子體覆蓋面積(4248.22 mm2)與覆蓋面積率(66.81%)。
以0.5×0.5 cm2配子體塊為培植體,利用1/2 MS培養基添加0.1 mg/L BA + 0.1 mg/L NAA + 0~20 mg/L Adenine處理,測試其誘導綠球體(Green Globular Body, GGB)之效果。所有處理2周後皆有綠球體產生,其中以腺嘌呤5 mg/L的處理最佳,每周的綠球體產生數皆高於其他處理,7週後平均每個配子體塊可產生2.89個綠球體。同樣以0.5×0.5 cm2配子體塊為培植體,使用1/2 MS培養基添加0-2 mg/L BA + 0-2 mg/L IAA的濃度處理組合進行綠球體誘導,結果以添加2 mg/L BA的處理能誘導最多的綠球體,培養1周即有綠球體產生,每個配子體塊8周後平均產生2.89個綠球體。
以0.5×0.5 cm2配子體塊為培植體直接誘導孢子體,利用不同MS巨量元素(1/2、1/4、1/8、1/16 MS(Macro))及活性碳濃度(0%、0.05%、0.1%)處理組合,進行測試。6周後,添加活性碳的各處理有孢子體產生,培養10周後,不同巨量元素及活性碳濃度組合處理之間對於配子體產生孢子體並無顯著差異,但其中以0.05%活性碳1/16 MS(Macro)處理,每個配子體塊平均可產生最多孢子體為1.78個。利用綠球體萌發為孢子體之試驗,綠球體培養1周後,於0.1%活性碳1/2 MS(Macro)處理即有孢子體產生,平均每個綠球體可產生0.22個孢子體,且此處理產生的孢子體數量於培養的10周內皆高於其他處理;以0%活性碳1/16 MS(Macro)處理孢子體產生數最少,培養10周每個綠球體平均產生不足1個孢子體。綠球體的生長情況與孢子體產生數量,隨著MS巨量元素及活性碳濃度上升而增加,因此,以1/2 MS(Macro)添加0.1%活性碳為最佳處理,培養10周後每個綠球體平均可產生15.78個孢子體。
從孢子至孢子體之生長過程,由配子體直接產生孢子體的數量較少且時間較長,因此可藉由配子體產生的綠球體進行誘導其萌發為孢子體,可望達到復育及商業化量產的目的。
The spores of Cyathea lepifera were treated with different combination of GA3 (0, 3 and 6 mg/L) and sucrose (0 and 5%) to test their germination. One week after sowing, spores were germinated in the medium supplemented with 3 mg/L and 6 mg/L GA3, and averaged 1.5 spores and 0.5 spores, respectively. The largest number of spore germination was in the 7th week after sowing and the coverage area of gametophyte nubble and coverage area percentage of gametophyte nubble increased rapidly. The spores cultured on 1/2 MS medium with 3 mg/L GA3 (5% sucrose), after 7 weeks of culture, had the highest number of germination (266), coverage area (4248.22 mm2) and coverage area ratio (66.81%). The spores cultured on 1/2 MS medium with 0 mg/L GA3 (0% sucrose) was poorest treatment it showed only 113 spores germinated.
The 0.5×0.5 cm2 gametophyte nubbles wre treated with different adenine (0, 1, 5, 10, 15 and 20 mg/L) in 1/2 MS medium with 0.1 mg/L BA + 0.1 mg/L NAA to induce green globular body. After 2 weeks of cultivation, all treatments produced green globular bodies and with 5 mg/L adenine added was the best treatment. The number of green globular bodies produced per week was higher than other treatments. After 7 weeks, it averaged 2.89 green globular bodies per gametophyte nubble. The explant 0.5×0.5 cm2 gametophyte nubble, treated with different combination of BA (0, 0.5, 1 and 2 mg/L) and IAA (0, 0.5, 1 and 2 mg/L) to induce green globular body were tested. The maximum number of green globular body was induced with 2 mg/L BA treatment. Green globular body were produced after 1 week of culture and averaged 2.89 green globular bodies per nubble after 8 weeks.
The 0.5×0.5 cm2 gametophyte nubbles were treated with different combination of macro nutrient (1/2, 1/4, 1/8 and 1/16 MS (Macro)) and active charcoal (0, 0.05 and 0.1%) to induce sporophyte formation. After 6 weeks, all the treatment added with activated carbon had sporophytes. After 10 weeks of cultivation, there was no significant difference in the production of sporophyte among different treatment combinations of macro nutrient and active charcoal. But the best treatment was under 0.05% of activated carbon with1/16 MS (Macro), which averaged 1.78 sporophytes per gametophyte nubble.
Using the green globular body (GGB) germinated to induce sporophyte, GGB were treated with different combination of macro nutrient (1/2, 1/4, 1/8 and 1/16 MS (Macro)) and active charcoal (0, 0.05 and 0.1%). Sporophytes were produced in 1/2 MS (Macro) with 0.1% active charcoal after 1 week of culture and averaged 0.22 sporophytes per green globular body. 1/2 MS (Macro) with 0.1% active charcoal is the best treatment; the number of sporophytes produced per week was higher than other treatments. The least sporophyte production was in the treatment with 0% activated carbon 1/16 MS (Macro) which produced less than 1 sporophyte on each green globular body in 10 weeks. Increasing MS macro-nutrients and active charcoal content helps the growth of green globular body and sporophyte formation rate. Therefore, 1/2 MS (Macro) with 0.1% activated carbon is the best treatment and which average 15.78 sporophytes per green globular body after 10 weeks of cultivation.
From the spores gerimation to induce to the production of sporophyte, using the gametophyte products sporophytes will get little sporophytes and take longer time. To achieve better commercial mass production of Cyathea lepifera, it can be produced though the green globular body, induced from gametophyte, then induce sporophytes formation.
中文摘要 I
Abstract III
謝誌 VI
圖目錄 X
表目錄 XII
壹、 前言 1
貳、 前人研究 3
一、 樹蕨種類介紹 3
二、 筆筒樹栽培及用途 3
(一) 筆筒樹之自然成長過程 3
(二) 栽培環境 4
(三) 用途 5
三、 蕨類生活史 6
四、 桫欏科植物之世代 7
五、 影響樹蕨有性繁殖之條件 8
(一) 孢子成熟度與萌芽之關係 8
(二) GA3與孢子萌芽之關係 8
(三) 孢子儲藏溫度與萌芽率之關係 9
(四) 光照與配子體發育之關係 9
(五) 碳源對配子體發育之影響 10
(六) 孢子萌芽及配子體發育 10
六、 樹蕨無性繁殖 11
(一) 分株繁殖 11
(二) 不定芽繁殖 11
(三) 托葉繁殖 12
(四) 球莖繁殖 12
(五) 組織培養 12
七、 活性碳之功能 15
八、 氨基酸(Amino acid)於組織培養中之功能 16
參、 材料方法 17
實驗一 GA3及蔗糖濃度對筆筒樹孢子萌芽之影響 17
一、 試驗植物材料 17
二、 試驗處理 17
三、 培養環境 18
四、 試驗紀錄 18
實驗二 筆筒樹配子體誘導綠球體 19
一、 試驗植物材料 19
二、 試驗處理 19
三、 培養環境 21
四、 試驗紀錄 22
實驗三 以配子體及綠球體產生孢子體的途徑測試 23
一、 試驗植物材料 23
二、 試驗處理 23
三、 培養環境 25
四、 試驗紀錄 25
五、 綠球體萌芽成孢子體之細胞分化過程 25
統計分析 29
肆、 結果 30
實驗(一) GA3及蔗糖濃度對筆筒樹孢子萌芽之影響 30
實驗(二)筆筒樹配子體誘導綠球體 42
(一) 添加腺嘌呤(Adenine)對筆筒樹配子體誘導綠球體及孢子體之影響 42
(二) 利用添加BA與IAA誘導配子體產生綠球體 54
實驗(三)以配子體及綠球體產生孢子體的途徑測試 68
(一) 筆筒樹配子體誘導產生孢子體 68
(二) 筆筒樹綠球體分化成孢子體 81
伍、 討論 97
一、 激勃素(GA3)及蔗糖濃度對筆筒樹孢子萌芽之影響 97
二、 筆筒樹配子體誘導綠球體 98
三、 以配子體及綠球體產生孢子體的途徑試驗 99
陸、 結論 101
柒、 參考文獻 103
作者簡介 110
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