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研究生:包瑞斯
研究生(外文):Boris Andres Bran Barrientos
論文名稱:苞舌蘭無菌播種及種子保存之研究
論文名稱(外文):Asymbiotic Seed Germination and Seed Conservation of Spathoglottis plicata
指導教授:方中宜
指導教授(外文):Fang, Jong-Yi
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:熱帶農業暨國際合作系
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:2013
畢業學年度:101
論文頁數:147
中文關鍵詞:無菌播種紫苞舌蘭幼苗生長種子保存
外文關鍵詞:asymbiotic germinationSpathoglottis plicataseedling growthseed conservation
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學號:M9922028
論文名稱: 苞舌蘭無菌播種及種子保存之研究
總頁數:147
學校名稱:國立屏東科技大學
系所別:熱帶農業暨國際合作系
畢業時間:101學年度第2學期 學位別:碩士學位
研究生:包瑞斯           指導教授:方中宜 博士

論文摘要內容:
台灣原生種紫苞舌蘭(Spathoglottis plicata)是一種栽培容易、花期長且花形和花色優美的植物。由於農業活動的過度發展,原生種之棲息環境受到破壞,因此急須發展出一套原生種遺傳資源的保存方法。本研究主要採用組織培養技術進行紫苞舌蘭的無菌播種、種苗生長、瓶苗馴化及種子保存的研究。種子採集自28天成熟的果莢並無菌播種在五種不同的培養基上,包括 ½ MS、蘭花播種培養基(OSSM)、BM-1陸生蘭培養基(BM-1)、Vacin and Went蘭花培養基(VW)及Knudson C蘭花培養基(KC)。結果顯示,種子在不同的培養基及光周處理下皆會發芽,而在黑暗下發芽速度比在光照下還快,但在光照的環境下,其萌發種子發育較快速(例如: stages 4-6)。在所有培養基中,種子播種於OSSM培養基黑暗及光照的環境處理及VW培養基光照的處理後11周有較好的發育情形(例如: stage 6)。在OSSM培養基中添加不同的細胞分裂素如6-benzylaminopurine (BAP) 和thidiazuron (TDZ) 及生長素如1-naphthaleneacetic acid (NAA) 和 2,4-dichlorophenoxyacetic acid (2,4-D) 組合,並觀察其種苗發育的情形,結果顯示種苗可在不含植物生長調節劑 (plant growth regulators; PGR)的條件下生長,而在含有PGR之培養基中其植株長度、葉片數、葉片重量、根數、根長及植株鮮重方面發現,以0.5 mg/L NAA的處理,其芽的長度、葉片重量、根
的數目及植株鮮重有最好的效果,而在0.5 mg/L NAA 以及BAP或TDZ的組合處理140天,能得到最多根的數目及最高的植株和葉片鮮重。將發育良好帶有葉片及根之植株移至介質為水苔及泥碳土的三吋盆中進行馴化,結果顯示實生苗可順利的在泥碳土介質中生長而水苔則否。將紫苞舌蘭之種子於低溫4和 -20˚C中儲藏,在低溫-20˚C中儲藏5周後其發芽率最高,而此處理可將種子保存2個月。最後,以不同的冷凍方法例如玻璃化冷凍法(vitrification)及密封脫水(encapsulation-dehydration)試圖長期保存苞舌蘭種子實驗中發現無法以液態氮來長期保存種子。但此研究可供未來紫苞舌蘭種源長期保存做為初步的參考。

Student ID: M9922028
Title of Thesis: Asymbiotic Seed Germination and Seed Conservation of Spathoglottis plicata
Total Page: 147
Name of Institute: Department of Tropical Agriculture and International Cooperation
Graduate Date: June, 2013 Degree Conferred: Master
Name of Student: Boris Bran Advisor: Fang Jong-Yi, Ph.D.
The Contents of Abstract in This Thesis:
Spathoglottis plicata, a native terrestrial orchid in Taiwan, is an attractive and easy to grow plant which can flower all year round in the garden. However, native orchid species are threatened as a consequence of environmental disturbances, succession of natural habitats, and overexploitation for horticultural purposes. Therefore, there is need to develop conservation procedures for preserving their genetic resources. The present research was conducted to study the in vitro asymbiotic seed germination, seedling growth, acclimatization and seed conservation of this orchid. Seeds of S. plicata from 28-day-old capsule were sown on five germination media, including half-strength Murashige and Skoog medium (½ MS), Orchid Seed Sowing Medium (OSSM), BM-1 Terrestrial Orchid Medium (BM-1), Vacin and Went Modified Orchid Medium (VW), and Knudson C Orchid Medium (KC). Germination occurred in all the media and photoperiods tested. Seeds subjected to darkness were found to germinate more rapidly than those exposed to light whereas seeds had a faster development to advanced seed development stages (i.e. Stages 4-6) when subjected to light.
IV
All the seeds grown on OSSM medium under light and dark regimes and those grown on VW medium under light condition proceeded to the advanced seed development stage (i.e. Stage 6) within 11 weeks of culture. In vitro seedling development were examined by subjecting the germinated seeds to OSSM medium supplemented with different combinations of cytokinins [i.e., 6-benzylaminopurine (BAP) and thidiazuron (TDZ)] and auxins [i.e., 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)]. Results showed that seedlings could develop on medium devoid of plant growth regulators (PGR) in the same way as those cultivated with PGR(s) in terms of their shoot length, leaf number, leaf width, root number and length, and seedling fresh weight. The treatment which contained 0.5 mg/L NAA was found to provide the highest shoot length, leaf width, root number and seedling fresh weight; whereas 0.5 mg/L NAA in combination with BAP or TDZ yielded the highest number of roots, seedling fresh weight and leaf production after 140 days. Plantlets with well-developed leaves and roots were hardened in 3-inch pots filled with either sphagnum moss or peat moss. It was observed that the in vitro plantlets can be successfully acclimatized using peat moss, but not with sphagnum moss. The viability of S. plicata seeds was tested following storage under low temperature conditions of 4 and -20°C. Maximum germination of stored seeds was observed at week 5 under -20°C. Consequently, it can be used for the seed conservation of S. plicata which last a period of 2 months. Finally, different cryopreservation methods (i.e. vitrification and encapsulation-dehydration) were used to test their possibility for long-term conservating S. plicata seeds and protocorms. However, none of the stored explants survived liquid nitrogen storage. This study presents the first step towards the development of a long-term storage procedure for S. plicata genetic resources.

Chinese Abstract.................... ............................................................................ I
English Abstract............. ................................................................................. III
Acknowledgements .......................................................................................... V
Table of Contents ............................................................................................ VI
List of Tables ................................................................................................ VIII
List of Figures ................................................................................................. IX
1. Introduction ................................................................................................... 1
2. Literature Review .......................................................................................... 4
2.1 Spathoglottis plicata ................................................................................ 4
2.1.1 Taxonomy .......................................................................................... 4
2.1.2 Plant morphology ............................................................................... 4
2.1.3 Geographical distribution .................................................................. 5
2.1.4 Propagation of Spathoglottis plicata .................................................. 6
2.2 In vitro germination of native orchid seeds ............................................. 9
2.2.1 Symbiotic germination ....................................................................... 9
2.2.2 Asymbiotic germination .................................................................. 12
2.2.3 Factors affecting asymbiotic germination of native orchid seeds ... 15
2.3 Conservation of native orchid seeds...................................................... 23
2.3.1 Short-term seed conservation ........................................................... 23
2.3.2 Long-term seed conservation ........................................................... 25
3. Materials and Methods ................................................................................ 28
3.1 Plant materials ....................................................................................... 28 3.2 Estimation of seed number .................................................................... 28
3.3 Seed sterilization ................................................................................... 29
3.4 Effect of different culture media on asymbiotic seed germination ....... 29 3.5 Microscopic analysis of seed development ........................................... 32
3.6 Effect of different PGRs on seedling development .............................. 32 3.7 Acclimatization of in vitro plantlets ...................................................... 33 3.8 Low-temperature storage of seeds ......................................................... 34
VII
3.8.1 Low-temperature storage without any pretreatment ........................ 34 3.8.2 Low-temperature storage with lithium chloride pretreatment ......... 34 3.9 Cryopreservation of seeds ..................................................................... 35
3.10 Culture conditions and data analysis ................................................... 36
4. Results.................. ....................................................................................... 38
4.1 Estimation of seed number .................................................................... 38
4.2 Effect of different culture media on asymbiotic seed germination ....... 41
4.3 Microscopic analysis of seed development ........................................... 61
4.4 Effect of different PGRs on seedling development .............................. 65
4.5 Acclimatization of in vitro plantlets ...................................................... 86
4.6 Low-temperature storage of seeds ......................................................... 91
4.6.1 Low-temperature storage of seeds without pretreatment ................ 91
4.6.2 Low-temperature storage with lithium chloride pretreatment and cryopreservation procedures .................................................... 98
5. Discussion .................................................................................................. 100
6. Conclusions ............................................................................................... 113
7. References ................................................................................................. 114
Biosketch of Author ...................................................................................... 145

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