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研究生:馮家華
研究生(外文):Kyaw Minn Hsan
論文名稱:芭菲爾鞋蘭及蝴蝶蘭之試管內形態發生
論文名稱(外文):In vitro morphogenesis of Paphiopedilum & Phalaenopsis
指導教授:陳彥澄陳彥澄引用關係
指導教授(外文):Jen-Tsung Chen
學位類別:碩士
校院名稱:國立高雄大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:71
中文關鍵詞:形態發生芭菲爾鞋蘭蝴蝶蘭植物生長調節劑癒傷組織類原球體
外文關鍵詞:morphogenesisPLBscallusPGRsPhalaenopsisPaphiopedilum
相關次數:
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本論文的第一部分試驗為芭菲爾鞋蘭之試管內形態發生,其材料來源為長期繼代之芭菲爾鞋蘭根部癒傷組織,試驗方法為測試癒傷組織在含有不同植物生長調節劑的培養基所導致之形態發生的反應,以便了解長期繼代癒傷組織的再生能力。試驗所用之基礎培養基為1/2MS添加20 g/l蔗糖(sucrose)、1 g/l蛋白腖(peptone)及3 g/l水晶洋菜(Gelrite)。試驗所用之植物生長調節劑為0.1、0.5、1 mg/l NAA、2,4-D和TDZ以及0.1、0.5、1 mg/l NAA或2,4-D組合0.1、0.5、1 mg/l TDZ,未添加植物生長調節劑之基礎培養基為控制組。培養150天後,癒傷組織呈現褐化死亡的狀態。癒傷組織之褐化情形在未添加植物生長調節劑的控制組最顯著,平均褐化率為 75%,其次為含有TDZ之培養基,平均褐化率為60%。培養基中含有植物生長素NAA及2,4-D能降低癒傷組織之褐化率。在NAA或2,4-D組合TDZ培養基中,癒傷組織之平均褐化率分別為35.33%和39.67%。在僅含有植物生長素NAA或2,4-D之培養基中,癒傷組織之平均褐化率分別只有20%和10%。此外,癒傷組織亦有增殖的情形,增殖情形在僅含有植物生長素NAA或2,4-D的培養基以及含有NAA或2,4-D組合TDZ培養基較明顯。培養5個月後,癒傷組織從鮮黃色轉為綠色,部分的癒傷組織外圍形成光滑白色團粒及吸收毛等結構,但無類原球體或體胚的形成。經由結果推測長期繼代之癒傷組織,其再生能力明顯下降。
第二部分的試驗為蝴蝶蘭之試管內形態發生,其材料為兩個月大與四個月大之蝴蝶蘭實生苗。培養於含有0.5 mg/l TDZ之液態基礎培養基,每隔兩個禮拜繼代一次,繼代三次即液態培養兩個月後更換至雙層培養基,並繼續培養兩個月。經培養四個月後,實生苗之基底部與葉片部位皆有類原球體的形成。在葉片部位形成之平均類原球體數目分別為22.6個與19.2個。將這些類原球體培養於再生培養基,結果顯示有百分之百的再生率。一般而言,植物組織培養是取植物的根、莖、葉或植株其他部分切割後作為培植體培養。本試驗中,培養材料為完全的實生植株,結果其葉片及基底部位仍可形成類原球體。生長在自然界的蘭花,唯有澤蘭在葉片部位偶而長出體胚,然後會落地生根。根據本試驗結果,未來可進一步研究其生理意義或應用於蘭科植物之種苗繁殖。此外,仍需進行形態解剖的研究以確認植株再生的途徑。
To study in vitro morphogenesis of Paphiopedilum, root-derived long-term callus of Paphiopedilum ‘Alma Gavaert’ was used to study the effects of plant growth regulators (PGRs) on in vitro morphogenesis and subsequent plant regeneration. Callus was cultured on basal medium supplemented with PGRs. Basal medium contains 1/2 MS medium, 20 g/l sucrose, 1 g/l peptone and 3g/l Gelrite. PGRs used in this study were 0.1, 0.5, 1 mg/l NAA, 2,4-D, TDZ and 0.1, 0.5, 1 mg/l NAA or 2,4-D combined with 0.1, 0.5, 1 mg/l TDZ; basal medium was used as the control. After 150 days of incubation, callus has been necrosis. Necrosis rate was highest in control, about 75%, and the next is medium contains TDZ, about 60%. Callus cultured in medium contains NAA or 2,4-D not only shows lower necrosis rate but also shows proliferation of callus. Though some callus turns green from yellowish in color, and there is whitish smooth granules formed at the peripheral regions of some callus, there is neither protocorm-like-bodies (PLBs) nor somatic embryos forming. Suggesting that long time subculture callus might have a low regeneration ability.
To study in vitro morphogenesis of Phalaenopsis, two months and four months old seedlings of Phalaenopsis aphrodite subsp. formosana were cultured in liquid basal medium containing 0.5mg/l TDZ, and subculture every two weeks. After two months of incubation in liquid medium, seedlings were transferred to two layer medium. Forming of PLBs at posterior region and leaf part of seedlings were observed. Average number of PLBs formed in two months and four months old seedlings is 22.6 and 19.2, respectively. Newly formed PLBs were then cultured in regenerate medium, and a 100% regeneration rate was obtained. In general, plant tissue culture is taking a part of tissue i.e; leaf, root or stem segment as explants and culture in medium. In this experiment, we cultured the whole seedlings and then obtained PLBs at posterior region and leaf part. In natural, just Malaxis Paludosa, one of orchidaceae sometimes forms embryos at leaf. We will study its significance and for the use in orchid’s mass propagation in future.
目錄

致謝 .............................................. i
縮寫表 ............................................ ii
目錄 .............................................. iii
表目錄 ............................................ vi
圖目錄 ............................................ ix

中文摘要........................................... 1
英文摘要........................................... 3

第一章 緒論 ..................................... 5
1.1蘭花之植物學特性 ............................. 5
1.2蘭花之試管內形態發生 ......................... 6
1.3形態發生之意義 ................................................ 7
1.4影響蘭科植物形態發生之因子 ................... 8
1.5蘭科植物之市場 ............................... 9
1.6參考文獻 ..................................... 9

第二章 芭菲爾鞋蘭之試管內形態發生 ............... 15
2.1前人研究及試驗目的 ........................... 15
2.2 材料與方法 ....................................................................................... 17
2.2.1 試驗材料 ........................................................................................ 17
2.2.2 基礎培養基 .................................................................................... 17
2.2.3 植物生長調節劑 ........................................................................... 17
2.2.4 芭菲爾鞋蘭癒傷組織之長期繼代 ............................................... 17
2.2.5 芭菲爾鞋蘭癒傷組織之試管內形態發生 ................................... 17
2.2.6 統計方法 ....................................................................................... 18
2.3 結果 ................................................................................................... 18
2.3.1 芭菲爾鞋蘭癒傷組織之長期繼代 ................................................ 18
2.3.2 芭菲爾鞋蘭癒傷組織之試管內形態發生 .................................... 18
2.4 討論 ................................................................................................... 20
2.5 參考文獻 ........................................................................................... 21

第三章 蝴蝶蘭之試管內形態發生 ............................................................ 34
3.1 前人研究及試驗目的 ....................................................................... 34
3.2 材料與方法 ....................................................................................... 35
3.2.1 試驗材料 ........................................................................................ 35
3.2.2 無菌播種 ........................................................................................ 35
3.2.3 播種培養基 .................................................................................... 35
3.2.4 以TTC溶液測試種子活力.............................................................. 36
3.2.5 培植體對蝴蝶蘭試管內形態發生之影響 .................................... 36
3.2.6 培養環境對蝴蝶蘭試管內形態發生之影響 ................................ 36
3.2.7 植物生長調節劑對蝴蝶蘭試管內形態發生之影響 .................... 37
3.2.8 形態發生所產生之類原球體的植株再生 .................................... 37
3.2.9 試驗條件 ........................................................................................ 37
3.2.10 統計方法 ...................................................................................... 38
3.3 結果 ................................................................................................... 38
3.3.1 種子活力測試 ................................................................................ 38
3.3.2 無菌播種 ........................................................................................ 38
3.3.3 蝴蝶蘭之試管內形態發生 ............................................................ 38
3.3.4 類原球體的植株再生 .................................................................... 40
3.4 討論 ................................................................................................... 41
3.5 參考文獻 ........................................................................................... 42












表目錄

表2-1. 芭菲爾鞋蘭實生苗之根部與葉片組織來源癒傷組織在每兩個月繼代過程中的增殖率 .............................. 25


表2-2. 芭菲爾鞋蘭實生苗之根部與葉片組織來源癒傷組織在每兩個月繼代過程中的褐化率 ................... 26


表2-3. 形態發生試驗中植物生長調節劑對根部癒傷組織的褐化之影響 ..... 27


表2-4. 形態發生試驗中植物生長調節劑對根部癒傷組織的增殖之影響 ..... 28


表2-5. 植物細胞生長素及植物細胞分裂素對芭菲爾鞋蘭實生苗之根部癒傷組織形態發生的影響 ............................. 29


表2-6. NAA組合TDZ對芭菲爾鞋蘭實生苗之根部癒傷組織形態發生的
影響 .......................................... 30


表2-7. TDZ組合2,4-D對芭菲爾鞋蘭實生苗之根部癒傷組織形態發生的影響 ................................................. 31


表3-1. 兩個月大與四個月大蝴蝶蘭實生苗在含有不同植物生長調節劑之固態培養基中培養兩個月後的存活率 .................. 46


表3-2. 兩個月大與四個月大蝴蝶蘭實生苗在含有不同植物生長調節劑之液態培養基中培養兩個月後的存活率 .............. 47


表3-3. 兩個月大與四個月大蝴蝶蘭實生苗在含有不同植物生長調節劑之雙層培養基中培養兩個月後的存活率 .................. 48


表3-4. 兩個月大蝴蝶蘭實生苗在含有不同植物生長調節劑之固態培養基中培養四個月後基底部與葉片部位的類原球體形成率 .... 49


表3-5. 兩個月大蝴蝶蘭實生苗在含有不同植物生長調節劑之液態培養基中培養2個月後換到雙層培養基培養2個月,共培養四個月後基底部與葉片部位的類原球體形成率 ....................... 50


表3-6. 兩個月大蝴蝶蘭實生苗在含有不同植物生長調節劑之雙層培養基中培養四個月後基底部與葉片部位的類原球體形成率 ..... 51


表3-7. 四個月大蝴蝶蘭實生苗在含有不同植物生長調節劑之固態培養基中培養四個月後基底部與葉片部位的類原球體形成率 .... 52


表3-8. 四個月大蝴蝶蘭實生苗在含有不同植物生長調節劑之液態培養基中培養2個月後換到雙層培養基培養2個月,共培養四個月後基底部與葉片部位的類原球體形成率 ...................... 53


表3-9. 四個月大蝴蝶蘭實生苗在含有不同植物生長調節劑之雙層培養基中培養四個月後基底部與葉片部位的類原球體形成率 .... 54
















圖目錄

圖2-1. 芭菲爾鞋蘭癒傷組織形態發生之研究 ................ 32


圖2-2. 芭菲爾鞋蘭癒傷組織在試管內的形態發生 ........... 33


圖3-1. 蝴蝶蘭之播種 ................................................................................. 55


圖3-2. 蝴蝶蘭之試管內形態發生 ............................................................. 56


圖3-3. 蝴蝶蘭之試管內形態發生之類原球體於葉片部位形成圖.a ....... 57


圖3-4. 蝴蝶蘭之試管內形態發生之類原球體於葉片部位形成圖.b ....... 58


圖3-5. 蝴蝶蘭葉片部位形成之類原球體的抽芽情形 ........ 59


圖3-6. 蝴蝶蘭之試管內形態發生試驗中植物生長調節劑對培植體的影響 ......................................... 60


圖3-7. 類原球體之植株再生 ............................. 61
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