臺灣博碩士論文加值系統

觀賞鳳梨組織培養參試有Guzmania cv.Cheery、Vriesea cv.Margo及Guzmania cv.Carine等三品種。本試驗嘗試以不同的培植體與培養方式進行組織培養誘導不定芽，以期提高增殖速率且建立完整之再生系統，並有助於體外育種與生物技術應用之發展。
(一)短縮莖培養：以龍鳳擎天鳳梨(Guzmania cv.Cheery)及金馬鸚歌鳳梨(Vriesea cv.Margo)以3~8 cm長吸芽切除葉片後(約0.5-0.8 cm)將短縮莖分切(1/4或1/6 對切)培養可打破頂芽優勢，並增加不定芽數量。培植體接種兩個月後，由葉腋分化成排之芽原體(bud primordia)，之後漸漸轉綠，再經兩個月之後形成稍微抽長而叢生之芽體。以1/2 MS培養基添加 NAA 1.0 mg/L + TDZ 0.5 mg/L，可誘導多量不定芽，在龍鳳擎天鳳梨(Guzmania cv.Cheery)平均可誘導39.2個芽體，最大芽體平均為0.73 cm;金馬鸚歌鳳梨(Vriesea cv.Margo) 平均可誘導31.3個芽體，最大芽體平均為0.55 cm，效果最好。1/2 MS培養基添加 NAA 1.0 mg/L + BA 1.0 mg/L亦可誘導多量不定芽，在龍鳳擎天鳳梨(Guzmania cv.Cheery)，平均可誘導19.3個芽體，最大芽體平均為 0.89 cm;金馬鸚歌鳳梨(Vriesea cv.Margo)平均可誘導28.2個芽體，最大芽體平均為 0.57cm，效果次之。由於添加細胞分裂素，由短縮莖所誘導的不定芽形成明顯的細胞分裂素效應(cytokinin-effects)，芽體多叢生而不易抽長。以1/2MS培養基添加 0.5 ~ 1.0 mg/L GA促進芽體抽長的效果最明顯，一個月的時間約可增長一公分，有助於得到單一植株，移出瓶外。
(二)葉片培養：以國王擎天鳳梨(Guzmania cv. Carine) 為材料，剝取吸芽1.5-3.0 cm長的幼嫩葉片為培植體。置於MS培養基添加2,4-D 1.0 mg/L，由葉片基部可誘導逆分化淡黃色癒合組織，再將此癒合組織移至1/2MS培養基添加NAA 1.0 mg/L繼代培養，增生鬆散癒合組織且再分化不定芽。
(三)花器培養：龍鳳擎天鳳梨及金馬鸚歌鳳梨在花序上選取長2.5-3.0 cm 小花，分取花萼、花瓣、花藥、子房作為培植體。龍鳳擎天鳳梨小花瓣培養，誘導逆分化癒合組織可達50%，子房形成量次之。金馬鸚歌鳳梨花器培養，癒合組織則以花瓣、花藥形成較容易。以龍鳳擎天鳳梨之幼嫩花瓣置於MS培養基添加 2,4-D 0.5 mg/L，可誘導逆分化淡黃色癒合組織，再將此癒合組織繼代培養於1/2MS培養基添加NAA 1.0 mg/L，增生鬆散癒合組織且再分化大量不定芽。取花瓣經癒合組織所誘導之間接不定芽1~2 cm大小，以1/2MS添加BA 1.0 mg/L配方液體培養20天可改善細瘦的形態，健化芽體。
(四)次生培植體增殖 :(1)葉片液體培養:以1/2MS添加 NAA 0.1 mg/L+ BA 0.1 mg/L效果最好，約兩星期就可看見葉片基部長出芽體，每棵誘導的芽體數平均為4.33個，主要是由發育較大的葉片所長出。(2)小芽體液體培養:約經30天可由植株基部增生側芽，以1/2 MS添加 BA 1.0mg/L較優，芽體數較多，生長勢良好，1/2 MS 添加NAA 0.1 mg/L + BA 0.1 mg/L效果次之。(3)叢生芽之誘導:以固體培養1/2 MS添加NAA 0.1 mg/L + BA 0.1 mg/L誘導，芽體較能保持正常生長。

The ornamental Bromeliads(Bromeliaceae ) included Guzmania cv. Cheery、cv. Carine and Vriesea cv.Margo were collected for study of micropropagation via caulogenesis.
1. Shoot tip culture : The young suckers(3 ~ 8 cm) required intensive pre-sterilization. The compressed shoot tip(0.5 - 0.8 cm) splitted into 4-6 sections as explant benefit for apical dominance breaking and increase the number of adventitious buds. Numerously adventitious bud appeared in the leaf axil and gradually turn to green two months after culture. Two more months later, the bud become slightly longer and continuous to proliferate new bud .The regeneration medium used 1/2 MS supplemented with NAA 1.0 mg/L + TDZ 0.5 mg/L, showed high efficiency in stimulation of caulogenesis. In Guzmania cv.Cheery, achieved 39.2 buds per explant, the largest bud size was 0.73 cm. In Vriesea cv.Margo, formed 31.3 buds, with the largest budding size on average being 0.55 cm. The combination of NAA 1.0 mg/L and BA 1.0 mg/L induced 19.3 and 28.2 buds In"Cheery"and "Margo"respectively. The cytokinins contributed to massive bud formation or proliferation, but the clustered bud sprouting was restricted. The extended growth and rooting could be approached by transferring into 1/2 MS medium added GA 0.5~1.0 mg/L and NAA 0.5 mg/L. The anatomical study exhibited that the adventitious buds were directly initiated from the epidermal and subepidermal tissue of the elongating axil , and capable to proliferate from the meristematic surface tissue.
2. Leaf culture : The young leaves 1.5 - 3.0 cm in length of suckers were excised as explant. Which were inoculated on MS medium contained 2,4-D 1.0 mg/L,for de-differentiation of pale-yellow callus from leaf bases. The frequency of callogenesis was about 5-10%. The condition of 1/2MS medium with NAA 1.0 mg/L efficiently showed to friable callus, and to re-differentiate adventitious buds.
3. Floral tissue culture : The florets 2.5 - 3.0 cm in length of Guzmania cv.Cheery and Vriesea cv.Margo were excised as explant and inoculated on dedifferentiation medium. It was demonstrated that the callogenic capability was variable among different floret parts. More than 50% callus were achieved in petal and ovary explants of Guzmania cv.Cheery.
The floral tissues in petals and anther of the Vriesea cv.Margo, showed more easily to induce callogenesis. The desirable floral parts inoculated on MS medium supplemented with 2,4-D 0.5 mg/L formed pale-yellow callus. Take these callus into 1/2 MS medium added with NAA 1.0 mg/L, oncomitantly stimulated friable callus proliferation and numerously adventitious buds regeneration. Subsequently, the advance growth and multiplication of the petal callus-derived buds could be improved under 1/2 MS liquid medium supplemented with BA 1.0 mg/L condition for 20 days. The rooting could be approached by transferring into 1/2 MS medium contained NAA 0.1mg/L after solid culture.
4. Secondary explant culture : (1) Leaf-bud liquid culture: The outer leaf-buds were excised from microshoot, and transferring into 1/2 MS liquid medium supplemented with NAA 0.1 mg/L + BA 0.1 mg/L, on shaker 110 rpm. The axillary bud was sprouting from leaf basal by 3 weeks after culture. The average 4.3 neomicroshoots achieved from each microshoot. (2)Microshoot liquid culture: The petal-derived adventitious buds were excised and inoculated 1/2 MS liquid medium contained BA 1.0 mg/L which proliferated 3-4 shoots from each microshoot by 4 weeks after culture.(3) Microshoot solid-medium culture: The petal-derived adventitious buds were transferring into 1/2 MS solid medium supplemented with NAA( 0.1-1.0 mg/L) and cytokinin( 0.1-0.5 mg/L). It was demonstrated that the combination of NAA 0.1 mg/L and BA 0.1 mg/L induced 3-5 lateral buds sprouting and appeared normal growth.

一、前言………………………………………………………….1
二、前人研究………………………………………………………3
(一)無菌播種………………………………………………………3
(二)短縮莖培養……………………………………………………3
(三)葉片培養………………………………………………………5
(四)花器培養………………………………………………………5
三、材料與方法……………………………………………………7
(一)參試材料………………………………………………………7
(二)取材與消毒……………………………………………………7
1、吸芽之短縮莖…………………………………………………7
2、幼葉…………………………………………………………7
3、花器……………………………………………………………8
(三)培養基之調配…………………………………………………8
(四)試驗內容………………………………………………………9
1、直接不定芽之誘導…………………………………………9
(1)短縮莖不定芽之誘導……………………………………9
(2)芽體抽長………………………………………………… 9
2、間接不定芽之誘導……………………………………………10
(1)葉片培養…………………………………………………10
a、癒合組織之誘導………………………………………10
b、不定芽的誘導…………………………………………10
(2)花器培養…………………………………………………11
a、癒合組織之誘導………………………………………11
b、不定芽的誘導………………………………………11
(3)芽體健化…………………………………………………11
3、胚性癒合組織之誘導………………………………………12
4、次生培植體增殖……………………………………………12
(1)葉片液體培養…………………………………………12
(2)小芽體液體培養…………………………………………12
(3)叢生芽之誘導…………………………………………… 13
5、小植株建立……………………………………………………13
(1)瓶內發根…………………………………………………13
(2)瓶外發根…………………………………………………13
(3)瓶苗健化及移植…………………………………………14
(五)形態發生石蠟切片法觀察……………………………………14
四、結果……………………………………………………………15
(一)直接不定芽的誘導…………………………………………… 15
1、短縮莖不定芽發生…………………………………………15
2、芽體抽長……………………………………………………18
(二)葉片及花器培養間接不定芽之誘導………………………… 18
1、葉片培養……………………………………………………18
(1)癒合組織的誘導……………………………………… 18
(2)不定芽的誘導………………………………………… 20
2、花器培養……………………………………………………21
(1)癒合組織的誘導……………………………………… 21
(2)花瓣衍生癒合組織不定芽的誘導…………………… 24
(3)子房逆分化之癒合組織誘導再分化之情形………… 25
(4)芽體健化……………………………………………… 26
(三)次生培植體增殖……………………………………………… 26
1、葉片液體培養再生…………………………………………26
2、單芽液體培養增生…………………………………………27
3、叢生芽之誘導………………………………………………27
(四)小植株建立 ……………………………………………………28
1、瓶內發根……………………………………………………28
2、瓶外發根……………………………………………………28
五、討論………………………………………………………………48
(一)短縮莖培養之影響因子……………………………………… 48
(二)葉片培養之再生……………………………………………… 51
(三）花器培養之探討………………………………………………52
1、誘導逆分化癒合組織之影響因子…………………………53
2、癒合組織誘導再分化不定芽之影響因子…………………54
3、芽體健化之探討……………………………………………55
(四)次生培植體增殖之探討……………………………………… 56
六、摘要…………………………………………………………… 58
中文摘要………………………………………………………… 58
英文摘要………………………………………………………… 60
七、參考文獻……………………………………………………… 62

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