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

參考文獻
1.阮育雄. 1997. 鳳梨花世界(六). 台灣花卉園藝 124：38-41。
2.李淑真 許圳塗. 1997.百香果(Passiflora edulis Sims)胚乳組織不定芽再生及三倍體植株建立. 中國園藝44:413-428 。
3.周邵蓮. 1989. 蜻蜓鳳梨的組織培養. 國立台灣大學研究所碩士論文. pp.35。
4.邱金春 林昭雄. 1987. 觀賞鳳梨組織培養繁殖之研究. 花卉生產改進研討會專集p.130-134. 桃園區農業改良場發 行。
5.邱金春. 1991. 蜻蜓鳳梨(Aechmea fasciata Baker)微體繁殖之研究. 中華農業研究40(3)：274-279。
6.林秀雄編撰. 1996. 民國八十六年台灣盆花調查報告. 台灣省政府農林廳編印. pp.76。
7.馬溯軒 許圳塗. 1988. 植物再生與繁殖及改良. 園藝作物組織培養之應用研討會專集. p.1-18. 國立台灣大學園藝 系. 台北。
8.馬溯軒 王淑娥. 1977. 鳳梨之組織培養繁殖. 中國園藝23: 107- 113。
9.馬溯軒. 1968. 鳳梨之切頂加速繁殖. 中國園藝14(1,2): 30-34。
10.蔡淑華著. 1973. 植物組織切片技術綱要. 茂昌股份有限公司. 台北. pp.72。
11.鍾仁彬 許圳塗. 1995.甘藍組織培養不定芽發生及再生模式. 中國園藝41:261-278。
12.DeWald, M.G.,G.A.Moore, W.B.Sherman, and M.H. Evans. 1988. Production of pineapple plants in vitro. Plant Cell Rep. 7:535-537.
13.Fitchet-Purnell, M. 1993. Maximum utilization of pineapple crowns for micropropagation. Acta Hort. 275:261-266.
14.Fitchet, M. 1990. Clonal propagation of queen and smooth cayenne pineapple. Acta Hort. 275:261-266.
15.Fleming,A.J.,Manded and I.Roth.1993.The patterns of gene expression in the tomato shoot apical meristem . Plant Cell. 5:297-309.
16.Gamborg,O.L.andG.C.Phillips.1995. Adventitious shoot proliferation. Plant Cell, Tissue and Organ Culture.p.55-65. Springer-Verlag, Berlin.
17.Hick,G.S.1980.Patterns of organ development in plant tissue culture and the problem of organ determination. Bot.Rev. 46:1-23.
18.Hick,G.S.1994.Shoot induction and organogenesis in vitro: A development prespective. In vitro Cell Dev.Biol. 30:10-15.
19.Hirimburegama, K.and L.P.J. Wijesinghe. 1992. In vitro growth ofAnanas comosus L. Merr (pineapple) shoot apices on different media. Acta Hort. 319:203-208.
20.Hosoki, T. and T. Asahira. 1980. In vitro propagation of Bromeliads in liquid culture. HortScience. 15(5)：603-604.
21.Huetteman A.C. and J.E. Preece. 1993. Thidiazuron: A potent cytokinin for woody plant tissue culture. Plant Cell, Tissue and Organ Culture. 33:105-119.
22.Jones, T. B. and T. Murashige. 1974. Tissue culture propagation of Aechmea fasciata, Baker and other Bromeliads. Proc. Inter. Plant Prop. Soc. 24：117-126.
23.Jollifffe, A. G. 1981. Propagation and cultural of Bromeliads. Proc. Intern. Plant Prop. Soc. 31：324-329.
24.Kiss, E., J.Kiss,G.Gyulai, and L.E.Heszky. 1995. A novel method for rapid micropropagation of pineapple. HortScience. 30:127-129.
25.Kukuczanka, K. and B. Czstka. 1989. Propagation of some species of the Bromeliaceae family cultured in vitro. Acta Hort. 251：167-172.
26.Mathew, V. H. and T. S. Rangan. 1979. Multiple plantlets in lateral bud and leaf explant in vitro cultures of pineapple. Sci. Hort. 11：319-328.
27.Mapes, M. O. 1973. Tissue culture of Bromeliads. Int. Plant Prop. Soc. 23：47-55.
28.Mekers, O. 1977. In vitro propagation of some Tillandsioideae(Bromeliaceae). Acta Hort. 78：311-320.
29.Mekers, O. 1984. In vitro culture techniques in a breeding program of Bromeliaceae. Eucarpia. Potplant Breeding. p.43-46.
30.Mekers , O. and J. G. Van Onsem. 1983. In vitro propagation of Vriesea cultivars in comparison with other ornamental Bromeliaceae. Acta Hort. 131：125-130.
31.Pierik, R .L. M. , H. H. M. Steegmans and J. Hendriks. 1984. The influence of naphthaleneacetic acid on the growth of in vitro cultivated seedlings of Bromeliaceae. Scientia Hort. 24：193-199.
32.Raman, K and R. I. Greyson. 1977. Graft unions between floral half meristems of different genotypes of Nigella damascena L. Plant Sci. Lett. 8: 367-373.
33.Skoog,F.and C.O.Miller.1965.Chemical regulation of growth and organ formation in plant tissue cultured in vitro. In :Bell,E. (ed) Molecular and Cellular Aspects of Development. Harper & Row. p481-494. New York.
34.Teng ,W.L. 1997. An alternative propagation method of Ananas through nodule culture. Plant Cell Rep. 16:454-457.
35.Van Dijck,R.,M. De Proft and J.De Greef. 1988. Role of ethylene and cytokinins in the initiation of lateral shoot growth in bromeliads. Plant Physiol. 86:836-840.
36.Zimmer, K. and W. Pieper. 1976. Methods and problems of clonal propagation of bromeliads in vitro. Acta Hort. 64： 25-29.