臺灣博碩士論文加值系統

本研究以巨獸鹿角蕨(Platycerium grande (Fee) Kunze)及長葉鹿角蕨(P. willinckii T. Moore.)孢子體幼葉誘導綠球體(green globular body; GGB)，再將其以均質化(homogenization)處理後進行幼孢子體的大量再生。將兩種鹿角蕨幼葉切成上半葉(遠軸端、葉尖端)及下半葉(近軸端、葉基部)，在添加5.37µM α-naphthaleneacetic acid (NAA)且濃度減半的Murashige and Skoog (MS)培養基中誘導GGB發生，以下半葉誘導效果明顯優於上半葉，其誘導率分別為27.12±3.7% (巨獸鹿角蕨)及58.3±5.8% (長葉鹿角蕨)。巨獸鹿角蕨GGB在增殖時，其速率隨NAA濃度提升而加快，但隨6-benzylaminopurine (BAP)濃度增加而遭抑制，孢子體幼葉之萌發在5.37µM NAA及3.33µM BAP組合中受明顯抑制，故此條件可用於維持GGB穩定增殖，而在2.69 µM NAA及4.44 µM BAP的組合中則有最佳的鮮重增加(× 5倍)。長葉鹿角蕨在培養基中NAA濃度增加時，GGB增殖明顯減緩，而增加BAP則無此效應，但同樣會抑制孢子體幼葉萌發，保持GGB增殖。GGB均質化後以每培養皿0.025g的接種量塗佈於添加不同濃度活性碳(activated charcoal; AC)的培養基中，產生幼孢子體數優於不添加AC之對照組。巨獸鹿角蕨在活性碳濃度0.5%時有最佳產出(264.3±28.3株/皿)，而長葉鹿角蕨則是在0.75%時最佳(129.1±10.8株/皿)。前者在混合介質(泥碳土:珍珠石:椰纖＝1:1:1)中有94%馴化存活率，而後者在受測各介質中皆能順利存活。

In this study, a mass in vitro propagation method for Platycerium grande (Fee) Kunze (Pg) and P. willinckii T. Moore. (Pw) was developed based on using juvenile leaves as explants to induce green globular body (GGB) followed by tissue homogenization to regenerate young sporophytes. The explants were first excised into distal and proximal half parts, and incubated on the half-strength Murashige and Skoog (MS) medium supplemented with 5.37 μM α-naphthaleneacetic acid (NAA) for GGB induction. The proximal half leaves were more response to the treatment giving a mean induction percentage of 27.12 ± 3.7% (Pg) and 58.3 ± 5.8% (Pw), respectively. The GGB proliferation in Pg was enhanced by increasing NAA concentration, but inhibited by 6-benzylaminopurine (BAP). The developmentof juvenile leaves of sporophyte was obviously depressed on medium containing 5.37μM NAA and 3.33μM BAP creating a stable phase for GGB proliferation. The proliferation rate (fresh weight increase) was best obtained on medium supplemented with 2.69 μM NAA and 4.44 μM BAP (× 5 fold). However, the GGB proliferation in Pw was highly inhibited by increasing NAA concentration, but not seen in higher BAP levels which also depressed the formation of juvenile leaves. Homogenized GGB tissue inoculated by 0.025 g per Petri dish on medium containing various levels of activated charcoal (AC) regenerated more sporophytes than those cultured on AC-free medium. The best sporophyte regeneration in Pg was at 0.5% AC (264.3 ± 28.3/dish) and 0.75% AC as for Pw (129.1 ± 10.8/dish). The former performed a 94% survival in a mixed medium (peat : perlite : coir = 1 : 1 : 1) during acclimatization and the latter achieved high survival in all tested media.

Ⅰ、前言----------------------------------------1
Ⅱ、前人研究
(Ⅰ)研究物種之介紹
1.巨獸鹿角蕨(P. grande)---------------------6
2.長葉鹿角蕨(P. willinckii)-----------------7
(Ⅱ)鹿角蕨傳統繁殖方式
1.孢子繁殖----------------------------------7
2.分株繁殖----------------------------------8
(Ⅲ)組織培養方式
1.不同器官的不定芽誘導------------------------8
2.癒傷組織 -----------------------------11
3.配子體-----------------------------------12
4.GGB-------------------------------------12
(Ⅳ)植株發根----------------------------------14
(Ⅴ)馴化流程----------------------------------16
Ⅲ、材料與方法
(Ⅰ)試驗材料-----------------------------------18
(Ⅱ)孢子表面殺菌處理----------------------------18
(Ⅲ)GGB誘導試驗 -----------------------------20
(Ⅳ)GGB形態觀察與組織石蠟切片--------------------20
(Ⅴ)GGB增殖試驗 -----------------------------22
(Ⅵ)均質化GGB接種試驗--------------------------23
(Ⅶ)馴化試驗----------------------------------25
(Ⅷ)培養條件----------------------------------25
(Ⅸ)資料處理與分析-----------------------------27
IV、結果
(Ⅰ)不同部位葉培殖體誘導GGB的差異------------------28
(Ⅱ) GGB的增殖情形-----------------------------32
(Ⅲ) GGB形態之石蠟切片觀察----------------------37
(Ⅳ)均質化GGB的幼孢子體再生試驗------------------40
(Ⅴ)馴化試驗-----------------------------------42
Ⅴ、討論----------------------------------------46
VI、參考文獻------------------------------------53

王承義、王穎 (2000) 鹿角蕨組織培養。中國林副特產3: 23-24。
王基成 (2015) 以綠球體組織培養繁殖筆筒樹。國立嘉義大學森林暨自然資源研究所碩士論文，63頁。
王清羿 (2006) 鹿角蕨的栽培和管理。北方園藝 1: 97。
王衛清、成曉、焦瑜 (2011) 鹿角蕨(鹿角蕨科)配子體發育及其瀕危機制探討。雲南農業大學學報 26: 293-297。
司亮、尤海波 (2009) 波斯頓蕨離體培養技術的研究。中國農學通報 25: 181-183。
全中和 (1999) 台灣山蘇花葉原體組織培養之研究。花蓮區農業改良場研究彙報 17: 53-62。
向燕、王皓、溫遠影 (2002) 鹿角蕨化學成份的研究。熱帶亞熱帶植物學報 10: 69-73。
何陽修 (2002) 鹿角蕨的種類及栽培。台灣花卉園藝 12: 40-46。
林證明、陳曄、韋麗娜、鍾音 (1998) 二叉鹿角蕨商品化育苗技術研究。江蘇林業科技25: 177-180。
姜長陽、寧淑香、于淼、王宇、宋立秀 (2003) 蕨菜癒傷組織高效再生體系的建立。園藝學報 30: 343-345。
徐豔 (2006) 觀賞蕨類植物種質超低溫保存研究。北京林業大學研究生院博士論文，166頁。
徐豔、石雷、劉燕、李東 (2004) 大葉黑桫欏孢子的無菌培養。植物生理學通訊40: 72。
張光飛、蘇文華 (2002) 水鱉蕨幼葉的離體快速繁殖。雲南大學學報24: 234-236。
莊孟晉 (1986) 八角蓮與鹿角蕨的組織培養。臺灣大學園藝研究所碩士論文，73頁。
許雯華 (2011) 深綠鹿角蕨和圓盾鹿角蕨組培苗之根培殖體再生現象。國立宜蘭大學園藝研究所碩士論文，59頁。
陳季呈 (2008) 利用組織培養技術繁殖波斯頓腎蕨種苗。花蓮區農業專訊66: 18-21。
陳俊銘 (2011) 18種鹿角蕨大揭密。18-21頁。張淑貞(編輯) 花草遊戲。城邦文化事業股份有限公司，台北市。119頁。
黃執纓 (2004) 二岐鹿角蕨組織培養的研究。生物學雜誌 21: 22-24。
黃智明 (1994) 名貴觀葉鹿角蕨。廣東園林 4: 26-28。
黃學林、李筱菊 (2000) 植物體細胞胚胎發生。科學農業 48: 120-127。
葉曉青、余建明、鄭衍明、童紅玉 (2012) 不同類型細胞分裂素對扇蕨不定芽誘導和植株再生的影響。江蘇農業學報8: 172-175。
葛佳、路程、伍丹丹、張光飛、蘇文華 (2010) 扇蕨綠色小球體的誘導和植株再生。北方園藝 1: 159-162。
趙秀芳 (2005) 蕨類植物的孢子繁殖技術。中國種業 2: 42-43。
劉黃碧圓 (1989) 不同培養基配方對糜角蕨幼苗葉片生長之影響。嘉義農專學報 20: 171-188。
蔣盛軍、宋希強、王勝培、劉慶昌 (2002) 蕨類植物組織培養研究進展。園藝學報 29: 651-656。
盧學甫 (2014) 吸附劑對馬來鹿角蕨均質化綠球體再生孢子體之影響。國立嘉義大學森林暨自然資源研究所碩士論文，68頁。
Agbo, M. O., C. O. Nnadi, N. N. Ukwueze and F. B. C. Okoye (2014) Phenolic constituents from Platycerium bifurcatum and their antioxidatant properties. Journal of Natural Products 7: 48-57.
Amaki, W. and H. Higuchi (1991) A possible propagation system of Nephrolepis, Asplenium, Pteris, Adiantum and Rumohra (Arachniodes) through tissue culture. Acta Horticulturae 300: 237-243.
Amaki, W. and H. Higuchi (1992) Micropropagation of Boston ferns (Nephrolepis species). pp.484-494. In Y. S. P. Bajaj, ed. Biotechnology in Agriculture and Forestry 20: High-Tech and Micropropagation IV. Springer-Verlag, Berlin. 497pp.
Ambrozic-Dolinsek, J. (1998) Regeneration studies of the fern Platycerium bifurcatum (Cav.) C. Chr. in tissue culture. Mater’s Degree Thesis, Department of Biotechnology and Systems Biology, University of Ljubljana, Ljubljana, Slovenia. 100pp.
Ambrozic-Dolinsek, J. and M. Camloh (1997) Gametophytic and sporophytic regeneration from bud scales of the fern Platycerium bifurcatum (Cav.) C. Chr. in vitro. Annals of Botany 80: 23-28.
Ambrozic-Dolinsek, J., M. Camloh and J. Zel (1999) Plant regeneration from scales of the fern Platycerium bifurcatum (Cav.) C. Chr. Phyton: Annales Rei Botanicae 39: 297-300.
Amoroso, C. B. and V. B. Amoroso (2003) Plantlet production of the Philippine Giant Staghorn fern (Platycerium grande (Fee) C. Presl) through spore culture. pp.491-495. In S. Chandra and M. Srivastava, eds. Pteridology in the New Millennium: NBRI Golden Jubilee Volume. Kluwer Academic Publishers, Dordrecht. 544pp.
Avila-Perez, M. C., L. White-Olascoaga and A. M. Arzate-Fernandez (2011) In vitro regeneration of leatherleaf fern (Rumohra adiantiformis (G. Forst.) Ching). American Fern Journal 101: 25-35.
Bertrand, A. M., M. A. Albuerne, H. Fernandez, A. Gonzalez and R. Sanchez-Tames (1999) In vitro organogenesis of Polypodium cambricum. Plant Cell, Tissue and Organ Culture 57: 65-69.
Birmeta, G. and M. Welander (2004) Efficient micropropagation of Ensete ventricosum applying meristem wounding: a three-step protocol. Plant Cell Reports 23: 277-283.
Bode, S. O. and O. O. Oyedapo (2011) Biological activities and phytoconstituents of the lower plant Platycerium angolense, Welwex Hook. Journal of Medicinal Plants Research 5(8): 1321-1329.
Camloh, M. and N. Gogala (1991) Platycerium bifurcatum adventitious bud and root formation without growth regulators in vitro. Acta Horticulturae 289: 89-90.
Camloh, M., N. Gogala and J. Rode (1994) Plant regeneration from leaf explants of the fern Platycerium bifurcatum in vitro. Scientia Horticulturae 56: 257-266.
Chiou, W.-L., D. R. Farra and T. A. Ranker (1998) Gametophyte morphology and reproductive biology in Elaphoglossum schott. Canadian Journal of Botany. 76(11): 1967-1977.
Chiou, W.-L., P.-H. Lee and S.-S. Ying (2000) Reproductive biology of gametophytes of Cyathea podophylla (Hook.) Copel. Taiwan Journal of Forest Science 15: 1-12.
Constantin, M. J., R. R. Henke and M. A. Mansur (1977) Effect of activated charcoal on callus growth and shoot organogenesis in tobacco. In Vitro Cellular and Developmental Biology – Plant 13: 293-296.
Cooke, R. C. (1979) Homogenization as an aid in tissue culture propagation of Platycerium and Davallia. HortScience 14: 21-22.
Dykeman, B. W. and B. G. Cumming (1985) In vitro propagation of the ostrich fern (Matteuccia struthiopteris). Canadian Journal of Plant Science 65: 1025-1032.
Ebert, A., F. Taylor and J. Blake (1993) Changes of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal. Plant Cell, Tissue and Organ Culture 33: 157-162.
Espinosa-Matias, S., B. Perez-Garcia, A. C. Mendoza-Ruiz and L. D. Gomez-Pignataro (2007) Gametophyte morphology of Platycerium andinum Baker and Platycerium wandae Racif. Acta Microscopica 16:176-177.
Fernandez, H., A. M. Bertrand and R. Sachez-Tames (1996) Micropropagation and phase change in Blechnum spicant and Pteris ensiformis. Plant Cell, Tissue and Organ Culture 44: 261-265.
Fernandez, H., A. Bertrand and R. Sanchez-Tames (1997) Plantlet regeneration in Asplenium nidus L. and Pteris ensiformis L. by homogenization of BA treated rhizomes. Scientia Horticulturae 68: 243-247.
Fridborg, G., M. Pedersen, L. Landstrom and T. Eriksson (1978) The effect of activated charcoal on tissue cultures: adsorption of metabolites inhibiting morphogenesis. Physiol Plant 43: 104-106.
Gallo-Meagher, M. and J. Green (2002) Somatic embryogenesis and plant regeneration from immature embryos of saw palmetto, an important landscape and medicinal plant. Plant Cell, Tissue and Organ Culture 68: 253-256.
Haccius, B. (1978) Question of unicellular origin of nonzygotic embryos in callus cultures. Phytomorphology 28: 74-81.
Hagiabad, M. S., Y. Hamidoghli and R. F. Gazvini (2007) Effects of different concentrations of mineral salt, sucrose and benzyladenine on Boston fern (Neprolepis exaltata Schott cv. Bostoniensis) runner tips initiation. Journal of Science and Technology of Agriculture and Natural Resources 11: 137-146.
Hennen, G. R. and T. J. Sheehan (1978) In vitro propagation of Platycerium stemaria (Beauvois) Desv. Horticultural Science 13: 245.
Hickok, L. G., T. R. Warne and R. S. Fribourg (1995) The biology of the fern Ceratopteris and its use as a model system. International Journal of Plant Sciences 156: 332-345.
Higuchi, H. and W. Amaki (1989) Effects of 6-benzylaminopurineon the organogenesis of Asplenium nidus L. through in vitro propagation. Scientia Horticulturae 37: 351-359.
Higuchi, H., W. Amaki and S. Suzuki (1987) In vitro propagation of Nephrolepis cordifolia Prsel. Scientia Horticulturae 32: 105-113.
Hoshizaki, B. J. (1972) Morphology and phylogeny of Platycerium species. Biotropica 4: 93-117.
Hoshizaki, B. J. (1975) A staghorn fern (Platycerium) hybrid. American Fern Journal 65: 99-101.
Hoshizaki, B. J. and M. C. Price (1990) Platycerium update. American Fern Journal 80: 53-69.
Jambor-Benczur, E., A. Csillag, A. Marta-Riffer and G. Csizmadia (1995) In vitro regeneration and propagation of Platycerium bifurcatum. Acta Agronomica Hungarica 43: 59-66.
Kunitake, H., T. Nakashima, K. Mori, M. Tanaka and M. Mii (1995) Plant regeneration from mesophyll protoplasts of lisianthus (Eustoma grandiflorum) by adding activated charcoal into protoplast culture medium. Plant Cell, Tissue and Organ Culture 43:59-65.
Kwa, S. H., Wee and P. P. Kumar (1995) Role of ethylene in the production of sporophytes from Platycerium coronarium (Koenig) Desv. Frond and rhizome pieces cultured in vitro. Journal of Plant Growth Regulation 14: 183-189.
Kwa, S. H., Y. C. Wee, T. M. Lim and P. P. Kumar (1997) Morphogenetic plasticity of callus reinitiated from cell suspension cultures of the fern Platycerium coronarium. Plant Cell, Tissue and Organ Culture 48: 37-44.
Liao, Y. K. and Y. H. Wu (2011) In vitro propagation of Platycerium bifurcatum (Cav.) C. Chr. via green globular body initiation. Botanical Studies 52: 455-463.
Lin, X.-F., W.-B. Zhang, H. Takano, S. Takio and K. Ono (2004) Efficient plant regeneration and micropropagation from callus derived from mature zygotic embryos of Larix gmelinii. Plant Technology 21: 159-163.
Linington, I. M. (1991) In vitro propagation of Dipterocarpus intricatus. Plant Cell, Tissue and Organ Culture 27: 81-88.
Linsmaier E. M. and F. Skoog (1965) Organic growth factor requirements of tobacco tissue cultures. Physiologia Plantarum 18: 100-127.
Loescher, W. H. and C. N. Albrecht (1979) Development in vitro of Nephrolepis exaltata cv. Bostoniensis runner tissues. Physiologia Plantarum 47: 250-254.
Martin, K. P., S. Sini, C.-L. Zhang, A. Slater and P. V. Madhusoodanan (2006) Efficient induction of apospory and apogamy in vitro in silver fern (Pityrogramma calomelanos L.). Plant Cell Reports 25: 1300-1307.
Murashige, T. and F. Skoog (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473-479.
Pence, V. C. (2015) Propagation and cryopreservation of Asplenium scolopendrium var. americanum, the American hart’s tongue fern. American Fern Journal 105: 211-225.
Pevalek-Kozlina, B. (1996) Effects of sucrose and agar concentration, and medium pH on staghorn fern (Platycerium bifurcatum (Cav.) C. Chr.) shoot multiplication. Horticultural Science 28:18-20.
Porembski, S. and N. Biedinger (2000) Epiphytic ferns for sale: influence of commercial plant collection on the frequency of Platycerium stemaria (Polypodiaceae) in coconut plantations on the Southeastern Ivory coast. Plant Biology 3: 72-76.
Richards, J. H., J. Z. Beck and A. M. Hirsch (1983) Structural investigations of asexual reproduction in Nephrolepis exaltata and Platycerium bifurcatum. American Journal of Botany 70(7): 993-1001.
Rodpradit, S., P. Thavipoke and K. Thammasiri (2003) Cryopreservation of young Platycerium ridleyi H. Christ. sporophytes by encapsulation/vitrification technique. pp.71-73. In: 29th Congress on Science & Technology of Thailand. The Science Society of Thailand under the Patronage of His Majesty the King. Khon Kean. 202pp.
Shukla, P. S. and P. B. Khare (2012) In vitro mass multiplication of a threatened tree fern, Cyathea spinulosa Wall. ex Hook. International Journal of Genetic Engineering and Biotechnology 3: 15-23.
Smith, R. H. (2013) Plant Tissue Culture. 3rd ed. Elsevier, Oxford. 188pp.
Soniya, E. V. and M. Sujitha (2006) An efficient in vitro propagation of Aristolochia indica. Biologia Plantarum 50: 272-274.
Stojicic, D. and S. Budimir (2004) Cytokinin mediated axillary shoot formation in Pinus heldreichii. Biologia Plantarum 49: 477-479.
Teng, W.-L. and M.-C. Teng (1997) In vitro regeneration patterns of Platycerium bifurcatum leaf cell suspension culture. Plant Cell Reports 16: 820-824.
Teng, W.-L. and M.-C. Teng (2000) The impact of a pulse treatment of penicillin-G and streptomycin sulphate on sporophyte regeneration of Platycerium bifurcatum. Plant Cell Reports 19: 345-350.
Thakur, R. C., Y. Hosoi and K. Ishii (1998) Rapid in vitro propagation of Matteuccia struthiopteris (L.) Todaro - an edible fern. Plant Cell Reports 18: 203-208.
Thentz, M. and C. Moncousin (1984) Micropropagation in vitro de Platycerium bifurcatum (Cav.) C. Chr. Rev. Horticole Suisse 57: 293-297.
Weatherhead, M. A., J. Burdon and G. G. Henshaw (1978) Some effect of activated charcoal as an additive to plant tissue culture media. Zeitschrift fur Pflanzenphysiologie 89: 141-147.
Wee, Y. C., S. H. Kwa and C. S. Loh (1992) Production of sporophytes from Platycerium coronarium and P. ridleyi frond strips and rhizome pieces cultured in vitro. American Journal of Botany 82: 75-79.
White, P. R. (1963) The cultivation of Animal & Plant Cells. 2nd ed. Ronald Press, New York. 515pp.
Winarto, b. and J. A. Teixeira da Silva (2012) Improved micropropagation protocol for leatherleaf fern (Rumohra adiantiformis) using rhizomes as donor explant. Scientia Horticulturae. 140: 74-80.
Zamora, P. M. and L. Co (1986) Guide to Philippine Flora and Fauna. Vol. II. Natural Resources Management Center and University of the Philippines, Quezon City, Philippines. 273pp.
Ziv, M. and A. Hader (1991) Morphogenic pattern of Nephrolepis exaltata cv. Bostoniensis in agar or liquid cultures: implications for micropropagation. Israel Journal of Botany 40: 7-16.