彩虹鳥蕉組織培養之研究

Abstract

赫蕉屬(Heliconia)植物目前被世界各國收集培育供觀苞或觀葉用的品種約在200種以上，其直立型或懸垂型花序具有多彩豔麗的苞片及如船形的構造。Heliconia psittacorum L.f.和雜交品種如‘Golden Torch’即因其鮮豔的花朵、直立且光滑的花莖及幾乎全年開花等特性，成為商業化栽培的主要切花用品種。本文以彩虹鳥蕉(Heliconia psittacorum cv. Rhizomatosa)為材料，探討利用組織培養大量繁殖種苗的可行性及檢定造成培植體污染之細菌種類，並尋求降低高污染率的方法。結果分述如下： 1. 使用Biolog GN/GP Microplate Identification System在赫蕉培植體內檢定出農桿菌屬細菌一種(Agrobacterium tumefaciens)、假單胞菌屬細菌二種(Pseudomonas diminuta, P. putida)、賽氏桿菌屬細菌一種(Serratia marcescens)、單胞菌屬細菌一種(Xanthomonas campestris)以及Sphingomonas pancimobilis。 2. 經0.5％次氯酸鈉溶液外部消毒15分鐘之根莖新芽培植體，在無菌操作檯上切除2-3片外覆鱗片葉後，再以相同濃度次氯酸鈉溶液消毒5分鐘，能顯著降低細菌污染。葉片培植體則以相同的方法外部消毒後，縱切並除去部份主葉脈，再二次消毒5分鐘，也能降低細菌污染。 3. 取自溫室無土介質[發泡煉石：樹皮碎塊(1：1 v/v) ]栽培區之培植體，受細菌污染比率顯著低於取自田間或溫室土壤栽培區者。 4. 子房和莖頂培植體先在已添加500 mg/l cefotaxime液體培養基中預處理3天，再繼代培養於固體培養基，能有效降低細菌污染且對培植體無毒害作用。若將子房浸漬於含100 mg/l streptomycin ＋ 50 mg/l gentamycin之液體培養基2天，再以固體培養基繼代培養，也可明顯降低細菌污染且不影響癒合組織誘導率。 5. 培植體在外部消毒前先以40.2-45.2℃熱水處理1小時，對降低細菌污染的效果不顯著。 6. 子房和葉片培植體接種於含億力殺菌劑1000 mg/l之培養基，對細菌污染率無明顯降低作用。 7. 子房及葉片培植體接種於含WPM基本鹽類及添加2 mg/l 2,4-D＋2 mg/l kinetin的培養基中，其癒合組織誘導率較使用MS或B-5基本鹽類培養基者高。但莖頂培植體則較適合接種於含MS基本鹽類及添加2 mg/l BA＋0.5 mg/l NAA的培養基。 8. 赫蕉花序抽苔第14-26天，花苞完全開展且花瓣開始脫落時期採取之子房培植體，培養後有較高癒合組織誘導率，且褐化死亡率也較低。此外，自植株頂端抽出4-8天且已達初展時期採取之葉片培植體，培養後也有較高癒合組織誘導率。 9. 莖頂培植體培養於含4-8 mg/l BA、0.5 mg/l NAA或含0.01 mg/l TDZ固體培養基45天後，再繼代於含相同組成分之液體培養基45天，其莖節生長情形優於全程使用液體或固體培養基者。

The genus Heliconia L. is represented by more than 200 species of tropical plants that constitute the family Heliconiaceae. The heliconia inflorescence is a colorful, multi-bracted structure which may be upright or pendent. Heliconia psittacorum L.f. and some of its hybrids (e.g., ‘Golden Torch') are particularly promising because of their attractive flowers, long straight clean peduncles, prolific year round flower production, and show considerable potential as commercial cut flower. The main objectives of the present investigation were- (1) to standardize a method of in vitro propagation of Heliconia psittacorum cv. Rhizomatosa by culturing terminal and axillary shoot tips of rhizomes; (2) to induce callus in leaf, ovary and shoot tip explants for its intended use in mutation breeding; (3) to identify and control bacterial contamination in explants cultured in vitro. The results are summarized as follows: 1. Six strains of bacteria were isolated and characterized from in vitro cultured explants of H. psittacorum cv. Rhizomatosa. All of them were Gram negative, and identified as Agrobacterium radiobacter, Pseudomonas diminuta, Pseudomonas putida, Serratia marcescens, Sphingomonas pancimobilis and Xanthomonas campestris. 2. Lower contamination rates were obtained in leaf and shoot tip explants when surface sterilized by a two-step method. In the first step, the explants were sterilized with 0.5％ sodium hypochlorite for 15 min outside the laminar flow. In the second step, before resterilization in the laminar flow, the midrib of the leaf explants and three leaves covering the shoot tip explants were removed and then sterilized with the same concentration of sodium hypochlorite used in the first step, for five minutes. The explants were washed 3-4 times with sterile distilled water after each sterilization step. 3. A lower rate of contamination was obtained in explants collected from donar plants grown in the medium without soil [HydropelletsR:tree bark (1:1 v/v) ] in the greenhouse. 4. Lower contamination rates were obtained in overy and shoot tip explants pretreated with liquid MS medium containing either 500 mg/l cefotaxime for 3 days or 100 mg/l streptomycin+50 mg/l gentamycin for 2 days. Pretreatment of explants with antibiotics did not inhibit either callus formation in leaf explants or growth of shoot tip explants. 5. Pre-treatment of explants (leaf, ovary, and shoot tip) with hot water (40.2-45.2℃) did not help in controlling contamination in in vitro cultures. 6. Addition of BenlateR, an antifungal agent, in the culture medium was not useful in controlling contamination in leaf and ovary explants. 7. WPM basal medium supplemented with 2 mg/l 2,4-D＋2 mg/l kinetin supported highest ﹪induction of callus in leaf and ovary explants compared to explants cultured on MS and B-5 medium. However, MS medium supplemented with 2 mg/l BA＋0.5 mg/l NAA supported growth of shoot tip explants. 8. Age of the explants (ovary and leaf) was found to be critical in callus formation. The ovary explants collected from 14-26 days old inflorescence produced maximum callus without necrosis or browning when cultured on WPM medium supplemented with 2 mg/l 2,4-D+2 mg/l kinetin. Higher ﹪induction of callus with low ﹪browning was observed in leaf explants taken after 4-8 days of emergence from the shoot tip. 9. Well developed plantlets of Heliconia psittacorum cv. Rhizomatosa were successfully obtained after 3 months of culture of shoot tip explants on MS medium supplemented with 4-8 mg/l BA 0.5 mg/l NAA. Addition of 0.01 mg/l TDZ in the medium was found to be benficial for rapid growth of plantlets from shoot tip explants.