透氣處理對香蕉組織培養苗生長、馴化與氣孔發育之影響

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(1)台灣農業研究 (J. Taiwan Agric. Res.) 64(4):243–252 (2015) DOI: 10.6156/JTAR/2015.06404.01. 研究報告. 透氣處理對香蕉組織培養苗生長、馴化與氣孔發育之影響陳涵葳 1 吳明哲 2 陳威臣 1,* 摘要陳涵葳、吳明哲、陳威臣。2015。透氣處理對香蕉組織培養苗生長、馴化與氣孔發育之影響。台灣農業研究 64(4):243–252。香蕉組培苗需要馴化後才能適應外界環境。在本試驗中組培瓶封口方式改以不織布材質取代鋁箔，以增加瓶內頂部空間的透氣效率，4 種透氣處理分別為：不透氣 6 wk (hermetically sealed for 6 weeks; S6V0)、不透氣 4 wk 後透氣 2 wk (4 weeks sealed followed by 2 weeks ventilation; S4V2)、不透氣 2 wk 後透氣 4 wk (2 weeks sealed followed by 4 weeks ventilation; S2V4) 及透氣 6 wk (ventilation for 6 weeks; S0V6)。在 S4V2、S2V4 與 S0V6 處理下，瓶內乙烯累積低於偵測極限，而完全密封之 S6V0 處理可測得高濃度乙烯累積 (203.3 nL L-1)，且 S6V0 處理之小植株具有較多的不定根。S2V4 與 S0V6 透氣處理之小植株其氣孔指數較完全密封處理者為高，氣孔密度亦隨透氣處理週數增加而提高。S6V0 與 S4V2 處理下的離體葉片氣孔可見永久開啟特徵，其氣孔閉合率分別為 6.67% 與 26.64%。香蕉組織培養苗成活的關鍵在於控制水分散失，因此瓶苗氣孔功能相當重要。在本試驗中透氣處理後組培瓶內無乙烯累積，且組培苗之氣孔功能正常、壯苗指數也比較完全密封處理為佳。本試驗之結果顯示，透氣處理對香蕉組培苗商業生產有正面助益。關鍵詞：透氣、香蕉組織培養、氣孔指數。. 前言組織培養大量繁殖技術對種苗商業生產極為關鍵，具有遺傳性狀穩定、無病毒，繁殖時間短、空間利用較為經濟等優點，目前廣泛應用於園藝、農藝及森林產業，未來此項產業之產值更將隨著關鍵技術之開發而成長。香蕉組織培養苗具有健康無病毒，適合集團栽培與機械化省工之優點，自 1983 年起推廣至 2012 年累積提供種苗 6,500 萬株 (Lee et al. 2013)；但香蕉組培瓶苗可見玻璃質化 (vitrification) 現象，偶有芽體異常、葉片捲縮、發根困難等情形，影響香蕉組培苗出瓶移植成活率 (Liu et al. 2006)。組培苗生長發育受組培瓶內部環境影響極大，瓶內環境常具下列特徵，包括無日夜溫差、高相對溼度、低光量子通量密度 (photosynthetic photon flux. * 1 2. density; PPFD)、極大的二氧化碳濃度變化、營養豐富碳源充足之培養基、以及毒性物質的累積。在這樣的條件下培植體本身蒸散速率降低，同時也伴隨光合作用效率低下、水分養分吸收速度緩慢的現象。培植體呼吸速率相對提高，常造成組培瓶苗生長遲緩，或是葉片型態異常 (Aitken-Christie et al. 1995)。覆盆子 (Rubus idaeus) 組培苗常表現高細胞含水量、喪失氣孔功能及低光自營 (photoautotrophic) 效率等特徵 (Donnelly et al. 1985)，而瓶內高相對濕度則易造成蘋果 (Malus spp.) 組培苗的玻璃質化，且常見角質與表皮細胞之不連續、葉肉柵狀組織與海綿組織發育不良、氣孔保衛細胞型態改變及細胞內葉綠素含量減少等現象 (Chakrabarty et al. 2005)。康乃馨 (Dianthus caryphyllus) 組培苗亦可見葉片缺乏臘質與氣. 投稿日期：2014 年 12 月 17 日；接受日期：2015 年 3 月 18 日。通訊作者：UCChen@tari.gov.tw 農委會農業試驗所生物技術組助理研究員。台灣台中市。農業科技研究院植物科技研究所研究員兼所長。台灣新竹市。.

(2) 244. 台灣農業研究第 64 卷第 4 期. 孔功能不健全的特性 (Majada et al. 2001)，增加植物組織培養容器之氣體通透性，可提升組培苗出瓶成活率 (Majada et al. 2000)。以多層藥包紙取代慣用之鋁箔封口，可增加瓶內氣體通透性，改善台灣雙鋸齒葉玄參 (Scrophularia yoshimurae) 組培苗葉片型態，增加組培苗出瓶存活率達 66.7% (Chen et al. 2006a)，並可提升高氏柴胡 (Bupleurum koai) 組培苗發根率與出瓶存活率 (Chen et al. 2006b)。本研究探討在不同透氣處理下，對於新北蕉瓶苗與出瓶馴化苗之生長發育影響，並從葉片氣孔發育情況，進一步瞭解透氣處理對氣孔生理之影響，期能提升新北蕉組培苗出瓶成活率及馴化速度，提供香蕉組培苗繁殖業者參考。. 材料與方法植物材料、透氣處理與生長指標量測本試驗所使用的新北蕉苗購自台灣香蕉研究所，經無菌處理與組織培養繼代繁殖後，挑選苗齡約 1 mo，大小一致且未發根之瓶苗進行試驗。無根瓶苗培養於 B3 固態培養基 (1/2 MS salts, 340 mg L -1 NaH 2 PO 4 •H 2 O, 160 mg L -1 adenine sulfate, 3% sucrose, pH 5.5, 0.7% agar)，於 25℃ ± 2℃、12 h 光照環境下培養。組培瓶口以雙層鋁箔覆蓋，並以保鮮膜包覆固定，透氣處理開始時，改以直徑 3 cm 之不織布圓片 (F-035，密度 0.13 g cm -3，鉅瑋，台中市，台灣) 取代雙層鋁箔，配合雙層透氣蓋 (中華民國專利第 349579 號) 固定於培養瓶上。本試驗 4 種處理分別為：不透氣 6 wk (hermetically sealed for 6 weeks; S6V0)、不透氣 4 wk 後透氣 2 wk (4 weeks sealed followed by 2 weeks ventilation; S4V2)、不透氣 2 wk 後透氣 4 wk (2 weeks sealed followed by 4 weeks ventilation; S2V4) 及透氣 6 wk (ventilation for 6 weeks; S0V6)。每處理 3 重複，每 1 重複內有 5 株新北蕉無根瓶苗。待為期 6 wk 的透氣處理結束後，抽取瓶內氣體進行分析。小植株出瓶後定植 3 吋盆放置溫室，利用透明塑膠袋覆蓋避免蒸散作用太過旺盛，3 d 後將塑膠袋取下，以出瓶當天為第 0 天，每 15 d 記錄株高。而其. 他生長指標，如鮮重、乾重、第一展開葉葉面積、假莖直徑以及根的數目、根長度、增殖倍數 (公式 1)，則分別在第 0 天與第 45 天量測，並於第 45 天馴化結束後計算出瓶成活率與壯苗指數 (公式 2)。增殖倍數 (multiplication rate) =. 6 wk 處理結束後小植株數目起始培植體數目. 壯苗指數 (vigor index) =. (1). 假莖直徑 × 乾重 (2) 株高. 組織培養瓶內乙烯測量新北蕉小植株出瓶前利用氣相層析儀 (gas chromatography, model “GC-430”, Varian Inc., Palo Alto, CA)，搭配火焰離子檢測器 (Flame Ionization Detector; FID) 進行瓶內乙烯含量測定。本試驗共有 4 種透氣處理，每個處理 3 重複，每重複以氣密針抽取瓶內上部空間 (head space) 氣體進行分析。氣體進樣量為 1 mL，毛細管柱 25 m × 0.53 mm × 0.7 μm (model “cp7584”, Varian Inc., Palo Alto, CA)，管柱溫度為 60℃，氣化室及檢測器溫度為 130℃，以氮氣為載流氣體，氣體流速設定 15 mL min -1，氫氣為燃燒氣體，流速設定 30 mL min -1，氣體分析時間為 2 min。資料使用 Galaxie Chromatography Data System (Varian Inc., Palo Alto, CA) 軟體進行分析。. 新北蕉小植株葉片氣孔型態、數目之觀測隨機選取 4 種處理之新北蕉小植株，取樣位置固定為植株第一完全展開葉的尖端靠近中肋部位，以雙面剃刀切取 0.5 cm × 0.5 cm 之葉片，用雙面膠固定於樣品載台，葉背朝上。對照組為出瓶馴化成活的新北蕉小植株 (after acclimation; Aa)，取樣位置與方法同前面敘述。開瓶取樣至樣品固定之時間為 20 min，以掃描式電子顯微鏡 (field emission gun scanning electron microscope, FEG-SEM, model “JSM-6330F”, JEOL, Japan) 進行觀測，並記.

(3) 245. 透氣處理對香蕉組培苗之影響. 錄氣孔直徑、單位面積氣孔數、氣孔指數 (公式 3)、表皮細胞數目與氣孔閉合率。. 結果顯示僅在完全密封處理 S6V0 之瓶內可偵測到 203.3 nL L -1，其餘處理瓶內乙烯均低於最低偵測極限 (表 1)。出瓶當天各處理之新北蕉小植株高度介於 2.2–2.4 cm 間，處理間無顯著差異；S0V6 處理之小植株根長度為 8.4 cm 顯著較長，根數目則以 S0V6 處理之 7.5 最少 (P < 0.05) (表 1)，S6V0 處理之小植株次生根分枝較多，顯示透氣處理週數增加，次生根與根毛發育較少但較長 (圖 1I–L)。新北蕉小植株第一完全展開葉面積及假莖直徑隨不同透氣處理而有顯著差異，S6V0 處理之小植株葉面積為 5.6 cm2，較透氣處理之 S4V2、S2V4 與 S0V6 為大 (P < 0.05)；小植株假莖直徑則以 6 wk 透氣處理之 S0V6 顯著較高 (P < 0.05)，達 0.65 cm。不同透氣處理之小植株乾物重並無顯著差異，但鮮重則隨著透氣處理之週數增加而遞減，各處理間差異顯著 (P < 0.05) (表 1)。透氣處理後小植株葉片質地薄、較硬挺，完全密閉處理之 S0V6 小植株葉片較為柔軟，偶出現小面積水浸狀區塊 (圖 1E–H)。增殖倍數係以不同透氣處理之新北蕉組培苗分株數來計算，S6V0 處理之增殖倍率為 1.4 較其他處理多，但不同透氣處理間差異未達顯著水準 (表 1)。. 氣孔指數 (stomatal index) =. 單位面積氣孔數 × 單位面積氣孔數 + 單位面積表皮細胞數 100%. (3). 試驗資料統計本試驗調查所得各項數據，以 Microsoft Excel 軟體進行試算，並以 SAS-EG (SAS Enterprise Guide, Version 4.1, 2006) 統計軟體進行變方分析 (analysis of variance; ANOVA)，再以最小顯著差異性測驗 (least significant difference test; LSD test)，比較 5% 顯著水準下各處理間的差異。. 結果不同透氣處理對瓶內乙烯含量與瓶苗生長之影響為瞭解透氣處理影響瓶內氣體交換效果，於小植株出瓶前偵測瓶內上部空間乙烯含量，. 表 1. 不同透氣處理對瓶內乙烯含量及新北蕉小植株生長參數之影響。 Table 1. Influence of different ventilation closure treatments on ethylene concentration and plant growth parameters in Musa spp. ‘Formosana’ plantletsz. Item [C2H4] (nL L-1)y Plant height (cm). z. S6V0 203.30 ± 4.09 2.42 ± 0.10 ax. S4V2. S2V4. ND. ND. S0V6 ND. 2.23 ± 0.20 a. 2.40 ± 0.20 a. 2.36 ± 0.25 a. Root length (cm). 6.56 ± 0.69 b. 7.50 ± 0.87 ab. 6.86 ± 0.96 ab. 8.40 ± 1.28 a. No. of roots. 9.00 ± 0.58 ab. 8.78 ± 1.35 ab. 9.44 ± 0.84 a. 7.44 ± 1.26 b. Leaf area (cm2). 5.64 ± 0.29 a. 4.68 ± 0.50 b. 4.95 ± 0.63 ab. 4.68 ± 0.54 b. Stem width (cm). 0.56 ± 0.04 b. 0.59 ± 0.02 a. 0.59 ± 0.02 a. 0.66 ± 0.04 a. Fresh weight. 3.58 ± 0.39 a. 3.08 ± 0.43 ab. 2.63 ± 0.31 b. 2.86 ± 0.18 b. Dry weight. 0.21 ± 0.02 a. 0.22 ± 0.02 a. 0.19 ± 0.02 a. 0.18 ± 0.02 a. Multiplication rate. 1.40 ± 0.69 a. 1.13 ± 0.23 a. 1.00 ± 0.00 a. 1.06 ± 0.12 a. Different ventilation treatments: (1) S6V0: hermetically sealed for 6 weeks, (2) S4V2: 4 weeks sealed followed by 2 weeks ventilation, (3) S2V4: 2weeks sealed followed by 4 weeks ventilation, and (4) S0V6: ventilation for 6 weeks. Ethylene concentrations were measured at the end of 6 weeks ventilation closure treatment just before deflasked. Values are the mean for 3 culture vessels ± standard deviation, ND represents the ethylene concentration that is lower than the detection limit. x Values are means ± standard deviation (n = 3). Means followed by the same letter within each row are not significantly different at 5% level by LSD test. y.

(4) 246. 台灣農業研究第 64 卷第 4 期. S6V0. S4V2. S2V4. S0V6. (A). (B). (C). (D). (E). (F). (G). (H). (I). (J). (K). (L). Stomatal aperture. In vitro plantlet. Root morphology. 圖 1. 不同透氣試驗處理對新北蕉小植株外表性狀之影響。 Fig. 1. Stomatal and morphological traits on Musa spp. ‘Formosana’ tissue culture under different ventilation closure conditions. S6V0, hermetically sealed for 6 weeks; S4V2, 4 weeks sealed followed by 2 weeks ventilation; S2V4, 2weeks sealed followed by 4 weeks ventilation; and S0V6, ventilation for 6 weeks. (A)–(D) Scanning electron micrographs of stomatal apertures from abaxial surfaces after 6 weeks ventilation closure treatment (Bars = 10 μm). (E)–(H) In vitro plantlet morphology after 6 weeks ventilation closure treatment (Bars = 1 cm). (I)–(L) Root morphology after 6 weeks ventilation closure treatment (Bars = 1 cm).. 不同透氣處理對新北蕉小植株出瓶馴化之影響出瓶馴化 45 d 後的新北蕉以 S0V6 處理之小植株最高達 10.8 cm (P < 0.05) (表 2)；小植株根部發育部分，以 S4V2 處理之根數目最少，各處理間根長度則無顯著差異 (表 2)；S4V2、 S2V4 與 S0V6 三組透氣處理之小苗經過 45 d 馴化後，葉面積為 56.1–58.9 cm 2，顯著較不透氣處理 S6V0 的 45.9 cm 2 大 (P < 0.05)，但. 不同處理間假莖直徑差異不顯著 (表 2)。生長量方面則以 S0V6 處理之小植株鮮重 14.4 g 最重 (P < 0.05)，但各處理間乾重變化無顯著差異 (表 2)；比較不同透氣處理的新北蕉小苗出瓶馴化後之壯苗指數，以 S2V4 處理之 106.2 最佳，S0V6 處理之 100.1 次之，處理間差異顯著 (P < 0.05) (表 3)。. 不同透氣處理對新北蕉氣孔發育之影響為進一步瞭解不同透氣處理下小植株生長.

(5) 247. 透氣處理對香蕉組培苗之影響. 表 2. 不同透氣處理之新北蕉小植株出瓶馴化 45 d 生長情形。 Table 2. Plant growth parameters in Musa spp. ‘Formosana’ plantlets grew with different ventilation closure treatmentsz. Item. S6V0. Plant height (cm). 9.61 ± 0.10 abx. S2V4. S0V6. 9.50 ± 0.34 b. S4V2. 10.16 ± 0.73 ab. 10.72 ± 0.96 a. Root length (cm). 21.33 ± 4.09 a. 21.16 ± 4.73 a. 21.78 ± 3.16 a. 22.86 ± 2.60 a. No. of roots. 14.44 ± 0.51 ab. 13.89 ± 0.51 b. 15.00 ± 0.33 a. 14.22 ± 0.38 ab. Leaf area (cm ). 45.99 ± 5.48 b. 57.45 ± 1.37 a. 56.13 ± 7.14 a. 58.97 ± 0.69 a. Stem width (cm). 0.86 ± 0.11 a. 0.88 ± 0.04 a. 0.88 ± 0.02 a. 0.90 ± 0.00 a 14.48 ± 0.55 a. 2. Fresh weight. 12.28 ± 1.33 bc. 11.41 ± 0.55 c. 13.48 ± 0.50 ab. Dry weight. 0.93 ± 0.21 a. 0.98 ± 0.11 a. 1.17 ± 0.18 a. 1.20 ± 0.09 a. Vigor indexy. 83.23 ± 14.16 c. 92.06 ± 10.38 bc. 103.16 ± 11.93 a. 100.99 ± 9.82 ab. z. Different ventilation treatments: (1) S6V0: hermetically sealed for 6 weeks, (2) S4V2: 4 weeks sealed followed by 2 weeks ventilation, (3) S2V4: 2weeks sealed followed by 4 weeks ventilation, and (4) S0V6: ventilation for 6 weeks. Vigor index = (stem width/plant height) × dry weight. x Values are means ± standard deviation (n = 3). Means followed by the same letter within each row are not significantly different at 5% level by LSD test. y. 表 3. 不同透氣處理對瓶內新北蕉小植株氣孔密度、大小與功能之影響。 Table 3. Influence of different ventilation closure treatments on density, size and function of stomata in Musa spp. ‘Formosana’ plantletsz. Item. S6V0. S4V2. S2V4. S0V6. Aa. 28.29 ± 3.54 cx. 90.78 ± 8.90 b. 141.47 ± 14.15 a. 135.58 ± 10.21 a. 114.35 ± 2.04 b. Epidermal cells density (mm-2) 721.50 ± 66.26 c 848.83 ± 48.63 b 1,092.86 ± 97.25 a. 1,029.20 ± 66.26 a. Stomata density (mm-2) Stomatal indexy Stomatal aperture length (μm) Stomatal closure (%). 721.50 ± 36.75 c. 3.81 ± 0.70 c. 9.65 ± 0.36 b. 11.49 ± 1.15 a. 11.68 ± 1.33 a. 12.21 ± 0.67 a. 32.61 ± 2.31 d. 36.27 ± 0.61 c. 39.21 ± 0.23 b. 38.41 ± 0.23 b. 42.97 ± 0.70 a. 6.67 c. 26.64 b. 100.00 a. 100.00 a. 100.00 a. z. Different ventilation treatments: (1) S6V0: hermetically sealed for 6 weeks, (2) S4V2: 4 weeks sealed followed by 2 weeks ventilation, (3) S2V4: 2weeks sealed followed by 4 weeks ventilation, (4) S0V6: ventilation for 6 weeks, and (5) Aa: after acclimation. Stomatal index = 100 × No. of stomata per unit area/(No. of stomata per unit area + No. of epidermal cells per unit area). x Values are means ± standard deviation (n = 3). Means followed by the same letter within each row are not significantly different at 5% level by LSD test. y. 差異之原因，以掃描式電子顯微鏡觀察處理後小植株葉片 (圖 1A–D)；不同透氣處理影響新北蕉小植株葉背氣孔分布密度，S6V0 處理之新北蕉小苗，葉背單位面積氣孔數目最少，每平方毫米僅 28.3 個氣孔，S0V6 及 S2V4 處理後小植株葉背氣孔數顯著增加，分別為 135.6 與 141.5 個，而完成馴化的新北蕉植株 (Aa) 氣孔密度則降為 114.4 (表 3)。不同透氣處理下單位面積之表皮細胞數目具有顯著差異，其數量變化約略與單位面積氣孔密度一致，以 S0V6 及 S2V4 處理最多 (P < 0.05)。氣孔指數為單位面積下氣孔數目與單位面積總細胞數目之比值，結果顯示 S0V6 與 S2V4 處理之氣. 孔指數與完成馴化的 Aa 小植株相近，數值介於 11.5–12.2 間，而密封時間較長的 S6V0 與 S4V2 處理，其氣孔指數分別為 3.8 與 9.7，顯著較透氣處理組低 (P < 0.05)。在氣孔型態方面，馴化完成的對照組 Aa 其氣孔直徑最大，為 43 μm，其餘各處理之氣孔直徑約在 32–38 μm 之間，隨著透氣週數增加，氣孔直徑亦有增大趨勢，處理間差異顯著 (P < 0.05)。以 SEM 觀測葉片樣品時，取樣至上機間隔固定 20 min，離體失水的情況會造成樣品氣孔之關閉，結果顯示馴化完成的對照組 Aa 與 S0V6、 S2V45 之葉片樣品，其氣孔均能感受環境失水調控氣孔關閉，而 S6V0 與 S4V2 處理之樣品.

(6) 248. 台灣農業研究第 64 卷第 4 期. 其氣孔閉合率分別為 6.67% 及 26.64%，顯示氣孔功能在不同透氣處理後具有顯著差異 (P < 0.05) (表 3)。. 討論利用降低瓶內濕度、增加光通量、瓶內通氣 (ventilation) 及改變培養基成分等處理，可促進組培苗進行光自營生長，達到瓶內馴化 (in vitro hardening) 目的，增加組培苗出瓶成活率。增加組培瓶內透氣可提升玫瑰、康乃馨、木瓜、花椰菜、高氏柴胡以及台灣雙鋸齒葉玄蔘組培苗生長勢，並使瓶苗葉片面積加大 (Sallanon & Maziere 1992; Chen et al. 1998; Lai et al. 1998; Zobayed et al. 1999; Chen et al. 2006a; Chen et al. 2006b)。本試驗結果顯示，透氣處理對於瓶內小植株根部生長及地上部生長無顯著影響，6 wk 密封處理之 S6V0 因苗株水分含量多鮮重最高，由表 1 與表 2 結果可知，馴化 45 d 之 S2V4、S0V6 透氣處理小植株株高增加較多，顯示透氣處理能減少出瓶後的生長停滯，壯苗指數以 S2V4 處理最佳，則顯示適時的透氣處理可提升小植株對外界適應力。Tanimoto et al. (1995) 及 Zhang et al. (2003) 之研究顯示，乙烯可調控根毛細胞 (trichoblast cell) 之形成，增加根毛細胞數目、縮短根毛細胞長度，提高單位長度內根毛密度；另有研究則指出，乙烯可誘導 auxin 生合成，兩者協同作用促進根部生長及根毛發育 (Osmont et al. 2007; Stepanova & Alonso 2009)。本研究結果顯示，繼代後即進行透氣處理之 S0V6，6 wk 後瓶內小植株根數目最少 (表 1)，而繼代後維持密閉環境至少 2 wk 以上之 S2V4、S4V2 與 S6V0，可能因瓶內累積繼代造成之創傷乙烯，誘導根源體發生產生較多的不定根，結果與上述研究相符。乙烯對不同物種培植體再生之影響差異大，例如乙烯可促進水稻莖的形成 (Cornejo-Martin et al. 1979)，提高番茄莖段再生力、提高增殖倍率 (Trujillo-Moya & Gisbert 2012)，但添加 5 mg L -1 硫代硫酸銀 (silver thiosulfate; STS) 於培養基中抑制瓶內乙烯作用，則可提高大岩桐 (Sinningia speciosa) 葉片培植體之芽體再生率. 達 40% (Chae et al. 2012)。不同透氣處理之新北蕉小植株增殖倍率於本研究中雖略有變化，但處理間差異不顯著，可能與瓶內氣體組成關聯性較低。植物組培苗出瓶馴化後，氣孔數目會因環境 CO2 濃度變化而改變，楓香 (Liquidambar styraciflua)、薔薇 (Rosa odorata × Rosa damascena)、藍莓 (Vaccinium corymbosum)、菸草 (Nicotiana tabacum) 與朝鮮薊 (Cynara scolymus)，小植株出瓶後氣孔數目減少 (Wetzstein & Sommer 1983; Johansson et al. 1992; Noé & Bonini 1996; Ticha et al. 1999; Brutti et al. 2002)，而櫻桃 (Prunus serotina) 與杜鵑 (Rhododendron spp.) 小植株氣孔數目，則於出瓶馴化後增加 (Waldenmaier & Schmidt 1990; Drew et al. 1992)。本研究結果顯示，新北蕉小植株出瓶馴化後氣孔數目增加，氣孔指數亦以完成馴化的 Aa 最高，隨著透氣處理週數增加小植株葉背氣孔指數隨之增加，S0V6 與 S2V4 之氣孔指數皆趨近於 Aa 小植株，S6V0 與 S4V2 處理組氣孔指數則顯著較低 (表 3)。 Woodward (1987) 指出植物氣孔數目與環境 CO2 濃度成反比，Royer (2001) 彙整 176 種 C3 植物已發表之氣孔密度與氣孔指數資料，發現隨著環境 CO2 濃度提高，大部分植物的氣孔密度與氣孔指數均降低，如銀杏 (Poole et al. 1996)、鼠尾草 (Knapp et al. 1994)、春小麥 (Yang & Wang 2001) 等植物都有此現象。另有研究指出，鴨跖草葉片在高濃度 CO2 環境生長時，其葉片氣孔指數無顯著變化 (Boetsch et al. 1996)，歐洲赤松的氣孔發生則對 CO2 不敏感，只有在高溫時氣孔指數才隨 CO2 增加而遞減 (Luomala et al. 2005)，Stace (1966) 更指出氣孔發育時物種遺傳效力大於環境因子作用。花椰菜 (Brassica oleracea) 培植體在自然換氣 (diffusive ventilation) 或強制換氣 (forced ventilation) 的培養條件下，其氣孔數目均顯著減少，瓶內 CO2 濃度量測結果顯示，增加氣體交換率可提高瓶內 CO2 濃度，降低組培苗氣孔密度 (Zobayed et al. 1999, 2001)。本研究之新北蕉小植株經透氣處理後氣孔密度反而增加，推測試驗初期偏向異營 (heterotrophic) 生長，.

(7) 249. 透氣處理對香蕉組培苗之影響. 至後期才轉變為混營 (mixophotoautotrophic) 生長或光自營生長。玫瑰培植體在 3% 蔗糖濃度培養基與 35 μmol m -2 s -1 光照下，可維持 3 wk 異營生長，瓶內 CO2 濃度達 1,000–1,500 mg L -1 (Wu & Chu 1999)，馬鈴薯 (Solanum tuberosum) 培植體不論在 1、2 及 3% 蔗糖培養基中，氣孔密度均以透氣處理組較完全密封處理組高 (Mohamed & Alsadon 2010)。泡桐 (Paulownia fortunei) 組織培養也顯示，3% 蔗糖培養基下培植體行混營生長，其葉片氣孔指數相對較高，不正常發育的氣孔數目亦較多 (Sha Vallikhan et al. 2003)。高相對濕度 (relative humidity; RH) 與低蒸氣壓差 (vapor pressure deficit; VPD) 為密閉組培環境的特性之一，蒸氣壓差為空氣水蒸汽壓力與葉片水蒸氣壓力的差值，在固定的溫度與大氣壓力下，VPD 與 RH 成反比。自然狀態下，植物蒸散作用受到環境溫度、陽光輻射量、風速、二氧化碳濃度以及 VPD 的影響，VPD 可視為植物葉片水分蒸發潛勢的指標。本試驗中 S6V0 與 S4V2 處理之樣品，其氣孔閉合率分別為 6.67% 及 26.64% (表 3)，顯示密閉環境下氣孔喪失原有功能，同樣的情形亦可見於康乃馨 (Ziv et al. 1987) 與大飛燕草 (Santamaria et al. 1993) 組培苗中。紫露草屬植物 (Tradescantia virginiana) 在移入高相對濕度環境中 4 d 後，氣孔對 ABA 與乾旱處理不敏感，無法調節正常關閉 (Rezaei Nejad et al. 2006)。紫露草屬植物在低 VPD 環境下，細胞內負責 ABA 分解之 8’-hydrogenase 活性提高，可在短時間內降低保衛細胞與植物細胞間系 ABA 含量 (Rezaei Nejad & van Meeteren 2008)，此外，在低 VPD 環境中，ABA 訊號傳遞之抑制因子 PYR/RCAR 與 PP2C 的結合被抑制，使得 ABA 訊號不能順利傳遞 (Fujii et al. 2009)。因此，長時間處於低 VPD 環境時，保衛細胞維持相對較低的 ABA 含量，且保衛細胞對於 ABA 濃度變化不敏感，使得氣孔失去正常功能，無法感應環境變化 (Montillet & Hirt 2013)。組織培養苗因為氣孔功能不健全，無法有效控制葉片蒸散作用合，是造成組培苗出瓶後乾枯失水的主要癥結 (Brainerd &. Fuchigami 1982)。綜合本研究結果顯示，增加新北蕉組培瓶內透氣可增加葉片氣孔密度、維持氣孔正常功能，並縮短出瓶馴化生長停滯，增加蕉苗出瓶成活率。. 引用文獻 Aitken-Christie, J., T. Kozai, and S. Takayama. 1995. Automation in plant tissue culture: General introduction and overview. p.1–18. in: Automation and Environmental Control in Plant Tissue Culture. (AitkenChristie, J., T. Kozai, and M. A. L. Smith, eds.) Kluwer Acad. Pub. Dordrecht. 574 pp. Boetsch, J., J. Chin, M. Ling, and J. Croxdale. 1996. Elevated carbon dioxide affects the patterning of subsidiary cells in Tradescantia stomatal complexes. J. Exp. Bot. 47:925–931. Brainerd, K. E. and L. H. Fuchigami. 1982. Stomatal functioning of in vitro and greenhouse apple leaves in darkness, mannitol, ABA, and CO2. J. Exp. Bot. 33:388–392. Brutti, C. B., E. J. Rubio, B. E. Llorente, and N. M. Apostolo. 2002. Artichoke leaf morphology and surface features in different micropropagation stages. Biol. Plant 45:197–204. Chae, S. C., H. H. Kim, and S. U. Park. 2012. Ethylene inhibitors enhance shoot organogenesis of Gloxinia (Sinningia speciosa). Sci. World J. 2012:1–4. Chakrabarty, D., S. Y. Park, M. B. Ali, K. S. Shin, and K. Y. Paek. 2005. Hyperhydricity in apple: Ultrastuctural and physiological aspects. Tree Physiol. 26:377–388. Chen, U. C., C. N. Hsia, D. C. Agrawal, and H. S. Tsay. 2006a. Influence of ventilation closures on plant growth parameters, acclimation and anatomy of leaf surface in Scrophularia yoshimurae Yamazaki- A medicinal plant native to Taiwan. Bot. Stud. 47:259–233. Chen, U. C., C. N. Hsia, M. S. Yeh, D. C. Agrawal, and H. S. Tsay. 2006b. In vitro micropropagation and ex vitro acclimation of Bupleurum kaoi- An endangered medicinal plant native to Taiwan. In Viro Cell Dev. Biol. Plant 42:128–133. Chen, U. C., Y. J. Shiau, C. C. Lai, and H. S. Tsay. 1998. Effects of medium composition and vessel closure on the hyperhydricity and rooting of carnation in vitro culture. J. Agric. Res. China 47:364–376. (in Chinese with English abstract) Cornejo-Martin, M. J., A. M. Mingo-Caste, and E. PrimoMillo. 1979. Organ redifferentiation in rice callus: effects of ethylene, carbon dioxide and cytokinins. Z. Pflanzenphysiol. 94:117–123. (in Spain with English abstract).

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(10) 252. 台灣農業研究第 64 卷第 4 期. Effect of Ventilation on Growth Parameters, Acclimation and Stomatal Development in Micropropagated Banana Plantlets Han-Wei Chen1, Min-Tze Wu2, and Uei-Chern Chen1,*. Abstract Chen, H. W., M. T. Wu, and U. C. Chen. 2015. Effect of ventilation on growth parameters, acclimation and stomatal development in micropropagated banana plantlets. J. Taiwan Agric. Res. 64(4):243–252.. Micropropagated banana (Musa spp. ‘Formosa’) requires discret management to acclimatize to the external environment. In this study, aluminum foil was replaced by non-woven fabric to improve ventilation efficiency of culture vessel headspace air condition. Different ventilation treatments of culture vessel were investigated, including hermetically sealed for 6 weeks (S6V0), 4 weeks sealed followed by 2 weeks ventilation (S4V2), 2 weeks sealed followed by 4 weeks ventilation (S2V4), and ventilation for 6 weeks (S0V6). No ethylene accumulation was detected in S4V2, S2V4, and S0V6 treatments; however, high ethylene level (203.3 nL L-1) under airtight vessels of S6V0 was noticed and promoted adventitious root formation on banana plantlets. The stomatal index in S2V4 and S0V6 seedlings was significantly higher than plantlets subjected to the airtight condition, and stomatal densities increased with increasing duration of ventilation. Permanently widely-open stomata in detached leaves were noticed in the S6V0 and S4V2 seedling, the stomatal closure rate were 6.67% and 26.64% respectively. Since the survival of tissue cultured banana plantlets may depend largely on the prevention of water loss, factors that enhance the stomatal functionality of in vitro seedling are important. Ventilation treatments in this study showed no ethylene accumulation, in which seedling performed normal stomatal response and better vigor index value than airtight cultivation vessels. The results would be useful for industrial production of in vitro banana seedlings. Key words: Ventilation, Banana tissue-culture, Stomatal index.. Received: December 17, 2014; Accepted: March 18, 2015. * Corresponding author, e-mail: UCChen@tari.gov.tw 1 Assistant Research Fellows, Biotechnology Division, Taiwan Agricultural Research Institute, Taichung, Taiwan, ROC. 2 Research Fellow and Director, Plant Technology Laboratories, Agricultural Technology Research Institute, Hsinchu, Taiwan, ROC..

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