蝴蝶蘭瓶苗下位葉黃化之成因及組培苗於礦物營養缺乏下之徵狀

摘要 (abstract) 本論文分為兩部分，第一部分探討蝴蝶蘭於組織培養子瓶階段下位葉黃化之成因，第二部分研究蝴蝶蘭瓶苗於礦物元素缺乏逆境下之病徵及生長所受之影響。以不同無菌操作方式及培養基組成探討瓶內乙烯累積與上述因子之相關性及其對蝴蝶蘭瓶苗生長之影響。朵麗蝶蘭下位葉易黃化品種 (Doritaenopsis Formosa Sunrise) 與較不易黃化品種 (Doritaenopsis Unimax Waltz) 在繼代操作過程中，矽膠瓶塞經過火處理1 天後，瓶內乙烯濃度維持在10-20 nL·L-1。瓶塞過火0、20 及 40 秒各處理間黃化程度無顯著差異，顯示瓶塞過火時間長短不是影響該兩品種下位葉黃化之主因。朵麗蝶蘭不論易黃化品種或不易黃化品種在繼代操作過程中，以25、40、60及80 oC 之解剖刀切除瓶苗根部後定植，各處理間瓶內乙烯濃度無顯著差異。不易黃化品種，在操作當天各處理間乙烯濃度均上升至100 nL·L-1 以上，1 天之後則下降至10-20 nL·L-1，但葉片無黃化現象；易黃化品種於各處理下均有黃化現象，但以80 oC 之處理最高。延遲繼代時間愈久，植株年齡愈大，蝴蝶蘭Phalaenopsis Brother Pico Bahama ‘E.G’瓶苗延遲3 個月後繼代之處理，於繼代操作當天其瓶內乙烯濃度顯著高於對照組 (正常時間繼代)，然而延遲繼代處理於培養3 個月期間並不會造成較嚴重的黃化現象。於培養基中添加不同比例的N6-benzyladenine / 1-naphthaleneacetic acid (BA / NAA)，5 mg·L-1 BA 處理下，Doritaenopsis Unimax Waltz 與Phalaneopsis Unimax Wish ‘CL271’黃葉數均為0.8，低於對照組 (分別為1.5 與1.0) 。NAA 對瓶苗黃葉數無顯著影響。在不同有機添加物之處理下，Phal. Brother Pico Bahama ‘E.G’瓶苗繼代培養3 個月後，以添加蘋果、香蕉或馬鈴薯之處理，其黃葉數達0.5 片以上，黃化程度高於對照組 (0.3 片)。培養基配方試驗中，Phal. Brother Pico Bahama ‘E.G’ 瓶苗繼代培養3 個月後，以Hyponex #1 (京都配方) 處理其瓶內乙烯濃度最高 (14 nL·L-1)，黃化程度最嚴重，平均每株有2.0 片黃葉；對照組僅有0.3 片。綜合上述，瓶塞過火、高溫解剖刀操作及延遲繼代時間等，均會增加瓶內乙烯濃度，但僅高溫解剖刀造成兩試驗品種的下位葉黃化。而培養基組成中，添加蘋果或香蕉等有機添加物，或使用Hyponex #1 配方，皆會使易黃化蝴蝶蘭產生較嚴重的黃化現象，且產生的乙烯也較高，顯示乙烯與黃化之間仍有相關。此外，BA 降低瓶苗下位葉黃化程度，可能與抑制乙烯的生成有關。臺灣蝴蝶蘭 (Phal. amabilis) 子瓶於養分缺乏逆境下6 個月後，各處理間的病徵表現明顯。缺氮植株根長顯著高於其他處理；缺磷植株新生葉片小，根尖生長停止；缺鉀植株矮小，中位葉有黃化現象；缺鈣植株新生葉壞疽，根部短小；缺鎂植株中位葉有壞疽及黃化現象；缺鐵植株新生葉片小，基部明顯黃化。大白花蝴蝶蘭 (Phal. Sogo Yukidian ‘V3’) 於養分缺乏逆境下培養6 個月後，缺氮植株下位葉黃化，地下部生長較地上部好；缺磷植株地上部生長量與對照組相近，根部較短；缺鉀植株矮小，葉片上有紫紅小斑點；缺鈣植株上位葉出現水浸狀，根部短小；缺鎂植株整體與對照組相似，所受影響較輕微；缺鐵植株根長及乾重均高於對照組，地下部生長較地上部良好。礦物元素缺乏逆境下6 個月後，各處理間的乾物重變化在試驗兩品種 (大白花蝴蝶蘭及臺灣蝴蝶蘭) 有相似趨勢。各元素缺乏處理的地上部乾重均低於對照組，以缺氮處理降低最多，分別為對照組的38.5%及52.3%，缺磷處理組降低較少，分別為對照組的22.2%及31.9%。在地下部乾重方面，兩品種均在缺氮及缺鐵處理組地下部乾重較對照組增加，缺氮處理於兩品種間分別增加36.3%及131%；缺鐵處理分別增加34%及88.5%。顯示礦物元素缺乏對蝴蝶蘭根部影響大於地上部。兩試驗品種於各元素缺乏處理組下，該元素含量為所有處理中最低；於根部構造方面，各元素缺乏處理並不影響根被層數。

關鍵字

乙烯；元素缺乏；組織培養

並列摘要

Abstract This thesis consists of two portions. The first portion investigates the causes of lower leaf yellowing in Phalaenopsis plantlets in vitro. The second portion investigates the symptoms of mineral nutrient deficiency in Phalaenopsis plantlets in vitro, as well as its effects on growth. Various aseptic procedures and media compositions were tested for correlation with ethylene accumulation in the tissue culture vessel of Phalaenopsis, and the effects of these factors on growth of the plantlets were studied. Plantlets of one cultivar susceptible to lower leaf yellowing in vitro (Doritaenopsis Formosa Sunrise) and one cultivar not susceptible to the disorder (Dtps. Unimax Waltz) were subcultured and the silicone stoppers of the tissue culture vessels were subjected to various durations of flaming before the vessels were sealed. Ethylene concentration in the vessel ranged 10-20 nL·L-1 1 day after flaming treatments. Flaming durations of 0, 20, or 40 seconds produced no significant difference in the extent of lower leaf yellowing of the plantlets, indicating that the duration for which the stoppers were flamed was not the main factor affecting lower leaf yellowing in the two cultivars. Plantlets of two Doritaenopsis cultivars, one susceptible and one not susceptible to leaf yellowing, were subcultured. Before planting into the culture medium, the roots of the plantlets were excised with a scalpel at a temperature of 25, 40, 60, or 80 oC. The various scalpel temperatures produced no significant difference in subsequent ethylene concentration inside the culture vessel. For the cultivar not susceptible to leaf yellowing, the ethylene concentration increased to higher than 100 nL·L-1 across all treatments on the day the plantlets were planted, and decreased to 10-20 nL·L-1 after 1 day. No leaf yellowing developed in this cultivar. In the susceptible cultivar, leaf yellowing developed across all treatments, but the greatest extent occurred when the scalpel temperature was 80 oC. Age of plantlet increased with longer delay before subculturing. Ethylene concentration within the culture vessel was significantly higher when plantlets of Phal. Brother Pico Bahama ‘E.G’ were subcultured after a 3-month delay, compared with control (i.e., subcultured to normal schedule). However, the delay did not result in more severe leaf yellowing over a 3-month culture period. Various BA-to-NAA ratios were added to the culture medium. Under 5 mg·L-1 BA treatment, the number of yellowed leaves on plantlets of Dtps. Unimax Waltz and Phal. Unimax Wish ‘CL271’ were 0.8, respectively, which was lower than that of control (1.5 and 1.0 for the two cultivars, respectively). NAA had no significant effect on leaf yellowing of the plantlets. The incorporation of organic additives, i.e., apple, banana, or potato into the culture medium resulted in number of yellowed leaf greater than 0.5 on Phal. Brother Pico Bahama ‘E.G’ plantlet after 3 months of culture, and the severity of leaf yellowing was greater than that of control (i.e., 0.3 yellowed leaf per plantlet). The effect of various base media was also investigated, and the medium based on Hyponex #1 resulted in the highest ethylene concentration in the culture vessel (14 nL·L-1) and the most severe leaf yellowing, with 2.0 yellowed leaves per plantlet against just 0.3 in the control group. Summing up the above, flaming stoppers, using scalpels while hot, and delaying subculture all increased ethylene concentration in the culture vessel, but only high scalpel temperature caused lower leaf yellowing in the two cultivars tested. As for medium composition, the addition of organic additives such as apple or banana, or using the medium based on Hyponex #1 both increased the severity of leaf yellowing in susceptible cultivars with concomitantly higher concentrations of ethylene. This result suggests that there is still a link between ethylene and leaf yellowing in Phalenopsis plantlets in vitro. In addition, BA reduced the extent of leaf yellowing, possibly through an inhibitory effect on ethylene biosynthesis. Plantlets of Phal. amabilis were subcultured and subjected to various mineral deficiency stresses. Within 6 months, mineral deficiency symptoms were obvious among treatments. Plantlets deprived of nitrogen had significantly greater root length compared with those in other treatments. Phosphorus deficiency resulted in small newly grown leaves and inhibited the growth of root tips. Under potassium deficiency, growth was stunted, resulting in small plantlets, and leaf yellowing developed on the middle leaves. Necrotic lesions developed on the newly grown leaves of plantlets deprived of calcium, and the roots of these plantlets were small and short. Under magnesium deficiency, necrotic lesions as well as leaf yellowing developed on the middle leaves. Iron deficiency resulted in small newly grown leaves with yellowing developing on their basal portion. Similarly, plantlets of the large white flowered hybrid Phal. Sogo Yukidian ‘V3’ were cultured under various of mineral deficiencies for 6 months. Lower leaf yellowing developed under nitrogen deficiency and the growth of roots was better than that of the shoot. Under phosphorus deficiency, shoot growth was similar to control, but the roots were shorter. Plantlets deficient in potassium were of small size, and small purple-red spots developed on their leaves. Under calcium deficiency, the upper leaves became vitrified and the roots were short and small. Plantlets deprived of magnesium were little affected and overall were similar to the control group. Plantlets deprived of iron had greater root length and dry weight compared with control, and had better root growth than shoot growth. The changes in dry weight after 6 months of various mineral deficiencies in vitro showed a similar trend between Phal. Sogo Yukidian ‘V3’ and Phal. amabilis. Shoot dry weights in the various mineral deficiency treatments were all smaller than control. The greatest reductions in shoot dry weight occurred under nitrogen deficiency, which, for the two Phalaenopsis tested, were 38.5% and 52.3% reductions compared with control, respectively. Smaller reductions in shoot dry weight occurred under phosphorus deficiency, which were 22.2% and 31.9% reductions compared with control, respectively. For both Phalaenopsis, root dry weight increased under nitrogen and iron deficiency treatments as compared with control. Under nitrogen deficiency treatment, root dry weight increased by 36.3% and 131%, respectively, while under iron deficiency treatment, it increased by 34% and 88.5%, respectively. This result shows that mineral deficiency had a greater effect on root growth than on the shoot growth of Phalaenopsis. It is showed a similar trend be between Phal. Sogo Yukidian ‘V3’ and Phal. amabilis. The mineral element contents in nutruient deficiency treatment is the lowest of all treatment, and the number of velament layers of root was not affected by mineral nutruient deficiency.