目前臺灣外銷蝴蝶蘭產業面臨極激烈之國際競爭壓力,因此如何快速培育健壯的苗株,供應市場的需求,在蝴蝶蘭產業上是首要的課題。蘭花組織培養苗之成長受內在生理及外在環境因子 (溫度及光度) 的影響極大,因此最適合及穩定之環境因子乃是促進其種苗發育生長之要素。本試驗目的為探討不同環境因子 (溫度及光照強度) 對臺灣白花蝴蝶蘭 (Phalaenopsis aphrodite subsp. formosana) 瓶苗瓶內CO2日韻律的影響,藉由分析各階段苗株 PEPC (Phosphoenolpyruvate carboxylase) 及Rubisco (Ribulose Bisphosphate Carboxylase) 活性以驗證固碳模式的轉變與瓶內CO2之日韻律濃度變化。組培苗培養期中可分為三階段:21天 (Stage I)、持續培養45天 (Stage II) 及再持續培養120天 (Stage III) 。植株在Stage I為典型C3型植物的固碳模式,培養至Stage II則較類似C3型型植物,苗株開始表現C3型及CAM型特性;持續培養120天後期苗則夜間CO2的釋放速率則明顯下降許多,CAM型的表現更加明顯。光強度試驗 (35、40、45、50、55、60 PPFD (Photosynthetic Photon Flux Density)) 結果:在培養中期 (Stage II) 除35 PPFD處理外皆有表現出類似CAM型行固碳模式,其中又以60 PPFD處理期瓶內CO2含量低於450 ppm 而更明顯。在植株Rubisco酵素活性顯示,Stage I及Stage II只有在暗期後1小時隨光強度的增加而提高,Stage III則顯示在50 PPFD光照下其酵素活性最高。而PEPC酵素活性在Stage I、Stage II及Stage III的暗期皆較光期下高,在Stage III時又以60 PPFD處理下最高。另植株葉面積隨光強度的增加而上升,且澱粉(50 PPFD)、可溶性糖 (60 PPFD) 及氮含量 (55 PPFD) 皆在較高光強度下最好。溫度試驗 (25/20℃、25/25℃、30/20℃、30/25℃、35/20℃、35/25℃) 結果顯示:在培養中期 (Stage II) 以35/20℃、25/25℃、30/25℃及35/25℃表現出類似CAM型行固碳模式,以35/20℃處理期瓶內CO2含量低於343 ppm 而更明顯,植株Rubisco初始活性在35/20處理下會提升,而Stage I及Stage II的暗期Rubisco酵素活性皆高於光期但在Stage III則相反。另植株在夜溫25℃下有較大的葉面積,然夜溫20℃則有較多的氮及澱粉含量。但結合光強度與日夜溫處理組合時 (55 PPFD + 30/25℃、55 PPFD + 35/25℃、60 PPFD + 30/25℃、60 PPFD + 35/25℃),則以結合30/25℃處理更早表現出類似CAM型行固碳模式,以60 PPFD + 30/25℃處理期瓶內CO2含量低於451 ppm 而更明顯。植株在Stage I及Stage II時以60 PPFD + 35/25℃有較高的總Rubisco酵素活性,而Stage III則以60 PPFD + 30/25℃高。另植株在高光照高溫更有助於蝴蝶蘭組培苗葉面積之增長,在Stage II以60 PPFD + 30/25℃處理下有較高碳水化合物含量。綜合上述結果,藉由驗證之各項結果更能證明蝴蝶蘭瓶苗隨著株齡成熟,固碳模式為一漸進式的過程由C3型轉CAM型。因此可以建議,當蝴蝶蘭繼代培養40天後以60 PPFD + 30/25℃處理可促進植株在瓶內之生長,並提供業者未來進行蝴蝶蘭組培幼苗環控栽培之參考。
Phalaenopsis industry is currently facing international competition, therefore, how to shorten the seedling period to fulfill the market demand is a primary issue. Light and temperature conditions may strongly affect the growth of the tissue culture seedlings. The objectives of this study were to explore the effects of environmental factors (temperature and light intensity) on CO2 rhythm of Phalaenopsis aphrodite subsp. formosana in vitro, and their PEPC (Phosphoenolpyruvate carboxylase) and Rubisco (Ribulose Bisphosphate Carboxylase) activities at different growth stages. Thus, it can be used to verify CO2 rhythm and carbon fixation pattern. The seedlings used can be clearly indentified into three stages: After cultured for 21 days (Stage I), 45 days (Stage II) and 120 days (Stage III). In Stage I it showed a typical C3 plant carbon fixation pattern. But, when plants continued to grow to Stage II they exhibited similar type C3-CAM plants. Two discrete CO2 absorption period, and more C3 to CAM carbon fixation pattern were found. CO2 rhythm changed as well as its concentration dropped. After 120 days cultured, seedlings showed significantly decreased CO2 concentration at night, and showed an typical CAM plant carbon fixation pattern. In light intensity experiment (35, 40, 45, 50, 55, 60 PPFD) at Stage II, all treatments showed similar C3-CAM carbon fixation pattern, however, 35 PPFD, and 60 PPFD have least CO2 concentrations (450 ppm) in vitro and were more obvious. The Rubisco activity increased with the increased of light intensity and improved only after 1 hour in dark at Stage I and stage II. In Stage III, they had the highest activity at 50 PPFD, while, at Stage I, Stage II and Stage III, lower PEPC activity in daytime than at night were evidence. And, the highest PEPC activity were 60 PPFD treatment. For carbohydrate contents, it was found that highest treatments were: starch (50 PPFD), soluble sugar (60 PPFD). The nitrogen concentrations showed that 55 PPFD were the best treatment. For temperature experiment, at Stage II, 35/20℃, 25/25℃, 30/25℃ and 35/25℃ treatments showed a similar CAM carbon fixation pattern, had least CO2 concentrations (343 ppm) in vitro and were more obvious. In three Stages, 35/20℃ treatment showed increase initial Rubisco activity. And, Stage I and Stage II showed lower Rubisco activity in daytime than at night, but Stage III showed the contrast result. Greater leaf area was found in 25℃ night temperature, but higher nitrogen and starch concentrations were found in 20℃ night temperature. In light intensity and day/night temperature combine experiment, day/night temperature 60 PPFD + 30/25℃treatment showed CAM carbon fixation pattern earlier. It had least CO2 concentrations (451 ppm) in vitro and exhibited more obvious CAM pattern. At Stage I and Stage II, 60 PPFD + 35/25℃ treatment showed highest total Rubisco activity. But, in Stage III, 60 PPFD + 30/25℃ treatment was highest. In Stage II, 60 PPFD + 30/25℃ treatment had higher carbohydrate concentrations. The above experiment results were in agreement with previous research on the CO2 circadian rhythm of seedling in vitro and showed the CO2 fixation pattern was transferring from C3 to CAM over three growth stages. Higher temperature with higher light intensity can promote Phalaenopsis in vitro seedling growth and fasten the transformation process. Therefore, 60 PPFD + 30/25℃ treatment could advance growth of Phalaenopsis aphrodite subsp. formosana in vitro seedling after subculture 40 days, and, can be recommended for commercial production of Phalaenopsis industry.
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