蝴蝶蘭長串花形誘導及轉錄體分析

本論文研究目的為蝴蝶蘭長串形花序技術之開發，利用不同濃度BA、處理間隔天數及氮肥比例，期望能促進蝴蝶蘭花序生長、誘導花苞持續發育，提高開花品質，並透過轉錄體分析，找出誘導蝴蝶蘭花序延長相關基因。以大白花Phalaenopsis Sogo Yukidian ‘V3’，及中型白花Phal. aphrodite subsp. formosana ‘KC1410’及‘Am9405’品種進行試驗。‘V3’植株每隔3及7天塗抹BA (50、100 mg L-1)於花序頂端可誘導花芽持續分化，花序分別增長至107.5及109.6 cm，花朵數增加一倍，但處理100 mg L-1 BA，使花朵壽命縮短約30天。‘KC1410’處理BA (25、50、100 mg L-1)及氮肥(N-P2O5-K2O 20-20-20、40-20-20)，結果顯示，高氮肥可促進營養生長，且花朵數隨著BA濃度提高而增加，平均肥對照組(7.1朵)與處理BA相比，分別增加至10.5、13.5及15.3朵；而高氮肥分別增加至12.8、13.3及16.5朵，但高濃度100 mg L-1 BA造成17.0%及22.6%花苞黃化。觀察‘KC1410’及‘Am9405’花梗抽出之縱切面，當花梗抽出至1 cm，尚無花原體形成，伸長至3 cm及5 cm，已有3-4個花原體形成。‘Am9405’間隔3、7、10、14天塗抹100 mg L-1 BA，間隔3天處理花朵數增加7.3朵；間隔14天僅增加4.0朵，黃苞率分別為17.9%、12.2%、4.2%及0%，故縮短BA處理天數，可增加花朵數，卻提高黃苞率。由轉錄體分析結果中挑選15基因進行qPCR驗證，共有4基因於BA處理後各時間點之表達趨勢相同。XLOC_060361 (cytokinin-specific binding protein 1)及XLOC_043601 (auxin-repressed protein)於第4小時表現量提高；而XLOC_016267 (cytokinin oxidase)及XLOC_033378 (MADS box protein 5)於48小時後逐漸增加，第7天時表達最高。上述基因可能因BA處理而調節細胞分裂素及生長素含量，因而誘導新花苞發育。總結試驗，施用適當濃度BA可誘導新花苞持續發育，配合肥培管理及處理間隔天數，可使花序延長及花芽形成，此過程受到許多基因調控，預期應用此技術可增加蝴蝶蘭開花株多樣化商品，提升花卉產業之經濟效益。

關鍵字

苄基腺嘌呤；肥料；花苞誘導；花朵壽命；高通量定序

並列摘要

The objective of this study was to exploit technique of long cascade inflorescence by treating different concentrations of N6-benzyladenine (BA) on the inflorescence tip, combined with nitrogen fertilizer on growth and flowering of Phalaenopsis orchids. It is expected to induce the development of more flower buds, and improve the flowering quality, there by increasing the ornamental value. The transcriptome was analyzed to understand up- and down-regulated genes during inflorescence elongation. Three white flower cultivars of Phalaenopsis were used for the experiments, including large white Phal. Sogo Yukidian ‘V3’, and two medium size white Phal. aphrodite subsp. formosana ‘KC1410’ and ‘Am9405’. The results showed that applying BA on the spike tip of the Phalaenopsis plants could induce more flower buds to further differentiation. Mature plants of Phal. Sogo Yukidian ‘V3’ were treating the spike tip with BA (50, 100 mg L-1) at 3 days and 7 days intervals, compared with the control, the inflorescence length of treated plants increased to 107.5 and 109.6 cm, respectively. The number of flowers was doubled. However, BA at 100 mg L-1 reduced the flower longevity up to 30 days. The effect of BA with different nitrogen levels (N-P2O5-K2O 20-20-20 and 40-20-20) application on Phal. aphrodite subsp. formosana ‘KC1410’. The results showed that increasing nitrogen concentration promoted vegetative growth. Average fertilizer together with increasing BA concentrations increased the number of flowers per spikes with control having 7.1 flowers while BA treated plants at 25, 50 and 100 mg L-1 levels increased to 10.5, 13.5 and 15.3 flowers respectively. High nitrogen fertilizer combined with BA treatment increased flower number from the control 6.8 to 12.8, 13.3 and 16.5. However, BA at 100 mg L-1 reduced flower longevity and increased bud yellowing. Both nitrogen levels combined with 100 mg L-1 BA treatment resulted in 17.0% and 22.6% bud yellowing, respectively. To examine the bud primordium development of Phal. aphrodite subsp. formosana ‘KC1410’ and ‘Am9405’, longitudinal section of young spikes at 1, 3 and 5 cm long was observed under microscope. No bud primordium was formed in 1 cm spike, while 3-4 bud primordia appeared when the spikes were 3 and 5 cm long. Application with 100 mg L-1 BA to ‘Am9405’ at different time intervals, the total number of flowers treated in 3 days interval was 13.6, higher than the untreated control of 7.3, while the 14 days interval treatment only increased by 4 flowers. However, the shorter the treatment interval, the higher the yellowing rate of flower buds, while no bud yellowing in the 14 days interval treatment. A total of 15 genes were selected from transcriptome and verified by qPCR. Four genes expressed in the same trend at each time point after BA treatment. Notably the expression of XLOC_060361 (cytokinin-specific binding protein 1) and XLOC_043601 (auxin-repressed protein) were increased after 4 hr. Both XLOC_016267 (cytokinin oxidase) and XLOC_033378 (MADS box protein 5) were up-regulated gradually after 48 hr and reached to maximum after 7 days. The above genes may participate in regulating cytokinins and auxins levels and thus promote flower-bud formation. Taken together, the application of BA could effectively induce the continuous development of new flower buds, and combining with fertilizer management and treatment interval days could prolong inflorescence development and flower bud formation. This developmental event could have been regulated by multiple. By using the technique developed in this study, it is expected to increase the diverse commodities of Phalaenopsis flowering plants and increase the economic benefits of the flower industry.