蝴蝶蘭花粉塊低溫貯藏、斑葉突變種生理與調控基因表現分析

Storage of pollina at sub-zero temperature and physiological and gene expression analysis of variegated mutants in Phalaenopsis orchids

袁世璋(Yuan Shih-Chang)

指導教授 ： 陳福旗

國立屏東科技大學/農學院/農園生產系所/博士(2020年)

https://doi.org/10.6346/NPUST202000124

摘要

蝴蝶蘭為台灣重要的出口花卉之一，為提升產業之國際競爭力，具有優良特新新品種之育成為首要之目標。然而，重要商業品種不一定隨時可得，若能長期貯藏其花粉塊，將可提供於未來雜交授粉之用。本研究探討花粉於不同低溫貯藏之可行性，且於貯藏期間定期解凍並授粉於檢定母本。以Phalaenopsis Little Gem Stripes之乾燥花粉塊分別於室溫、4℃、-20℃及-80℃進行長期貯藏，並以TTC染色法檢測花粉活力、Alexander染液染色法觀察貯藏花粉之花粉管體外萌發率及利用人工授粉檢測其授粉成功率、並觀察花粉管於母本花柱萌芽情形。結果顯示，當花粉塊分別貯藏於上述四種溫度下4週，仍具有活力並可成功授粉於母本Phal. Sogo Vivien ‘F858’。花粉塊貯藏於室溫下4週即喪失活力；而貯藏於4℃下40週及貯藏於-20℃、-80℃下96週仍可保有50%-75%之活力，且亦可成功授粉於母本Phal. Sogo Vivien ‘F858’、結實及產生種子。綜合上述，蝴蝶蘭花粉塊可短期貯藏於4℃，而於-20℃及-80℃可長期貯藏。
另外探討蝴蝶蘭斑葉之生理及可能參與之基因表現分析。以台灣白花蝴蝶蘭(Phal. aphrodite subsp. formosana)及小花品種Phal. Sogo Vivien ‘F858’的斑葉突變株為供試材料，其葉片上分布黃綠不均之斑紋。結果顯示，兩斑葉蝴蝶蘭之黃色區域其葉綠素及類胡蘿蔔素含量皆較綠色區域及野生型低。在Phal. aphrodite斑葉突變株之黃色區域，其葉綠體僅存在於靠近表皮之葉肉細胞中； Phal. Sogo Vivien ‘F858’斑葉突變株之黃色區域僅於近表皮之葉肉細胞有較少數之葉綠體。利用穿透式電子顯微鏡觀察兩斑葉突變之葉綠體發育，在Phal. aphrodite黃色區域可觀察到發育不全的基粒堆疊(stacked grana)及質體球(plastoglobules)，而Phal. Sogo Vivien ‘F858’之黃色區域並未觀察到類似構造。進一步由相關之葉綠素代謝、類胡蘿蔔素生合成途徑、葉綠體發育及光合作用等共挑選14個基因進行定量即時聚合酶鏈鎖反應 (quantitative real-time PCR)分析。葉綠素降解基因CLH和RCCR在兩斑葉蝴蝶蘭之黃色區域表現量大量增加，推測兩斑葉品種之黃色區域低葉綠素b含量，可能為葉綠素降解相關基因大量表現之緣故。另外，兩斑葉品種之黃色區域其PsbO表現量較綠色區域高，可能因而使基粒堆疊受阻進而導致光系統II (photosystem II)之D1和D2蛋白合成受抑制；且可能會抑制葉綠體相關基因(GLK)及光系統II等相關基因之表現。

關鍵字

花粉管萌發 ； 短期貯藏 ； 長期貯藏 ； 黃色區域 ； 葉綠素b ； 葉綠體發育 ； 葉綠素降解相關基因 ； 光系統II

並列摘要

Phalaenopsis orchid is one of the important floral crops in Taiwan for export. Exploiting novel cultivars is critical for keeping the competitiveness in the global orchid market. Pollen of elite cultivars may not be available at hand, therefore, properly stored pollens provide an opportunity for further cross-hybridization and selection of novel varieties. The objective of this study was to evaluate the storage capability of Phlalaenopsis Little Gem Stripes pollinia at different temperatures, including room temperature, 4℃, -20℃, and -80℃ for up to 96 weeks. The viability of pollen was assessed by several methods, including TTC staining, in vitro germination, and hand pollination during and after storage. The results revealed that pollen stored at all four temperature regimes for 4 weeks remained viable and capable of successful pollination to Phal. Sogo Vivien ‘F858’. Pollen lost viability after 4 weeks storage at room temperature. Pollen remains viable after 40 weeks at 4℃, and also after 96 weeks at both -20 and -80℃ of storage, with the viability between 50%-75%. Stored pollen could be successfully pollinated to female parent at all storage conditions and produce capsule and viable seeds. Taken together, Phalaenopsis pollinia can be kept at 4℃ up to 40 weeks for short-term storage. For long-term storage, pollen can be kept at both -20 and -80℃.
The objective of this study focused on physiology and expression analysis of candidate genes in variegated Phalaenopsis orchids. Leaf variegation in Phal. aphrodite subsp. formosana and Phal. Sogo Vivien ‘F858’ showing irregular yellow stripes were observed in the orchid nurseries. The chlorophyll and carotenoid contents were lower in the yellow sectors of variegated Phalaenopsis orchids than in green sectors and wild-type. The chloroplast is only present in the sub-epidermal mesophyll cells in the yellow sectors of variegated Phal. aphrodite, while only fewer chloroplast was present in the sub-epidermal mesophyll of the yellow sectors of Phal. Sogo Vivien ‘F858’ when observed under light microscope on leaf cross-sections. Observation under transmission electron microscopy (TEM) revealed the presence of some underdeveloped stacked grana and plastoglobules in the yellow sectors of Phal. aphrodite but not in the yellow sectors of Phal. Sogo Vivien ‘F858’. A total of 14 probable candidate genes were selected and analyzed by quantitative real-time PCR, including chlorophyll metabolism, carotenoid biosynthesis, chloroplast development, and photosynthesis-related genes. The chlorophyll degradation-related genes, CLH and RCCR, were up-regulated in the yellow sectors of both variegated Phalaenopsis orchids. The lower chlorophyll content in the yellow sectors, especially chlorophyll b, probably was due to the activation of chlorophyll degradation-related genes in both genotypes. In addition, the results further revealed that the up-regulated PsbO activity might inhibit the grana stacking in the yellow sectors of both genotypes and block the synthesis of the D1 and D2 proteins in the PSII complex as further revealed by down-regulated chloroplast development-related gene GLK, and the PSII complex-related genes.

並列關鍵字

Pollen germination ； Short-term storage ； Long-term storage ； Yellow sectors ； Chlorophyll b ； Chloroplast development ； Chlorophyll degradation-related genes ； Photosystem II

參考文獻

Abdelgadir, H. A., S. D. Johnson, and J. Van Staden. 2012. Pollen viability, pollen germination and pollen tube growth in the biofuel seed crop Jatropha curcas (Euphorbiaceae). S. Afr. J. Bot. 79: 132-139.

Alexander, M. P. 1969. Differential staining of aborted and nonaborted pollen. Stain Technol. 44: 117-122.

Aluru, M. R., H. Bae, D. Wu, and S. R. Rodermel. 2001. The Arabidopsis immutans mutation affects plastid differentiation and the morphogenesis of white and green sectors in variegated plants. Plant Physiol. 127: 67-77.

Aluru, M. R., J. Zola, A. Foudree, and S. R. Rodermel. 2009. Chloroplast photooxidation-induced transcriptome reprogramming in Arabidopsis immutans white leaf sectors. Plant Physiol. 150: 904-923.

Barnabas, B., G. Kovacs, A. Abranyi, and P. Pfahler. 1988. Effect of pollen storage by drying and deep-freezing in the expression of different agronomic traits in maize (Zea may L.). Euphytica 39: 221-225.