蝴蝶蘭屬間雜交、黃葉病抗病性及香氣分析

Abstract

蝴蝶蘭(Phalaenopsis)為台灣重要的經濟花卉作物，蝴蝶蘭與異屬蘭花間之雜交，有潛力引入新性狀，開發具抗病性、香氣與特殊花色等優良性狀之新品種。本研究針對蝴蝶蘭育種過程中常見的問題，包括蝴蝶蘭體外花粉離體培養、異屬雜交障礙克服、優化無菌播種培養基、後代抗病性測試與香氣育種進行一系列之探討。 花粉活力測試能篩選具有稔性之父本，可藉由花粉染色或離體培養進行。本研究發現蘭科植物的花粉活力染色結果易高估其實際的萌發能力及花粉管生長之情形，而離體培養可以更直觀調查花粉之萌發與花粉管生長。然而花粉培養之培養基需針對不同物種進行優化，測試之花粉狀態也會影響培養結果。試驗中以Brewbaker and Kwack培養基為基礎，針對蝴蝶蘭花粉調整蔗糖、H3BO3以及Ca(NO3)2·4H2O濃度，並測試培養溫度、花粉發育階段、花粉發育溫度、花粉密度之影響。培養基配方以0%蔗糖、125 mg·L-1 H3BO3、100 mg·L-1 Ca(NO3)2·4H2O得到最佳之花粉生長結果。在25°C環境，並以鬆蕾期取樣、未受高溫影響之花粉塊配置懸浮液進行培養為最佳培養條件。本研究亦首次證實蝴蝶蘭具有花粉群聚效應，提高離體培養之花粉密度有助於花粉萌發生長。 進一步觀察蝴蝶蘭Phal. Hualien Pink Apple ‘Hualien No. 1-Pink Apple’與異屬蘭花(包含狐狸尾蘭屬Rhynchostylis gigantea var. alba、槽舌狐狸尾蘭屬Holcostylis Pink Yawi)之自交、正反交親和性與稔實性。蝴蝶蘭自交後花粉管可順利生長並成功結實；狐狸尾蘭自交後花粉能順利生長惟81.2%胚發育異常。蝴蝶蘭與狐狸尾蘭雜交後形成的胚有91.7%為異常胚，該組合播種300個種子以上結果發芽數不到10個。試驗亦測試1或3 μM萘乙酸(1-naphthaleneacetic acid, NAA)對柱頭腔閉合之影響，雖能使柱頭腔閉合增加花粉停留於子房內之發育時間，但無法提升花粉活力或克服受精後之雜交障礙。未來異屬雜交之操作，應更著重於品種親和性選配，以提升雜交後代之育成率。 雜交後代種子無菌播種技術優化方面，本研究測試培養基型態及添加細胞分裂素(6-benzylaminopurine, BA)對發芽率提升之影響。蝴蝶蘭Phal. Hualien Pink Apple ×self、槽舌蝶蘭(Phal. Little Gem Stripes ×Holcoglossum amesianum) ×Phal. Hualien Pink Apple、腎藥蘭Renanthera monachica ×self的種子，在液態培養基中均優於固態基質之萌發率，在低發芽率的腎藥蘭上也有顯著的提升。蝴蝶蘭及槽舌蝶蘭以0.1-0.5 mg·L-1 BA表現較佳，而腎藥蘭則偏好不添加BA之培養基。 本研究以自行分離之Fusarium phalaenopsidis C.L. Wang & W.C. Tsao蝴蝶蘭黃葉病株Ph4、Ph5、Ph15及參考菌株TJP 2178 10作為接種菌株，建立蝴蝶蘭黃葉病的抗病評估系統。結果顯示Phal. Hualien Pink Apple抗病性相對較佳，可作為抗病親本。除評估常見蝴蝶蘭品系，亦針對異屬雜交後代作抗病比較，發現部分後代對黃葉病抗病能力明顯提升，顯示異屬遠緣雜交為抗病育種有效途徑，能符合育種策略並提升產業競爭力。 在香氣育種與香氣成分分析方面，在雙親本皆有香氣表現的雜交組合中，有57.1%的後代表現出香味，而僅單親具香氣者，後代低於50%表現香味。當以Phal. schilleriana為父本時，也能傳遞香氣，顯示出香氣不僅是母系的遺傳。進一步分析異屬雜交中香氣的遺傳，發現雜交後代的狐狸尾蝶蘭Rhynchonopsis Karson’s Viola保留了Rhynchostylis coelestis的氣味特徵，而Rhnps. Low Elaine則顯現了雜種優勢，其揮發性化合物的種類與總量均高於親本。本研究評估屬間雜交後代之香氣代謝物與遺傳表現，為香花育種研究提供了基礎資訊。 綜上所述，本研究探討蝴蝶蘭異屬雜交技術，優化花粉離體培養與種子無菌播種技術，並建立雜交後代抗病及香氣性狀之篩選與應用方式。未來可以更精確評估因花粉萌芽困難所造成的雜交障礙，以及抗病、香氣等商業優良性狀加速育種，強化台灣蝴蝶蘭在全球市場的競爭力與產業永續發展。

Phalaenopsis is an important economic floriculture crop in Taiwan. The intergeneric hybrid between Phalaenopsis and other orchid genera has the potential to introduce new traits and develop novel varieties with enhanced disease resistance, fragrance, and unique colors. This research investigates successive processes during breeding, including in vitro pollen culture in Phalaenopsis, overcoming intergeneric hybrid barriers, optimization of culture medium for asymbiotic germination, and phenotypic assessment of the progeny, including disease resistance and fragrance performance. The pollen viability test can evaluate the fertility of the paternal parent through viability staining or in vitro pollen culture. Our results indicate that the staining methods overestimate actual germination and pollen tube growth. In contrast, in vitro culture allows direct observation of pollen germination and tube development. However, the medium required species-specific optimization, and the conditions of the pollen would also affect germination. This study optimized the concentrations of sucrose, H3BO3, and Ca(NO3)2·4H2O for Phalaenopsis pollen based on Brewbaker and Kwack medium, and examined the effects of culture temperature, pollen developmental stage, pollen developmental temperature, and pollen density. The optimal medium contained 0% sucrose, 125 mg·L-1 H3BO3, and 100 mg·L-1 Ca(NO3)2·4H2O, with culture at 25°C using pollen from flower buds at the loose-bud stage, prior to heat exposure. Notably, this study is the first to report the pollen population effect in Phalaenopsis, where higher pollen density significantly promoted germination and growth in vitro. Compatibility and fertility of the crosses were evaluated between Phal. Hualien Pink Apple ‘Hualien No. 1–Pink Apple’ and intergeneric orchids including Rhynchostylis gigantea var. alba and Holcostylis Pink Yawi. Self-pollination in Phalaenopsis led to successful pollen tube growth and seed development. In Rhynchostylis, self-pollination facilitated pollen tube formation but led to 81.2% abnormal embryo development. Crosses between Phalaenopsis and Rhynchostylis exhibited 91.7% abnormal embryo, and the sowing of over 300 seeds from this cross resulted in fewer than 10 germinated seedlings. Application of 1 or 3 μM naphthaleneacetic acid (NAA) facilitated stigma closure and prolonged pollen retention, but did not overcome infertile pollen or post-fertilization barriers. These findings suggest the need for careful parental compatibility selection and suggest embryo rescue techniques as a potential strategy for overcoming hybridization barriers. To optimize seed germination protocols, various culture media types and cytokinin concentrations (6-benzylaminopurine, BA) were tested. Seeds of Phal. Hualien Pink Apple ×self, the intergeneric hybrid (Phal. Little Gem Stripes ×Holcoglossum amesianum) ×Phal. Hualien Pink Apple, and Renanthera monachica ×self, all germinated better in liquid media than solidified media, with particularly significant improvement in the low-germinating Renanthera. Optimal BA concentrations for Phalaenopsis and its intergeneric hybrids ranged from 0.1 to 0.5 mg·L-1, whereas Renanthera preferred BA-free media. For disease resistance assessment, Fusarium phalaenopsidis C.L. Wang & W.C. Tsao isolates Ph4, Ph5, Ph15, and the reference strain TJP 2178 10 were used to establish a disease evaluation system for Phalaenopsis yellow leaf disease. Phal. Hualien Pink Apple showed relatively strong resistance and is considered a potential parent for disease resistance breeding. Some intergeneric progenies exhibited enhanced resistance, indicating that wide hybridization is an effective approach for developing disease-resistant cultivars, aligning with the aim of sustainable production. In crosses where both parents were fragrant, 57.1% of progeny expressed fragrance, while this proportion was below 50% when only one parent was fragrant. Use of Phal. schilleriana as the male parent, also inherited fragrance to the progeny, indicating a non-maternal inheritance pattern. In intergeneric hybrids, Rhynchonopsis Karson’s Viola retained the scent profile of Rhynchostylis coelestis, while Rhnps. Low Elaine exhibited hybrid vigor, with increased diversity and total quantity of volatile compounds compared to either parent. This study provides preliminary insights into the genetic and metabolic basis of fragrance expression in intergeneric hybrids, laying the groundwork for future breeding of fragrant cultivars. In summary, this research explores intergeneric hybridization techniques in Phalaenopsis, optimizing pollen culture and seed germination protocols, and establishing evaluation systems for disease resistance and fragrance traits in hybrid progeny. These results provide a foundation for overcoming hybridization barriers and accelerating breeding for disease resistance and fragrance characteristics, thereby enhancing the global competitiveness and sustainability of the Phalaenopsis industry in Taiwan.