利用形態及RAPD標誌鑑別原生石斛蘭之研究

Abstract

本論文係以原生於台灣地區，包括屏東里龍山之長爪石斛(Dendrobium chameleon)等14種，菲律賓地區原生之鴿石斛(D. crumenatum)等2種及大陸地區原生之鐵皮石斛(D. candidum)等3種，共計19種之原生石斛蘭為試驗材料，並以產自台東大武之白花蝴蝶蘭(Phalaenopsis aphrodite)組培苗為遠端對照，進行植物外觀形態及生育特性調查，並以分子層次之逢機增殖多形性DNA片段(RAPD)標誌鑑別原生石斛蘭種原，以瞭解種間親緣關係之遠近及不同地域之生長環境對同種石斛蘭之遺傳變異情形。 試驗結果顯示：石斛蘭著生的位置與接受的光照強弱會影響其生育的表現，如果植株生長在岩石上，其假球莖會變得較小，若生長在樹林中，則假球莖發育較佳，葉片數增多，兩者間外部形態大小之差異可以達一倍以上。不同種間之區別，以花朵大小及顏色外觀之差異較易辨識。若要由植株形態來判定，可以根據葉的伸展角度、假球莖顏色、節間大小及新芽形狀與生長樣態來區別。花瓣色澤以金草石斛之橙色系最鮮豔，其次為紅鸝石斛、櫻石斛、紅花石斛的粉色系，鴿石斛的亮白色及黃花石斛的黃色系等皆有可觀之處；單朵花綻放天數以黃花石斛的15天最長，其次為櫻石斛的11天，細莖石斛、白花石斛的10天；鴿石斛及鬚唇暫花蘭的花朵均只有不到1天的壽命。 在參試的19種原生石斛蘭中，發現容易自交結實的有金草石斛、黃花石斛、鬚唇暫花蘭等3種，而燕石斛、細莖石斛、櫻石斛、連珠石斛等4種之自交結實率則約有五成左右；然鴿石斛、紅鸝石斛、櫻石斛、白花石斛等以異花授粉則可有效提高結實率。 以OPERON之隨機引子組OPE-01~20進行RAPD分析，結果顯示：原生石斛蘭種間的條帶圖譜相同的地方少，種間變異性相當大且無法得到種間一致性或是石斛蘭屬間特有的條帶，然而在不同來源之相同種間條帶圖譜的一致性就顯的相當高。繪製出之親緣關係樹狀圖顯示：本試驗中的原生石斛蘭可以分為六群，分別是第Ⅰ群─鴿石斛(菲律賓及綠島兩種)，第Ⅱ群─紅花石斛(菲律賓及蘭嶼兩種)、長爪石斛、三星石斛，第Ⅲ群─白花石斛、大陸鐵皮石斛、黃花石斛、紅鸝石斛(谷關、杉林溪、大陸雲南三種)，第Ⅳ群─金草石斛、櫻石斛，第Ⅴ群─小雙花石斛、細莖石斛，第Ⅵ群─鬚唇暫花蘭、尖葉暫花蘭、流蘇金石斛。就中第Ⅰ群之鴿石斛較遠端對照組之白花蝴蝶蘭相似度更低，理當獨立自成一屬；第Ⅱ群之紅花石斛、長爪石斛、三星石斛以及第Ⅴ群之雙花石斛、細莖石斛等依相關資料顯示亦有獨立成屬的條件。同種但不同地域生長者其遺傳相似度雖較他種接近，但仍存在有差異性，外觀形態也有些許不同；以紅鸝石斛為例：產於台灣杉林溪、谷關以及來自大陸雲南三者，以台灣自產者親緣相似度高於產自大陸者，故地域的遠近也會影響同種間之親緣相似度。

Genetic diversity and relatedness were assessed among 19 Dendrobium species such as D. chameleon, D. erumenatum, D. candidum, etc., using 10 randomly amplified polymorphic DNA (RAPD) primers. Of these Dendrobium studied, 14 are native in Taiwan, 2 from the Philippinges and 3 from mainland China. The phalaenopsis aphrodite seedlings derived from tissue culture were included for comparison. The plant morphology and biological traits were also investigated to study ecological effects on genetic composition. The plant growth was influenced by its epiphytic site and intercepted light intensity. Grown on rocks, the plant has smaller pseudobulb and less leaves than that grown in woods. The latter may be two times of the size of the former. Different Dendrobium species could be distinguished by the color,size and shape of flower. Other morphological traits could also be used to separate different species, such as angle of leaf growth, color of pseudobulb, internode length and growth habit. The brilliant orange color of D. clavatum is the most beautiful, follower by pink series of D. falconeri, D. linawianum and D. miyakei. The bright white color of D. crumenatum. and yellow series of D. tosaense are also valuable. The flowering period of individual flower was longest in D. tosaense (15 days) , followed by D. linawinanum (11 d) and D. leptocladum and D. moniliforme (10 d), The shortest flowering period was observed in D. crumenatum and Flickingeria comata with life span less than one day. Self-pollination was found successful in D. clavatum , D. tosaense and F. comate. About 50% of fruit set from self- pollination was obtained in D. equitans, D. leptocladum, D. linawianum and Epigeneium nakaharaei. Cross pollination enhanced fruit set in D. crumenatum, D. falconeri, D. linawinanum and D. moniliforme. Twenty Operon primers (OPE-01~20) were used to screen for polymorphism. The results showed interspecific divergency. The Dendrobium species surveyed were classified into six groups. D. crumenatum from both Green Island and the Philippines as a group, D. miyakei(from both the Philippines and Orchid Island), D. chameleon and Epigeneium sanseiense in group Ⅱ, D. moniliforme, D. candidum, D. tosaense, and D. falconeri (from Ku-kuan, Sun-Link —Sea & Yun-Nan) in group Ⅲ, D. clavatum and D. linawianum in group Ⅳ, D. somai and D. leptocladum in groupⅤ, Flickingeria comata, F. tairukountia and F. finbriata in group Ⅵ. Low similarity value detected from D. miyakei suggested it as another genus. Species in group Ⅱ and Ⅴ may also be treated as independent genera. Genetic diversity as well as morphological difference existed between accessions of the same species from different regions. As in D. falconeri, accessions from Ku-kuan and Sun-Link —Sea are genetically more similar in between than to that from Yun-Nan, China. Geographical distance might affect intra specific similarity.