Download Application of Biotechnology in Phalaenopsis Industry

Approaches and Perspectives towards Molecular Breeding of
Orchids
Fure-Chyi Chen, G. Mangai Kasthuri, Yi-Jung Tsai, Jian-Zhi Huang, Wen-Li Lee,
Roy Yuan-Hung Luo, Su-Feng Chiang, Ya-Huei Chen, Teen-Chi Cheng, Manju M.
George
National Pingtung University of Science & Technology, Taiwan
Abstract
Orchids are the largest family of flowering plants and consist of more than 800
genera and 25,000 species of which many are of commercial importance. Phalaenopsis
and Oncidium are two major and popular orchids grown for commercial production.
The commodity of these orchids includes potted plant and cut flower production.
Novelty is the driving force in ornamental plant industry. The novel forms (i.e.) variant
cultivars or new flower colors, shapes, plant and inflorescence architecture, longer shelf
life, fragrance modifications, disease and pest resistance are partly achieved through
conventional sexual hybridization and can be amended by advancement and
applications of gene transfer or genetic transformation mediated either by
Agrobacterium tumefaciens or particle bombardment for the development of cultivars
with novel or aesthetic properties as traditional breeding process aimed at genetic
modification is always limited by long reproductive cycle and cross incompatibility.
This article summarizes personal views about Phalaenopsis and Oncidium research,
the current state of art towards molecular breeding of orchids with reference to color
mutants, flower color manipulation, control mechanism of flowering etc., and future
perspectives on molecular breeding of economic orchids species.
Introduction
Orchidaceae is one of the largest families of flowering plants, which consists of
more than 800 genera and 25,000 species of which many are of commercial importance.
Phalaenopsis and onicidium are the two major and popular orchids grown for
commercial production as cut flower and potted plants. Potted orchid plants have been
the prime importance for agro industries world wide in recent years (Griesbach, 2002).
Traditional breeding methods (i.e. continuous crossing and selection) have paved the
way for the breeders to create new varieties that have desirable traits viz. color, shape,
fragrance, plant architecture, vase life and resistance to disease and pests but this kind is
the limited gene pool of any single species.
Molecular approaches have opened a new way for the genetic transformation of
plants to create novel traits, such as flower color modulations, floral formation, plant
and inflorescence architecture, fragrance etc. Potential application of this technology for
orchid improvement is being pursued in many institutions. Phalaenopsis and onicidium
orchids are being systematically produced as high value cash crops in Taiwan. The
creation of mutant cultivar has become an important factor in the market for commercial
orchid production. Novelty is the driving force in ornamental plant industry. Hence
molecular genetic modification can be adopted in addition to traditional breeding for the
emergence of plants with novel aesthetic properties.
Transgenic plants are produced via Agrobacterium-mediated and other direct DNA
transfer methods like PEG, electroporation, microprojectile bombardment, and
microinjection. In orchids protocorms from germinated seeds or protocorms-like-bodies
(PLBs) derived from shoot tip or leaves are the most easily obtained materials that are
capable of regenerating plants. The routine transformation procedure for orchids via
either Agrobacterium-mediated or microprojectile bombardment for introducing genes
with horticultural and economically important traits, such as virus disease resistance, is
being started. Several studies on successful transformation of Phalaenopsis, Oncidium,
Cymbidium, and Dendrobium have been reported. However, long period of selection
and regeneration, and low recovery of transgenic plants have hindered the efficient
transformation of these recalcitrant orchid species.
In this report, we try to elaborate the potential application of genes with valuable
horticultural traits reported in other crop plants in orchids. Approaches and strategies for
obtaining important genes are also discussed.
Targets for molecular flower breeding of orchids
Flower color modification
Flower color of higher plants is de to the production of pigments, including
flavonoids, carotenoids, and betalains (Christinet et al., 2004; Trezzini & Zrÿd, 1991;
Winkel-Shirley, 2001). Flovonoids contribute wide spectrum of colors in plants,
including red, blue, yellow and purple pigments. Six subgroupds of the flavonoids are
widespread in plants, including chalcones, flavones, flavonols, flavandiols,
anthocyanins, and condensed tannins (or proanthocyanidins)( Winkel-Shirley, 2001).
The biosynthesis of anthocyanin pigments and flavonol copigments made flowers
showy and function to recruit pollinators and seed dispersers. Direct modification of
anthocyanin production has been achieved in several plant species, such as petunia and
carnation (Mol et al., 1999). Wildtype carnation was modified to produce blue colors
after introducing a heterologous flavonoid 3′,5′-hydroxylase gene (Mol et al., 1999;
Fukui et al., 2003). Transgenic carnation with selected color shades has been introduced
in Japan market. This points the possibility of color modification through gene
technology in the non-food flower crops such as orchids.
The blue petals of the morning glory (Ipomoea tricolor cv. Heavenly Blue) change
from purplish red to blue during flower opening. The color shift was due to an increase
of the vacuole pH from 6.6 to 7.7 in the cells of epidermal layers (Yamaguchi et al.,
2001; Yoshida et al. 1995). The increase of vacuolar pH in the petals during flower
opening was due to active transport of Na+ and/or K+ from the cytosol into vacuoles by
the Na+/H+ exchanger (NHX1). Immunochemical and Northern blot analyses have
confirmed the correlation of the petal color change with the NHX1 (Yamaguchi et al.,
2001; Yoshida et al., 2005). The manipulation of color shift to blue may have the
potential application in phalaenopsis color modification using the Na+/H+ exchanger
from morning glory, Doritis pulcherrima (blue flowers), or other plant species using
genetic transformation technology. Interestingly, the metal ion molybdenum (Mo)
sequestration in the plant vacuoles was shown to bind with anthocyanins to make the
petals blue (Hale et al., 2001). This may also open an alternative choice for transgenic
blue phalaenopsis.
Flavonoid biosynthetic gene expression is regulated by the cooperation of R2R3
MYB and basic helix-loop-helix (bHLH) transcription factors, which activate
anthocyanin biosynthesis (Hernandez et al., 2004). The MYB transcription factors from
Arabidopsis, Gerbera, petunia and tomato have been shown to function in transgenic
plants producing anthocyanins in petals as well as other tissues (Borevitz et al., 2000;
Elomaa et al., 2003; Mathews et al., 2003; Mol et al., 1998; Ramsay et al., 2003).
ANTHOCYANIN1 (AN1) of petunia is a bHLH transcription factor required for the
synthesis of anthocyanin pigments (Spelt et al., 2002). Transgenic plants expressing
AN1 can activate the expression of the dfrA gene encoding the enzyme dihydroflavonol
4-reductase (Spelt et al., 2000). Similarly, overexpression of the tomato MYB
transcription factor ANT1 in tobacco and tomato up-regulated the expression of
anthocyanin biosynthesis genes in transgenic plants (Mathews et al., 2003). An
alternative approach for enhancing flower color is by the manipulation of the
flavonoid-binding protein. Petunia AN9 is a glutathione S-transferase responsible for the
sequestration of anthocyanins into vacuoles to make deeper flower color (Mueller et al.,
2000). The maize BZ2 gene also encodes glutathione S-transferase which is responsible
for the anthocyanin accumulation in the vacuoles (Marrs et al., 1995). bz2 mutants led
to the accumulation of anthocyanins in the cytoplasm where they were oxidized into
brown color (Alfenito et al., 1998).
An unusual flower pigment, betalains, has recently been studied in some detail.
The genes related to betalain biosynthesis have been cloned. One of them, DODA,
encoding 4,5-extradiol dioxygenase, was isolated and characterized from Portulaca
grandiflora at molecular level. The gene could function in a mutant white Portulaca
petal to complement the production of yellow pigments in the bombarded cells
(Christinet et al., 2004). This finding may have a potential application of the DODA
gene in color modification for orchids.
Many yellow-flower plants accumulate carotenoids by carotenoid-binding protein
located in the chromoplasts (Vishnevetsky et al., 1999a). A chromoplast-specific
carotenoid-associated protein (CHRC) was identified and cloned from cucumber corolla
(Vainstein et al., 1994; Vishnevetsky et al., 1996). The promoter of the CHRC gene was
fused to the reporter gene and bombarded into cucumber petals and other tissues, the
result showed unique expression of the CHRC promoter in the cucumber petal
(Vishnevetsky et al., 1999b). Three carotenoid-associated protein, or plastid
lipid-associated protein (PAP), were also isolated from Brassica rapa, with the PAP2
most abundant in the yellow petals (Kim et al., 2001). This result prompted us to clone
the homologous gene, fibrillin, from the yellow-flowered Oncidium. A full length
fibrillin cDNA has been cloned from the labellum (lip) of the Oncidium flowers. To
create white or pale yellow flowers of Oncidium, the RNA interference strategy will be
used to transform Oncidium PLBs with the expectation of knockout of the carotenoid
accumulation in flowers to make them white. A light colored orchid cut flower may
have the potential market in Japan.
Inflorescence architecture
Inflorescence branching is an important trait in Oncidium cut flowers. At least 6
branches in a flower stalk are regarded as top grades for domestic and export markets.
During the hot and humid summer time in subtropical areas like Taiwan, the
inflorescence branching is reduced to minimal degree, with probably only one single
branchless main stem present. During winter to spring time, the inflorescence branching
is enhanced. The control mechanism for this inflorescence architecture in Ondicium
orchid is currently unknown.
Observations and experiments from other plant species have contributed to our
understanding of the mechanisms of shoot branching. Through the study of mutants in
shoot branching of Arabidopsis and pea, several genes were implied to play a key role
for the shoot branching phenotype (Morris et al., 2001). In pea, reduced cytokinin
content in root xylem sap was observed in some branching mutants but not in others. A
novel graft-transmissible signal involved in the control of shoot branching was proposed
(Foo et al., 2001, 2005; Morris et al., 2001; Turnbull et al., 2002). A mobile
branch-inhibiting signal may have been produced in the wildtype plant, and could be
transmitted through grafting to the branching mutant max4 of Arabidopsis (Sorefan et
al., 2003). The gene encodes an auxin-inducible polyene dioxygenase family protein
(Sorefan et al., 2003). A carotenoid cleavage dioxygenase gene (Dad1/PhCCD8 ) was
identified in petunia shoot and root. The CCD enzyme regulates the biosynthesis a
graft-transmissible compound that inhibits branching. In the dad-1 mutant, the
Dad1/PhCCD8 was mutated and reduced the mobile signal so that more shoot
branching was observed (Snowden et al., 2005). The graft-transmissible
branch-inhibiting hormones were shown to be related to the plastidic dioxygenase that
cleaved multiple carotenoids in arabidopsis (Booker et al., 2004; Schwartz et al., 2004).
Another gene, MAX2, controlling shoot lateral branching has been cloned and shown to
be an F-box leucine-rich repeat family of proteins (Stirnberg et al., 2002).
Currently, a CCD homolog from Oncidium Gower Ramsey has been cloned from
the flower buds in this lab (Chiang, S. F., Y. J. Tsai & F. C. Chen, unpublished).
Expression level of the CCD in the flower stem as well as other tissues of the Oncidium
plants will be determined to check its relation with inflorescence branching. Transgenic
approach with CCD knockout by RNA interference strategy is being tested in order to
obtain free-branching Oncidum cut flowers. As there is several color mutants of the
Oncidium Gower Ramsey, namely the wildtype, the albino clone ‘White Jade’, and the
orange mutant ‘Sunkist’, they are valuable plant materials for molecular genetic studies
regarding to the carotenoid pigments changes due to their uniform genetic background.
They vary in carotenoid contents in the flowers. The mutants will offer us the
opportunity to examine the relationship of carotenoid biosynthesis and accumulation,
cloning and comparison of the corresponding genes, and subsequent transgenic studies
to elucidate their functions in flower pigmentation of Oncidium orchids.
Fragrance manipulation
Consumers are always allured by orchids with scents. Many orchid species emit
fragrance during the process of flowering. Many Phalaenopsis species, such as violacea,
bellina, equestris, amboinensis, leuddemaniana, schilleriana, and parishii, will emanate
mixtures of volatile components to attract insects and small animals, probably for the
purpose of pollination by these vectors, or as a defense mechanism (da Silva et al., 1999;
Dudareva et al., 2004; Kaiser, 1993; Nishida et al., 2004; Pichersky & Gershenzon,
2002). The main components in the P. violacea (bellina) are linalool and geraniol
(Kaiser, 1993). It is rather difficult to isolate genes corresponding to the target
component from Phalaenopsis flowers. The gene encoding linalool synthase has been
isolated and characterized from other plant species, such as Artemisia, Clarkia, basil
(Cseke et al., 1998; Dudareva et al., 1996; Iijima et al., 2004b; Jia et al., 1999). A
geraniol synthase gene has also been isolated from the basil (Iijima et al., 2004a). The
linalool synthase gene of the Clarkia has been introduced into tomato genome.
Transgenic tomato fruit produced more S-linalool and 8-hydroxylinalool in ripening
fruits (Lewinsohn et al., 2001). Similarly, three monoterpene synthase genes from
lemon have been introduced into tobacco with increased fragrance in tobacco flowers
(Lucker et al., 2004). These results suggest us the potential use of heterologous genes
for introducing into orchid genomes to make the orchid flowers more fragrant. Recently,
we have cloned a linalool synthase-like gene from cDNA subtracted library of the
Phalaenopsis flowers. It will be characterized at molecular as well as biochemical levels
to confirm its role in scent production of Phalaenopsis.
Flowering control
Flowering behavior varies in different orchids. Some species responds to
photoperiod and some to growth regulators (Goh& Arditti, 1985). In Phalaenopsis, a
four-week of night temperature at 15-20℃ and day temperature at 25℃ will induce the
spiking of inflorescence (Lee & Lin, 1984; Sakanishi et al., 1980). Light intensity
during the low temperature induction period may affect the spiking and quality of the
phalaenopsis plants (Konow & Wang, 2001; Sakakibara et al., 1995; Sugiyama et al.,
2001). It has become a standard practice to control flowering of the Phalaenopsis plants
in the orchid nurseries worldwide.
There are two aspects of flowering control. One is how to control flowering in
schedule production. The other one is how to prevent spiking of the mature plants in
order to export to foreign growers for forcing. To prevent or delay spiking of mature
phalaenopsis plants, growers usually raise greenhouse temperature up to 28-30℃ during
the growing period. However, many hybrids still may spike at some degree during the
high temperature vegetative growth stage. In order to understand the mechanism of
inflorescence induction and subsequent floral meristem development, it is necessary to
isolate genes up- or down-regulated during the flowering induction period. Regulation
of gene expression has been intensively studied in the model plant, Arabidopsis
(Simpson et al., 1999) and many monocot species. The results indicated conservation of
similar genes among different plant species (He & Amasino, 2005). One of the key
genes related to phase change from vegetative to reproductive stage is the LEAFY (LFY)
gene. We have recently cloned the LFY gene from a Phalaenopsis hybrid. Its function
involved in flowering will be studied by transformation of the gene into an Arabidopsis
mutant with defect in LFY. The promoter of the Phalaenopsis LFY will also be studied
to elucidate its role in flower induction and plant development.
Orchid Transformation Technology
Genetic transformation of several orchids either through Agrobacterium or particle
bombardment-mediated techniques has been reported, including Cymbidium,
Dendrobium, Oncidium and Phalaenopsis (Anzai et al., 1996; Belarmino & Mii, 2000;
Chai et al., 1994, 2002; Knapp et al., 2000; Kuehnle & Sugii, 1992; Liau et al., 2003a, b;
Men et al., 2003a, b; Yang et al., 1999; Yu et al., 2001). As regeneration protocol for
each orchid species varies, to achieving genetic transformation of the target orchid
hybrids requires a reliable and efficient regeneration system while avoiding somaclonal
variation in the transgenic plants. Despite of many reports on tissue culture and
subsequent plant regeneration, the protocol may not work during the antibiotic selection
procedure due to their sensitivity to the chemicals, and needs to be optimized for each
species.
Perspectives
Orchids have become daily consumption produce of most developed or developing
countries. Conventional breeding works have contributed to the avalanche new hybrids
every year. Biotechnology or molecular biology techniques will play an important role
for the improvement of commercial traits of orchid hybrids when combined with tissue
culture system. Isolation and characterization of genes regulating plant growth and
development of orchid species will help us understand their function. We optimistically
foresee the orchid hybrids with novel traits through molecular breeding in the near
future.
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蘭花分子育種的策略及展望
陳福旗、G. Mangai Kasthuri、蔡宜容、黃建誌、李文立、羅元宏、姜素芬、陳雅
惠、鄭汀琦、Manju M. George
國立屏東科技大學
摘要
蘭花為開花植物中最大的家族，總共超過 800 個屬及 25,000 個以上的原種，
其中不少蘭類具有經濟栽培價值。蝴蝶蘭及文心蘭為主要的栽培蘭花類，可供商
業生產，其產品型式包括盆花及切花。新奇性為觀賞植物產業成長的動力。利用
傳統的雜交育種，可以創造出新的株型、新花色、花型、花序排列及分叉性、花
朵壽命 (切花及盆花)、香氣的增強、抗病蟲性等。但是由於有些特性難以在實生
後代選拔，或種原中不具標的特性，因此利用農桿菌或基因槍媒介之基因轉殖技
術，可以創造新的特性，此種特性為傳統遺傳改良難以達成的，特別是雜交育種
的有性世代長，而且許多雜交組合具有不親和性或雜交障礙。因此結合傳統的雜
交育種及生物技術，可以進行蘭花的分子育種。
本文嘗試從個人的觀點，介紹蝴蝶蘭及文心蘭的研究，以及討論可以利用分
子育種進行改良的特性，例如由花色突變、改變花色的策略、開花之調節等；最
後討論經濟栽培蘭花利用分子育種的展望。
前言
蘭科植物為開花植物中最大的家族，總共超過 800 個屬及 25,000 個以上的原
種，其中不少蘭類具有經濟栽培價值。蝴蝶蘭及文心蘭為主要的栽培蘭花類，可
供商業生產，其產品型式包括盆花及切花。近年來，蘭花盆花已成為全球重要的
農產業(Griesbach, 2002)。傳統育種方法（即連續雜交和選拔）已為想要創造具優
良性狀，如：花色、花型、香氣、株型、花朵壽命和病蟲害抗病性等新品種的育
種者鋪好路了，但這種方法所創造的種原，僅侷限在各自物種內使用。
分子育種遺傳轉殖，已經為植物創造新奇性狀，如改變花色、花朵形成、株
型、花序排列和香氣等，開啟一條新方法，許多研究單位已經利用分子技術進行
蘭花育種。在台灣，蝴蝶蘭和文心蘭已經是企業化生產的高經濟作物，創造突變
新品種在蘭花商業生產市場上，已成為一重要因子；新奇性為觀賞植物產業成長
的動力。因此，除了傳統育種方法，分子遺傳改造可以用來育成新奇優良的品種。
以農桿菌轉殖和其他 DNA 直接導入，如 PEG、電穿孔、基因槍、微注射等轉
殖方法，可以產生轉殖植株。由蘭花種子發芽而來的原球體，或由莖頂或葉片誘
導而來的擬原球體是最容易取得且可以再生植株的材料，以農桿菌或基因槍法，
將園藝或重要經濟性狀如病毒病抗性已開始進行，也有許多文獻已報導能成功轉
殖 蝴 蝶 蘭 (Phalaenopsis), 文 心 蘭 (Oncidium), 蕙 蘭 (Cymbidium) 和 石 斛 蘭
(Dendrobium)， 然而，篩選和再生所需時間長，且轉殖植株的再生率低，都是阻
礙這些蘭花轉殖效率的原因。
本篇報導嘗試說明，由其他作物選殖之有價值的園藝性狀基因，應用在蘭花
的可能性，也將討論選殖及獲得重要基因的策略。
蘭花分子育種之目標
花色修飾
高等植物的花色是由於類黃酮 (flavonoids), 類胡蘿蔔素 (carotenoids), 和 甜
菜素 (betalains) 等色素產生造成的 (Christinet et al., 2004; Trezzini & Zrÿd, 1991;
Winkel-Shirley, 2001)，類黃酮對植物顏色的貢獻非常廣泛，包括紅、藍、黃和紫色 ，
類黃酮又可分為 6 類 ，包括 chalcones, flavones, flavonols, flavandiols, anthocyanins,
和 聚 合 的 tannins ( 或 proanthocyanidins) ( Winkel-Shirley, 2001) 。 花 青 色 素
(anthocyanins) 和黃酮醇 (flavonol) 的生合成使花朵亮麗耀眼，而能吸引授粉昆蟲
及種子散播動物。在許多物種已能利用生物技術直接修飾花青素生合成，如矮牽
牛 及 康 乃 馨 (Mol et al., 1999) ， 野 生 型 的 康 乃 馨 導 入 其 他 物 種 的 flavonoid
3′,5′-hydroxylase 基因，而可產生藍色的花朵 (Mol et al., 1999; Fukui et al., 2003)，
康乃馨篩選出之特別花色轉殖品種，已在日本市場上市了，這顯示經由基因轉殖
技術修飾花色的分子育種，應用在非食用的花卉如蘭花是可行的。
牽牛花 (Ipomoea tricolor cv. Heavenly Blue) 的藍色花瓣，在開花過程由紫紅
色轉變為藍色，顏色的改變是因為表皮層的細胞液泡 pH 值由 6.6 上升至 7.7
(Yamaguchi et al., 2001; Yoshida et al. 1995)，開花過程花瓣細胞液泡 pH 值的增加，
是由於細胞質的 Na+ 和/或 K+ 經由鈉離子質子轉運蛋白(Na+/H+ exchanger, NHX1)
以主動運送方式，傳送到液泡，以組織化學染色和北方雜合分析結果，也確認花
瓣顏色改變與 NHX1 的相關性 (Yamaguchi et al., 2001; Yoshida et al., 2005)。利用
牽牛花、朵麗蘭 Doritis pulcherrima (藍色花)或其他植物物種的 Na+/H+ exchanger
這種顏色改變的模式，也許可以應用在蝴蝶蘭基因轉殖上，使花色改變為藍色。 有
趣的是，在植物細胞液泡內的金屬離子鉬(Mo)被發現會與花青色素結合，而使花
瓣呈現藍色 (Hale et al., 2001)，這或許是育出藍色轉殖蝴蝶蘭的另一選擇。
Flavonoid 生合成基因的表達，受植物體內 R2R3 MYB 和 basic helix-loop-helix
(bHLH) 轉錄因子的共同調節，而能活化花青色素的生合成 (Hernandez et al.,
2004)。由阿拉伯芥 Arabidopsis, 非洲菊 Gerbera, 矮牽牛和番茄分離的 MYB 轉錄
因子，轉殖至其他植物已經顯示可以在花瓣和其它組織產生花青色素(Borevitz et
al., 2000; Elomaa et al., 2003; Mathews et al., 2003; Mol et al., 1998; Ramsay et al.,
2003)。矮牽牛的 ANTHOCYANIN1 (AN1) 是一種花青色素生合成所需的 bHLH
轉錄因子 (Spelt et al., 2002)，轉殖植物表達 AN1 後，可以活化產生 dihydroflavonol
4-reductase 酵素的 dfrA 基因的表達 (Spelt et al., 2000)，相同地，在轉殖煙草和番
茄中，過量表達番茄 MYB 轉錄因子 ANT1 ，會使花青素生合成基因表達增加
(Mathews et al., 2003)。另一個加強花色的方法是操縱 flavonoid 結合蛋白，矮牽牛
的 AN9 蛋白是一種 glutathione S-transferase 酵素，它能將花青色素隔離侷限在液
泡內，而使花色更深 (Mueller et al., 2000)，玉米的 BZ2 是 glutathione S-transferase
酵素的基因，它能將花青素累積在液泡內 (Marrs et al., 1995)，bz2 突變會導致花
青素累積在細胞質內，而使之氧化變為褐色 (Alfenito et al., 1998)。
最近一種少見的花朵色素甜菜素(betalains) 被仔細研究發表，betalain 生合成
相關基因已被選殖,，其中之一 4,5-extradiol dioxygenase 的基因 DODA，已從馬齒
莧 Portulaca grandiflora 花朵選殖出來，分子層次的特性也被研究完成，突變的白
花馬齒莧花瓣，經過基因槍轉殖 DODA 之細胞，可產生黃色素 (Christinet et al.,
2004)，由這個結果顯示，也許 DODA 基因可以應用在蘭花花色調控。
許多黃花植物以類胡蘿蔔素結合蛋白(carotenoid-binding protein)，將胡蘿蔔素
累積在雜色體內 (Vishnevetsky et al., 1999a)，從胡瓜花冠鑑定及選殖出一種雜色體
專一的胡蘿蔔素結合蛋白 (CHRC) (Vainstein et al., 1994; Vishnevetsky et al.,
1996)，將 CHRC 基因的啟動子接合報導基因，以基因槍轉殖到胡瓜花瓣和其他組
織，結果顯示 CHRC 基因的啟動子只會在胡瓜花瓣專一表達 (Vishnevetsky et al.,
1999b)。有 3 種胡蘿蔔素結合蛋白或質體脂質-結合蛋白(PAP)已從蕪箐 Brassica
rapa 選殖出，其中 PAP2 在黃色花瓣含量最高 (Kim et al., 2001)，這個結果促使我
們由黃花文心蘭花朵選殖到相似的基因 fibrillin。我們由黃花文心蘭的唇瓣，選殖
fibrillin 互補 DNA 的全長，我們將嘗試以 RNA 干擾技術(RNAi) 的策略，將 fibrillin
轉殖到文心蘭的擬原球體，希望能抑制花朵胡蘿蔔素的累積，而創造白花或淺黃
花的文心蘭，淺色系的蘭花可能在日本切花市場具有潛力。
花序構造
花序的分枝性是文心蘭切花重要的性狀，國內和外銷市場的頂級規格花，一
支花莖必須至少有 6 個分枝，在台灣等亞熱帶地區，酷熱與潮濕的夏季，花序分
枝減至最少，可能只有主花序而無分枝 (俗稱一條龍)，目前對文心蘭花序構造的
控制機制仍不清楚。
由其他植物種類的觀察與試驗報告，有助於我們對枝梢分枝機制的了解，從
阿拉伯芥與豌豆分枝性突變株的研究，似乎有許多基因在枝梢分枝外表性狀上，
扮演重要的角色 (Morris et al., 2001)，有些分枝突變種豌豆根部木質部汁液的細胞
分裂素含量減少，但有些突變種並無此現象。一種可藉由嫁接傳送訊息物質，與
控制分枝有關的假說被提出 (Foo et al., 2001, 2005; Morris et al., 2001; Turnbull et
al., 2002)，野生型的阿拉伯芥可以產生具移動性的分枝抑制訊息物質，並可經由嫁
接而傳送至分枝突變種 max4 (Sorefan et al., 2003)，max4 基因可以解碼轉譯出一種
可由生長素誘發的 polyene dioxygenase 家族蛋白質 (Sorefan et al., 2003)。從矮牽牛
的莖與根部鑑定出一種類胡羅蔔素裂解 dioxygenase 酵素基因(Dad1/PhCCD8 )，
CCD 酵素可以調節嫁接傳導抑制分枝物質的生合成，在 dad-1 突變株中，
Dad1/PhCCD8 基因發突變，可移動之訊息傳導物質減少，所以分枝增加 (Snowden
et al., 2005) ， 阿 拉 伯 芥 中 嫁 接 傳 導 之 分 枝 抑 制 賀 爾 蒙 被 認 為 是 由 plastidic
dioxygenase 將多種 carotenoids 裂解的產物 (Booker et al., 2004; Schwartz et al.,
2004)。另外一個控制枝梢分枝的基因 MAX2，也已被選殖，其為 F-box 富含 leucine
重複的蛋白質家族 (Stirnberg et al., 2002)。
近來，本實驗室已從文心蘭 Gower Ramsey 花苞，選殖出類似 CCD 的基因
(Chiang, S. F., Y. J. Tsai & F. C. Chen, 未發表)，我們將測定文心蘭花莖和植株其他
組織的 CCD 表現量，以了解它與花序分枝性的關係；並完成以 RNAi 策略進行轉
殖，測試壓制 CCD 的功能，以獲得分枝性強的文心蘭切花。由於一般的文心蘭
Gower Ramsey 已有許多花色的突變，有白變種 ‘白玉’ (‘White Jade’) 和橘色變種
‘香吉士’ (‘Sunkist’)，由於它們除了 carotenoid 色素的差異外，其他遺傳背景都相
同，所以這些品種是研究分子遺傳非常有價值的植物材料，它們的差異僅在花朵
類胡蘿蔔素的含量，所以我們正在進行測試類胡蘿蔔素生合成和累積的關係，選
殖並比較相關的基因，並進行後續的轉殖研究，以釐清這些基因在文心蘭花朵色
素形成的作用。
花朵香氣
消費者總是會被蘭花的香氣所吸引而購買蘭花，許多蘭花種類在開花過程都
會散發出香味，許多蝴蝶蘭屬的種，如 violacea、 bellina、equestris、amboinensis、
leuddemaniana、schilleriana 和 parishii 都會散發出揮發性的混合物質，以吸引昆
蟲和小動物來進行授粉，或做為一種防禦機制 (da Silva et al., 1999; Dudareva et al.,
2004; Kaiser, 1993; Nishida et al., 2004; Pichersky & Gershenzon, 2002)。P. violacea
(bellina) 香氣的主要成份是 linalool 和 geraniol (Kaiser, 1993)，要從蝴蝶蘭花朵分
離出目標成分的基因是相當困難的，由其他物種如艾草 Artemisia、Clarkia 和羅勒
的沉香醇 (linalool) 合成基因已被選殖出來，相關特性也以建立完成 (Cseke et al.,
1998; Dudareva et al., 1996; Iijima et al., 2004b; Jia et al., 1999)。羅勒的香葉醇
(geraniol) 合成基因也被選殖出來了(Iijima et al., 2004a)，山字草（Clarkia）的 linalool
合成基因已被轉殖導入番茄基因體內，轉殖的完熟番茄果實可以產生更多的
S-linalool 和 8-hydroxylinalool (Lewinsohn et al., 2001)；相似地，由檸檬選殖的 3
個單萜類(monoterpene)合成基因，也已經轉殖到菸草，而增加菸草花朵的香味
(Lucker et al., 2004)，由這些結果使我們認為，可將其他物種的基因轉殖導入蘭花，
以增加蘭花的香氣。最近我們已經從蝴蝶蘭花朵的扣除 cDNA 庫選殖到擬似
linalool 合成基因，將對這個基因進行分子及生化特性分析，以確認它在蝴蝶蘭香
氣產生所扮演的角色。
開花控制
不同種類蘭花的開花習性各不相同，有的受光週影響，有的受生長調節劑調
節 (Goh& Arditti, 1985)。蝴蝶蘭在 15-20℃夜溫及 25℃日溫下 4 週，就會誘導花梗
抽出 (Lee & Lin, 1984; Sakanishi et al., 1980)，低溫誘導花芽時期的光強度，也許
會影響蝴蝶蘭植株的抽梗與品質 (Konow & Wang, 2001; Sakakibara et al., 1995;
Sugiyama et al., 2001)，蝴蝶蘭植株開花的調控已成為全球蘭園的標準措施。
蝴蝶蘭開花控制包括兩方面，一是如何依生產規劃時間控制開花，另一方面
是如何在運輸過程之前，抑制外銷成熟株的抽梗，以利於外國當地進行催花栽培。
栽培者通常在蘭株營養生長期將溫室溫度提高至 28-30℃，以抑制或延遲成熟株的
抽梗，但有許多雜交種在高溫營養生長期，仍有部分植株會抽梗，為了瞭解花序
誘導和後續花芽生長組織發育的機制，必須分離出在開花誘導期的調控基因。模
式植物阿拉伯芥(Simpson et al., 1999)和許多單子葉物種開花誘導的基因表達調
控，已經研究相當清楚，結果顯示不同植物物種相似基因序列具有保守性 (He &
Amasino, 2005)，其中調控植物由營養相轉變至生殖相的重要基因為 LEAFY
(LFY) ，我們最近已經從一蝴蝶蘭雜交種選殖到 LFY 基因，我們會將選殖到的蝴
蝶蘭 LFY 基因，轉殖至 LFY 基因缺乏的阿拉伯芥突變株，以研究它在開花控制
的功能，我們也將研究蝴蝶蘭 LFY 基因的啟動子，以了解它在開花誘導與植株發
育所扮演的角色。
蘭花轉殖技術
許多蘭花如蕙蘭、石斛蘭、文心蘭和蝴蝶蘭，以農桿菌或基因槍轉殖方法進
行基因轉殖的研究已被發表(Anzai et al., 1996; Belarmino & Mii, 2000; Chai et al.,
1994, 2002; Knapp et al., 2000; Kuehnle & Sugii, 1992; Liau et al., 2003a, b; Men et al.,
2003a, b; Yang et al., 1999; Yu et al., 2001)，由於各種蘭花再生方法不同，要達成目
標蘭花雜交品種的基因轉殖，必須建立可信與有效率的再生系統，避免轉殖植株
在組培過程產生芽體變異，雖然組織培養和後續植株再生技術已有許多報告，但
這些方法可能因為培植體在抗生素篩選過程對化學藥劑敏感，而無法成功培養，
且針對每個物種或品種都須要研發不同的方法。
未來展望
蘭花已成為多數已開發或開發中國家的日常消費商品，傳統育種工作每年也
育成數量如雪花般多的新品種。生物技術或分子生物技術在組織培養技術配合
下，將會在蘭花雜交種商業性狀改進上，扮演重要的角色，分離與鑑定調節蘭花
生長與發育的基因，將有助我們了解它們的功能，在不久的將來，我們樂觀地期
盼將以分子育種方法育成具新奇性狀的蘭花新品種。
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中譯：種苗改良繁殖場
李美娟