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 & Zrd, 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 flavonoidbinding 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 lipidassociated 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 branchinhibiting 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 grafttransmissible 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 branchinhibiting 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 Fbox 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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G. Mangai Kasthuri Manju M. George

800 25,000 ( )

800 25,000 (Griesbach, 2002)

 DNA PEG (Phalaenopsis), (Oncidium), (Cymbidium) (Dendrobium)

(flavonoids), (carotenoids), (betalains) (Christinet et al., 2004; Trezzini & Zrd, 1991; WinkelShirley, 2001) 6 chalcones, flavones, flavonols, flavandiols, anthocyanins, tannins ( proanthocyanidins) ( Winkel-Shirley, 2001)(anthocyanins) (flavonol) (Mol et al., 1999) flavonoid 3,5-hydroxylase (Mol et al., 1999; Fukui et al., 2003) (Ipomoea tricolor cv. Heavenly Blue) pH 6.6 7.7 (Yamaguchi et al., 2001; Yoshida et al. 1995) pH Na+ / K+ (Na+/H+ exchanger, NHX1) NHX1 (Yamaguchi et al., 2001; Yoshida et al., 2005) Doritis pulcherrima () Na+/H+ exchanger (Mo) (Hale et al., 2001) Flavonoid R2R3 MYB basic helix-loop-helix

(bHLH) (Hernandez et al., 2004) Arabidopsis, Gerbera, MYB (Borevitz et al., 2000; Elomaa et al., 2003; Mathews et al., 2003; Mol et al., 1998; Ramsay et al., 2003) ANTHOCYANIN1 (AN1) bHLH (Spelt et al., 2002) AN1 dihydroflavonol 4-reductase dfrA (Spelt et al., 2000) MYB ANT1 (Mathews et al., 2003) flavonoid AN9 glutathione S-transferase (Mueller et al., 2000) BZ2 glutathione S-transferase (Marrs et al., 1995)bz2 (Alfenito et al., 1998) (betalains) betalain , 4,5-extradiol dioxygenase DODA Portulaca grandiflora DODA (Christinet et al., 2004) DODA (carotenoid-binding protein) (Vishnevetsky et al., 1999a) (CHRC) (Vainstein et al., 1994; Vishnevetsky et al., 1996) CHRC CHRC (Vishnevetsky et al., 1999b) 3 - (PAP) Brassica rapa PAP2 (Kim et al., 2001) fibrillin fibrillin (RNAi) DNA RNA fibrillin 6 ()

 (Morris et al., 2001) (Foo et al., 2001, 2005; Morris et al., 2001; Turnbull et al., 2002) max4 (Sorefan et al., 2003)max4 polyene dioxygenase (Sorefan et al., 2003) dioxygenase (Dad1/PhCCD8 ) CCD dad-1 Dad1/PhCCD8 (Snowden et al., 2005) plastidic dioxygenase carotenoids (Booker et al., 2004; Schwartz et al., 2004) MAX2 F-box leucine (Stirnberg et al., 2002) Gower Ramsey CCD (Chiang, S. F., Y. J. Tsai & F. C. Chen, ) CCD RNAi CCD Gower Ramsey (White Jade) (Sunkist) carotenoid violacea bellina equestris amboinensis leuddemaniana schilleriana parishii (da Silva et al., 1999; Dudareva et al., 2004; Kaiser, 1993; Nishida et al., 2004; Pichersky & Gershenzon, 2002) P. violacea (bellina) linalool geraniol (Kaiser, 1993) Clarkia Artemisia (linalool) (Cseke et al., 1998; Dudareva et al., 1996; Iijima et al., 2004b; Jia et al., 1999) (geraniol) (Iijima et al., 2004a) Clarkia linalool S-linalool 8hydroxylinalool (Lewinsohn et al., 2001) 3

(monoterpene) (Lucker et al., 2004) cDNA linalool (Goh& Arditti, 1985) 15-20 25 4 (Lee & Lin, 1984; Sakanishi et al., 1980) (Konow & Wang, 2001; Sakakibara et al., 1995; Sugiyama et al., 2001) 28-30 (Simpson et al., 1999) (He & Amasino, 2005) LEAFY (LFY) LFY LFY LFY LFY

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