Applications of Biotechnological Approaches in the Product and Breeding of Phalaenopsis Orchids

Abstract

Phalaenopsis orchids native to the tropics are called “Moth Orchids”. It is one of the most commercially popular orchids because of its beautiful, colorful, and long-lasting variety of flowers. Biotechnology used in the production and breeding of Phalaenopsis was reviewed in this chapter. In the commercial production of Phalaenopsis, biotechnologies, such as methods of aseptic sowing and tissue culture, have been used for a long time. Recently, molecular phylogenetic analysis of original species and molecular breeding by the transformation of Phalaenopsis has been actively studied. The role of biotechnology in the Phalaenopsis orchid industry is significant, and the development of the technology in this field will bring further benefits to researchers, producers, and fancier of Phalaenopsis orchids.

1. Introduction

The genus Phalaenopsisconsists of approximately 60 species and the various traits of hybrids are due to easy interspecific and intergeneric crossing compared to other higher plants. Germination and propagation of Phalaenopsisin nature (or under natural conditions) are very difficult since their seeds contain no endosperm storing nutrients for germination. Therefore, “micropropagation” (mass proliferation) by aseptic culture technologies has been used for the research and industrial production of Phalaenopsisfor a long time before. In recent years, molecular breeding by biotechnology has been extensively studied. In this chapter, we will review the latest knowledge of classification, proliferation methods, and molecular breeding of Phalaenopsisby biotechnology.

2. Phalaenopsisand related genera

2.1 Classification

The genus Phalaenopsis(Orchidaceae) is classified as subfamily Epidendroideae, tribe Vendeae, and subtribe Aeridinae[1]. The native species of Phalaenopsisare distributed throughout northern Australia to southern India, China, and Taiwan in tropical Asia. Cultivars called moth orchids (Phalaenopsisand Doritaenopsis) are mainly generated by crossing native species of genus Phalaenopsisand genus Doritis(Doritis pulcherrima). Because Phalaenopsisis amenable to artificial crossing with other species and even other genera, such as Doritis, Ascocentrum,and Vanda, many of the cultivars have been produced as interspecific and intergeneric hybrids [2].

2.1.1 Morphological classification

PhalaenopsisOrchids have been classified morphologically by unique features, such as pollen. Phalaenopsisare epiphytic orchids, which live sticking to trees. They are monopodial plants with a short stalk and three to six widely and fleshy leaves. Their flowers consist of sepal, petal, lip, and column, which are flower structures particular in Orchids. The genus Phalaenopsisare defined by Blume in 1825 and has been classified by many taxonomists mainly based on morphological features of flower structure [3] and the number of pollens [2, 4] and based on cytogenetic features, [5, 6] such as a number of chromosomes, chromosome shape, and permissibility of crossing.

Christenson [7] defined the genus Phalaenopsiswhich consists of 62 original species. He divided the genus into five subgenera by morphological classification. Of these, two subgenera also were subdivided into four sections. He integrated the genus Doritis, which has been treated as an independent genus by other taxonomists, into the genus Phalaenopsis(section Esmeralda) in the broad sense (Table 1). He systematically described species characteristics, habitat, history of discovery, etc. in this work. Currently, his work is one of the most referenced in the classification of Phalaenopsisorchids.

Genus	Subgenus	Section	Species
Phalaenopsis	Proboscidioides	Proboscidioides	lowii
	Aphyllae	Aphyllae	taenialis
			braceana
			minus
			wilsonii
			stobartiana
			haiananensis
			honghenensis
	Parishianae	Parishianae	appendiculata
			gibbosa
			parishii
			lobbii
	Polychilos	Polychilos	mannii
			cornu-cervi
			borneensis
			pantheriana
		Fuscatae	cochlearis
			viridis
			fuscata
			kunstleri
		Amboinenses	pulchra
			violacea
			bellina
			micholizii
			fimbriata
			floresensis
			robinsonii
			gigantea
			fasciata
			doweryensis
			luteoka
			modesta
			maculata
			javanica
			mariae
			amboinensis
			luddemanniana
			venosa
			reichenbachiana
			pallens
			bastianii
			hieloglyphica
		Zebrinae	inscriptioshinensis
			speciosa
			tetraspis
			corningiana
			sumatrana
	Phalaenopsis	Phalaenopsis	philippinensis
			stuartiana
			amabilis
			aphrodite
			sanderiana
			schilleriana
		Deliciosae	chibae
			deliciosa
			mysorensis
			buyssoniana
		Esmeralda	pulcherrima
			regnieriana
		Stauroglottis	equestris
			celebensis
			lindenii

Table 1.

Classification of Phalaenopsis. Created with reference to Christenson [7].

2.1.2 Molecular phylogenetic classification

Differences in opinion on the importance of morphological features, such as pollen numbers caused disagreement among taxonomists. Therefore, molecular phylogenetic analyses based on DNA information independent from morphology have been actively studied. Molecular phylogenetic analysis [8, 9, 10, 11] supports Christenson’s proposal that the closely related genus Doritisand Kingidiumshould be included in the genus Phalaenopsis(section Esmeraldaand subgenus Aphyllae, respectively). However, because there were many consequences that classifications under subgenera do not match his classifications, reexaminations were proposed. Although many results that genus Doritisis classified into genus Phalaenopsisare shown, some taxonomists proposed that genus Doritisshould be used, considering the established Phalaenopsisintergeneric hybrids of Doritaenopsisin the past.

Recently, researchers reported that distantly related genera Lesliea, Nothodoritis, Ornithochilus, Hygrochilus, etc. should be included in the genus Phalaenopsis[11, 12]. In the genus Phalaenopsissubgenus Hygrochilus, a new species, Phal. pingxiangensiswere discovered in China [13]. Due to proposals for revision of classification criteria based on the molecular phylogeny of Phalaenopsisand related genera, the classification of Phalaenopsisorchids will become more diverse than ever.

2.2 Cultivars

The registration system for new cultivars of Orchids was established by Sander (Sander’s Complete List of Orchid Hybrids [14]), and now the Royal Horticultural Society in the United Kingdom (RHS) is taking over the system. Thus, the history of hybridization of orchid cultivars (horticultural varieties) can be traced to their original species. Today, the database of Sander’s list makes it easy for us to search for the ratio of each original species constituting orchid hybrids.

Major cultivars on the current market of moth orchids are divided into two groups (standard or novelty) (Figure 1). Standard types include traditional cultivars with white, pink, semi-alba (white flower with a red lip), and striped big flowers. Novelty types include cultivars with new colorful flowers, such as red, orange, yellow, multiple flowers, flowers of dots (spotted) or mottle (harlequin), and flowers with fragrance. Phylogenetic analysis of recent most popular cultivars revealed their original species composition as ancestors of these hybrids [15]. In standard cultivars, original species of subgenus Phalaenopsiswere the most important ancestors. Most white flower hybrids were the progeny of Phal. amabilis, Phal. aphrodite, Phal. schilleriana. The pink flower hybrids were progeny of Phal. amabilis, Phal. schillerianaand Phal. sanderiana. Phal. equestrisand Phal. stuartianawas important for the creation of semi-alba and striped hybrid cultivars, respectively. In novelty cultivars, original species in such subgenus Polycilosother than subgenus Phalaenopsiswere important ancestors. Of spotted/harlequin cultivars, the genetic contribution in the generation of red spots of the famous hybrid Phal. Golden Poker “Brother” was 25, 18.75, 12.5, and 6.25% from Phal. gigantea, Phal. leuddemanniana, Phal. Amboinensis,and Phal. faciata, respectively.

Figure 1.

Examples of cultivar types (standard, novelty).

3. Micropropagation

Phalaenopsisorchids are very difficult to germinate in nature because their seeds have no endosperm with nutrients for germination and to vegetatively propagate such as the method of bulb division. Therefore, propagation thorough aseptic culture has been desired. This section reviews the mass propagation methods using the tissue culture technic (micropropagation) and its problems in Phalaenopsis.

3.1 Aseptic sowing method

The aseptic sowing method greatly affected the industrial production of PhalaenopsisOrchids. A method of aseptically germinating the Phalaenopsisseeds with no endosperm on a medium that can artificially supply nutrients was developed. Many new cultivars have been created and produced in this method using various mediums, such as Knudson medium [16, 17], Vacin and Went (VW) medium [18], Murashige and Skoog (MS) medium [19], and Hyponex (Kano) medium [20]. However, the characteristics and quality of mature plants derived from seedlings are tending to vary genetically. Therefore, the development of a method to propagate moth orchids vegetatively using tissue culture and the production of clonal plantlets with the same traits has been desired.

3.2 Micropropagation

Protocorm-like bodies (PLBs) are generically used in the micropropagation of Phalaenopsis. PLBs are cell masses similar to protocorm, which is the state of enlarged embryos during orchid seed germination. PLBs are somatic embryos induced from somatic cells of Orchids [21]. Since a PLB can form a number of new secondary PLBs on the surface by culturing on an appropriate medium, the proliferation efficiency is very high (Figure 2) and then they grow to plantlets. On the other hand, callus induction is difficult in PhalaenopsisOrchid and embryogenic callus (EC) induction was first reported by Sagawa [22]. Although studies on proliferation using, such as EC, have also been conducted [23, 24, 25], the methods using PLBs are still mainstream in Phalaenopsismicropropagation because PLB is easier to grow to plantlet than a callus. To date, PLB induction methods using a variety of plant tissue have been established, as shown in Figure 3.

Figure 2.

PLB proliferation. a: PLB. b: Secondary PLBs formed on the original PLB.

Figure 3.

The process of micropropagation inPhalaenopsis. Micropropagation ofPhalaenopsisorchids is performed using PLB derived from various tissues.

3.2.1 Flower stalk culture

Flower stalk culture is firstly performed in a vegetative propagation system of Phalaenopsisfor PLB induction. In other Orchidaceae plants, PLB induction from shoot apex (shoot apical meristem) has been established. However, in monopodial Phalaenopsisorchids, varieties of alternate culture methods have been studied since only one shoot apex can be obtained from one strain and the removal of the shoot apex means the disappearance of the mother plant. Thus, flower stalk buds were firstly used for vegetative propagation of Phalaenopsisorchids [26]. Flower stalk culture is a method for obtaining the plantlets from dormant buds on the flower stalk. Although vegetative propagation systems that do not damage mother plants have been established by many researchers [27, 28, 29, 30], the propagation efficiency of this method is still lower because only one plantlet can be obtained from one flower stalk bud. Therefore, reproduction of shoots from these plantlets [31, 32] or PLB induction from these shoots/plants (as described below) was conducted in practice.

3.2.2 PLB induction from plantlets

PLB induction using leaf segments of plantlets obtained by flower stalk culture has been studied in detailed conditions, such as medium, plant growth regulator, plantlets condition, temperature, lighting intensity, and subculture interval, and practically used since early times by Tanaka et al. [33, 34]. Also, many PLB induction methods are being studied because the leaves are easy to obtain and use as explants throughout the year [35, 36]. Hyponex, VW, and 1/2 strength MS medium are often used in this culture method. Since PLB induction from leaves is adventitious, the use of plant growth regulators, such as α -naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP) is essential. Highly active Thidiazuron (TDZ) instead of BAP is often used. Recently, efficient induction by leaf thin-section culture [37] and PLB induction using original species of Phal. bellina[38] and Phal. cornu-cervi[39], which are difficult to induce the PLB, have been studied.

Roots on plantlets are also easy to use without losing the mother strains and ideal tissue for PLB induction [40]. Park et al. [41] reported that highly efficient PLB induction from root tip on a modified MS medium supplemented with 2.3 mM TDZ. On the other hand, although it is necessary to sterilize, PLB induction is also possible from the aerial roots exposed to the air of potted mature plants [42].

3.2.3 Direct PLB induction

PLB also can be directly induced from flower stalk tissue on the mother strain. Internode segments from flower stalks were cultured for PLB induction. PLBs were formed at the bottom of the section with 50–80% after transferring the segments to a culture medium. Thomale GD medium supplemented with 10% coconut milk, 5 mg/l NAA, and 20 mg/l BAP was effective for PLB formation [43]. PLBs were also induced on the VW medium as a basic medium. Green PLBs with high proliferative efficiency were induced from the shoot apex of flower stalk bud with one or two leaf primordia on ND medium (NDM) supplemented with 0.1 mg/l NAA and 1 mg/l BAP [44].

3.2.4 PLB proliferation

The proliferation efficiency of PLBs induced from the tissues remarkably increases by adding cutting treatment. The upper part (tip) is apt to differentiate the shoot and the middle and bottom (base) parts tend to form new secondary PLBs on dividing PLBs [33, 45]. Protocorms with the trimmed base were formed secondary PLBs efficiently [46]. The survival rate tends to decrease with the division of PLBs. However, the PLB proliferation rate could be increased without decreasing the survival rate by partially incising the top of PLBs after removing the tip part of the PLB (partial incision treatment) [47]. Enoki and Takahara [48] developed a highly efficient PLB proliferation system by combining this treatment with elongated PLBs showing skotomorphogenesis in the dark.

3.3 Problems with micropropagation

3.3.1 Browning and death

Browning and death during tissue culture are critical problems for plant species, such as Orchids, including Phalaenopsis, fruit trees, etc. Although tissue culture technologies with cutting are essential for micropropagation of Orchids, these plant species are very sensitive to injury. Injured tissues elute a large amount of secondary metabolites, such as phenol-like substances into the medium [49] and it is thought that oxidative condensation of these substances destroys the physiological balance of the plant and then causes the death of tissues. There is a positive correlation between the exudation of phenolic compounds to medium and the survival rate of tissue explants in Mango [50]. In Phalaenopsis, phenolic compounds exudation causes poor regeneration from cultured plant tissues [34].

This phenomenon is reaction called wound responses, which are known in many plant species. Injury on plants causes plant defense system to production of antibacterial active substances, such as phenolic compounds or their own programmed cell death by hypersensitivity reactions due to production of reactive oxygen species, to prevent wounds from additional infection of fungi or insects [51]. Browning and death will occur in the tissue culture since these reactions may be excessive in Phalaenopsisorchids. Of these reactions, phenol is synthesized by phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), etc. in phenylpropanoid synthesis pathway. In fact, enzyme activities including PPO are higher in browning tissues of Phalaenopsis[52]. Therefore, activated charcoal adsorbing phenol [53, 54], antioxidants such as ascorbate acid (vitamin C) [55], L-2-aminooxy-3-phenylpropionic acid (AOPP, inhibitor of PAL) [56], and cycloheximide (inhibitor of PPO) [57] were added to the medium in tissue culture of Orchids. A semisynthetic PhalaenopsisShoot Reproduction (PSR) medium was developed that relieves the effects of phenolic compounds and enhances the survival rate of the explants of Phalaenopsis[31].

Recently, transcriptome analysis of Phalaenopsisduring tissue browning provided comprehensive information on genes involved in browning and death other than the phenylpropanoid synthesis pathway [58]. However, the complex molecular mechanisms of browning and death are still unclear in Phalaenopsisorchids. Further elucidation of this molecular mechanism will make it possible to propose some more effective solutions to browning and death, and contribute to the commercial production of Phalaenopsis.

3.3.2 Interspecific and varietal differences

In the difficulty of micropropagation such as flower stalk [31], PLB [59], and callus [23] cultures of Phalaenopsis, there are large interspecific and varietal differences. This is probably because the moth orchid is a generic name for hybrids produced from various original species shown in Table 1. In fact, the ease of micropropagation in Phalaenopsiscultivars is due to characteristics of the original species involved in the creation of the cultivars [60], and thus there are few micropropagation methods that can be applied to all cultivars. Therefore, it is important to evaluate and estimate the proliferation difficulty of the original species in the development of the micropropagation method. Choice of proliferation methods based on the original species composition of the cultivars on Sander’s list and information about their propagation difficulties from these investigations will be necessary for breeding of Phalaenopsiscultivars.

4. Molecular breeding

Various transformation methods have been studied as tools for molecular breeding. To date, a number of high-quality cultivars have been produced by traditional crossbreeding since interspecific and intergeneric hybrids are easy to obtain in Orchids, compared to other plant groups. However, it takes a lot of time and labor in improvement by traditional breeding, because the vegetative growth periods and reproductive cycle of the Phalaenopsisorchids are very long. Furthermore, genetic resources for new traits which are important in commerce have limitations found within only Phalaenopsisand closely related, crossbreeding possible genera. The transformation methods are one of the molecular breeding methods capable of solving these problems. In this section, we summarize the transformation methods and the application examples in practice.

4.1 Genetic transformation methods used in Phalaenopsis

4.1.1 Major methods

Genetic transformation methods are powerful tools for introducing useful genes of other plant species into target plant species. Transformation is advantageous in breeding because it can modify only specific traits of target plant species. Crossbreeding with the aim of improvement of only a particular trait is not suitable for Phalaenopsisorchids that have long reproductive cycles because multiple backcrossing at various times is required. To date, two transformation methods of Agrobacterium-mediated transformation (AT) and particle bombardment (PB) have been mainly used in Phalaenopsisorchids. The former is a method utilizing Agrobacterium tumefaciens(synonym: Rhizobium radiobacter) having the property of infected plant cells and sending their own genes into the infected plant genome (Figure 4). This gene part, the T-DNA region, is replaced with a useful target gene to be introduced by molecular biology techniques in practice. In this method, transgenic plants are obtained by the process of infection of Agrobacteriumto explants, gene transfer by co-cultivation, sterilization of Agrobacterium, selection of transformed cells, and regeneration from the transformed cells to plants. The latter is a method of directly shooting gene-coated gold particles into cells using a gun device.

Figure 4.

Agrobacterium-mediated transformation.

Many AT methods rather than PB have been studied in the examination of efficient transformation conditions in Phalaenopsis(Table 2). The first reported transformation in Phalaenopsisorchids was using the PB method by Anzai (1996) [70]. Belarmino and Mii (2000) [61] reported the first transformation of PhalaenopsisOrchids by AT. Thereafter, the success of transformation by AT was reported one after another [62, 63, 64, 65, 66, 67, 68, 69]. Although PB has advantages, such as easy operation, and can be applied to a wide range of plant species and tissues, there is the largest bottleneck in the high cost of equipment and maintenance. The AT has lower maintenance costs and higher transformation efficiency than PB. In addition, gene silencing would occur less frequently and the later inheritance pattern of the transformed cultivar is also simple since a smaller number of copies of the gene are introduced in AT than in PB. The AT method has the disadvantage that it is difficult to use in monocotyledonous plants. However, the use of AT method in monocotyledonous plants, including Phalaenopsisorchids has also increased due to improved methods, such as the discovery of inducers for gene transfer into monocotyledonous plants in rice [81].

Method	Explant	Marker genes	Reporter/Target genes	References
Reporter genes
AT	Callus	hpt, nptII	gus	[61]
AT	PLBs	hpt	gus, GFP	[62, 63, 64]
AT	PLBs	nptII	gus	[65]
AT	protocorm	BP/KNAT1, nptII	GFP	[66, 67]
AT	protocorm	hpt	GFP	[68]
AT	protocorm	hpt, nptII	gus	[69]
PB	PLBs	bar, nptII	gus	[70]
Target genes
AT	Callus	hpt, nptII	Wasabi defensin gene	[71]
AT	Callus	nptII	LTP	[72]
AT	PLBs	hpt	PaFT	[73, 74]
AT	PLBs	nptII	GAFP-NPI genes	[75]
AT/PB	PLBs	hpt	CP, pflp	[76, 77]
PB	flower	—	F3’5’H, CYP78A2 gene	[78, 79]
PB	PLBs	—	PeUFGT3	[80]

Table 2.

Examples of the transformation of Phalaenopsis.

Abbreviations: AT, Agrobacterium-mediated transformation bar bialaphos resistance BP/KNAT1, Arabidopsis class 1 KNOX; CP, CymMV coat protein; F3’5’H, flavonoid-3, 5-hydroxylase; GAFP, Gastrodia Antifungal Protein; GFP, green fluorescent protein; gus, β-glucuronidase; hpt, hygromycin phosphotransferase; LTP, lipid transfer protein; NPI, Neutrophils Peptide-I; nptII, neomycin phosphotransferase II; PaFT, Phal. amabilis Flowering locus T; PB, particle bombardment; PeUFGT3, Phal. equestris UDP glucose: flavonoid 3-O-glucosyltransferase; pflp, sweet pepper ferredoxin-like protein; PLB, protocorm-like body.

4.1.2 Target explants

The key to successful transformation depends on the ability of the tissue to regenerate since Agrobacteriumparticularly tends to infect cells that are active in cell division and since desired good cultivars cannot be created if the regeneration from the transformed cell to the mature plant is impossible. PLBs are often used as the target tissues for transformation rather than callus because a series of regeneration processes from PLBs to plantlets has already been established in Phalaenopsisas shown in Figure 3. The protocorms are also sometimes targeted to perform crossbreeding in parallel with transformation.

4.1.3 Marker and reporter genes

Selectable marker genes with target/reporter genes are introduced into target explants. In general, an antibiotic resistance gene such as neomycin phosphotransferase II(nptII) (kanamycin resistance) or hygromycin phosphotransferase(hpt) (hygromycin resistance) is used as marker genes [82]. Since transformation in practice would not occur in all cells of target tissues, it is possible by culturing the explants infected with AT on a medium containing antibiotics responsible for marker gene to select and propagate only the transformed and survived cells, and then to regenerate the transformed plantlets.

At the stage of examining optimal transformation conditions, reporter genes are used instead of the desired target gene to be introduced. β-glucuronidase(gus) and green fluorescent protein(GFP) genes are popular as reporter genes [83]. Both genes are useful in calculating transformation efficiency because the success or not of transformation can be visually recognized in the introduced cells at an early stage in Phalaenopsis. The GUS-transformed cells exhibit blue color by giving a substrate solution from the outside. The GFP-transformed cells emit green fluorescence when exposed to ultraviolet rays. Although GFP is convenient because it does not need a substrate and transformed cells are not destroyed unlike the use of the GUS solution, there is a problem that it is difficult to distinguish green fluorescence from tissue color in the case of green color tissues.

4.2 Applications for breeding

In recent years, molecular breeding of moth orchids using useful target genes derived from other species and gene functional analysis of moth orchid itself using genetic transformation technique have been performed in practice (Table 2). Traits, such as new flower color, plant-pathogen resistance, and cold tolerance, which are important in commercial cultivation, are poor in genetic resources within the genera Phalaenopsisand Doritaenopsis. It is difficult to introduce such a trait to Phalaenopsiscultivars through conventional breeding methods. Therefore, molecular breeding using transformation methods have been studied.

4.2.1 Flower traits

In many flower plants, including Phalaenopsis, flower traits such as flowering time and new colors are important for breeding. To accelerate the floral transition and shorten the reproductive cycle of Phalaenopsis, transformants were obtained by overexpression of FT(Flowering locus T), a floral transition-related gene derived from Phal. amabilisby AT method [73, 74]. Overexpression of FT is known to be involved in early flowering by promoting floral transition in Arabidopsis thalianaand other species. Currently, functional analysis of this gene for flowering has been continued in the transformed Phalaenopsis.

Regarding the flower color traits, functional analysis of pigment synthesis-related genes of Phalaenopsisitself using the transformation method has been performed. Hsu’s group introduced flavonoid-3, 5-hydroxylase(F3’5’H) derived from Phalaenopsisinto the petal of Phalaenopsis, confirming that the flower color changed from pink to magenta [78]. In addition, they revealed by the same method that new CYP78A2 in the Cytochrome P450 (CYP 450) group of Phalaenopsis, which is specifically expressed in the pollen tube, is also involved in anthocyanin pigment synthesis [79]. Functional analysis of UDP glucose: flavonoid 3-O-glucosyltransferase(PeUFGT)-suppressed transformants in Phal. equestrisalso proved that this gene plays a crucial role in the anthocyanin synthesis pathway [80]. Cultivars with a blue flower, which are rare in nature, have been produced by transformation technology in many flower plants without blue pigment synthesizing ability. The creation of a blue rose by the introduction of exogenous F3’5’Hwhich is the key gene for the synthesis of delphinidin as blue pigment gave a great influence all over the world [84]. In addition to the previous blue carnation, blue chrysanthemums have also been produced in recent years by the same method. In Phalaenopsis, the first genetically engineered blue moth orchid using the same method was created by the group of Mii of Chiba University and Ishihara Sangyo Kaisha, Ltd. in Japan [85], and was exhibited for the first time in Japan in 2013.

F3’5’Hitself exists in Phalaenopsis, although there is no report on the presence of delphinidin in Phalaenopsis. Furthermore, the presence of varieties of the original species Phal. violaceaand Dor. pulcherrimaexhibiting blue color has been known since the olden days and Dtps. Kenneth Schubert as the world first’s blue moth orchid has been already produced by crossbreeding these original species. The moth orchid produced by the above transformation method and this cultivar is still not perfectly blue. It is known in many flower plant species that complex mechanisms due to some factors, such as pH, metal complex, and intramolecular stacking of anthocyanin, other than the kind of anthocyanin pigments are involved in the determination of blue flower color [86]. Although Griesbach [87, 88] revealed that some of these factors are involved in the blue flower color of Phalaenopsisby crossing test and chemical analysis of the hybrid and original species described above, the detailed molecular mechanisms which determine flower color are not clarified so far. Why does not the Phalaenopsisorchid with bright blue flowers still exist? Further elucidation of the molecular mechanism of blue coloration of the original species of Phalaenopsismay lead to perfect bluing of Phalaenopsisby molecular breeding using methods other than the introduction of pigment synthesis gene.

4.2.2 Plant defense

Disease resistance breeding is one of the important tasks in the breeding of Phalaenopsis. Infection of plant pathogens (bacteria and viruses) to plants causes serious damage to producers in the actual farm field. Recently, conferring pathogen resistance into Phalaenopsisby introducing foreign genes derived from other species is attempted. In transformed Phalaenopsiswith GAFP (GastrodiaAntifungal Protein)—NPI (Neutrophils Peptide-I) genes, the disease resistance to Colletotrichum gloeosporioidescausing anthrax disease was confirmed in vitroand in vivo[75]. The introduction of the Wasabi defensin gene derived from Wasabia japonicainto Phalaenopsisincreased the resistance to Rrwinia carotovoracausing soft rot disease [71]. The research group of Chan et al. [76, 77] reported that double transformation with Cymbidium mosaic virus (CymMV) coat protein (CP) and sweet pepper ferredoxin-like protein (pflp) genes confer dual resistance to CymMV and Erwinia carotovorainto Phalaenopsis. In the future, Phalaenopsiswith further multiple resistances to pathogens might be produced.

4.2.3 Cold tolerance

The breeding of low-temperature stress tolerance is a serious issue in the moth orchids which are tropical plants. In general, Phalaenopsisorchids have poor cold tolerance and the structure of the cell membrane degenerates at 15 degrees or less, and it suffers irreversible damage from low temperature. The lipid transfer protein (LTP) gene is involved in the transfer of monomers, such as wax and cutin, and the stabilization of plasma membrane. The expression of this gene is known to confer various biotic (such as fungi) and abiotic (such as cold) stress tolerance upon plants [89, 90]. In fact, the introduction of LTP derived from rice (Oryza sativacv. IAPAR9) into the callus of Phal. amabilisgave the regenerated transformed plants strong cold tolerance with growing healthy leaves at 10°C/7°C (day/night) [72].

5. Conclusion

The utilization of biotechnology such as micropropagation by tissue culture and transformation methods has played a very important role in the commercial production and breeding of Phalaenopsisorchids. The further development of such technologies in this field and the acquisition of new knowledge by many studies utilizing these technologies will contribute to the Phalaenopsisorchid industry.