*3.2.1 Flower stalk culture*

Flower stalk culture is firstly performed in a vegetative propagation system of *Phalaenopsis* for PLB induction. In other Orchidaceae plants, PLB induction from shoot apex (shoot apical meristem) has been established. However, in monopodial *Phalaenopsis* orchids, 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 *Phalaenopsis* orchids [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 [27–30], the propagation efficiency of this method is

**Figure 2.** *PLB proliferation. a: PLB. b: Secondary PLBs formed on the original PLB.*

*Applications of Biotechnological Approaches in the Product and Breeding of* Phalaenopsis*… DOI: http://dx.doi.org/10.5772/intechopen.104597*

**Figure 3.** *The process of micropropagation in* Phalaenopsis*. Micropropagation of* Phalaenopsis *orchids is performed using PLB derived from various tissues.*

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 *Phalaenopsis* orchids. 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

*Applications of Biotechnological Approaches in the Product and Breeding of* Phalaenopsis*… DOI: http://dx.doi.org/10.5772/intechopen.104597*

PPO) [57] were added to the medium in tissue culture of Orchids. A semisynthetic *Phalaenopsis* Shoot 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 *Phalaenopsis* during 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 *Phalaenopsis* orchids. 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 *Phalaenopsis* cultivars 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 *Phalaenopsis* cultivars.

#### **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 *Phalaenopsis* orchids are very long. Furthermore, genetic resources for new traits which are important in commerce have limitations found within only *Phalaenopsis* and 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

**Figure 4.** Agrobacterium*-mediated transformation.*

*Phalaenopsis* orchids 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 *Phalaenopsis* orchids. 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 *Agrobacterium* to explants, gene transfer by cocultivation, 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.

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 *Phalaenopsis* orchids was using the PB method by Anzai (1996) [70]. Belarmino and Mii (2000) [61] reported the first transformation of *Phalaenopsis* Orchids by AT. Thereafter, the success of transformation by AT was reported one after another [62–69]. 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 *Phalaenopsis* orchids has also increased due to improved methods, such as the discovery of inducers for gene transfer into monocotyledonous plants in rice [81].

#### *4.1.2 Target explants*

The key to successful transformation depends on the ability of the tissue to regenerate since *Agrobacterium* particularly 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

*Applications of Biotechnological Approaches in the Product and Breeding of* Phalaenopsis*… DOI: http://dx.doi.org/10.5772/intechopen.104597*


*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 ferredoxinlike protein; PLB, protocorm-like body.*

#### **Table 2.**

*Examples of the transformation of* Phalaenopsis*.*

processes from PLBs to plantlets has already been established in *Phalaenopsis* as 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 *Phalaenopsis* and *Doritaenopsis*. It is difficult to introduce such a trait to *Phalaenopsis* cultivars 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*. *amabilis* by AT method [73, 74]. Overexpression of FT is known to be involved in early flowering by promoting floral transition in *Arabidopsis thaliana* and 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 *Phalaenopsis* itself using the transformation method has been performed. Hsu's group introduced *flavonoid-3, 5-hydroxylase* (*F3'5'H*) derived from *Phalaenopsis* into 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*. *equestris* also 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'H* which 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'H* itself 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*. *violacea* and *Dor*. *pulcherrima* exhibiting blue color has been known since

#### *Applications of Biotechnological Approaches in the Product and Breeding of* Phalaenopsis*… DOI: http://dx.doi.org/10.5772/intechopen.104597*

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 *Phalaenopsis* by 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 *Phalaenopsis* orchid with bright blue flowers still exist? Further elucidation of the molecular mechanism of blue coloration of the original species of *Phalaenopsis* may lead to perfect bluing of *Phalaenopsis* by 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 *Phalaenopsis* by introducing foreign genes derived from other species is attempted. In transformed *Phalaenopsis* with GAFP (*Gastrodia* Antifungal Protein)— NPI (Neutrophils Peptide-I) genes, the disease resistance to *Colletotrichum gloeosporioides* causing anthrax disease was confirmed *in vitro* and *in vivo* [75]. The introduction of the Wasabi defensin gene derived from *Wasabia japonica* into *Phalaenopsis* increased the resistance to *Rrwinia carotovora* causing 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 ferredoxinlike protein (pflp) genes confer dual resistance to CymMV and *Erwinia carotovora* into *Phalaenopsis*. In the future, *Phalaenopsis* with 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, *Phalaenopsis* orchids 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 sativa* cv. IAPAR9) into the callus of *Phal*. *amabilis* gave 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 *Phalaenopsis* orchids. 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 *Phalaenopsis* orchid industry.
