Research On Orchid Biology and Biotechnology: Plant and Cell Physiology July 2011

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Research on Orchid Biology and Biotechnology
Article in Plant and Cell Physiology · July 2011

DOI: 10.1093/pcp/pcr100 · Source: PubMed
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Research on Orchid Biology and Biotechnology
Special Issue – Review

Yu-Yun Hsiao1,2, Zhao-Jun Pan1, Chia-Chi Hsu1, Ya-Ping Yang1, Yi-Chin Hsu1, Yu-Chen Chuang1,
Hsing-Hui Shih1, Wen-Huei Chen2, Wen-Chieh Tsai2,3,* and Hong-Hwa Chen1,2,3,*
1
Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan
2
Orchid Research Center, National Cheng Kung University, Tainan 701, Taiwan
3
Institute of Tropical Plant Sciences, National Cheng Kung University, Tainan 701, Taiwan
*Corresponding authors: Hong-Hwa Chen, E-mail, hhchen@mail.ncku.edu.tw; Fax, +886-6-235-6211; Wen-Chieh Tsai,
E-mail, tsaiwc@mail.ncku.edu.tw; Fax, +886-6-274-2583
(Received May 1, 2011; Accepted July 18, 2011)
Orchidaceae constitute one of the largest families of angio- common ancestor of extant orchids lived in the late
sperms. They are one of the most ecological and evolution- Cretaceous (76–84 Mya) as dated by a fossil orchid and its
ary significant plants and have successfully colonized almost
Downloaded from http://pcp.oxfordjournals.org/ at national cheng kung unversity on November 1, 2011

pollinator (Ramirez et al. 2007). The radiation of the orchid
every habitat on earth. Because of the significance of plant family probably took place in a comparatively short period as
biology, market needs and the current level of breeding compared with that of most flowering plant families, which
technologies, basic research into orchid biology and the ap- suggests that their speciation rates are presumed to be excep-
plication of biotechnology in the orchid industry are con- tionally high (Gill 1989).
tinually endearing scientists to orchids in Taiwan. In this Associated with the enormous number of Orchidaceae spe-
introductory review, we give an overview of the research cies is extraordinary floral diversification. Orchids are renowned
activities in orchid biology and biotechnology, including for an abundance of types, with a seemingly unending array of
the status of genomics, transformation technology, flower- strange and often fantastic variations, and represent a highly
ing regulation, molecular regulatory mechanisms of floral advanced and terminal line of floral evolution in the monocoty-
development, scent production and color presentation. ledons. This spectacular diversification has been linked to the
This information will provide a broad scope for study of specific interaction between the orchid flower and pollinator
orchid biology and serve as a starting point for uncovering (Cozzolino and Widmer 2005), sequential and rapid interplay
the mysteries of orchid evolution. between drift and natural selection (Tremblay et al. 2005), the
Keywords: Oncidium Orchidaceae Orchid biology role of obligate orchid–mycorrhizal interactions (Otero and
Orchid biotechnology Phalaenopsis. Flanagan 2006), and Crassulacean acid metabolism and epi-
phytism (Silvera et al. 2009). In addition to the prosperity of
Abbreviations: ANS, anthocyanidin synthase; BA, benzylami- their ecological manipulations, orchids have several unique re-
nopurine; BAC, bacterial artificial chromosome; BES, BAC end productive strategies that contribute to their success. These
sequence; bHLH, basic helix–loop–helix; CymMV, Cymbidium include mature pollen grains packaged as pollinia,
mosaic virus; DMAPP, dimethylallyl diphosphate; EST, ex- pollination-regulated ovary/ovule development, synchronized
pressed sequence tag; GDP, geranyl diphosphate; GDPS, ger- timing of micro- and mega-gametogenesis for effective fertil-
anyl diphosphate synthase; GGPP, geranyldiphosphate; GUS, ization and the release of thousands or millions of immature
b-glucuronidase; IPP, isopentenyl diphosphate; LD, long days; embryos (seeds without endosperm) in mature pods (Yu and
MEP, methylerythritol phosphate; PLB, protocorm-like body; Goh 2001).
PTGS, post-transcriptional gene silencing; PYS, phytoene syn- Taiwan is one of the orchid cultivation and hybridization
thase; SD, short days; SSR, simple sequence repeat; VIGS, centers of the world, and the fine quality of Taiwan orchid
virus-induced gene silencing. hybrids has attracted consumers worldwide. Some of the Editor-in-Chief’s choice
most popular potted flowering plants in the floriculture
market are Phalaenopsis hybrids. Their stylish, elegant appear-
Introduction ance and extended longevity have boosted popularity among
With an estimated >25,000 species, orchids are the most spe- growers and consumers. Hybrids within the genus Oncidium are
cies rich of all angiosperm families. They show a wide diversity also some of the top-traded cut flowers and differ from other
of epiphytic and terrestrial growth forms and have successfully commercial genera by their predominant yellow coloration
colonized almost every habitat on earth. The most recent commonly accented with reds. Both the government and
Plant Cell Physiol. 52(9): 1467–1486 (2011) doi:10.1093/pcp/pcr100, available FREE online at www.pcp.oxfordjournals.org
! The Author 2011. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: journals.permissions@oup.com
Plant Cell Physiol. 52(9): 1467–1486 (2011) doi:10.1093/pcp/pcr100 ! The Author 2011. 1467
Y.-Y. Hsiao et al.
entrepreneurs have high hopes for the orchid industry and transition rates and less potential for further endoreduplica-
intend it to become another commercial success story like tion, and endoreduplication stops when the flower fresh
the IT industry. weight stops increasing (Lee et al. 2004). Furthermore, low tem-
Because of the thriving and prosperous orchid breeding and perature (15 C compared with the normal 25 C) can decrease
industry, plant scientists in Taiwan are well placed to study the growth and endoreduplication transition rates in P. aphro-
orchid biology and develop orchid biotechnology to apply to dite and Oncidium varicosum (Lee et al. 2007).
the orchid industry. In this article, we have reviewed the scien-
tific activities in orchid research including the status of genom- BAC end sequences (BESs) analysis
ics, transformation technology, flowering regulation and Two bacterial artificial chromosome (BAC) libraries (BamHI
molecular regulatory mechanism of floral development, scent and HindIII) constructed from P. equestris contain 120,000
production and color presentation. clones, for an 8.4-fold coverage. An overview of the
Phalaernopsis genome was performed by generating pair-end
sequences from 2,920 BAC clones, and a total of 5,535 BESs were
Genomics generated, representing 4.5 Mb, or about 0.3% of the

Karyotypes of Phalaenopsis orchids Phalaenopsis genome. Sequence homology searches of the
BESs revealed that 641 (11.6%) were predicted to represent
A better understanding of the karyotypes and DNA contents of
protein-encoding regions, and 1,272 (23.0%) contained repeti-
orchid will aid in the development of new cultivars of orchids.
tive DNA. Most of these repetitive DNA sequences were gypsy-
All Phalaenopsis species have the same chromosome num-
and copia-like retrotransposons (41.9 and 12.8%, respectively),
ber (2n = 2x = 38), but their genomes vary considerably in
size. Analysis of karyotypes of nine Phalaenopsis species and and only 10.8% were DNA transposons. Furthermore, 950 po-
Doritis pulcherrima by Feulgen- and 40 ,6-diamidino-2- tential simple sequence repeats (SSRs) were discovered.
phenylindole (DAPI)-stained somatic metaphase chromosomes Dinucleotides were the most abundant repeat motifs; AT/TA
from root tips revealed P. aphrodite, P. stuartiana, P. equestris, dimer repeats were the most frequent SSRs, representing 253
P. cornu-cervi and P. lueddemanniana with small and uniform (26.6%) of all of the identified SSRs. Microsynteny analysis re-
chromosomes (1–2.5 mm), and all are metacentric or submeta- vealed that more of the BESs mapped to the whole-genome
centric. Phalaenopsis venosa, P. amboinensis, and P. violacea sequences of poplar than to those of grape or Arabidopsis, and
have bimodal karyotypes, with large and small chromosomes, even fewer to the rice genome. This work can facilitate the
and most are subtelocentric or acrocentric (Kao et al. 2001). analysis of the Phalaenopsis genome, which will help clarify
The distribution of heterochromatin in late prophase/early the relationship between orchids and other plant species
metaphase chromosomes indicates that in general, large (Hsu et al. 2011).
chromosomes contain more heterochromatin than do small
ones: Phalaenopsis species containing small chromosomes The chloroplast genome
show heterochromatin clustering around centromeres, whereas The entire chloroplast genome of P. aphrodite subsp. formosana
species containing large chromosomes possess large hetero- has been sequenced, with the generation of 148,964 bp se-
chromatin regions at both ends of the large metacentric/sub- quences, which encode 110 different known genes, including
metacentric chromosomes and the ends of the long arms of 74 protein-coding genes, four rRNA genes, 30 tRNA genes and
subtelocentirc/acrocentric chromosomes (Kao et al. 2001). two conserved reading frames of unknown function (Chang
Further investigation of genome organization and relationships et al. 2006). A comparison of nucleotide substitutions between
of Phalaenopsis species by genomic in situ hybridization (GISH) P. aphrodite and the grasses indicates that only the plastid ex-
revealed that species with large genomes had much more re- pression genes have a strong positive correlation between
petitive sequences, in both type and amount (Lin et al. 2005). non-synonymous (Ka) and synonymous (Ks) substitutions
per site, which provides evidence for a generation time effect,
Genome size analysis mainly across these genes (Chang et al. 2006). Furthermore, the
Flow cytometry has proven to be an efficient and reliable transcripts of 74 protein-coding genes from the chloroplast
method for analyzing plant genomes. The nuclear DNA con- genome of P. aphrodite subsp. formosana were used to study
tents from 18 Phalaenospsis species and D. pulcherrima were extensively the pattern of RNA editing in chloroplasts. A total
estimated by flow cytometry; 2C values ranged from 2.74 pg for of 44 editing sites are identified in the 24 transcripts of
P. sanderiana to 16.61 pg for P. parishii (Lin et al. 2001). The P. aphrodite chloroplast genes, and all are of the C-to-U con-
ploidy levels during orchid flower development were analyzed version type (Zeng et al. 2007). In addition, the entire chloro-
by combining a logistic growth model with an endoreduplica- plast genome of P. equestris has been sequenced and was found
tion model of Schweizer (Lee et al. 2004). Young floral buds to be very similar to that of P. aphrodite subsp. formosana
have lower ploidy levels than do old flowers, which suggests (C.C. Chang et al. unpublished data).
that the ploidy level increases with development. The results On the basis of the above information, the chloroplast
also indicated that cells with a higher ploidy level have lower genome of O. Gower Ramsey was sequenced by PCR and
1468 Plant Cell Physiol. 52(9): 1467–1486 (2011) doi:10.1093/pcp/pcr100 ! The Author 2011.
Orchid biology and biotechnology
Sanger-based ABI sequencing, and revealed 146,484 bp se- tissues, including developing seed, protocorm, vegetative tissue,
quences, which encode 133 genes. A total of 7,042 bp sequences leaf, cold-treated plantlet, pathogen-treated plantlet, inflores-
amplified from eight regions of the genome were used to iden- cence and flower buds (Fu et al. 2011, Hsiao et al. 2011). The
tify the relationships at the species level between the 15 EST sequences collected in OrchidBase were obtained through
Oncidiinae hybrids, which were supported by high bootstrap both deep sequencing with ABI 3730 and NGS Roche 454 and
values (Wu et al. 2010). Illumina/Solexa. The OrchidBase is freely available at http://lab
.fhes.tn.edu.tw/est and provides researchers with a high-quality
Expressed sequence tags (ESTs) genetic resource for data mining and efficient experimental
A subtractive EST library was constructed from the pseudobulb studies of orchid biology and biotechnology.
of O. Gower Ramsey, which plays a key role in water, carbohy- Another orchid transcriptomic database, Orchidstra (http://
drate and other nutrition support during floral development, orchidstra.abrc.sinica.edu.tw), was constructed from the
and 1,080 subtractive ESTs were obtained. Most ESTs were 233,924 unique contigs of the transcriptome sequences of
annotated as being involved in carbohydrate metabolism, in P. aphrodite subsp. formosana by use of a Roche 454 and
mannose, pectin and starch biosynthesis, transportation, and Illumina/Solexa platform, and the genes of tissue-specific ex-

stress-related and regulatory function (Tan et al. 2005). pression were categorized by profiling analysis with RNA-Seq
To study gene expression in Phalaenopsis reproductive (Su et al. 2011).
organs, a cDNA library was constructed from mature flower Oncidium cDNA libraries for six different organs, including
buds of P. equestris; 5,593 ESTs were sequenced and assembled leaves, pseudobulbs, young inflorescences, inflorescences,
into 3,688 unigenes (including 732 contigs and 2,956 singletons) flower buds and mature flowers, were generated from 50,908
(Tsai et al. 2006). The Phalaenopsis flower bud cDNA library contig sequences by use of the Roche 454 platform and were
contains a significant proportion of ESTs encoding enzymes of constructed into the OncidiumOrchidGenomeBase (http://
primary and secondary metabolism, then subcellular organiza- predictor.nchu.edu.tw/oogb/) (Chang et al. 2011).
tion-, transcription- and signal transduction-related genes. In All this EST information will be very useful for gene anno-
addition, a cDNA library has been constructed from scented tation in genomic sequencing, specificity of orchids, and organ-
P. bellina flower buds with the column removed; 2,359 ESTs ization of the orchid genome. The plentiful collection of ESTs
were sequenced and assembled into 1,187 unigenes (including and BESs in Phalaenopsis makes them reasonable candidates for
499 contigs and 688 singletons) (Hsiao et al. 2006). The set of orchid whole-genome sequencing. The two native Phalaenopsis
floral scent-producing enzymes in the biosynthetic pathway sepecies in Taiwan, P. equestris and P. aphrodite subsp. formo-
from glyceraldehyde-3-phosphate to geraniol and linalool is sana, are usually used as parents for breeding and have a rela-
recognized through these ESTs and distinguished by comparing tively small genome size, of 3.37 pg/2C and 2.80 pg/2C,
their expression patterns in P. bellina and a scentless species, respectively (1.6 109 bp and 1.3 109 bp, respectively). The
P. equestris (Hsiao et al. 2006). A similar strategy was adopted basic studies and genomics information collected have laid
for Vanda Mimi Palmer principally to mine any potential the groundwork for P. equestris to serve as a model orchid
fragrance-related EST-SSRs as markers in the identification of plant for whole-genome sequencing.
fragrant vandaceous orchids endemic to Malaysia (Teh et al.
2010).
Phalaenopsis ESTs derived from cDNA-amplified fragment
Virus-induced gene silencing (VIGS) for functional
length polymorphism (cDNA-AFLP) or randomly amplified validation of genes in orchids
polymorphic cDNAs (cDNA-RAPD) were also reported. These Because of the long life cycle and inefficient transformation
methods were used to systematically screen a large number of system of orchids, the study of functional genomics of orchid
differentially expressed cDNA fragments in the wild type as well genes is not feasible. Alternatively, VIGS analysis involving a
as somaclonal variants (Chen et al. 2005, Hsu et al. 2008). symptomless Cymbidium mosaic virus (CymMV) for gene silen-
Several differentially expressed transcripts related to flower de- cing was developed for Phalaenopsis orchids (Lu et al. 2007). In
velopment and flower color were identified. Despite the limited Phalaenopsis plants inoculated with CymMV transcripts con-
number of ESTs generated by these two methods, they are still taining 500 nt of PeMADS6, an orchid floral organ identity
valuable for further studying their roles in orchid flower devel- B-class GLOBOSA/PISTILLATA-like gene, the transcription
opment and flower color regulation. level of PeMADS6 and the B- and C-class MADS-box genes
Recently, the OrchidBase has collected the transcriptome was reduced by up to 97.8% and the flower morphology was
sequences from Phalaenopsis cDNA libraries and assembled affected. This in vivo experiment demonstrates an efficient way
them into 84,617 non-redundant transcribed sequences to study gene functions involved in the reproductive stage of
(including 8,501 contigs and 76,116 singletons) (Fu et al. orchid plants with a long life cycle.
2011). The OrchidBase contains the transcriptome sequences Further application of this tool for Phalaenopsis func-
derived from 11 Phalaenopsis orchid cDNA libraries, which were tional genomics has been made possible by knocking down
constructed from different species, including P. aphrodite PeUFGT3 via VIGS in a Doritaenopsis hybrid derived from
subsp. formosana, P. equestris and P. bellina, and from different a cross of Dtps. ‘I-Hsin Lucky Girl’ Dtps. ‘I-Hsin Song’.
Y.-Y. Hsiao et al.
The PeUFGT3-suppressed Doritaenopsis hybrids exhibited vari- same time demonstrates that the target gene is turned on after
ous levels of fading of flower color, which was well correlated introducing new DNA into a cell. Three major methods for gene
with the extent of reduced PeUFGT3 transcriptional activity transformation include Agrobacterium tumefaciens-mediated
(Chen et al. 2011). transformation, microprojectile bombardment and direct gene
transformation. Agrobacterium tumefaciens-mediated trans-
formation is commonly used in dicots, whereas microprojectile
Transformation technology bombardment and direct gene transformation are mainly used
in monocots. Recently, several A. tumefaciens-mediated trans-
Orchids are used for cut flowers and potted plants, and con-
formation events for monocotyledonous ornamental plants,
situte an important ornamental industry in Taiwan because of
including Phalaenopsis, Oncidium, Dendrobium, Anthurium
their excellent properties, including long-lasting, fascinating
and iris, have been reported (Mishiba et al. 2005, Sanjaya and
flowers. Thus, the improvement of traits, such as flower
Chan 2007).
shape, color, fragrance, longevity, architecture, and disease
Li and co-workers (2005) developed a highly efficient proto-
and stress resistance, and creation of novel variations are im-
col for efficiently producing transgenic plants of O. ‘Sherry Baby

portant to increase the commercial value of orchids. Traditional
cultivar OM8’. PLBs pre-treated with 0.5 M sucrose for 2 h were
breeding can alter traits and produce new variants but is limited
transformed by particle bombardment with the pflp gene: trea-
in the use of germplasm of the same or closely related species.
ted PLBs showed 3- to 4-fold increased single-cell embryogen-
Because of processing the long juvenile periods and reproduct-
esis and 14.8-fold increased GUS expression compared with
ive cycles, farmers in orchid nurseries usually expend high cost,
untreated PLBs. Hence, sucrose-pre-treated Oncidium PLBs
much effort and long-term culture to modify traits or produce
can increase single-cell embryogenesis and efficiency of trans-
new hybrids.
formation (Li et al. 2005).
Orchid transformation Disease resistance
In past two decades, many researchers have devoted efforts to Maintaining or increasing the yield of orchid variants through
the study of gene transformation of orchids to improve orchid classical breeding is difficult because productivity is limited by
traits or create new orchid variants. Several factors are con- infection of the viral and bacterial pathogens that cause orchid
sidered essential for the gene transformation method of diseases, particularly by Odontoglossum ringspot virus (ORSV),
orchids. How to choose what kinds of explants and regener- CymMV and Erwinia carotovora (Chia et al. 1992). To solve this
ation capacity is important to increase the efficiency of trans- problem, efforts have been made to establish a genetic trans-
genic orchid plants. Using unsuitable explants such as callus formation system in orchids for resistance to orchid diseases.
cultures as target materials for transformation may cause chi- In 2003, You and co-workers successfully developed a novel
meric transgenic plants and often confuse the analysis because selection marker, the sweet pepper (Capsicum annuum L.)
of difficulty in maintaining single-cell embryogenesis or a high ferredoxin-like protein (pflp) gene, for O. ‘Sherry Baby cultivar
incidence of somaclonal variation (Ishii et al. 1998, Chen et al. OM8’ transformation by A. tumefaciens and particle bombard-
1999). In general, protocorm-like bodies (PLBs) have been ment. The pflp gene is a disease resistance gene, which encodes
generally used as target tissues because of their higher regener- a ferredoxin-like protein to decrease infection by E. carotovora
ation capacity for gene transformation of orchids, especially pathogen for soft rot disease. The authors further used
in Oncidium, Dendrobium, Cymbidium and Phalaenopsis. In E. carotovora as a selection agent to screen transformants,
addition, protocorms could also be used as target tissues thereby obtaining transgenic plants without antibiotic se-
(Mishiba et al. 2005). So far, PLBs have been used as target lection. The selection efficiency of PLBs transformed by
tissues for orchid transformation in Taiwan with Oncidium E. carotovora was greater than by hygromycin. This selection
orchid ‘Sherry Baby cultivar OM8’ (Liau et al. 2003, You et al. marker can contribute to the transformation selection system.
2003, Li et al. 2005) and Phalaenopsis orchid ‘TS340’ (P. Taisuco In addition, transgenic orchid plants with a pflp gene can
Kochdiam P. Taisuco Kaaladian) (Liao et al. 2004, Chan et al. enhance resistance to E. carotovora.
2005) as transgenic explants. Liao and co-workers (2004) established a gene transform-
Selectable marker genes can be used to monitor transgenic ation system for Phalaenopsis orchids for resistance to viral
events and manually separate transformants from non- disease. The authors used the concept of post-transcriptional
transformants on medium containing the selective agent. gene silencing (PTGS) at the small interfering RNA (siRNA)-
Most orchid selection markers are antibiotic resistance genes mediated RNA level. The constructs with a CymMV coat
encoding resistance proteins, such as neomycin phosphotrans- protein (CP) cDNA fragment and a nos terminator placed
ferase II, hygromycin phosphotransferase II and phosphinothri- downstream of a maize ubiquitin promoter were transformed
cin acetyl transferase. In addition, to obtain more successful into Phalaenopsis orchid ‘TS340’ by particle bombardment.
transformants, two reporter genes are widely used as selection Transgenic orchid plants showed enhanced protection against
markers: green fluorescent protein and b-glucuronidase (GUS) CymMV infection because of RNA-mediated resistance
fusion genes. Expression of these marker genes detected at the through a PTGS mechanism (Liao et al. 2004). Furthermore,
the authors attempted to confer both viral and bacterial disease Blanchard and Runkle 2006) and Zygopetalum (Lopez et al.
resistance on Phalaenopsis orchids by double transformation. 2003). The promotion of flowering in these orchid genera by
CymMV CP and Pflp cDNA were transformed into PLBs of exposure to low temperature suggests that flowering in other
Phalaenopsis ‘TS340’ by A. tumefaciens transfection. orchid species could also be regulated by temperature.
Transgenic orchid plants showed enhanced resistance to The ambient temperature treatment of warm day and cool
CymMV and E. carotovora infection. This is the first report night (28 C day/20 C night) significantly enhances the transi-
describing a transgenic Phalaenopsis orchid with dual resistance tion to an inflorescence branch (spike) for P. aphrodite within
to phytopathogens (Liao et al. 2004). 3 weeks (Chen et al. 2008). Uniform spiking can be reached in
Phalaenopsis orchids through temperature control with a 25 C
day/20 C night for 4–5 weeks. However, a cool temperature is
Flowering regulation required for continuing development of the inflorescence
branch. The branch will become a vegetative shoot if it encoun-
Flowering mechanism in Phalaenopsis and ters high temperature.
Oncidium At low temperature, blocking the sunlight with shields or

The control of flowering time has been widely studied by gen- keeping the plantlet under shelves to prevent exposure to sun-
etic analyses in several plant species, especially Arabidopsis, and light could not promote inflorescence branches in Phalaenopsis
a genetic network including four main pathways—photo- (Liu et al. 2010). These results suggest that accumulation of
period, vernalization, autonomous and gibberellinss (Blazquez sugar production through photosynthesis is important for
et al. 2001)—has been developed. The photoperiod pathway orchid flowering. Flowering is also promoted when the plants
senses seasonal changes in daylength, whereas the vernalization are exposed to short photoperiods, although at low tempera-
pathway monitors the prolonged exposure to low temperature. tures. The effects of daylength on protein synthesis and flower-
The gibberellin pathway promotes flowering under non- ing in D. pulcherrima have been studied: 2–3 cm spikes were
inductive photoperiods, and the autonomous pathway per- initiated under 9 h short-day (SD) conditions for 30 d with day/
ceives the plant development status to mediate flowering night temperature of 30 C/20 C, and spikes grew to 7–10 cm
(Shen et al. 2011). under SD conditions for 45 d (Wang et al. 2002). A cell
Usually, plants flower only after they reach a certain stage of division-related protein, p21, is more expressed in the SD con-
growth (i.e. maturity). For many orchids, 4–7 years are required dition than under the long-day (LD) condition (17 h) (Wang
to finish the juvenile stage and flower from seed (Goh and et al. 2003). Even though most Phalaenopsis species are SD
Arditti 1985). However, certain commercial hybrids can plants, there are several species of Phalaenopsis, such as P.
flower within 36 months from seeding (Hew and Yong 1997). bellina and P. violacea, which flower in summer, suggesting
Based on the study of orchid flowering regulation, various they are LD plants.
predominating pathways prevail for different species in In addition to ambient temperature, plant growth regulators
Phalaenopsis orchids. For example, autonomous, temperature (PGRs) also play important roles in inflorescence induction. A
(cool or ambient temperature) or hormone (cytokinin) path- wide range of PGRs, including gibberellins, auxins, cytokinins
ways may play an important role for P. aphrodite subsp. and ABA, affect flowering in orchids. However, different experi-
formosana. mental conditions and types of orchids may have various ef-
Reproductive development in the flowering shoot of fects. For Phalaenopsis, cytokinins [e.g. benzylaminopurine
Phalaenopsis orchids begins with the transition of the dormant (BA)] stimulate flowering, and auxin suppresses the BA effect;
meristem from producing vegetative structures to producing gibberellin is not effective when applied alone but when added
inflorescence branches, floral bracts and finally flowers. in combination with BA seems to accelerate the BA effect
Phalaenopsis orchids develop at least two undifferentiated slightly (Goh and Yang 1978, Hew and Clifford 1993).
bud primordia at each node, and then these buds will partially Cytokinins (e.g. BA) stimulate flowering in monopodial (e.g.
develop and become dormant to wait for proper conditions Phalaenopsis) and sympodial orchids (e.g. Dendrobium) (Goh
for flowering (Rotor 1959, Wang et al. 2002). When the weather and Arditti 1985). For Dendrobium (Lee and Koay 1986),
becomes appropriate, the upper bud elongates and emerges Doritaenopsis and Phalaenopsis (Blanchard and Runkle 2008),
through the epidermis of the stem and develops into an inflor- GA3 did not stimulate the BA effect. However, Doritaenopsis
escence (Wang 1995). Usually, the inflorescence emerges from and Phalaenopsis sprayed with BA and kept at 29 C did not
the fourth node below the apical leaf (Sakanishi et al. 1980). initiate an inflorescence. The promotion of flowering by BA
Flower bud initiation occurs after the spike has reached about application seems to suggest that cytokinins play a part in
5 cm long if the environment is favorable. Several previ- regulating the inflorescence initiation of Doritaenopsis and
ous studies of regulation of flowering in orchids are mainly Phalaenopsis, but its promotion depends on certain conditions,
of the effects of low temperature or daylength. Temperature and BA application is not an effective replacement for an in-
has been reported to control flowering in several orchid gen- ductive low temperature (Blanchard and Runkle 2008).
era such as Dendrobium (Rotor 1952), Miltoniopsis (Lopez Although gibberellin is not effective at inducing flowering, it
and Runkle 2006), Phalaenopsis (Sakanishi et al. 1980, increases flower spike length and flower size. When a plant with
Y.-Y. Hsiao et al.
an inflorescence <10 cm is subsequently grown at 28 C for that FT may be the target for OMASD1. In addition, four AP1/
extended periods, a spike can form a vegetative air plantlet AGL9 functional MADS-box genes, OMADS6, OMADS7,
instead of flower buds, buds may abort, or the stem may elong- OMADS10 and OMADS11, have also been characterized in O.
ate indefinitely without open flowers (Sakanishi et al. 1980, Gower Ramsey. OMASD6 is an SEP3 ortholog that is expressed
Wang 1995). The blockage of flower development under high in sepal, petal, lip and carpel, but rarely expressed in stamen.
temperature can be rescued by applying gibberellin exogenous- OMADS11 is a SEP1/2 ortholog with an expression pattern simi-
ly (Su et al. 2001). Dormant buds of Phalaenopsis contain a lar to that of OMADS6. OMADS7 is an AGL6-like gene whose
relatively high level of free ABA, whereas no detectable free expression pattern is nearly identical to that of OMADS6.
or bound ABA was found in flowering shoots (Wang et al. OMADS10 is a putative AP1 ortholog that is expressed only in
2002). A decrease in free ABA in buds may be associated with vegetative leaf, lip and carpel of mature flowers. The similar
bud activation and the development of flowering shoots. expression patterns of OMADS6, 11 and 7 indicate that their
Another important orchid in the Taiwan floral industry is transcriptional regulation is highly conserved in orchids during
Oncidium, a thin-leaf, epiphytic sympodial orchid with an evolution. Ectopic expression of these genes in Arabidopsis
enlarged bulb-like structure called a pseudobulb. The pseudo- shows different phenotypes. Overexpression of OMADS6, 11

bulb serves for water, mineral and carbohydrate storage that or 7 causes extremely early flowering, whereas 35S::OMADS10
supports both vegetative growth and reproduction (Herold and only causes moderate early flowering. Flower organ conver-
Lewis 1977, Zimmerman 1990, Stern and Morris 1992). The sions have also been observed in transgenic Arabidopsis, includ-
phase switch from vegetative to reproductive stage is defined ing carpelloid sepals and staminoid petals found in 35S::
by the bolting period from the pseudobulb base. The transition OMADS6 and carpelloid sepals produced in 35S::OMADS7.
to bolting (flowering) requires a radical change in the apical However, flower organ conversions are not observed in
meristem of the auxillary shoot apex of Oncidium. Once the 35S::OMADS11 or 35S::OMADS10 transgenic flowers, indicating
shoot meristem becomes committed to the new development that there might be a functional diversification in these genes in
program for bolting, it is considered the reproductive stage the regulation of flower transition and formation (Chang et al.
(Shen et al. 2009). Usually, Oncidium orchids flower twice per 2009).
year from March to May, and from September to November. The orthologs of FT and TFL1, OnFT and OnTFL1, have also
Cool nights are not required for Oncidium to produce flowers. been isolated and characterized from O. Gower Ramsey (Hou
Unlike Phalaenopsis, the regulation of Oncidium flowering is and Yang 2009). The mRNA of OnFT is detected in axillary buds,
more via the autonomous pathway, and it is tightly linked to leaves, pseudobulbs and flowers. Their expression remains at a
the nutrition status of the pseudobulb. lower level during the vegetative stage and increases during the
A large amount of mucilage composed mainly of mannan reproductive stage. In flowers, OnFT is expressed more in young
was found in the pseudobulb during the early inflorescence flower buds than in mature flowers and is expressed mainly in
stage of O. Gower Ramsey (Wang et al. 2006). The ratio of sepals and petals. Although they are highly expressed in axillary
mannose to total sugar is 96.4% and outcompetes that of buds during vegetative stages, they are insufficient for the
other sugars such as arabinose, galactose and glucose by 1.5, flower transition, indicating that the production of the pseudo-
0.9 and 1.2%, respectively. Mannan will decrease gradually and bulb before flower transition is also necessary. The expression of
convert to starch with the emergence of the inflorescence. The OnFT is regulated by photoperiod, with the lowest expression at
starch synthesized at the developing inflorescence stage will daybreak and the highest from the eighth to the 12th hour of
eventually be degraded at the floral development stage the light period. This pattern is slightly different from FT in
(Wang et al. 2008). Arabidopsis, with the highest level at the 16th hour of the
light period and the lowest at dawn under LD (Corbesier
et al. 2007, Fujiwara et al. 2008), or Hd3a of rice and PnFT of
Molecular mechanisms of flowering initiation in
Pharbitis, which have the highest expression at dawn under SD
orchids (Ishikawa et al. 2005, Hayama et al. 2007, Tamaki et al. 2007).
Unlike Arabidopsis, in which research on flowering time has The difference may be due to Oncidium being a light-neutral
been done by examining the phenotypes of mutant plants, plant (Hew and Yong 1997). OnTFL1, on the other hand, is
the only available strategy to study flowering time in orchid is expressed only in axillary buds and pseudobulbs and is not
to search homolog genes identified in other species and to regulated by light. OnTFL1 can suppress the flower transition
overexpress or silence these genes to examine the phenotype during the vegetative stage with its high expression in axillary
changes. So far, several genes involved in flowering time regu- bud. Since the development of the pseudobulb is essential for
lation have been identified in Oncidium. OMADS1, an AGL6-like Oncidium, OnTFL1 also plays an important role in controlling
gene isolated from Oncidium, is able to up-regulate the expres- the length of the vegetative stage because of its high expression
sion of flowering time genes FT and SOC1, and the flower meri- in pseudobulbs. Flowering patterns are affected when overex-
stem identity genes LFY and AP1 in transgenic Arabidopsis (Hsu pressing OnFT and OnTFL1 in Arabidopsis. When OnFT is over-
et al. 2003). The late-flowering defect in gi-1 or co-3 can be expressed, the expression of AP1 is up-regulated and a strong
compensated by ectopically expressing OMADS1, indicating correlation between the level of AP1 and the expression of
OnFT is found, indicating that OnFT regulates flower transition A-class genes
similarly to Arabidopsis FT. Overexpression of OnFT in
At present, no nomenclature system exists for the MADS-box
Arabidopsis ft-1 mutants promotes flowering, but 35S::OnFT/
genes. Many of them were named according to their order
ft-1 transgenic plants still flower later than wild-type plants,
of identification. A-class MADS-box genes are placed in the
suggesting that OnFT cannot completely complement
AP1/AGL9 clade of MADS-box genes by phylogenetic analysis
Arabidopsis FT in regulating flowering time. On the other
(Theissen 2001). The A-class SQUAMOSA (SQUA) MADS-box
hand, delayed flowering and the production of more rosette
gene lineage within the eudicots is recognized as two subclades
leaves are observed in transgenic Arabidopsis overexpressing
(euAP1 and euFUL clades), whereas the non-core eudicots and
OnTFL1, and altered leaf and shoot morphology are also de-
monocots have sequences similar only to euFUL genes and are
tected. AP1 is observed to be down-regulated in both
classified in the paleoAP1 subclade (Litt and Irish 2003). Two
35S::OnTFL1 and 35S::OnTFL1 /tfl1-11, indicating that OnTFL1
functions are attributed to AP1: specification of (i) floral meri-
can prohibit flower transition and the development of floral
stem and (ii) perianth identity in Arabidopsis (Irish and Sussex
meristem by negatively regulating AP1, similarly to Arabidopsis
1990, Bowman et al. 1993).
TFL1 (Hou and Yang 2009).

So far, two A-class MADS-box genes, ORAP11 and ORAP13,
So far, flowering genes have been identified and character-
have been identified from P. hybrida cv. Formosa rose (Chen
ized in Oncidium, but studies of flowering time genes in
et al. 2007), one (OMADS10) from O. Gower Ramsey (Chang
Phalaenopsis are deficient. Although genes such as CO and
et al. 2009) and four (DOMADS2, DthyrFL1, DthyrFL2 and
FLC cannot be detected, several genes including AP1, FT, LFY
DthyrFL3) from Dendrobium (Yu and Goh 2000, Skipper et al.
and SOC1 have been found in OrchidBase or in Orchidstra.
2005). Based on results of the expression patterns and over-
These include vernalization insensitive 3 (PaVRN3-1 and
expression in transgenic plants, orchid A-class MADS-box genes
PaVRN3-2) and vernalization 2 (PaVRN2) in the vernalization
may be involved in the transition of flowering and floral organ
pathway. PaGI and PaFT were also identified. In addition, five
development. However, unlike its homolog AP1 in Arabidopsis,
autonomous pathway genes, PaFY, PaFVE, PaSVP, PaSOC1 and
orchid A-class MADS-box genes may not be associated with the
PaFCA, have also been identified from Phalaenopsis EST libraries
development of the first two whorls.
and were characterized under cool-night treatment (F.M. Tsao
In Arabidopsis, another A-class gene without a MADS-box
et al. unpublished data). With the information provided by the
region is APETALA2 (AP2). AP2 encodes a transcription factor
orchid database, more flowering time genes can be isolated for
with two continuous AP2 domains. AP2 enables A-class func-
functional analysis, and their role in the control of flowering in
tion in Arabidopsis and represses AGAMOUS (AG) in the first
orchid can be clarified.
and second floral whorls (Jofuku et al. 1994). Only one AP2-like
gene, named DcOAP2, from Dendrobium has been reported (Xu
et al. 2006). Transcripts of DcOAP2 were detected in all the
Molecular mechanisms of flower development floral organs, as was AP2 in Arabidopsis. However, the concur-
According to the classical view, the orchid flower is composed rence of the expression of DcOAP2 and DcOAG1, a putative
of five whorls of three segments, each including two perianth AG-like gene in all floral organs, implies that the mechanism
whorls, two staminal whorls and one carpel whorl. This struc- underlying the regulation of AG orthologs may be different
ture also conforms to the general flower structure of many from that in Arabidopsis (Xu et al. 2006).
other monocotyledonous families. Within the monocots, only
well-known crop species such as rice and maize have been B-class genes
studied thoroughly. However, their highly reduced flowers Both the developmental and biochemical aspects of B-class
make them unsuitable for general floral development studies. genes required to specify the identity of petals in whorl 2 and
All expected whorls in the flowers are present in orchids, stamens in whorl 3 appear to be conserved in many core eudi-
and their highly sophisticated flower organization offers an op- cots and monocots (Ambrose et al. 2000, Theissen et al. 2000,
portunity to discover new variant genes and different levels Whipple et al. 2004). So far, several members of APETALA3
of complexity within morphogenetic networks. Thus, the (AP3)-like and PISTILLATA (PI)-like genes have been isolated
Orchidaceae provide a rich subject for investigating evolution- from P. equestris and O. Gower Ramsey. These genes include
ary relationships and developmental biology to verify the val- four AP3-like genes and one PI-like gene identified in P. equestris
idity of the ‘ABCDE model’ in monocots and how MADS-box and three AP3-like genes and one PI-like gene isolated from O.
genes are involved in defining the different highly specialized Gower Ramsey (Hsu and Yang 2002, Tsai et al. 2004, Tsai et al.
structures in orchid flowers. Many orchid MADS-box genes 2005, Chang et al. 2010). In addition, two AP3- and one PI-like
related to floral development have been isolated (Table 1), gene were also identified in D. crumenatum (Xu et al. 2006). All
and we focus on the discussion of the roles of MADS-box these AP3-like genes in orchids are members of the paleoAP3
genes in P. equestris, O. Gower Ransey and C. ensifolium, be- lineage. The paleoAP3 genes identified in orchids were subdi-
cause these three species are major research targets in Taiwan vided into four subclades (Tsai et al. 2004, Mondragón-
(Fig. 1). Palomino and Theissen 2008, Pan et al. 2011). PeMADS2 and
1474
Table 1 MADS-box genes in orchids
Clade B-class C-class D-class AP1/AGL9
Y.-Y. Hsiao et al.
Subclade AP3A1 AP3A2 AP3B1 AP3B2 PI AP1 LOFSEP SEP3 AGL6

Phalaenopsis PeMADS3 PeMADS4 PeMADS2 PeMADS5 PeMADS6 PeMADS1 PeMADS7
equestris
Phalaenopsis PhalAG1 PhalAG2 ORAP11 ORAP13
hybrid
Oncidium OMADS9 OMADS5 OMADS3 OMADS8 OMADS4 OMADS2 OMADS10 OMADS11 OMADS6 OMADS1 OMADS7
Gower
Ramsey
Dendrobium DcOAP3B DcOAP3A DcOPI DcOAG1 DcOAG2 DcOSEP1
crumenatum
Dendrobium grex DOMADS2 DOMADS3 DOMADS1
Dendrobium DthyrPI DthyAG1 DthyAG2 DthyrFL1 DthyrFL2 DthyrFL3
thyrisiflorum
Cymbidium CeMADS1 CeMADS2
ensifolium
Plant Cell Physiol. 52(9): 1467–1486 (2011) doi:10.1093/pcp/pcr100 ! The Author 2011.
Habenaria HrDEF HrGLO1 HrGLO2
radiata
Spiranthes SpodoDEF1 SpodoDEF3 SpodoDEF4 SpodoDEF2 SpodoGLO1
odorata
Gongora galeata GogalDEF3 GogalDEF1 GogalDEF2 GogalGLO1
Phragmipedium PhlonDEF3 PhlonDEF4 PhlongDEF2 PhlonDEF1 PhlonGLO1
longifolium
Vanilla planifolia VaplaDEF3 VaplaDEF2 VaplaDEF1 VaplaGLO1
Orchis italica OrcPI


Fig. 1 Floral diagrams of Phalaenopsis, Oncidium and Cymbidium. (A) Phalaenopsis. (B) Oncidium. (C) Cymbidium. (D) Diagram showing orchid
flower organs (adapted from Tsai et al. 2005). Orchid flowers have three sepals and three petals. One of the petals is morphologically different in
structure and is known as the labellum or lip. The male and female reproductive parts are fused in a structure, the gynostemium or column, in
the center of the flower. The pollen grains stick together to form the pollinia located at the upper tip of the column under the anther cap.
OMADS5 belong to clade AP3B1 (the same as clade 1 proposed (Pan et al. 2011). After analyzing expression patterns of
by Mondragón-Palomino and Theissen 2008). OMADS3 and B-class genes by PCR-based methods and in situ hybridization,
PeMADS5 are included in clade AP3B2 (the same as clade 2). the ‘Homeotic Orchid Tepal (HOT) model’ was proposed to
PeMADS3 and OMADS9 are classified in clade AP3A1 (the illustrate the regulation of perianth morphogenesis in orchids.
same as clade 3) and PeMADS4 is in clade AP3A2 (the same In this model, PI and AP3B clades determine the formation of
as clade 4). sepals. The combination of PI and both AP3A1 and AP3B clades
Expression and protein–protein interaction studies suggest controls the lateral petal formation at the inflorescence stage,
that the specific combination of duplicate gene expression and whereas PI and AP3A1, AP3A2 clade genes and AGL6-like genes
protein –protein interactions is responsible for the develop- contribute to lip morphogenesis in the floral bud stage (Pan
ment of different floral organs (Tsai et al. 2008a, Tsai et al. et al. 2011). The HOT model is somewhat similar to the ‘orchid
2008b, Chang et al. 2010). However, the proposed models code’ (Mondragón-Palomino and Theissen 2008) but more pre-
derived from studies of Phalaenopsis and Oncidium to explain cisely points out the effect of PeMADS4 (AP3A2 clade) in deter-
lip development are diverse. From study of Phalaenopsis, the mining lip morphogenesis at a relevant later floral development
PeMADS4 gene (clade AP3A2 or 4) is suggested to play a critical stage.
role in determining lip development because its transcripts are The overexpression of paleoAP3 genes from Oncidium,
specifically detected in lip and in peloric mutant lip-like petals Phalaenopsis and Dendrobium under the control of the
(Tsai et al. 2004, Tsai et al. 2008a). However, OMADS5 (clade Cauliflower mosaic virus 35S promoter was examined in
AP3B1 or 1) may have a suppression effect on cell proliferation. Arabidopsis (Hsu and Yang 2002, Tsai and Chen 2006, Xu
Lack of expression of OMADS5 is necessary for the development et al. 2006). Consistently, the flower morphology of the trans-
of the lip or lip-like structures (Chang et al. 2010). Very recently, genic Arabidopsis plants overexpressing the orchid paleoAP3
B-class genes were collected from 11 species in four orchid genes was indistinguishable from that of wild-type plants. The
subfamilies except Apostasioideae to give a picture of absence of any morphological changes in the floral organs of
the floral organ development controlled by B-class genes transgenic plants suggested that sequence diversification
Y.-Y. Hsiao et al.
between orchid paleoAP3 and Arabidopsis AP3 genes causes classified in the C-lineage of AG-like genes, whereas OMADS2
some functional differences when placed into heterologous was in the D-lineage genes. Phylogenetic analysis showed
plants. that duplication of C-class genes may have occurred in
Only one PI-like gene was reported in P. equestris, D. crume- Epidendroideae. Our current understanding of these two para-
natum and O. Gower Ramsey: PeMADS6, DcOPI and OMADS8, log functions in Cymbidium suggests that after the duplication
respectively (Tsai et al. 2005, Xu et al. 2006, Chang et al. 2010). event, the lineages underwent functional diversification to pro-
All of these three genes are expressed in all floral organs. duce distinct functional repertoires. CeMADS1 may initiate the
However, OMADS8 is also expressed in vegetative roots and development of fused male and female reproductive organs
leaves, whereas both PeMADS6 and DcOPI are not expressed and be involved in floral meristem determinancy. However,
in vegetative tissues. In addition, PeMADS6 expressed in the CeMADS2 may play a maintenance role to complete gynoste-
undeveloped ovary suggests that the expression of PeMADS6 mium morphogenesis and have a redundant function in floral
in the ovary has an inhibitory effect on the development of the meristem determinancy (Wang et al. 2011). Good examples of
ovary (Tsai et al. 2005). Ectopic overexpression of PeMADS6, functional diversified genes are the rice C-class genes OsMADS3
DcOPI or OMADS8 in Arabidopsis demonstrated that both and OsMADS58 (Yamaguchi et al. 2006). The specification of

share the angiosperm PI function (Tsai et al. 2005, Xu et al. C function in rice is divided so that OsMADS3 is required for
2006, Chang et al. 2010). stamen identity, whereas OsMADS58 is mainly involved in floral
In conclusion, the expression patterns of B-class genes in meristem determinacy and carpel morphogenesis (Yamaguchi
orchid floral organs nicely fit the ‘modified ABC model’ in et al. 2006). Together these genes fulfill the functions that in
that the expression of the B-class genes is extended to whorl Arabidopsis are accomplished by a single AG gene.
1 in plants possessing nearly identical morphology of sepals and According to the expression patterns, OMADS4 is probably
petals (van Tunen et al. 1993). The paleoAP3 genes are highly the CeMADS1 ortholog in Oncidium. However, OMADS4 ap-
duplicated in orchids. Diversification and fixation of both of pears not to be functionally equivalent to CeMADS1.
these gene sequences and expression profiles might be ex- Transgenic Arabidopsis with overexpressed OMADS4 shows
plained by subfunctionalization and even neofunctionalization. an early flowering phenotype. This phenotype is not the
The driving force behind the specialized labellum and diversi- same as ectopic expression of CeMADS1 in Arabidopsis present-
fied orchid flowers may be linked to the duplication and fast ing the primary inflorescence apices terminated with a cluster
evolution rate of paleoAP3 genes. The final piece of the puzzle of flower buds. The protein behavior of OMADS4 is also of
for the unique evolutionary trajectory of orchid B-class genes is interest. OMADS4 and D-class protein OMADS2 can form
still hidden in the primitive Apostadioideae. Further investiga- homodimers and can form heterodimers with each other
tion of the B-class genes in Apostadioideae will undoubtedly (Hsu et al. 2010). Interaction between C- and D-class proteins
shed light on the mystery of the great diversity of orchid was still not observed in eudicots or in rice or maize.
flowers. Investigating the interaction behavior between C- and D-class
The models proposed to explain orchid floral development proteins in Phalaenopsis and Dendrobium is of interest, because
were reasoned from comparing expression patterns of B-class C- and D-class genes have been isolated from these two species
genes between wild-type and peloric flowers in orchids. Peloric (Skipper et al. 2006, Song et al. 2006, Xu et al. 2006).
flowers are actinomorphic mutants with lip-like petals (Tsai The expression pattern of the D-class gene OMADS2 is simi-
et al. 2004). Evidence that epigenetic mechanisms play a role lar to that of OMADS4. Flowering in Arabidopsis with over-
in peloric flowers includes methylation pattern variation (un- expressed OMADS2 has a phenotype similar to that of
published data). Because altered B-class gene expression is linked overexpressed OMADS4, except that 35S::OMADS2 transgenic
to the peloric flower of orchids, it will be of interest to isolate Arabidopsis flower earlier and produce curled leaves. Co-
cycloidea-like genes to study their roles on orchid flower zygo- expression in the stigmatic cavity and ovary, the ability to inter-
morphy and the regulation relationship between B-class genes. act with each other and the similar phenotypes when ectopi-
cally expressed in Arabidopsis suggest that OMADS2 and OMADS4
C- and D-class genes have a close relationship in regulating the formation of stigmatic
cavity and ovary in O. Gower Ramsey (Hsu et al. 2010).
According to the genetics ABCDE model, C-class genes specify
stamen and carpel development and function in meristem de-
termination because C-class mutants are indeterminate in E-class genes
whorl 4 and form a new C-class mutant flower instead of car- E-class MADS-box genes are placed into the SEPALLATA (SEP)
pels. The development of the column in orchids, which involves clade. The clade is characterized by a duplication coinciding
whorls 3 and 4, would be an interesting subject to elucidate the with the origin of the angiosperms that produced the SEP3
evolution of C-class genes. More recently, one C-class gene and (AGL9) and LOFSEP or AGL2/3/4 (SEP1, SEP2, SEP4) subclades
one D-class gene were isolated from O. Gower Ramsey, and two (Zahn et al. 2005). E-class genes are required for floral organ
C-class genes were isolated from C. ensifolium (Hsu et al. 2010, identity in all four floral organs and for floral determinacy (Ditta
Wang et al. 2011). OMADS4, CeMADS1 and CeMADS2 were et al. 2004, Kaufmann et al. 2005). In addition, members in the
AGL6 subclade are placed in the AP1/AGL9 group between the Pichersky and Gang 2000). In orchids, pollinators play an im-
SQUA-like and SEP-like subclade. Phylogenetic analysis of portant role in orchid floral diversification, which is advanta-
MADS-box genes and/or proteins has shown that AGL6-like geous to the evolution of a successful family. Large quantities of
genes are sister to the SEP-like genes (Zahn et al. 2005). pollen in masses are spread by bees, moths, flies and birds, and
Recently, an AGL6-like gene, OsMADS6, was found to have func- the floral scents serve as attractants for species-specific pollin-
tions in regulating floral organ identity and meristem fate in rice ators (van der Pijl and Dodson 1969). Floral signals from distinct
(Ohmori et al. 2009, Li et al. 2010). modalities elicited both attraction and copulation behavior,
So far, two E-class MADS-box genes and two AGL6-like genes as seen by continuous volatiles attractive to patrolling males
have been identified from O. Gower Ramsey (Hsu et al. 2003, and pollination by mimicking the pheromone and posture
Chang et al. 2009): OMADS11 in the LOFSEP subclade, OMADS6 of female thynnine wasps in Chiloglottis orchids (Australian
in the SEP3 subclade, and OMADS1 and OMADS7 in the AGL6 orchids) (Schiestl et al. 2003, Mant et al. 2005). An enormous
subclade (Chang et al. 2009). OMADS1 is expressed in apical variety of orchids pollinated by bees, wasps, flies and bumble-
meristem and in the lip and carpel of flowers, but not in vege- bee species cover a whole range of scents, from rosy-floral
tative tissues. Ectopic-expressed OMADS1 in Arabidopsis and ionone-floral to aromatic-floral and spicy-floral (Arditti

showed an early flowering phenotype and homeotic conversion 1992).
of flower organs, such as carpelloid sepals and staminoid petals
(Hsu et al. 2003). Interestingly, 35S::OMADS1 transgenic Difficulties in orchid scent research
Eustoma grandiflorum plants flowered significantly earlier and The global flower industry thrives on novelty. Domestication of
produced more flowers than did non-transgenic plants
wild species in conjunction with traditional breeding has long
(Thiruvengadam and Yang 2009). The other three genes were
been the principle path for generation of novel flowers in the
discussed in the section ‘Flower regulation’ and are not men-
industry. For orchid, traits such as flower color, shape and fra-
tioned here. Four E-class genes (OM1, DOMADS1, DOMADS3
grance are primary novel markers because they are key deter-
and DcOSEP1) have also been identified from Dendrobium (Lu
minants of consumer choice. However, many modern
et al. 1993, Yu and Guo 2000, Xu et al. 2006): DOMADS3 in the
floricultural varieties have lost their scent with traditional
LOFSEP subclade, and the other three genes in the SEP3 sub-
breeding programs. Breeders of orchids in cut-flower and orna-
clade. In addition, two E-class genes have also been identified
mental markets have focused on producing plants with im-
from P. equestris (our unpublished data). In general, the fact
proved vase life, shipping characteristics and visual aesthetic
that the expression pattern of E-class genes overlaps with that
values (i.e. color and shape).
of ABCD genes in orchids suggests that the higher order
Phalaenopsis Alliance is undoubtedly the most widely grown
MADS-box complexes are involved in all floral organ develop-
orchid in the world. Despite the sympatric speciation of orchids
ment in orchids as for Arabidopsis E-class genes. However,
being linked to differences in floral odors, large genome size,
whether orchid E-class genes play a role on floral determinacy
long life cycle and regeneration time of these plants, together
still needs to further investigated. Further investigation of these
with inefficient transformation systems, the investigation of
gene functions in orchids will provide useful information in
orchid scent biology is difficult. Furthermore, several scented
understanding the evolutionary roles of E-class MADS-box
and scentless species are cross-incompatible, which restricts the
genes in orchid flowering and/or flower development.
production of scented offspring by traditional breeding tech-
These findings are in line with current thoughts on how
niques. In some successful cases of cross-breeding, the progeny
major evolutionary changes in the genetic basis of organ iden-
have a diluted scent or have lost the ability to produce scent. To
tity were established by gene duplication and the separation of
date, the biosynthetic pathways of orchid flower fragrance have
functions. However, we cannot be sure that all the genes within
not been well understood. Little was known about the enzymes
each family have been isolated in orchids. Recently established
and genes controlling scent production in monocotyledons
orchid EST databases providing plentiful gene information, and
such as orchids.
integrated bioinformatic tools offer opportunities for finding
A-, B-, C-, D- and E-function paralogous genes or additional
genes related to flower development. The effort of many scien-
Orchid floral scent biosynthesis pathway and
tists will lead to a better understanding of the molecular and related genes
genetic mechanisms of orchid floral control. Phalaenopsis bellina, classified in the subgenus Polychilos, is
native to Malaysia, and numerous commercial varieties have
been bred because of the orchid’s pleasant fragrance. In add-
Flower scent ition, the species has some native tetraploid species to breed
scented commercial Phalaenopsis orchids and therefore is an
Significance and distribution of the orchid floral important parent for breeding scented cultivars. Floral scent is a
scent composite characteristic determined by a complex mixture of
Floral scent volatiles and pigments have evolved to attract low molecular mass volatile molecules and dominated by
insect pollinators and enhance fertilization rates (Kaiser 1993, monoterpenoid, sesquiterpenoid, phenylpropanoid and
Y.-Y. Hsiao et al.
benzenoid compounds, and fatty acid derivatives. The floral areas of plant biology, has come from the use of molecular
scents in P. bellina are rich in monoterpenes, geraniol and lina- and biochemical techniques. A strategy combining chemical
lool and their derivatives (Hsiao et al. 2006). They include analysis, genomics and bioinformatics was adopted to uncover
geraniol, nerol, 2,6-dimethyl-octa-3,7-diene-2,6-diol, 2,6- the scent biosynthesis pathway and the relevant genes in
dimethyl-octa-1,7-diene-3,6-diol, 3,7-dimethyl-2,6-octadienal, P. bellina flower (Hsiao et al. 2006; Fig. 2). According to volatile
geranic acid and 2,6-dimethyl-octa-2,6-diene-1,8-diol analysis, monoterpenoids are major compounds of scent
(Table 2). In contrast, no monoterpenoid derivatives were (Table 2) and therefore are probably biosynthesized in these
emitted in scentless P. equestris flowers; fatty acid derivatives, flowers.
phenylpropanoids and benzenoids were the major volatiles. From chemical and bioinformatics analyses, we deduced a
These compounds are barely detectable by the human nose. monoterpene biosynthesis pathway of 15 steps in the P. bellina
Research into plant scents has been hampered mainly by flower leading from glyceraldehyde-3-phosphate to geraniol,
the invisibility of this character, its dynamic nature and com- linalool and their derivatives (Hsiao et al. 2006; Fig. 3).
plex mixtures of components that are present in very Comparisons of the most abundant ESTs by calculating
small quantities. Most progress in scent research, as in other the enrichment factor for different transcripts in the floral

dbESTs of P. bellina and P. equestris facilitates the provisional
identification of scent metabolism genes. The enrichment
Table 2 Major classes of volatiles emitted by P. bellina and factor is obtained by dividing the proportion of a certain tran-
P. equestris script in the scented species by that in the scentless species.
Class of volatiles Amount Geranyl diphosphate synthase (GDPS), epimerase (EPI), lipox-
(ng flower1 h1) ygenase and diacylglycerol kinase show 15-, 10-, 10- and 8.5-fold
P. bellina P. equestris enrichment, respectively. Expression of the scent-related
(scent) (scentless) genes was confirmed by RNA blot hybridization (Hsiao et al.
Monoterpenes 382.8 ± 16.1 2006).
Linalool 105.4 ± 15.3 ND
Critical enzyme for scent biosynthesis
Linalool derivaties 39.6 ± 20 ND
trans-Geraniol 163.4 ± 1.6 ND Terpenoids belong to a large family of plant secondary metab-
Geraniol derivaties 34.5 ± 5.4 ND
olites, and their corresponding alcohols possess useful proper-
ties such as fragrance, flavor, insecticidal properties and
Sequesterpene ND 5.3 ± 2.1
characteristics that make them useful as pharmaceutical
Phenylpropanoid 38.6 ± 17.1 109.0 ± 14
agents. All monoterpenes are derived from the same substrate,
Benzenoid 40.2 ± 20.8 33.2 ± 7
GDP (C10), which is catalyzed by GDPS, a member of the
Fatty acid derivatives 3.3 ± 1.8 330.5 ± 35.0 short-chain trans-prenyltransferase family, via the condensa-
tion of dimethylallyl diphosphate (DMAPP) with isopentenyl
Construction of cDNA library of P. bellina

flower bud (remove column)
assembly and editing
1187 unigene database

(499 contigs and 688 singletons)
P. bellina P. equestris
Comparison of floral ungiene database in P. bellina

Data mining/literature and P. equestris
mapping with scent metabolism EST filtering:
keywords P. eqestris floral ungiene database (E-value <0.1)
Arabidopsis and Oryza floral ungiene (E-value <10-7)
Scent metabolism pathway in Scent metabolism related genes in

P. bellina P. bellina
Fig. 2 Strategy of identification of the Phalaenopsis scent metabolism pathway and scent candidate genes involved in the deoxyxylulose-5-
phosphate–geraniol–linalool pathway.
Fig. 3 Putative metabolic pathway from pyruvate and glyceraldehyde-3-phosphate to scent synthesis and related enzymes in P. bellina. DXPS,
deoxyxylulose-5-phosphate synthase; DXPR, deoxyxylulose-5-phosphate reductoisomerase; DMEC, 4-diphosphocytidyl-2-C-methyl-D-erythritol
2-phosphate cyclase; EPI, epimerase; GDPS, geranyl diphosphate synthase; NADPHDH, NADPH dehydrogenase (adapted from Hsiao et al. 2006).
diphosphate (IPP). GDPS is differentially expressed in The full-length cDNA of P. bellina GDPS (PbGDPS) was iso-
the scented species. All monoterpenes are formed from lated from a P. bellina floral cDNA library (Hsiao et al. 2006) and
GDP, which is synthesized from DMAPP and IPP (Tholl et al. sequenced. The PbGDPS lacks an aspartate-rich motif for scent
2004). production in the flower of P. bellina. Instead, a glutamate-rich
Y.-Y. Hsiao et al.
sequence (EAEVE) was identified in the SARM motif site to be hybrids was discovered between a species with small chromo-
able to catalyze the formation of both GDP and farnesyl di- somes and one with large chromosomes. Doubling the chromo-
phosphate (FDP; C15) by site-directed mutagenesis (Hsiao et al. somes of diploid scent species to form tetraploid species is
2008). Spatial expression analyses revealed that PbGDPS is a urgently needed to accelerate the improvement of scented
flower-specific gene. Temporal expression analyses showed commercial Phalaenopsis orchids.
the highest expression of PbGDPS during flower development
concomitant with the maximal floral emission of monoter-
penes at day 5 post-anthesis, which suggests that PbGDPS Flower color
plays a crucial role in scent production/emission in orchid
flowers. Further evidence of the involvement of PbGDPS in Flower pigments, including anthocyanins, carotenoids, beta-
floral scent emission came from the analysis of PbGDPS expres- lains and Chl, contribute to varied flower color patterns. In
sion in various orchid species with different scent-producing addition to attracting pollinators, these pigments play roles in
abilities (Hsiao et al. 2008). Expression of GDPS was also ana- photosynthesis and the response to UV radiation in vegetative
lyzed in two varieties of P. equestris. The scented flowers of P. tissues (Davies 2004). Anthocyanins, carotenoids and Chl are

equestris ‘W-72’ emit volatiles that are not monoterpenes, as do major pigments found in Orchidaceae, and anthocyanins have
those of the scentless species P. equestris. GDPS was expressed the broadest distribution. In this section, we discuss floral color
at lower levels in the scented offspring than in the parental line, in Phalaenopsis and Oncidium.
which is associated with the lower production of linalool in the
Anthocyanin biosynthesis pathway
former. In contrast, no GDPS expression was detected in P.
equestris or the scentless offspring D. Kenneth Schubert Enzyme and transcription factors involved in the anthocyanin
(Hsiao et al. 2008). Thus, PbGDPS may play a key role in the biosynthesis pathway have been well characterized in different
regulation of scent production in P. bellina flowers. species, such as grapes (He et al. 2010) and Arabidopsis thaliana
Floral scent research has mainly concentrated on the isola- (Martens et al. 2010), and have been reviewed extensively
tion and characterization of enzymes and genes involved in the (Grotewold 2006). The pathway starts with the condensation
final steps (terpene synthase) of scent biosynthesis. However, of coumaroyl-CoA and malonyl-CoA via a polyketide folding
scent production might not only be regulated by the level of mechanism by chalcone synthase (CHS) to form the intermedi-
these enzymes; the direct precursor (GDP) of the scent com- ate, chalcone, the primary precursor for all classes of flavonoids.
pounds could also contribute to the regulation (Guterman After the action of chalcone isomerase (CHI), chalcone is mod-
et al. 2006, Nogues et al. 2006). Meanwhile, several terpene ified to its isomer naringenin, which is further hydroxylated by a
synthases (TPSs) were identified from the P. bellina flower, group of cytochrome P450-dependent monooxygenases to
which has a dual function to catalyze monoterpene and sesqui- produce flavanones. Cytochrome P450-dependent monooxy-
terpene products. In addition, the MYB transcription factors genases include flavonoid 3-hydroxylase (F3H), flavonoid
were also identified from Phalaenopsis TF-Oligo microarray, 30 -hydroxylase (F30 H) or flavonoid 30 50 -hydroxylase (F30 50 H)
and they showed a similar expression pattern to PbGDPS, indi- for dihydrokaempferol, eriodictyol or pentahydroxyflavanone,
cating the possibility of MYB transcription factors being respectively. These three flavanones can be modified again by
involved in terpenoid biosynthesis regulation (unpublished the catalysis of F3H, F30 H and F30 50 H to produce dihydroflava-
data). nols, dihydrokaempferol, dihydroquercetin and dihydromyrice-
tin. Dihydroflavonol 4-reductase (DFR) further reduces these
dihydroflavonols to colorless leucoanthocyanidins, which are
Metabolic engineering of flower scent catalyzed by anthocyanidin synthase (ANS) to their corres-
To date, successful transformations have been developed for ponding anthocyanidins, pelargonidin, cyanidin and
several cut flowers, including commercial roses, chrysanthe- delphindin.
mums and carnations, but for most varieties work is still in A putative F30 50 H gene has been isolated from a
progress. Overall, it is clear that genetic manipulation of floral Phalaenopsis flower petal cDNA library (Su and Hsu 2003).
scent is possible but will require a more rational design based This gene contains a P450 conserved motif, Phe-X-X-Gly-X-
on the correct choice of species. If we hope to create new scent Arg-X-Cys-X-Gly (where X is a non-conserved amino acid),
varieties of Phalaenopsis, we need to understand scent compo- that is homologous to a published P450 found in pollen
nents, the pathways involved, key enzyme regulation, tubes of other Phalaenopsis. Bombardment of the F30 50 H gene
flux-controlling steps, possible feedback control, judicious use in Phalaenopsis petals altered the cell color from pink to ma-
of promoters and empirical testing in this candidate variety. genta as compared with those transformed with an antisense
For genetic engineering, most of the scented F1 plants are fragment, so the putative F30 50 H influences anthocyanin syn-
sterile, even if a few of these interspecific hybrids are obtained. thesis. Four color-related genes in Oncidium, i.e. OgCHS, OgCHI,
A high frequency of bivalents was observed in hybrids of both OgANS and OgDFR, have been identified (Chiou and Yeh 2008).
species with small chromosomes and between both species These genes are expressed during floral development. Among
with large chromosomes, but a low frequency of bivalents in them, OgCHI and OgDFR show especially low expression in
yellow lip tissue but greater expression in the red part of Carotenoids
Oncidium flowers. Transformation of OgCHI and OgDFR to- The carotenoid biosynthesis pathway is well established
gether by particle bombardment complements the absence
(Christopher et al. 2010, Lu and Li 2010). Carotenoids are C40
of anthocyanin synthesis in lip tissue. Several CHS, CHI, F30 H
isoprenoids synthesized via the methylerythritol phosphate
and ANS genes isolated from theP. equestris flower buds dbEST
(MEP) pathway in plastids. The MEP pathway starts with the
showed close correlation to red color formation in Phalaenopsis
formation of a basic C5 unit, IPP and DMAPP. Three molecules
(unpublished data), and the function of these genes need to be
of IPP are condensed with one molecule of DMAPP to form C20
further characterized.
diphosphate, and geranylgeranyl diphosphate (GGPP;
The anthocyanidins undergo further modification for hydro-
Dudareva et al. 2004), the common precursor of all caroten-
philicity, such as glycosylation, methylation and acylation, and
oids. Phytoene synthase (PSY) catalyzes the condensation of
become stabilized as vacuolar anthocyanins (Grotewold 2006).
two molecules of GGPP into phytoene, which is further satu-
UDP-glucose: anthocyanidin:flavonoid glucosyltransferase
rated by two enzymes, phytoene desaturase and z-carotene
(UFGT) is responsible for O-glycosylation of anthocyanidins
desaturase, into red-colored lycopene. Poly-cis-configured phy-
or anthocyanins. A UFGT isolated from the P. equestris flower

toene is transformed into all trans-form phytoene by
buds dbEST, PeUFGT3, showed high expression in red cultivars
z-carotene isomerase (Z-ISO) and carotenoid isomerase.
(Chen et al. 2011). Knockdown of the expression of PeUFGT3 by
Lycopene "-cyclase (LCYE) and/or lycopene b-cyclase (LYCB)
RNA interference resulted in various levels of fading color in
catalyzes the cyclization of lycopene and is an important
Phalaenopsis, so PeUFGT3 may be associated with red color
branch point of this pathway. The combination of LYCE and
formation in Phalaenopsis.
LYCB yielded a-carotene (b,"-carotene), whereas LYCB alone
produces b-carotene. a-Carotene and b-carotene are hydroxy-
Regulation in the anthocyanin biosynthesis lated by the action of b-hydroxylase (HYB) to generate
pathway orange-colored lutein and zeaxanthin, respectively. Lutein is
MYB transcription factors are involved in regulating flavonoid the most abundant form of carotenoids detected in leaf
and anthocyanin biosynthesis (Sablowski et al. 1994, Allan et al. tissue. Zeaxanthin epoxidase (ZEP) further epoxidates zeax-
2008). They contain a MYB DNA-binding domain composed of anthin to violaxanthin. The final step of the carotenoid biosyn-
51–52 amino acid residues to form a helix–turn–helix docking thesis pathway is the formation of neoxanthin by neoxanthin
in a major groove of the DNA (Dubos et al. 2010). They are synthase (NXS). Chiou et al. (2010) isolated several
classified into three groups on the basis of repeat numbers, carotenoid-related genes in three Oncidium cultivars, and
including MYB-related factors with one repeat, R2R3 MYB four genes exhibited varied expression: OgHYB and OgZEP
with two repeats and MYB3R factors with three repeats (Ito showed higher expression in yellow Gower Ramsey than in
2005). The first plant MYB gene, C1, a R2R3 MYB factor isolated orange Sunkist, whereas OgZDS and OgLCY were more active
from Zea mays, is related to anthocyanin biosynthesis (Paz-Ares in Sunkist, thus resulting in lower level of violaxanthin, 9-cis-
et al. 1987). Extensive studies indicate that most R2R3 MYBs violaxanthin and neoxanthin, and accumulation of b-carotene
interact with basic helix–loop–helix (bHLH) factors to control in Sunkist as mentioned above and orange color formation in
anthocyanin accumulation. Examples are the R/B gene family in Sunkist flowers. The white color pattern might result from the
maize, which interacts with C1 (Mol et al. 1998), and TT8 in up-regulated expression of OgCCD1 (carotenoid cleavage dioxy-
Arabidopsis, which, with TT2, controls flavonoid metabolism in genase 1). Both CCD and 9-cis-epoxycarotenoid dioxygenase
the seed coat (Nesi et al. 2000). A motif [D/E]Lx2[R/ catalyze the oxidative cleavage of carotenoids to produce a
K]x3Lx6Lx3R on the R3 domain is related to bHLH interaction diversity of apocarotenoids and are involved in the catabolism
(Zimmermann et al. 2004). Analysis of a R2R3 MYB isolated of carotenoids, helping to regulate the level of carotenoids,
from Oncidium, OgMYB1, suggested that it is critical for the generating substrates for ABA synthesis and signaling molecules
red color pattern formation in floral organs. OgMYB1 contains (Lu and Li 2010). Methylation assay of the OgCCD1 promoter in
bHLH-interacting motifs and is expressed in red lip crests but white Jade and yellow Gower Ramsey indicated a higher level of
not yellow lip tissues. Bombardment of OgMYB1 in lip tissue of methylation in Gower Ramsey. The function of OgCCD1 was
Oncidium flowers regenerated red pigment spots in yellow lip demonstrated by bombardment of OgCCD1 in the yellow lip
tissue (Chiou and Yeh 2008). tissue of Gower Ramsey, which resulted in a snow-white spot
In contrast to the uniform color performance of tepals dis- (Chiou et al. 2010).
cussed above, several Phalaenopsis cultivars are spotted flowers, Carotenoids are synthesized de novo in plastids, but they
especially for the harlequin flowers, which show denser color- accumulate in chloroplasts in leaves for function in photosyn-
ation with fused dark spots (Chen et al. 2004). This pigment thesis and are present in chromoplasts of flowers and fruits.
pattern is different from the central and large spots shown on Chloroplasts and chromoplasts differ in sequestering caroten-
Clarkia gracilis subsp. Sonomensis (Gottlieb and Ford 1988), oids (Christopher et al. 2010). Carotenoids integrate with
suggesting the regulation mechanism is probably dissimilar, Chl-binding proteins in chloroplasts. In contrast, they are asso-
and could be an interesting subject. ciated with polar lipids and carotenoid-associated protein, such
Y.-Y. Hsiao et al.
as CHRC and CHRD, to retain stability and reach a considerable culture technologies for Phalaenopsis and Oncidium are feas-
level in chromoplast membrane (Vishnevetsky et al. 1999). ible, and new genetically engineered varieties with charac-
Chiou et al. (2008) identified OgCHRC and its promoter ters of adjustable flowering and new varieties of colors
(Pchrc) in Oncidium. OgCHRC is specifically expressed in flowers. are promising areas. Genetic engineering of novel flower
Transient expression of Pchrc in different species by bombard- colors is now a practical technology, as typified by commer-
ment resulted in Pchrc-GUS mimicking the expression pattern cialization of a transgenic blue rose and blue carnation
of OgCHRC, expressed in conical papillate cells of adaxial epi- (Nishihara and Nakatsuka 2011). Blue Phalaenopsis and red
dermis of lip tissues, with accumulation of anthocyanins and Oncidium cannot be bred by traditional means. Through
carotenoids and higher expression in monocots. The tissue spe- basic research into the formation and regulation of floral
cificity of Pchrc could provide a convenient and useful tool in color of Phalaenopsis and Oncidium, genetically modified
orchid breeding biotechnology, such as OgCHI and OgDFR ec- orchid flowers with designed colors will be genetically engin-
topic transformation (Chiou and Yeh 2008). eered, propagated by micropropagation and then distributed
Compared with the well-studied regulation mechanism of worldwide.
the anthocyanin pathway, little is known for the carotenoids. Although most of the Phalaenopsis spp. are scentless, some

Several studies have shown that PSY expression is directly nega- do have a pleasant scent. However, many modern Phalaenopsis
tively regulated by phytochrome-interacting factor (PIF), a re- cultivars have lost the ability for scent emission with traditional
pressor of photomorphogenesis in A. thaliana (Toledo-Ortiz breeding programs. In order to revive fragrance, molecular
et al. 2010, Meier et al. 2011). The situation in Phalaenopsis breeding by genetic engineering for a gene network is currently
and Oncidium is under investigation. underway for generating the scented P. aphrodite subsp. for-
mosana. The prospective applications for restoring scent in
Phalaneopsis follow the wishes of the consumer. Further
study of key enzymes, such as GDPS and terpene synthase,
Perspectives
involved in the scent biosynthesis pathway, are necessary for
At present, the orchid industry in Taiwan faces several prob- using metabolic engineering to improve the production of
lems waiting to be resolved. The breeding process is too slow (3 scent precursors to emit high-level scents. In general, con-
years for one generation), and no marker systems are avalaible sumers accept genetically modified flowers more readily than
for screening desirable varieties. Thus, orchid breeding is not foods; therefore, many genetically modified floricultural plants
efficient. A regular supply of Oncidium flowers to the market all are at advanced stages of development.
year round has been constrained by lack of effective means for A much more urgent issue faced by the orchid industry is
flowering modulation. Blue Phalaenopsis flowers, red Oncidium the decrease in or the early detection of somaclonal variations
flowers, and perfect flower shape with a pleasant fragrance are occurring during mass clonal micropropagation. Evidence that
the highly expections of consumers. In addition, poor quality of epigenetic mechanisms play a role in somaclonal variation in-
planting materials due to somaclonal variations generated by cludes activation of transposons and retrotransposons, puta-
micropropagation constrains the full expansion of the orchid tive silencing of genes, and methylation pattern variation of the
industry. genome. Understanding epigenetic regulation will have import-
For an important floral crop, both physical and genetic maps ant implications in the orchid industry while increasing the
are needed for developing markers linking economic traits. consistency of floral products. Eventually, directed manipula-
However, there are no maps available for orchids. Recently, tion of epigenetic regulators will open the way to epigenetic
we began a preliminary attempt to build the physical map of engineering in orchids.
P. equesrtris. So far, about 20,000 BAC clones have undergone With the draft of the whole genome sequence of P. equestris
fingerprinted contig analysis, and several of them were in progress, the genetic blueprint of orchids will soon be avail-
assembled to be contiguous. Nevertheless, more vigorous able. In addition to providing a basic understanding of the
inputs in terms of budget and workflow will be needed to genetic basis of orchids, this information will be useful in
complete the 120,000 BAC clones for 8.4-fold coverage to re- having significant effects on floral crop design. This information
solve a good-quality physical map. However, constructing the includes identifying genes that could improve crop value and
genetic map of Phalaenopsis orchids is hampered by their long our understanding of the evolution of the unique orchid biol-
life cycles and slow growth rate. Currently, a colleague at ogy. Application of these resources through the common lan-
National Taiwan University has initiated the genetic mapping guage of nucleotide sequences will greatly enhance insights into
of Phalaenopsis. Hopefully, more updated statistics and avail- orchid biology and biotechnology.
able software may help resolve this problem with progeny from
a few generations.
The next challenging problems to be solved are molecu-
Funding
lar networks of flowering and the regulation mechanism of
flower color formation. These two fields are under study in This work was supported by The National Science Council,
Taiwan. On the application side, transformation and tissue Taiwan [grants NSC98-2321-B-006-004-MY3 and
NSC99-2311-B-006-004-MY3 to H.H.C. and W.C.T., Chen, D., Guo, B., Hexige, S., Zhang, T., Shen, D. and Ming, F. (2007)
respectively]. SQUA-like genes in the orchid Phalaenopsis are expressed in both
vegetative and reproductive tissues. Planta 226: 369–380.
Chen, J.T., Chang, C. and Chang, W.C. (1999) Direct somatic embryo-
genesis on leaf explants of Oncidium Gower Ramsey and subse-
Acknowledgments quent plant regeneration. Plant Cell Rep. 19: 143–149.
We thank Tun-Yu Chen and Hao-Chian Chien (Institute of Life Chen, T.C., Wu, W.L. and Chen, W.H. (2004) Development of harlequin
Sciences, National Cheng Kung University, Taiwan) for provid- flower derived from somaclone mutants of Phalaenopsis Golden
ing orchid flower photographs, and Dr. Chang-Sheng Kuoh Peoker ‘Brother’. In Proceedings of 8th Asia Pacific Orchid
Conference (APOC8). pp. 128–137. Tainan.
(Department of Life Sciences, National Cheng Kung
Chen, W.H., Hsu, C.Y., Cheng, H.Y., Chang, H., Chen, H.H. and Ger, M.J.
University, Taiwan) for making the line drawing of the orchid
(2011) Downregulation of putative UDP-glucose:flavonoid 3-O-glu-
flower. cosyltransferase gene alters flower coloring in Phalaenopsis. Plant
Cell Rep. 30: 1007–1017.
Chen, W.H., Tseng, Y.C., Liu, Y.C., Chuo, C.M., Chen, P.T., Tseng, K.M.
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Construction of cDNA library of P. bellina

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