September 2021. Horticultural Plant Journal, 7 (5): 380–392.

Horticultural Plant Journal

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A review for the breeding of orchids: Current achievements and

prospects
Chengru Li, Na Dong, Yamei Zhao, Shasha Wu, Zhongjian Liu∗, and Junwen Zhai∗
Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization at College of Landscape Architecture,
Fujian Agriculture and Forestry University, Fuzhou, China
Received 16 September 2020; Received in revised form 13 November 2020; Accepted 10 February 2021
Available online 27 February 2021

A B S T R A C T

Orchids are horticultural plants with significant ornamental and economic value. Increasing market circulation and rising trade in orchids
is forcing breeders to develop varieties with unique characteristics, including flower color, morphology, and resistance using a range of ap-
proaches, including traditional and molecular breeding. Advances in high-throughput technologies have generated extensive data sets with
greater sequencing depths and broader coverage, providing the potential for discovering new genes/pathways that give rise to key traits. Several
attempts have been made to use emerging molecular and omics methods to accelerate the breeding process in certain of the commercially
valuable orchids. This review consolidates current approaches and achievements in orchid breeding and discusses their future applications for
improving the resistance, ornamental, and other valuable characteristics of these plants.

Keywords: Orchidaceae; Multi-omics; Breeding methods

1. Introduction a highly specialized labellum (ornamented with an appendage or

Mating systems can have a profound influence on the ge-
netic development of plants and animals. However, whereas
the sex system of animals tends to be relatively simple and
hermaphroditism is rare, the differences between sexes in land
plants are often difficult to characterize, which, as botanists as-
sert, may be attributable to the fact that the sexes co-exist in
individual plants (Barrett, 2002). In the current literature, mat-
ing systems are described in two different ways, each with dif-
ferent implications for sexual differences that may or may not
occur (Shuster, 2019). One describes the mating systems of plants,
fungi, and protists, the other describe the mating systems of ani-
mals. Selfing, partial selfing, random mating, inbreeding, and out-
breeding are all examples of mating systems described in terms
of genetic relationships that may occur between breeding pairs
(Shuster, 2019).
The zygomorphic flower, one of the most valuable parts of an
orchid, is composed of three sepals, two petals (similar to sepals),
not), a basal spur or nectary or not, and a gynostemium fused by
the style and at least part of the androecium (Rudall and Bate-
man, 2002).
Orchids are traded around the world as cut flowers and pot-
ted plants, and the market circulation is increasing along with
a rise in the amount of trade (Hinsley et al., 2018). In addition,
being rich in polysaccharides, alkaloids and other chemical com-
ponents, orchids are also used in the medical, food and beverage
industries (Wang et al., 2020). Orchids not only have great eco-
nomic and ornamental value, but also have special cultural sig-
nificance. In Chinese classical literatures, the orchid is known as
one of the “Four Gentlemen among the Flowers”, along with the
Chinese plum, chrysanthemum, and bamboo (Zhao, 2009).
With the development of economic globalization, the mar-
ket demand for orchids in quantity and variety has increased
year by year, forcing scientists and breeders to develop new va-
rieties with novel appearances, improved resistance, and qual-
ity characteristics (Kamboj, 2020). Traditional breeding, although

∗
Corresponding authors.
E-mail addresses: zjliu@fafu.edu.cn; zhai-jw@163.com
Peer review under responsibility of Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese
Academy of Agricultural Sciences (CAAS)

https://doi.org/10.1016/j.hpj.2021.02.006
2468-0141/Copyright © 2021 Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy
of Agricultural Sciences (CAAS). Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
A review for the breeding of orchids: Current achievements and prospects
time-consuming, is still the mainstream approach to orchid
breeding. However, some desired traits, such as the mottled
flowers/foliage of a single individual, cannot be attained by hy-
bridization and mutation due to limitations and drawbacks of
the traditional approach. In recent years, transgenic molecular
breeding has been extensively employed by introducing the de-
sired target genes into orchids using Agrobacterium-mediated
transformation and particle bombardment methods, which has
led to considerable progress in horticultural character improve-
ment (Mii, 2012; Filippo, 2017). The rapid development of DNA-
based molecular marker technology provides plant breeders with
new opportunities to employ molecular marker-assisted selec-
tion (MAS) in breeding and shows great potential for increasing
the effectiveness of plant breeding (Wijerathna, 2015; Das et al.,
2017). This review consolidates the merits and drawbacks of con-
ventional breeding. It focuses mainly on recent advances and
challenges in molecular breeding of orchids and discusses the fu-
ture prospects for accelerating breeding programs.
2. Breeding technology and methods
2.1. Crossbreeding
The family Orchidaceae is the most diverse among flowering
plants, comprising more than 28 000 species with multiple breed-
ing strategies and characteristics. Acianthera aphthosa, a repre-
sentative species with self-pollinated flowers, has fewer seeds
lacking embryos than cross-pollinated flowers (Pansarin et al.,
2016). However, Dendrobium with both self-compatibility and self-
incompatibility, accounts for nearly half of all self-compatibility
orchids (Niu et al., 2018). Hybridization, both natural and arti-
ficial, has the effect of integrating the excellent characteristics
of the two parents within the hybrid offspring. Phalaenopsis in-
termedia, a cross between P. aphrodite and P. rosea, first described
in 1853, is one of the oldest natural hybrids, whereas Calanthe,
the first artificial orchid hybrid, recorded by Dominy in 1856, was
cultivated from a cross between Calanthe masuca and Calanthe fur-
cata (de Chandra et al., 2019). However, although crossbreeding is
a simple and effective technique for cultivating orchid hybrids,
there are several factors that must be considered when perform-
ing crossbreeding, including the fertility of the hybrid combina-
tion, qualitative analysis of target traits, and the selection of su-
perior hybrid offspring (Su et al., 2019). The F1 progenies derived
from two parents with contrasting target traits (such as a par-
ent with long flowering time but small flowers in size and the
other with large flowers in size but short flowering time) usually
exhibit large phenotypic differences. For example, Ionmesa pop-
corn ‘Haruri’ produces flowers that differ notably from those of its
parents. However, in the case of Cymbidium, it has been reported
that hybrid seeds, particularly those of distant hybrids, are dif-
ficult to culture owing to distant genetic relationship, with the
degree of difficulty increasing in the order intraspecific < intra-
generic < intergeneric (Zhang et al., 2001). Luo et al. (2012) also
reported that there are various obstacles that impede the pro-
cess of hybridization, including parent incompatibility and post-
fertilization embryo abortion, resulting in the failure of distant
hybridization. However, whether this situation is applicable to all
orchid species remains to be studied. As a key part of traditional
breeding, seed germination is directly related to the efficiency
381
and success of crossbreeding. Therefore, in-depth study of the
developmental characteristics and germination mechanisms of
distant hybrid seeds is particularly necessary to establish an ef-
ficient germination system.
When hybrid seeds are obtained, a suitable cultivation ap-
proach is needed to keep the population stable or growing. In this
regard, propagation in vitro is one of the most important breeding
methods for orchids, as orchid seeds are difficult to reproduce in
the natural environment. Seed maturity, culture conditions, and
culture media are key factors affecting the efficiency of in vitro
propagation. In vitro propagation studies have been carried out on
numerous orchid species, including those in the genera Cymbid-
ium, Phalaenopsis, Dendrobium, Oncidium, Dactylorhiza, and Calanthe
alliance (Kanchanapoom et al., 2014; Bae et al., 2015; Ram et al.,
2016; Teixeira et al., 2016; Bezerra et al., 2019; Gao et al., 2020). At
present, innovations of the hybrid grex and a shortening of the
breeding cycle, are the main targets of propagation in vitro, and
significant progress has been made in attaining these targets.
2.2. Mutation breeding
Mutation breeding, including both natural and artificial mu-
tations, is well suited for ornamental plant breeding because
many species can be propagated easily, facilitating the produc-
tion of spontaneous and induced mutants (Yamaguchi, 2018). Mu-
tation breeding has a number of advantages, including a high
mutation rate, breaking of the relationship of traits, effective im-
provement of individual traits, and shortening the breeding cycle
(Toker et al., 2007). Over the years, mutation breeding has been
used to produce orchids with unique phenotypic traits, higher
content of medicinal ingredients, and greater adaptability and re-
sistance (de Chandra et al., 2019).
Polyploidization is a common method of mutation breeding.
Currently, polyploid breeding has been successfully conducted in
many orchid species, including Cymbidium (Wang et al., 2011b),
Dendrobium (Li and An, 2009; Zhang et al., 2011), Oncidium (Cui
et al., 2010a), and Phalaenopsis (Cheng, 2011; Cui et al., 2010b). Yin
et al. (2010) treated the hybrid with colchicine to obtain tetraploid
plants, which developed thicker leaves, roots, and rhizomes, as
well as a deeper stem color, and a slower growth rate. Jin et al.
(2012) treated the protocorm of Dendrobium huoshanensis hybrid
with nitric oxide (NO) donor sodium nitroprusside to improve
the alkaloid content of medicinal Dendrobium produced by tis-
sue culture. More recently, Guo et al. (2016) irradiated D. catenatum
seedlings with an artificial simulation of UV-B radiation, resulting
in an increase in the content of total polysaccharides, flavonoids,
alkaloids, and other major secondary metabolites.
The high heterozygosity in orchids can increase the appar-
ent mutation rate and produce a series of excellent mutation
types in a short cycle. However, unpredictable mutations can oc-
cur throughout the genome, and thus deleterious mutations may
occur and typically only single changes are obtained (Su et al.,
2019). In addition, the feasibility of mutation breeding depends
on factors such as the explant type, an appropriate genotype, the
induction mutation method, and the optimal dose (Kharkwal
et al., 2004). However, there are currently no related reports of
chemical mutations caused by alkylating agents such as ethyl
methane sulfonate, a nucleobase analog such as 5-bromouracil,
or an antibiotic such as azaserine. Making full use of the
382
Fig. 1 Anatomy of the normal and mutant of Calanthe nipponica
a: dorsal sepal, b: petal, c: lateral sepal, d: lip, e: ovary, f: spur, g: gynandrium.
advantages of crossbreeding and mutation breeding is construc-
tive for the cultivation of new varieties.
2.3. Selection breeding
Compared to crossbreeding, selection breeding uses the nat-
ural variation of existing varieties as the original material for
selection in the breeding process (Osadchuk, 2020). When selec-
tion breeding is performed, three particularly important genetic
parameters must be considered: heritability, genetic correlations
between traits, and interactions between genotypes and the en-
vironment (Boudry, 2009; Falconer and Mackay, 1996). The three
parameters should be used to deal with the relationship between
heritability, variation, and selection given that plant phenotypes
are determined by the environment as well as by the genetic ma-
terial. A new Phalaenopsis cultivar ‘SM 333’ was bred by hybridiza-
tion, selection and in vitro propagation (Park et al., 2015). Luo et al.
(2019) developed a new Oncidium variety called ‘Jinhui’ by means
of plant somaclonal mutation, selected strains screening, molec-
ular identification, tissue culture, and multi-point testing. In
addition, in the field investigation, we found three strains of
Calanthe nipponica with pure yellow sepals and petals, whereas its
normal color is purple-brown (Fig. 1). We do not know whether
the mutation is heritable, but if it is, it would provide excellent
selective breeding material.
2.4. Molecular marker-assisted breeding
Molecular marker-assisted breeding (MMAB), the application
of molecular biotechnologies for practical breeding and selec-
tion, has the advantages of being fast, accurate, and free from
the influence of environmental conditions (Jiang, 2015). Among
the many types of molecular markers available to scientists and
breeders, the following are the most relevant in terms of preva-
lence and potential: restriction fragment length polymorphism
(RFLP), amplified fragment length polymorphism (AFLP), sim-
ple sequence repeat (SSR), and single nucleotide polymorphism
Chengru Li et al.
(SNP). The first three have been widely used in the breeding of
orchids with good results thus far. Li et al. (2014) constituted a
set of markers (genic-SSR) in Cymbidium ensifolium that can be
applied to multiple orchid species for the evaluation of genetic
relationships and traits mapping studies. These marker types, in
combination with functional annotations provided by unigenes,
will assist with the location of candidate genes with specific
functions. Simple sequence repeat transcriptome sequencing of
the roots of Paphiopedilum concolor has provided valuable insights
into the mechanisms of root growth and development as well as
root secondary metabolism-related genes and molecular marker-
assisted research (Li et al., 2015). Wu et al. (2017) used SSR to iden-
tify genes related to flower color, flower shape, and resistance
in Phalaenopsis, providing an important reference for genetic en-
gineering breeding of Phalaenopsis and the Orchidaceae in gen-
eral. The genetic diversity of different species of Phalaenopsis was
analyzed using gene-specific single nucleotide amplified poly-
morphism markers to evaluate the effectiveness of predicting
flower color, supporting the breeding of new Phalaenopsis varieties
(Sudarsono et al., 2017). Integrating the Phal. aphrodite genome
with an SNP-based genetic linkage map and verifying it by op-
tical mapping has not only provided an unprecedented resource
for improving the breeding efficiency of horticultural orchids, but
has also contributed important research on the adaptation ge-
nomics of epiphytes for future reference (Chao et al., 2018). Lu
et al. (2018) reported the first high-density SNP integrating ge-
netic map with good coverage in the Dendrobium genome, some
QTL sites associated with polysaccharides were revealed, which
has laid the foundation for mapping of other medically relevant
traits. The assembly, characterization, and development of the
EST-SSR of the Bletilla striata transcriptome has laid a solid foun-
dation for functional gene-mining and genome research of B. stri-
ata and promoted phylogenetic research and breeding (Xu et al.,
2018). RNA-sequencing, expression quantitative trait locus (eQTL)
analysis of stem length and diameter, and construction of high-
density genetic maps of Dendrobium (D. nobile × D. wardianum) has
A review for the breeding of orchids: Current achievements and prospects
set the foundation for fine eQTL mapping and MMAB of Dendro-
bium (Li et al., 2019). Wang et al. (2019) used specific-locus ampli-
fied fragment sequencing (SLAF-seq) to investigate the variation
of SNPs and insertion-deletions in somaclones of Onicidium ‘Mil-
liongolds’ regenerated by protocorm-like bodies (PLBs) after 10
years of in vitro culture. The results suggested that SLAF-seq is
an efficient technology for genome-wide analysis of somaclonal
variations for most species without reference genome sequences.
Although molecular marker technology has been widely used
in orchids, it has mainly focused on phylogenetic and genetic
relationship research, with only a few studies on the combina-
tion of phenotypic traits and molecular marker technologies. In
addition, other technology (Genome-Wide Association Studies)
has been applied in studies on chrysanthemum, grape, cabbage,
tomato, and tea (Chong et al., 2019; Huang et al., 2019; Xing et al.,
2019; Dominguez et al., 2020; Xiong et al., 2020; Zhao et al., 2020),
but only to a limited extent in orchids. Therefore, additional re-
searches should be conducted on these aspects to provide more
accurate genetic information for orchid breeding.
2.5. Transgenic breeding
Transgenic technology transfers the desired target genes to
host plants and inhibits or enhances gene expression in a pro-
grammable manner (Zhu et al., 2018). Transgenic technologies
can also enhance ornamental value by modifying changes in
plant genomes (Kishi-Kaboshi et al., 2018). Compared to tra-
ditional breeding strategies, transgenic methods have greater
potential for creating novel phenotypes. Introduction of new
properties such as new colors or disease resistance in orchids
via mutation breeding or crossbreeding is often difficult, but
can be achieved relatively easily using transgenic technology
(Nirmala et al., 2006; Mii and Chin, 2010). Plant genetic trans-
formation methods generally include Agrobacterium-mediated
transformation, particle bombardment, use of pollen-tube path-
ways, electrophoresis, and polyethylene glycol. Among these, the
Agrobacterium-mediated and particle bombardment methods are
most commonly used in orchid breeding (Mii and Chin, 2010).
Successful orchid transformations were first reported in Vanda
(Chia et al., 1990) and Dendrobium (Kuehnle and Sugii, 1992; Nan
and Kuehnle, 1995) and mediated through particle bombardment.
At present, effective transformation systems have been estab-
lished for some important commercial orchids, including Pha-
laenopsis (Hsieh et al., 1997; Tong et al., 2019), Vanda (Shrestha
et al., 2007), Cymbidium (Chin et al., 2007), Dendrobium (Xian et al.,
2017; Chen et al., 2018), Cattleya (Zhang et al., 2010) and Erycina
pusilla (Li and Chan, 2018). Griesbach and Hammond (1993) intro-
duced an anthocyanin synthesis gene into the powders of Doritis
pulcherrima by electrophoresis and obtained transient expression
in flowers, which presented as a change in color of the petals.
Yang et al. (1999) used the particle bombardment method to
transfer a plasmid with NPTII and GUS marker genes into Cymbid-
ium orchids to obtain transgenic plants resistant to kanamycin.
Wang et al. (2006) used RAPD molecular markers to identify genes
related to scent. Agrobacterium carrying NPTII, the plasmid carry-
ing the GUS marker gene, was transferred into the genus Cymbid-
ium, resulting in transgenic plants resistant to hygromycin (Chin
et al., 2007). Transgenic technology has become an important
means of developing new orchid cultivars and has led to signifi-
383
cant achievements with regard to floral attributes, plant architec-
ture, and biotic and abiotic tolerance, as reviewed by Chai and Yu
(2007), Jaime and de Teixeira (2013), and Mii and Chin (2010). This
review describes only the most recent reports of useful traits that
have been introduced into orchids (Table 1).
Over the past ten years, transgenic research in the Or-
chidaceae has achieved rapid development, especially post-
2015, and the transgenic plants produced belong mainly to
the mainstream ornamental orchid genera including Cymbidium,
Phalaenopsis, Dendrobium, and Oncidium. Regarding the use of
transgenic technology, the efficacy of Agrobacterium-mediated
and particle bombardment methods is well established, whereas
the ovary injection and pollen-tube pathway methods are less
commonly used. As far as the target genes are concerned, more
transformations of resistance genes have occurred, possibly be-
cause orchids are prone to mosaic virus, soft rot, and other dis-
eases. Studies on flower color and flowering target genes are less
common, with even fewer studies focused on plant growth and
development, and anti-senescence. Therefore, we need to em-
phasis on the above three main aspects.
2.6. Genome editing breeding
In recent years, genome editing technology, an important tool
for functional genomics and biotechnology research, has become
an accurate breeding method for plant species improvement (Ma
et al., 2015, 2016). The clustered regularly interspaced short palin-
dromic repeats (CRISPR)/CRISPR-associated (Cas) system is one
of the most popular genome editing techniques and is currently
revolutionizing the field of molecular biology. To date, few studies
have reported on genetic editing of orchids using the CRISPR/Cas9
method (Chandler and Sanchez, 2012; Watanabe et al., 2017,
2018).
The crucial requirement of genome editing is the accessi-
bility of accurate genomic data as well as gene functions (Tsai
et al., 2017). The deficiency of orchid genomic information has
significantly limited breeding efficiency; however, several orchids
have been whole-genome sequenced, including Phal. equestris
(Cai et al., 2015), D. catenatum (Zhang et al., 2016), Apostasia
shenzhenica (Zhang et al., 2017), and Gastrodia elata (Yuan et al.,
2018). In the absence of genomes, a growing number of stud-
ies emphasize that cDNA sequencing is available for discovering
candidate genes or metabolic pathways associated with flower
traits (Hsu and Yang, 2002; Tsai et al., 2004; de Paolo et al.,
2014; Teixeira et al., 2014). A substantial increase in the num-
ber of transcriptomes of many orchids is also feasible, includ-
ing Phalaenopsis, E. pusilla, Cymbidium sinense, and Orchis italica
(Huang et al., 2015; Lin et al., 2016; Su et al., 2018; He et al.,
2019; Maria et al., 2019). To obtain symbiotic relationships be-
tween orchids and fungi and the molecular mechanisms of or-
chid seed germination, transcriptome data were derived from Ser-
apias vomeracea (Perotto et al., 2014), Cymbidium hybridum (Zhao
et al., 2014), Anoectochilus roxburghii (Liu et al., 2015), and G. elata
(Tsai et al., 2016). In order to manage the gene sequences ex-
pressed in orchids, databases have been developed, such as Or-
chidBase (Fu et al., 2011; Tsai et al., 2013; Niu et al., 2016), Orchid-
stra (http://orchidstra.abrc.sinica.edu.tw) and Oncidium Orchid
Genome Base (http://predictor.nchu.edu.tw/oogb/) (Chang et al.,
2011). These expression sequence tag data sets are valuable for
384 Chengru Li et al.

Table 1 Transgenic research on Orchidaceae

Species Transgenic Genes Type Post-transgenic phenotype References
methods
C. sinense Agrobacterium CsFT Flowering Flowering early Huang et al., 2017
tumefaciens
Dendrobium RNAi-induced DseCHS-B Flower color Impaired anthocyanin accumulation and Ratanasut et al.,
Sonia ‘Earsakul’ DseDFR reduction of endogenous mRNAs of the 2015
corresponding targets
D. Chao Praya Particle DOFT DOFTIP1 Flowering DOFT promotes both pseudobulb formation Wang et al., 2017
Smile bombardment and flowering
E. pusilla Agrobacterium MSRB7 Resistance Higher resistance Lee et al., 2015
tumefaciens
Oncidium Agrobacterium OMADS1 Flowering Flowering significantly earlier, more flowers Thiruvengadam
tumefaciens and pseudobulbs et al., 2012
RNAi-induced PSY Growth and Some biosynthetic pathways (carotenoid, Liu et al., 2014;
development gibberellic acid, abscisic acid and Liou et al., 2017
chlorophyll) were interfered, predominant
defects in plant growth and development
Agrobacterium OnFd OnFNR Resistance Higher resistance to soft rot Wu, 2017
tumefaciens
Particle pCB199 plasmid Resistance Significant decrease in CymMV expression Krittiya et al.,
bombardment in transgenic plants 2018
Agrobacterium EIN2 Anti-senescence The expression of EIN2 was lower than the Shi, 2018
tumefaciens control
Agrobacterium PR1 Resistance The expression of PR1 washighly increased Guo, 2018
tumefaciens
RNAi-induced PSY Flower color New varieties of Oncidium orchids with Liu et al., 2019
white flowers
Phalaenopsis Particle EgTCTP Growth and The time required for initiation of primordial Kantamaht et al.,
bombardment development shoots in the transformed PLBs was much 2012
shorter
Agrobacterium fluorescence Resistance Hygromycin resistance Hsing et al., 2016
tumefaciens marker gene
eGFP
Pollen-tube cbf1 Resistance Higher seed setting rate Yu, 2017
pathway and ovary
injection
Agrobacterium CHS Flower color Fading color Meng et al., 2018
tumefaciens
Agrobacterium CHS and DFR Flower color The color of transgenic Phalaenopsis is Meng et al., 2018
tumefaciens significantly lighter than the control group
P. ‘Purple Gem’ Ovary injection GUS and Kan Resistance Higher resistance than the control group Zhang et al., 2018
P. ‘Sogo Vivien’ Agrobacterium AtRKD4 KNAT1 Growth and Somatic embryogenesis and shoot Mursyanti et al.,
tumefaciens development development were induced 2018
P. Sogo Yukidian Agrobacterium bHTH, MYBx-like Flower color PebHLH1 and PebHLH2 improve PeMYB Hsu et al., 2019
‘V3’ tumefaciens functions in anthocyanin accumulation in
Phalaenopsis flowers and present different
preference
P. aphrodite Agrobacterium Pha21 Resistance Pha21 can respond to multiple Chang et al., 2019
tumefaciens defense-related plant hormones and plays a
positive role in virus resistance

the identification of orchid-specific genes, gene annotation for
genome sequencing, and assembly of orchid genomes (Tsai et al.,
2017).
The development of next generation sequencing has ushered
into the omics era. Flower shape, flower color, flowering period,
resistance, and other major functional genes have been uncov-
ered, providing unrestricted targets for genome editing for de-
picting the gene functions, which can sequentially assist in en-
gaging the CRISPR/Cas technology to cultivate better orchids.
In addition to genomic data, the availability of genetic trans-
formation and regeneration process platforms is also critical
for gene editing techniques. The transformation of D. catenatum
is relatively mature and the highest transformation efficiency
of 56.5%. Addition of 0.001% (v/v) TritonTM X-100 increased the
transformation efficiency by up to 2-fold. (Chen et al., 2018). The
transformation system of Phal. equestris has been established, but
the regeneration time is longer (2–3 years). Tong et al. (2019) con-
structed a high-efficiency CRISPR/Cas-based editing of Phalaenop-
sis orchid MADS genes by using two CRISPR/Cas strategies, intro-
ducing three MADS target sites (MADS44, MADS36, and MADS8)
together in one vector (pYLMADS8_36_44; Ma et al., 2015), or
separately in individual vectors (P1300_MADS8, P1300_MADS36,
and P1300_MADS44; Lin et al., 2018) to produce single-guide
RNAs to generate multiple mutants (single, double, and triple)
in Phal. equestris MADS-box genes. D. catenatum is a popular
medicinal plant rich in polysaccharides with various pharma-
ceutical properties, including immunomodulation, anti-oxidant,
and anti-fatigue effects. Recently, it has been reported that, by
knocking out several lignocellulose biosynthesis genes, includ-
ing C3H, C4H, 4CL, CCR, and IRX, the success rate of genome
editing was shown to be 100%, but there was no associated de-
crease in lignocellulose content in the knockout plants (Kui et al.,
2016). Although Agrobacterium-mediated transformation can ef-
fectively promote the CRISPR/Cas9 genome editing system in the
A review for the breeding of orchids: Current achievements and prospects
D. macrophyllum genome, the transformation efficiency is only
0.66% (Setiawati et al., 2020), and therefore, in future, it will be
necessary to enhance the efficacy of the CRISPR/Cas9 technique
for each gene target.
Compared to other crops, the application of genome editing
technology in ornamental plants, including orchids, chrysanthe-
mums, and roses has not been well developed due to the rela-
tively small economic benefits of ornamental species and market
demands. The lack of specialized transformation systems, inef-
ficiency of existing transformation systems, and lack of under-
standing of some trait-regulatory networks have also hindered
development of genome editing breeding in these ornamental
plants. Therefore, development of appropriate expression sys-
tems and target sequence configurations for each type of orna-
mental plant is needed, and once genome editing is being con-
ducted, it will be necessary to take the law into consideration and
improve the target traits in legally compliant ways.
3. Breeding hotspots of orchids
3.1. Flower morphology
Flower morphology is crucial for successful pollination. The
genetic processes controlling flower development in some valu-
able ornamental orchids have been studied, and the genes
involved in the formation of specific flower organs have been
identified. The MADS-box genes play an essential role in the
emergence of flower structures during plant evolution (Urbanus
et al., 2009; Murai, 2013; Lu et al., 2019; Zhou et al., 2019; Li et al.,
2020). Previous studies have suggested that A-class genes play
a vital role in sepals and promote flowering; B-class genes are
expressed in all floral organs, consistent with the floral quar-
tet model, and C/D-class genes are crucial for clarifying the de-
velopment of the gynostemium and are activated by pollination
(Becker and Theiβen, 2003; Tsai et al., 2004; Wang et al., 2011a).
E-class and other genes are thought to jointly control the four
whorls of the orchid floral structure, with the AGL6-like gene
expressed in sepals, petals, stelidia, and the gynostemium in
the first, second, third, and fourth whorls of the orchid flower
(Huang et al., 2016; Dirks-Mulder et al., 2017; Pramanik et al.,
2020). In addition, other MADS-box genes have been studied in
orchids, such as SVP and FLC (Hartmann et al., 2000; Ratcliffe
et al., 2001). Notably, some genera in the Orchidaceae, such
as Angraecum, Calanthe, Dendrobium, Orchis, and Neottianthe have
flowers with a spur which is an outgrowth attached to the lip.
Research is needed to explore the convergent evolution of this
morphological feature with pollinators, particularly in relation
to the molecular mechanism of development. Studies on the
molecular genetic processes controlling flower development re-
main inconclusive, and the combination of forward genetics and
reverse genetics may provide opportunities for research in this
field.
3.2. Flower color
Developing attractive flower color variations is a major ob-
jective of ornamental plant breeding because of its strong in-
fluence on consumers’ choice. In addition, flower color is im-
portant for attracting pollinators to complete pollination, and
385
it also plays an essential role in photosynthesis in plant tis-
sues and responses to ultraviolet radiation (Davies, 2004). An-
thocyanins, carotenoids, and chlorophylls are the main pigments
found in orchids, with anthocyanins the most widely distributed
(Hsiao et al., 2011). Although diverse flower colors have been ob-
served in orchids during their long history of cultivation, flowers
with violet and blue coloration are lacking due to a deficiency in
flavonoid 3 5 -hydroxylase (F3 5 H) activity (Hsu et al., 2015). Ac-
tivity of the F3 5 H enzyme is key for delphinidin biosynthesis in
most blue flowers (Su et al., 2019). Hsu and Chen (2017) identi-
fied and the structural and regulatory genes that control orchid
color as well as the anthocyanin biosynthetic pathway. The lat-
ter includes three PeMYBs (PeMYB2, PeMYB11, and PeMYB12), and
three structural genes: PeF3H5, PeDFR1, and PeANS3. Using the
Agrobacterium-mediated method, the interference vectors: PhDFR,
PhCHS, PhF3 H, and IhF3 5 H, Ma3 5 H, IhDFR, ThDFR, MaDFR, and
other genes related to flower color were transformed into Pha-
laenopsis, and the interference of PhCHS and PhDFR gene vectors
on petal color produced significantly lighter petals than in the
control group (Meng et al., 2018). Liu et al. (2019) constructed a
chimeric hpRNA T-DNA vector targeting PSY mRNA. Petal-specific
RNAi silencing of the phytoene synthase gene reduced lutein lev-
els and produced new varieties of Oncidium orchids with white
flowers.
Phalaenopsis is one of the most popular cultivated orchids
in the world and, as of 2018, the Royal Horticultural Society
had registered 92 native and 34 112 hybrids of Phalaenopsis, but
only 18 native species are frequently used in breeding, These
belong to the subgenus Phalaenopsis and the subgenus Polychi-
los (Huang et al., 2012). In the subgenus Phalaenopsis, Phal. am-
abilis and Phal. aphrodite are mainly used for breeding white
and large flowers of more than 10 cm in flower size, whereas
Phal. sanderiana, Phal. schilleriana, and Phal. stuartiana are used
for breeding red and large-flower hybrids (Baburek, 2009). In ad-
dition, Phal. equestris is frequently used as a breeding parent
because of its multi-flower traits, Phal. lindenii introduces the
red-stripe feature, and Phal. pulcherrima gives dark-red-to-purple
colors. Members of the subgenus Polychilos are the most impor-
tant ancestors for yellow-to-orange flowers (Hsu et al., 2018). At
present, Phalaenopsis plants with unusual petal patterns such as
spots, stripes, and different colors are increasingly being sought.
At the same time, harlequin flowers with clown-like spots and
extremely complicated color patterns have appeared on the
market.
Oncidium is another popular ornamental orchid. Oncidium
Sharry Baby is a cross between Onc. Jamie Sutton and Onc. Hon-
olulu, also known as the chocolate orchid, not for its colors, but
for its fragrance. This cross is a true icon in the orchid world.
Ionmesa popcorn ‘Haruri’ is a cross between I. utricularioides and
Gomesa flexuosa, the parents belonging to Ionmesa and its related
genus Gomesa, respectively. Compared with the parents, which
show rich colors on a single individual, the cross has intensive
flowers and slightly better growth conditions (Fig. 2).
3.3. Flower scent
Besides floral color and shape, fragrance is one of the key de-
terminants of consumer preferences for orchids. Plant volatile or-
ganic compounds (VOCs) are complex mixtures of low melting
386
Fig. 2 Ionmesa popcorn ‘Haruri’ and its parents
A: I. utricularioides; B: Gomesa flexuosa; C: I. popcorn ‘Haruri’ (I. utricularioides × G. flexuosa).
point, low molecular weight, and lipophilic molecules (Dudareva
and Pichersky, 2006). play vital roles in pollinator attraction,
defense, and interaction with the environment (Paulus and
Gack, 1990; Borg-Karlson et al., 1993; Piechulla and Pott, 2003;
Knudsen et al., 2006; Kessler et al., 2008; Raguso, 2008; Dudareva
et al., 2013). Based on their sources and functions, the VOCs of
flowers can be divided into three categories: terpenes, phenyl-
propanoids/benzenoids, and fatty acid derivatives (Dudareva
et al., 2013).
In recent years, with the development of omics technology,
genes (such as TPS and JMT) encoding candidate enzymes for
plant VOC biosynthesis and regulatory pathways have been iso-
lated (Xu et al., 2019; Yu et al., 2020). Uncovering the biosynthetic
pathway and regulatory mechanism of floral scent production
is not only necessary for a better understanding of the func-
tion of the related genes, but also for developing new varieties
with ideal traits through molecular breeding methods (Ramya
et al., 2017). Because orchids have a long life span and complex
and large genomes, little is known about the pathways responsi-
ble for floral scents; however, some terpene pathways have been
reported in orchids (Hisao et al., 2006, 2008). Recently, a grow-
ing number of studies have reported that several types of tran-
scription factors (TFs), including basic helix-loop-helix (bHLH),
alkaline leucine zipper, ethylene reaction factor, NAC, MYB, and
WRKY family members are involved in the regulation of terpene
biosynthesis (Ramya et al., 2017). ODORANT1, a member of the
R2R3-MYB TF family, is the first MYB TF identified in flowers
which regulates genes involved in floral scent production through
the shikimate and phenylpropanoid pathways (Kim et al., 2016).
The TF PbbHLH4 regulates the geranyl diphosphate synthase gene
for the synthesis of monoterpenoids in Phalaenopsis bellina (Zvi
et al., 2012). Cymbidium Sael Bit MYB1 is regarded as a regulator of
phenylpropanoid/benzenoid genes in floral scent profiles (Ramya
et al., 2017).
3.4. Flowering time
Through genetic analysis of several plants (especially Ara-
bidopsis thaliana), the control of flowering time has been ex-
tensively studied, and a genetic network including photope-
Chengru Li et al.
riod, vernalization, autonomy, and gibberellin has been identi-
fied (Blazquez et al., 2001). Flowering time is the main deter-
minant of commercially successful plants, and the cultivation
of early flowering varieties helps to meet the needs of con-
sumers’, including use of orchids to celebrate particular holi-
days. Some studies have suggested that FLOWERING LOCUS T (FT)
and AP1-like genes could promote the transition from vegetative
growth to reproductive growth (Mandel et al., 1992; Kobayashi
et al., 1999). Thiruvengadam et al. (2012) used Agrobacterium-
mediated transformation to transfer OsMADS1 into Oncidium
plants and found that transgenic plants presented early flower-
ing, and produced more flowers and pseudobulbs than the con-
trol group. Chang et al. (2009) used the same method to transfer
the OMADS10 gene into A. thaliana, which also showed early flow-
ering after transformation. More recently, transfer of Dendrobium
Chao Praya Smile DOAP1 into A. thaliana presented early flower-
ing and early termination of inflorescence meristem into flower
meristem (Wang et al., 2017). In addition, Gao (2017) cloned FT
and MOTHER OF FT genes from Phalaenopsis and transferred them
to Arabidopsis, showing early flowering and delayed flowering, re-
spectively.
3.5. Abiotic and biotic stress tolerance
Biotic stresses such as disease and insect pests, as well as
certain abiotic stresses are important factors that hinder the
industrialization of orchids. To improve the yield and quality-
related attributes of orchids, breeders should consider breed-
ing for resistance to biotic factors and tolerance of abiotic fac-
tors. For example, plants of the Oncidium series were transformed
by the pflp gene encoded by antimicrobial ferritin improved re-
sistance to the soft rot disease factor Erwinia carotovora (Liau
et al., 2003). In addition, Mii and Chin (2010) have cultivated
a series of transgenic ornamental orchids, which contain use-
ful genes (such as GST and PR1) for disease resistance, stress
resistance, flower color, flower pattern, and plant height. The
Phalaenopsis protocorm transgenic system was successfully con-
structed by Agrobacterium tumefaciens using the Phalaenopsis pro-
tocorm as the receptor material, pCAMBIA1301 (containing the
GUS reporter gene and hygromycin resistance gene hpt) as the
A review for the breeding of orchids: Current achievements and prospects
expression vector (Yu, 2015). Yu (2017) used the pollen-tube path-
way and ovary injection methods to transfer the resistance gene
cbf1 into Phalaenopsis and found that the former had a higher seed
setting rate. It was found that both OnFd and OnFNR have sig-
nificant effects on soft rot, suggesting that these two genes may
play an important role in the resistance of Oncidium to soft rot
(Wu, 2017). Agrobacterium-mediated transformation was used to
transfer the PR1 gene (an important downstream gene in plant-
acquired resistance) into PLBs of Oncidium for transient expres-
sion. The transformed plants exhibited higher resistance (Guo,
2018).
In addition to the above transgenic hotspots, transgenic stud-
ies of orchids have also been conducted in relation to growth
and development. A phytoene synthase-RNAi construct was de-
livered into PLBs of Oncidium hybrid orchids, and the transgenic
orchids showed down-regulated levels of the PSY and geranyl
synthase genes. In addition, endogenous levels of gibberellic acid
and abscisic acid in the dwarf transgenic orchids were lower than
in wild type plants (Liu et al., 2014). Two genes, AtRKD4 (a key
gene for embryonic differentiation) and KNAT1 (a key gene for
bud apical meristem differentiation), can be used to induce or-
chid embryogenesis and bud development using Agrobacterium-
mediated insertion of the orchid genome (Semiarti, 2018).
This strategy effectively increases the yield of native and
hybrid orchids through somatic embryogenesis and shoot
development.
Although orchid researchers have made some advancements
in the establishment and optimization of the genetic transfor-
mation system and the transformation of target genes, there
are still many problems to be solved. The first is that currently,
most transgenic orchids remain in the exploration of transfor-
mation systems because the genetic transformation efficiency
of orchids is relatively low. Moreover, there are few reports on
breeding and gene function. These limitations make it difficult
to apply transgenic breeding of new germplasm. Therefore, im-
proving transformation efficiency is a key to the study of or-
chid transgenic technology in the future. Secondly, the trans-
genic false positive rate of orchids is relatively high. Therefore,
reducing the false positive rate is one of the keys to research
on transgenic technology of orchids. Thirdly, at present, studies
on the genetic transformation of orchids are mainly focused on
commercial orchids, such as Phalaenopsis, Dendrobium, and Oncid-
ium. Therefore, the coverage of research on the genetic transfor-
mation of orchids should be expanded. Finally, protocorm-like
bodies are thought to be ideal transforming receptor materials
for orchids and have a high regeneration rate during transfor-
mation. Such PLBs have been successfully used as transforming
receptors in Cymbidium (Yang et al., 1999), Oncidium (Liau et al.,
2003; Li et al., 2005), Phalaenopsis (Liao et al., 2004; Chan et al.,
2005), and Dendrobium (Uddain et al., 2015). However, the choice
of the best receptors remains controversial and needs to be
explored.
4. Orchid breeding prospects
With a rise in the demand for increased orchid quality and
yield, the breeding of new, attractive cultivars with unique col-
ors and/or morphology, and comprehensive resistance to vari-
ous stresses is urgently needed. Plant breeders, in general, are
387
required to do all they can to speed up the breeding process to
produce new cultivars of the many famous ornamental flowers
such as orchids, chrysanthemums and roses. However, it is worth
noting that whether this is accomplished by traditional breeding
or molecular breeding, genetics is the foundation.
Forward genetics studies genetic changes from phenotypic
changes, by identifying the sequence variation responsible for
a phenotypic trait (Ji, 2013). In application of forward genetics,
however, there is a misalignment of scientific research and prac-
tice because field workers find appropriate mutants but do not
know how to make use of them, whereas laboratory researchers
are constrained by not having suitable mutants to study.
In contrast to forward genetics, reverse genetics studies phe-
notypic changes from genetic changes based on massive or big
data. As a result of advancements and low-cost sequencing tech-
nology, omics data from orchids, including genomic, transcrip-
tome, and metabolome sequencing,are rapidly accumulating.
These data will provide a reference for transgenic breeding and
genome editing breeding, which will lay the foundation for or-
chid molecular breeding. However, one of the most obvious draw-
backs of reverse genetics is that when implemented, many genes
related to the desired traits may be found, and it is therefore dif-
ficult to narrow the target gene range. Given this challenge, there
is a need to integrate genomic with other omics data, and breed-
ing data with phenotypic data. Combining production, learning,
and research, will contribute to identification of genes/pathways
associated with important traits (Langridge and Fleury, 2011).
Hybrid orchids are the offspring of crosses between two
individuals with different traits. Intraspecific, intragenic, and
intergenic hybrids have been obtained in orchids, indicat-
ing traditional breeding is still the most commonly used for
the current orchid breeding. Intergeneric crosses are widely
conducted for orchids and a large number of cross grexes
have been performed, including bi-generic, tri-generic, tetra-
generic, penta generic. For example, Vandoritis = Vanda × Doritis
and Epilaeliocattleya = Epidendrum × Laelia × Cattleya, Kirchara =
Cattleya × Epidendrum × Laelia × Sophronitis. Although intergeneric
hybridization of orchids is necessary to overcome the cross-
incompatibility and zygotic abortion after hybridization, it may
be one of the best approaches for breeding new varieties. Muta-
tion breeding can also be used to obtain wide variations in flower
color, morphology, and size by overcoming incompatibility and
sterility. Accordingly, combining hybridization and mutation
breeding would represent an effective strategy for realizing the
full potential of hybridization in orchid breeding.
With respect to molecular breeding, the identification of gene
function is a key prerequisite, although currently there is no
stable transformation system available for orchids. At present,
transformation is mainly achieved using Agrobacterium-mediated
procedures, particle bombardment, and gene silencing. Further-
more, although plants in important ornamental genera such as
Phalaenopsis and Dendrobium have undergone gene editing, the
overall efficiency is low, and consequently, it remains necessary
to strengthen research on CRISPR/Cas9 to facilitate control of the
key phenotypes of orchids. In addition, although there are cur-
rently little genomic data for orchids, increasing transcriptomic
data are becoming available, which will contribute to the investi-
gation of important traits such as flower color, floral morphology,
and floral scents. Consequently, it is anticipated that molecular
388
breeding will emerge as the main approach for the breeding of
orchids.
In summary, regardless of whether we study the genes of key
traits from a basis of forward or reverse genetics, or whether tra-
ditional breeding, selective breeding, or molecular breeding are
used to obtain excellent offspring with target traits, each ap-
proach has its advantages and disadvantages, and if employed in-
dependently is unlikely to accelerate the breeding process. Con-
sequently, a range of methods and research ideas should be inte-
grated to facilitate the cultivation of orchids with unique flower
morphologies, novel colors, and rich flower scents.
Acknowledgments
We are grateful to this work for the funding of Scientific
Research Project of Shanghai Landscaping & City Appearance
Administrative Bureau (Grant No. G202401), Disciplinary Profes-
sional Construction Project of College of Art & College of Land-
scape Architecture (Grant No. YSYL-bdpy-2) and The National
Natural Science Foundation of China (Grant No. 31870199).
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