plants
Review
Advances and Perspectives for Polyploidy Breeding in Orchids
Pablo Bolaños-Villegas 1,2,3,* and Fure-Chyi Chen 4
1 Fabio Baudrit Agricultural Research Station, University of Costa Rica, La Garita District,
Alajuela 20101, Costa Rica
2 Lankester Botanical Garden, University of Costa Rica, Dulce Nombre District, Cartago 30109, Costa Rica
3 Faculty of Food and Agricultural Sciences, Rodrigo Facio Campus, School of Agronomy,
University of Costa Rica, Montes de Oca County, San Jose 11503, Costa Rica
4 General Research Service Center, National Pingtung University of Science and Technology, #1 Shuefu Road,
Neipu township, Pingtung 91201, Taiwan; fchen@mail.npust.edu.tw
* Correspondence: pablo.bolanosvillegas@ucr.ac.cr
Abstract: The orchid market is a dynamic horticultural business in which novelty and beauty
command high prices. The two main interests are the development of flowers, from the miniature
to the large and showy, and their fragrance. Overall organ size might be modified by doubling
the chromosome number, which can be accomplished by careful study of meiotic chromosome
disjunction in hybrids or species. Meiosis is the process in which diploid (2n) pollen mother cells
recombine their DNA sequences and then undergo two rounds of division to give rise to four haploid
(n) cells. Thus, by interfering in chromosome segregation, one can induce the development of
diploid recombinant cells, called unreduced gametes. These unreduced gametes may be used for
breeding polyploid progenies with enhanced fertility and large flower size. This review provides
an overview of developments in orchid polyploidy breeding placed in the large context of meiotic
chromosome segregation in the model plants Arabidopsis thaliana and Brassica napus to facilitate
molecular translational research and horticultural innovation.





 Keywords: orchid breeding; polyploidy; meiosis; fertility; flower size
Citation: Bolaños-Villegas, P.; Chen,
F.-C. Advances and Perspectives for
Polyploidy Breeding in Orchids.
Plants 2022, 11, 1421. https:// 1. Introduction
doi.org/10.3390/plants11111421
Orchids, including Phalaenopsis (2n = 2x = 38) [1] (Figure 1A), also called the moth or-
Academic Editors: Tomás Naranjo chid, are among the most popular ornamental potted plants currently traded around
and Pilar Prieto the world [2]. Other famous genera traded are Cymbidium, the Chinese ‘lan’ orchid
(2n = 2x = 40) [3] (Figure 1B and C); Dendrobium (2n = 2x = 38) [4] (Figure 1D); Paphiope-
Received: 9 February 2022
Accepted: 17 March 2022 dilum (2n = 2x = 26), the famous lady slippers [5] (Figure 1E); Oncidium (section Oncidium,
Published: 27 May 2022 2n = 2x = 42), the dancing-lady orchids [6] (Figure 1F); and Cattleya (2n = 2x = 40) [7,8]
(Figure 1G), among many others.
Publisher’s Note: MDPI stays neutral Rational breeding of commercial orchid cultivars may have been initiated in 1896 in
with regard to jurisdictional claims in Hawaii with the introduction of Dendrobium superbum from the Philippines [4], followed
published maps and institutional affil- by a formal research program initiated at the University of Hawaii in 1950 that involved
iations. a cooperation with the University of Hiroshima in Japan [4,9]. This program focused
on cytogenetics, cross-compatibility, and meiotic behavior, and quickly concluded that
polyploidy is key to breeding superior hybrids in Dendrobium and Vanda [4,9] while also
Copyright: © 2022 by the authors. establishing that genome homology is crucial to breeding Phalaenopsis [10]. Since the 1980s,
Licensee MDPI, Basel, Switzerland. large-scale orchid production has shifted to Taiwan and has been accompanied by research
This article is an open access article into in vitro seed germination, micropropagation, and the study of meiosis and pollination
distributed under the terms and to overcome hybridization barriers, especially in Dendrobium and Phalaenopsis [11]. This
conditions of the Creative Commons is a serious horticultural operation worth 200 million USD per year in Taiwan and about
Attribution (CC BY) license (https:// 85 million USD in Thailand [12].
creativecommons.org/licenses/by/ To remain competitive, new orchid cultivars must be developed continuously to meet
4.0/). the market demands [6]. Usually, both wild species and commercial cultivars are chosen as
Plants 2022, 11, 1421. https://doi.org/10.3390/plants11111421 https://www.mdpi.com/journal/plants
Plants 2022, 11, 1421 2 of 18
parent plants in breeding programs that rely on interspecific hybridization and selection of
progenies [2] because sexual reproduction may increase heterosis (see Table 1) and diversity
of traits [3], including flower longevity [7]. Nonetheless, high infertility is often the result
of breeding these advanced orchid hybrids [13]. It is believed that about 60% of pollination
events fail to induce fruit development in most orchid genera [11]. The reported causes are
many, varying from untimely tapetal degeneration in Oncidesa [6] to premature chromatid
separation and formation of lagging chromosomes during metaphase I in Aranda [14].
Misplaced bivalents with no clear position along the spindle, also called pseudo-bivalents,
have also been reported in Vanda semi-terete diploid hybrids [9]. Traditionally, fertility
can be restored in these orchid hybrids by doubling the number of chromosomes with
antimitotic agents such as colchicine and oryzalin during tissue culture [13]; the result is an
allotetraploid [15] (see Table 1). Ideally, the process induces disomic pairing of homologous
chromosomes during meiosis I and the creation of balanced gametes [13], but unfortunately,
Plants 2022, 11, x FOR PEER REVIEW
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which may reduce fertility [16].
 
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Atupnpgr oCxoimunattye, sbcya lAe mbaigr:o 2H.5sicemh ((BA,C,C),– iEn, GT)a,in1acnm Co(Bu,nFt)y. ,P ahnodt obsyt Pak. eBnolbayñoFs.-VCi.llCeghaesn (F(A) ,aDt ,tEh,eG L)ainn-
Pkiensgtetur nGgarCdoeunn itny ,CbayrtAagmo,i gaoll Hins 2ie0h22(.B ,C), in Tainan County, and by P. Bolaños-Villegas (F) at the
LankeTstoe rrGeamrdaeinn icnoCmaprteatgioti,vaell, inne2w02 2o.rchid cultivars must be developed continuously to 
meet the market demands [6]. Usually, both wild species and commercial cultivars are 
chosen as parent plants in breeding programs that rely on interspecific hybridization and 
selection of progenies [2] because sexual reproduction may increase heterosis (see Table 
1) and diversity of traits [3], including flower longevity [7]. Nonetheless, high infertility is 
often the result of breeding these advanced orchid hybrids [13]. It is believed that about 
60% of pollination events fail to induce fruit development in most orchid genera [11]. The 
reported causes are many, varying from untimely tapetal degeneration in Oncidesa [6] to 
premature chromatid separation and formation of lagging chromosomes during meta-
phase I in Aranda [14]. Misplaced bivalents with no clear position along the spindle, also 
called pseudo-bivalents, have also been reported in Vanda semi-terete diploid hybrids [9]. 
Traditionally, fertility can be restored in these orchid hybrids by doubling the number of 
chromosomes with antimitotic agents such as colchicine and oryzalin during tissue cul-
ture [13]; the result is an allotetraploid [15] (see Table 1). Ideally, the process induces di-
somic pairing of homologous chromosomes during meiosis I and the creation of balanced 
gametes [13], but unfortunately, chromosomes may undergo structural rearrangements 
under the influence of colchicine, which may reduce fertility [16]. 
  
 
Plants 2022, 11, 1421 3 of 18
Table 1. Processes involved in meiotic polyploidization and karyotype evolution in orchids and plants in general.
Term Description
Aneuploidy Loss or gain of chromosomes relative to the normal chromosome complement [17]
Allopolyploid Organism that combines two genomes or more deriving from related species [18]
Autotetraploid A tetraploid plant formed directly from the merger of two unreduced gametes provided by the same diploid individual and formed via FDR [18]
Allotetraploid A tetraploid formed from the merger of two unreduced gametes provided by different diploid plants and formed by FDR [18]
Amphihaploid A haploid derived from unbalanced meiotic segregation in a hybrid [19]
Autopolyploid Organism that combines two genomes or more deriving from the same species [18]
Bivalent Pair of homologs physically linked at the end of metaphase I [18]
Centric fission The break within the centromere of a single chromosome producing two whose raw ends can fuse after replication. Telomeric sequences may be added to the
termini and two stable chromosomes are formed. Fission increases chromosome number and karyotype symmetry [20]
Chiasmata The physical manifestation of reciprocal exchanges of DNA between non-sister chromatids (e.g., crossovers). Chiasmata maintain pairs of homologs linked at the
end of metaphase I (as bivalents). At least one is required per bivalent to obtain well-balanced gametes and avoid aneuploidy [18]
Chromosomal Inversion A structural change in chromosomes formed when two breaks are caused and the segment between them realigns and rejoins in the opposite orientation. To
enable effective pairing in meiosis an inversion loop is formed. If a chiasma is formed within the loop the chromatids will form a dicentric bridge and an acentric
fragment that is lost [18].
Cytokinesis Also known as cytoplasm division. It is the formation of a cell wall in plant cells at the telophase stage. Cellular organelles such as mitochondria are partitioned
between the new daughter cells [18].
Descending dysploidy Evolutionary decrease in the base chromosome number (x). Also called polyploid drop and viewed as the mechanism that turns polyploids into functional
diploids [21]
Dyad A pair of cells instead of the usual four cells resulting from abnormal meiosis [4]
First Division Restitution (FDR) A process in which a plant gamete is formed due to a defect in meiosis I. In these gametes there is neither recombination nor disjunction, thus parental
heterozygosity is fully retained [19].
Fractionation The loss of one or the other copy of a newly duplicated gene [22]
Gene Dosage Relative expression of a gene product [18]. Polyploids avoid imbalances in gene dosage by retaining duplicates and allowing them to eventually develop new
functions [23].
Genome Dominance A process antagonistic to heterosis in which a subgenome gains relevance through dosage and functionalization and that may involve changes in chromatin
marks and expression [23].
Heterosis The unevolved retention of genes or cis regulatory elements as homoeologous pairs, it is antagonistic to dominance [23]. It is linked to vigor in wide hybrids and
allopolyploids. It may manifest as the acceleration of somatic growth including the enlargement of organs [23].
Plants 2022, 11, 1421 4 of 18
Table 1. Cont.
Term Description
Homoeologous Non-homologous, often used to refer to meiotic pairing between non-homologous chromosomes. Homoeologous pairing reduces fertility [18].
Homologous Recombination Exchange of DNA sequences with the same linear arrangement of genes between a paternal and maternal chromosome copy, also called a homologous pair [24].
Karyotype Chromosome complement of an individual plant or species as described by number and morphology [4]
Micronuclei Aberrant nuclei formed by the unequal distribution of chromosomes in daughter cells, often caused by unpaired chromosomes and common in tumor cell lines
and meiotic cells of orchid hybrids [16,18].
Multivalent Association during meiosis of more than two chromosomes whose homologous regions are synapsed by pairs [4]. Multivalent formation is detrimental to
meiotic chromosome segregation and reduces fertility [18].
Ploidy The basic chromosome set, often defined as ’x’. Thus, 2 x indicates that the organism has two basic sets of chromosomes. It is different from the number of
chromosomes in zygotic cells defined as ’n´. For instance, bread wheat is a hexaploid in which 2n = 6x = 42, where x = 7 [18].
Polyhaploid Haploid individual resulting from polyploid parents [4]
Post-Polyploid Diploidization A process of evolutionary modifications that transform a polyploid genome into a quasi-diploid one. It is mediated by homoeologous recombination leading to
(PPD) structural chromosomal changes including reduction of chromosome number [21].
Robertsonian Fusion The fusion of two non-homologous chromosomes that gives rise to single chromosome with a single centromere [18,20]. As a result, the chromosome number is
reduced, and the karyotype symmetry is increased [20].
Second Division Restitution A process in which a plant gamete is formed due to a defect in meiosis II. The exclusive separation of recombined homologs causes the formation of partially
(SDR) homozygous unreduced gametes [17].
Somaclonal Variation Variation caused during tissue culture of orchids. Mutant plants can be distinguished by their morphological and physiological traits. It may be detected in the
diploid karyotype of Phalaenopsis, but the specific chromosomal rearrangements are difficult to examine [25].
Tetrad The four haploid products of meiosis [4]
Tissue Culture General term for the aseptic growth of tissues, calls and organs in vitro [4]
Univalents Homologs that fail to pair and form chiasmata between them at metaphase I. Their behavior at anaphase I is unpredictable and may not engage in successful
division [18].
Unreduced Gamete A gamete with the somatic chromosome complement [24].
Whole Genome Duplication Events of whole genome doublings (tetraploidizations) or whole genome triplications that lead to the formation of autotetraploid, allotetraploid and hexaploid
(WGD) plants [22].
Plants 2022, 11, 1421 5 of 18
Polyploid orchids, including autopolyploids (which arise from a single species) (see
Table 1) have desirable traits such as large flowers with great substance, round conformation
and intense coloration, and thicker stems and leaves [13]. In Cymbidium, newly synthesized
sexual allopolyploids (see Table 1) such as ‘Yutao’ show increased width and thickness of
sepal, petal, and lips, and flowers are rounder and produce more fragrance; therefore, they
have increased commercial value [3]. These polyploids are bred from naturally produced
unreduced gametes (see Table 1), and these plants are considered better than those obtained
from somatic polyploidization because of the resulting genetic diversity and heterosis [3].
As in the case of Dendrobium (see Figure 1A), the horticultural value of these unreduced
gametes is enhanced by the following facts [4]: (1) pollination of a single orchid flower
can potentially produce half a million to a million seeds, so a hybrid with 1% fertility can
give rise to 5000 to 10,000 viable seeds, and fertility as low as 0.01% may still produce 50
to 100 viable seeds; (2) endosperm tissue is lacking in most seeds, so embryo–endosperm
antagonism does not exist; and (3) orchid embryos are cultured in aseptic nutrient culture,
which allows for better survival of hybrids as compared with the normal seeding of other
plants.
Evolutionarily speaking, polyploid organisms are often more resilient to extreme
environments and cataclysms because of their increased genetic variation and the buffer-
ing effect of their duplicated genes [21,26]. For instance, whole genome duplications
(WGDs) (see Table 1) may have contributed to gene diversification and fine-tuning of
orchid ovule initiation and closure of the stigmatic cavity, as in the case of the DROOPING
LEAF/CRABS CLAW (DL/CRC)-like genes in Phalaenopsis equestris and Dendrobium catena-
tum [27]. Additionally, in the genome of P. equestris, a large WGD event was associated with
the Cretaceous-Paleogene extinction about 66 million years ago. This WGD is believed to
have been followed by intense radiation that enabled the Orchidaceae to become the second
largest angiosperm plant family with its remarkable diversity in flower morphology [28].
In nature, orchid autotetraploids (see Table 1) of Gymnadenia conopsea exhibit high
pollination and fruiting success [29], possibly because of changes in flower scent, changes
in pollinaria morphology, larger flowers, and low inbreeding depression [29]. Thus, a
higher reproductive fitness of tetraploids than diploids may enable them to become more
common [29]. In fact, genome duplication events are assumed to be beneficial for or-
chid breeders in search of new morpho-types and improvements in size, substance, and
form [30].
This review gives an overview of meiotic mechanisms and breeding techniques that al-
low for polyploidization of plant gametes to highlight opportunities for molecular breeding
in orchids for the accelerated creation of a new generation of elite hybrids.
2. Polyploidy Breeding in Orchids
Allopolyploids are usually created by hybridizing related species (allo = different);
thus, the resulting individual may have divergent genomes combined within its own
chromosome complement [31]. Merging genomes from different species provides genome
variation and novel opportunities to diversify, with the added advantage that gene re-
dundancy may mask recessive deleterious alleles by dominant ones [31]. Additionally,
the expression of genes required for chromatid cohesion and meiosis may be enhanced,
as observed in the Arabidopsis suecica allopolyploid [22]. The relative abundance of mei-
otic multivalents (more than three chromosome pairs) (see Table 1) has been used as a
cytological factor to distinguish auto- and allopolyploids. For instance, the prevalence
of multivalent pairing at metaphase I may point to homology between chromosome sets
and thus autopolyploidy [32]. However, in contrast, a high formation of bivalents (see
Table 1) at diakinesis may result from pairing between non-homologous (homoeologous)
(see Table 1) parental chromosome sets, which may indicate allopolyploidy [32], although
this behavior is not absolute [32]. In the actual case of orchid breeding, the development of
cultivars with multiple spikes involves crossing species such as Phalaenopsis micholitzii (with
multiple short spikes) and Phalaenopsis tetraspis (long spikes) [33]. The resulting hybrid,
Plants 2022, 11, 1421 6 of 18
P. Tzu-Chiang Tetralitz, develops up to five spikes [33]. In many cases, tissue culture (see
Table 1) induces spontaneous polyploidization of hybrids [33], but selection is laborious,
and crosses must be redone often to retain stability of traits in the progenies. Early gen-
erations of synthetic allopolyploids show quick and broad reorganization of the merged
genomes, including chromosome rearrangements and changes in chromosome number as
well as epigenetic modifications, such as transposon activation, chromatin modifications,
and altered methylation patterning [32]. Indeed, chromosome rearrangements are often
observed in meiocytes of presumptive orchid allopolyploids [34], along with micronuclei
(see Table 1) in tetrads. These micronuclei are common in human cancer cells and arise
from hypomethylation in peri-centromeric DNA, dysfunctional kinetochore assembly, poor
organization of the spindle, or uncoordinated expression of anaphase checkpoint genes [35].
3. Chromosomal Pathways for Meiotic Polyploidization
Although several reproductive mechanisms may create polyploid plants, most plants
are formed by the random production of diploid (2n) gametes [36]. However, despite the
huge biological and agricultural significance of forming diploid gametes, the molecular
mechanisms that lead to the formation are not well understood [36]. Cytologically, 2n
gametes possess the somatic chromosome number because of meiotic defects, which leads
to a mitosis-like/non-reduced division with dyads (2n) (see Table 1) formed alongside triads
(3n) and normal haploid tetrads (n) [37] (see Table 1), a phenomenon called meiotic nuclear
restitution [37]. Most of these 2n gametes result from a few basic processes of nuclear
restitution: (1) omission of meiosis I (also called first division restitution [FDR]) (see
Table 1), (2) omission of meiosis II (also called second division restitution [SDR]) (see
Table 1), (3) defects in spindle organization and (4) incomplete cytokinesis [37,38] (see
Table 1).
In eukaryotes, meiotic cell division halves the chromosome number in gametes via a
single DNA replication followed by two events of chromosome segregation [39]. During
meiosis I, homologous chromosomes should pair and synapse and exchange genetic infor-
mation via recombination. Then the crossover sites, called chiasmata (see Table 1), form
physical links between the two homologs and ensure (1) proper placement of the bivalent
in the spindle and (2) proper segregation of homologs during anaphase I [40]. To achieve
this, sister kinetochores from each homolog attach to microtubules extruding from the same
spindle pole (e.g., monopolar kinetochore attachment), and cohesion is removed at the
chromosome arms but not centromeres. Then during meiosis II, chromosome segregation in
the two new haploid nuclei proceeds in an equational fashion. Chromatid centromeres are
attached bipolarly to the microtubules up to anaphase II, when cohesion is finally removed
to ensure that chromatids segregate into four haploid daughter cells [40].
4. Synapsis, Chromosome Segregation and Meiotic Non-Reduction
Meiotic division and gametophytic ploidy (see Table 1) are tightly regulated processes
at the molecular level, and many of these regulators have been successfully character-
ized [38]. For instance, mutations in the Arabidopsis thaliana gene DYAD/SWITCH1 (SWI1;
At5g51330) (see Table 2) functioning in cohesion regulation [41] and its maize and rice
homologs, both named AMEIOTIC1 (GRMZM5G883855 and Os03g44760) [42], lead to the
abrogation of synapsis during meiosis I and rather turn it into a mitotic-like cycle [43].
However, in the Arabidopsis mutants parallel-spindle 1 or Jason, disturbed orientation of the
spindle leads to a similar effect [43]. These features are often referred to as examples of
FDR [43] and are somewhat common in amphihaploid- (see Table 1) and polyhaploid-wide
(see Table 1) F1 hybrids in which homology is very low and meiotic pairing does not
occur [39]. Thus, some researchers consider that this type of meiotic non-reduction is better
described as asynaptic- or univalent-dependent [39].
Plants 2022, 11, 1421 7 of 18
Table 2. Genes from model plant A. thaliana reportedly involved in meiotic restitution, crossover formation and chromatid alignment of interest for breeding
polyploid orchid hybrids.
Gene Name Functional Features in the Literature and Databases 1,2,3 Original Locus ID According Homologs of Interest in the Orchid
to TAIR and NCBI 1,2 Database Orchidstra 2.0 4 Horticultural Use
Encodes a protein with a HORMA-domain for recognition of
chromatin states linked to DNA double-strand breaks, a
peptidase domain, a Winged Helix (WH) DNA-binding domain Phalaenopsis aphrodite
and a SWIRM-domain for mediation of protein–protein transcript Heat-stress mediated disruption of
ASY1NAPTIC1 (ASY1) interactions in the assembly of chromatin-protein complexes. At1g67370 PATC148994 synapsis and recombination. Formation
ASY1 Participates in assembly of chiasmata and homologous (E-Value: 1.32 × 10−142) of clonal gametes [44]
chromosome pairing during meiosis. Mutants may show few
chiasmata per nuclei.
CYCA1;2/TARDY Encodes a N-terminal cyclin-like protein that may regulate Phalaenopsis modesta transcript
ASYNCHRONOUS MEIOSIS cyclin dependent kinases (CDKs). Encodes a core cell cycle gene At1g77390 PMTC011254 Formation of 2n gametes through SDR
(TAM) involved in meiosis II progression. Mutants develop dyads. (E-Value: 7 × 10−94) [45]
Encodes a protein that features a catalytic
phosphatidylinositol-specific phospholipase C X-domain that is
DYAD/SWITCH1 (SWI1) probably involved in signal transduction. Protein intervenes in Phalaenopsis lueddemanniana Development of unreduced gametes by
chromatid cohesion establishment, in chromosome structure At5g51330 transcript PLTC046384
during male and female meiosis, and in axial element (E-Value: 4 × 10−84) FDR [43]
formation.
ARABIDOPSIS THALIANA Encodes a protein with a conserved Mitogen-Activated Protein Oncidium Gower Ramsey transcript
MAP KINASE 4 (MAPK4) (MAP) kinase site. Required for male-specific meiotic At4g01370 OGTC022747 Formation of recombinant, SDR-type
cytokinesis. The mRNA is cell-to-cell mobile. (E-Value: 6 × 10−13) unreduced gametes [46]
ARABIDOPSIS THALIANA Encodes a kinase protein with an ATP-binding site. Phalaenopsis aphrodite transcript Formation of recombinant, SDR-type
MAP KINASE KINASE 6 (MKK6) Phosphorylates MAPK4. Required for male meiotic cytokinesis. At5g56580 PATC137812
(E-Value: 5 × 10−152) unreduced gametes [46]
Encodes a protein that features a core MutS, domain for DNA
ARABIDOPSIS MUTS mismatch repair. It is involved in homologous chromosome Phalaenopsis schilleriana transcript Reduction of homoeologous
HOMOLOG 4 (MSH4) segregation and meiotic mismatch repair during recombination. At4g17380 PSTC034674 recombination in polyploid hybrids,
(E-Value: 9 × 10−36
Mutants show low chiasmata formation. ) improved chromosome segregation [47]
Encodes a protein that features a WH-like DNA-binding
MMS and UV Sensitive 81 domain for branch migration and transcriptional repression, Phalaenopsis lueddemanniana Reduction of homoeologous
(MUS81) and an ERCC4 domain for cleaving branched structures At4g30870 transcript recombination in polyploid hybrids,
generated during DNA repair, replication, and recombination. PLTC011379 (E-Value: 2.82 × 10−69) improved chromosome segregation [47]
Mutants show few bivalents.
Encodes a protein with a kinesin motor domain and a P-loop Cymbidium ensifolium
NPK1-ACTIVATING KINESIN NTPase domain. It is required for cytokinesis in pollen. In transcript Formation of recombinant, SDR-type
2/TETRASPORE (NACK2/TES) mutants, all four microspore nuclei remain within the same At3g43210 CETC000340 unreduced gametes [48]
cytoplasm after meiosis. (E-Value: 6 × 10−38)
Plants 2022, 11, 1421 8 of 18
Table 2. Cont.
Gene Name Functional Features in the Literature and Databases 1,2,3 Original Locus ID According Homologs of Interest in the Orchid
to TAIR and NCBI 1,2 Database Orchidstra 2.0 4 Horticultural Use
Encodes a protein with a Serine/Threonine kinase domain.
NPK1-RELATED PROTEIN Regulates microtubule organization. May regulate formation of Phalaenopsis schilleriana Formation of recombinant, SDR-type
KINASE 3 (NP3) the intersporal callose wall after male meiosis. Mutants may not At3g06030 transcript PSTC039874
complete meiotic cytokinesis. (E-Value: 2 × 10−41) unreduced gametes [46]
Encodes a protein from the Polychome protein. It may work as
OMISSION OF SECOND a negative regulator of the activity of the anaphase-promoting Phalaenopsis schilleriana transcript
DIVISION 1/GIGAS CELL 1 complex/cyclosome (APC/C) ubiquitin ligase. Mutants At3g57860 PSTC034707 Formation of 2n gametes through SDR
(OSD1/GIG1) produce diploid gametes by skipping the second meiotic (E-Value: 7 × 10−75) [45]
division.
The respective protein contains an armadillo (ARM)-like fold,
PRECOCIOUS DISSOCIATION consisting of a multi-helical fold comprised of two curved Phalaenopsis schilleriana transcript
layers of alpha helices that allow for proteins and nucleic acids. At1g77600 PSTC040504 Stabilization of meiosis in hybrid
OF SISTERS 5B (PDS5B). The Arabidopsis genome contains five orthologues that are (E-Value: 9 × 10−30) polyploids [22]
required for proper chromosome segregation at anaphase I.
Encodes a RAD21-protein which may assemble as a
RECOMBINATION hetero-tetramer that enables opening of SMC-kleisin rings. It is Phalaenopsis aphrodite transcript Heat-stress mediated disruption of
8/SYNAPTIC 1 (REC8/SYN1) involved in chromosome condensation, pairing and segregation At5g05490 PATC192174 synapsis and recombination. Formation
during meiosis. Responsible for cohesion between replicated (E-Value: 9 × 10−44) of clonal gametes. Formation of
sister chromatids. unreduced gametes by FDR [44,49].
Encodes a factor known as the Replication Protein
A-70kDa-DNA-binding subunit. Contains an
REPLICATION PROTEIN A 1C Oligonucleotide/Oligosaccharide Binding motif, or OB fold, a Phalaenopsis bellina transcript Reduction of homoeologous
(RPA1C) five-stranded beta-sheet coiled to bind single-stranded DNA. At5g45400 PBTC027818 recombination in polyploid hybrids,
This protein regulates DNA unwinding during replication, (E-Value: 2.70 × 10−128) improved chromosome segregation [47]
recombination, and repair. Mutants show incomplete synapsis
and meiotic chromosome fragmentation.
May encode a member of the Structural Maintenance of
Chromosomes (SMC) family of proteins. These proteins share a
five-domain structure, with globular N- and C-terminal
STRUCTURAL MAINTENANCE domains separated by a coiled-coil segment with a globular Phalaenopsis equestris transcript
OF CHROMOSOMES 3/TITAN 7 “hinge” domain. The N-terminal domain contains a ‘Walker A’ At2g27170 PETC035815 Stabilization of meiosis in hybrid
(SMC3/TTN7) nucleotide-binding domain, while the C-terminal domain (E-Value: 3.57 × 10−147) polyploids [22]
contains a ‘Walker B’ motif and an ATP-binding cassette (ABC).
SMC3 localizes to the axial elements of pachytene chromosomes.
Heterozygous mutants show reduced cohesion along the arms.
STRUCTURAL MAINTENANCE
OF CHROMOSOMES 1/TITAN 8 May encode a SMC protein. Works together with SMC3 during Phalaenopsis bellina transcript Stabilization of meiosis in hybrid
(SMC1/TTN8) the establishment of proper meiotic cohesion. At3g54670 PBTC023989
(E-Value: 3.25 × 10−122) polyploids [22]
Plants 2022, 11, 1421 9 of 18
Table 2. Cont.
Gene Name Functional Features in the Literature and Databases 1,2,3 Original Locus ID According Homologs of Interest in the Orchid
to TAIR and NCBI 1,2 Database Orchidstra 2.0 4 Horticultural Use
STRUCTURAL MAINTENANCE
OF CHROMOSOMES May encode a protein part of the SMC5/6 complex. This Phalaenopsis equestris transcript
6B/HYPERSENSITIVE TO complex promotes sister chromatid alignment and homologous PETC039012 Stabilization of meiosis in hybrid
MMS, IRRADIATION AND recombination after DNA damage. Mutants produce unreduced At5g61460 (E-Value: polyploids [22]
−111
MMC; MIM (SMC6B/MIM) gametes. 6.95 × 10 )
May encode a myosin heavy chain-related protein. It may
SYNAPTONEMAL COMPLEX feature two coiled-coil domains and several sites for polar, basic, Phalaenopsis lueddemanniana Heat-stress mediated disruption of
PROTEIN 1A (ZYP1A) and acidic residues. It is involved in chromosome synapsis At1g22260 transcript PLTC005471 synapsis and recombination. Formation
during meiosis I and localizes at the synaptonemal complex (E-Value: 3.64 × 10−24) of unreduced gametes by FDR [44]
(SC), Mutants show improper or non-homologous synapsis.
Abbreviations: FDR, first meiotic restitution; SDR, second meiotic restitution. 1 The Arabidopsis Information Resource (TAIR). Available online: https://www.arabidopsis.org (accessed
on 6 March 2022). 2 The National Center for Biotechnology Information (NCBI). Available online: https://www.ncbi.nlm.nih.gov/nucleotide/ (accessed on 6 March 2022). 3 Universal
Protein Resource (UniProt). Available online: https://www.uniprot.org/ (accessed on 6 March 2022). 4 Orchidstra 2.0, A Transcriptomics Resource for the Orchid Family. Available
online: http://orchidstra2.abrc.sinica.edu.tw/orchidstra2/index.php (accessed on 13 March 2022).
Plants 2022, 11, 1421 10 of 18
And what is synapsis? At the early stages of meiosis, homologous chromosomes find
each other within the tight confines of the nucleus and then become fully aligned in a pro-
cess called homologous chromosome pairing [50]. Once chromosome stretches are paired,
those same regions will be held together by a scaffold of proteins called the synaptonemal
complex (SC). This tight alignment is called synapsis and allows for recombination, in which
information is exchanged between the parental homologs and generates crossovers that pro-
mote faithful chromosome segregation during meiosis I [50]. Arabidopsis genes that regulate
establishment of the cohesion (e.g., the entrapment of DNA) between homologous chromo-
somes can affect the assembly of synaptonemal complex. Such genes include STRUCTURAL
MAINTENANCE OF CHROMOSOMES 5 (SMC5; At5g15920), STRUCTURAL MAINTE-
NANCE OF CHROMOSOMES 6 A/B (SMC6A/B; At5g07660, At5g61460) and PRECOCIOUS
DISSOCIATION OF SISTERS 5 (PDS5A/E; At5g47690, At1g77600, At4g31880, At1g80810,
and At1g15940). However, whether these mutants show signs of FDR during meiosis is
unclear. Another gene that regulates synapsis and cohesion is SYNAPTIC1, also known
as RECOMBINATION 8/SYNAPTIC 1 (REC8/SYN1; At5g05490) [49] (see Table 1). This
gene has a severe impact on meiotic synapsis by affecting the correct polymerization of the
SC [43], and its absence during meiosis I leads to illegitimate interhomolog recombination
and catastrophic chromosome fragmentation [43]. Univalents are also produced owing
to failure to synapse, but their chromosomes are extremely tangled, and distinguishing
between them is difficult [51]. Thus, because the phenotype is so extreme, it might be
necessary to develop weak alleles to induce an FDR-like phenotype that might be useful
for orchid breeding.
For the formation of 2n gametes through SDR, several Arabidopsis genes have been
linked to the omission of meiosis II, including OMISSION OF SECOND DIVISION 1 (OSD1;
At3g57860, also known as GIGAS and UVI4-Like) (see Table 2), a key negative regulator of
the anaphase-promoting complex/cyclosome (APC/C) that may control the turnover of
cyclins to elicit the exit from mitosis or meiosis [45,52]. Another gene is the plant A-type
cyclin gene CYCA1;2/TARDY ASYNCHRONOUS MEIOSIS (TAM; At1g77390) (see Table 2),
which is essential for the transition between the first and second meiotic division and
whose mutations cause exit from meiosis after prophase [45]. In Arabidopsis, mutants
for TAM develop diploid gametes [45], whereas mutants for OSD1 develop triploid or
tetraploid gametes [45]. Combining both mutations led to the production of tetraploid
spores, and by adding mutant alleles for meiotic recombination (spo11-1) and for segregation
(rec8/syn1), meiosis is completely abrogated in the tam or osd1 backgrounds. This results
in the now legendary Mitosis into Meiosis (MiMe) phenotype [45] that was introduced in
rice to produce apomictic progenies identical to the mother plant [53]. This might be an
interesting approach to mass-reproduce valuable orchid hybrids, perhaps by CRISPR-Cas–
mediated transformation, as was shown recently in Taiwan with the orchid model species
P. equestris [54]. Moreover, the Orchidstra 2.0 transcriptomic database developed by the
Academia Sinica (Taipei) allows for identifying orthologues for up to 17 orchid species,
including P. equestris [55]. Hence, it might be technically feasible to perform genome editing
and test induction of unreduced gamete formation or MiMe-like phenotypes.
A third gene that is epistatic to both OSD1 and TAM is SUPPRESSOR WITH MORPHO-
GENETIC EFFECTS ON GENITALIA7 (SMG7; At5g19400) [56]. This gene may operate as a
regulator of the first to second meiotic division transition, probably by down-regulating
or inducing the degradation of CDKA;1 [56]. Pollen mother cells in the smg7 mutants
that arrest during anaphase II do not seem to form any pollen, as seen by Alexander Red
staining [56]. Perhaps yet-identified alleles of interest may be present in orchids.
5. Cytokinesis, Heat Shock and Polyploidy
Cytokinesis is another key cellular process that may lead to the production of poly-
ploid gametes [38]. In plants, meiotic cytokinesis is timed to occur after chromosome
segregation, but the process varies somewhat between dicots and monocots [46]. In dicots
such as Arabidopsis, meiotic cell walls are synthesized after the separation of the sister
Plants 2022, 11, 1421 11 of 18
chromatids, which occurs at the final stages of meiosis II, called simultaneous cytokine-
sis [46]. Nevertheless, in monocots such as rice and maize, meiotic cytokinesis involves
the synthesis of a cell wall after each round of chromosome segregation. Hence, a dyad is
formed at the end of meiosis I, whereas tetrads are formed after meiosis II [46]. This type
is called successive cytokinesis. Mitogen-activated protein kinases (MAPKs) are common
signal transduction factors that control meiotic cytokinesis [38], and they operate in a
relay fashion, a signaling cascade that may involve MAPK kinase kinases, MAPK kinases
(MKKs), and MAPKs, which are activated sequentially by phosphorylation at conserved
activation sites [57]. Of these, the interaction between Arabidopsis NPK1-ACTIVATING
KINESIN 2/TETRASPORE (NACK2/TES, At3g43210), NPK1-RELATED PROTEIN KINASE
3 (ANP3, At3g06030), MKK6 (At5g56580), and MAPK4 (At4g01370) (see Table 2) have been
shown to mediate male meiotic cytokinesis [46,57]. In the case of the tes mutants, all tetrads
share the same cytoplasm and initiate male gametogenesis together, thus leading to the
formation polyploid sperm, as seen by DAPI staining [48]. This situation is presumably due
to defects in the assembly of the radial microtubule system (also called the phragmoplast),
which leads to severe microtubule accumulation in nuclear surfaces and total failure to es-
tablish cytoplasmic domains [48]. Of note, mutants for suppressors of gibberellin signaling,
specifically rga-24 and gait6, show similar alterations at telophase II, thus leading to the
formation of diploid gametes in Arabidopsis (3.3%). Spraying with 100 µM GA3 caused the
same phenotype (3%) in the Ler phenotype [38].
Work in A. thaliana and Brassica napus has shown that heat, cold, and drought stress
may affect the expression or activity of MAPKs [57]. Remarkably, in Arabidopsis, heat
stress (36–38 ◦C for 24 h) caused defects in cytokinesis at metaphase I, namely reduced
abundance of microtubule fibers, failure to form a bipolar spindle, formation of multiple
mini-phragmoplasts at anaphase I, and total failure to form a radial microtubule system at
the tetrad stage [46]. Moreover, fluorescent in situ hybridization with a centromere probe
suggested that during meiosis I, homologous recombination or crossover formation is
impaired, because only univalents are observed [46] (see Table 1).
Cold stress is effective in inducing the formation of 2n gametes in Arabidopsis [36].
With cold shock treatment of 4 to 5 ◦C for up to 40 min followed by sampling seven
days later, 6% to 38% of flowers showed enlarged pollen grains. The sperm nuclei in
these pollen grains consistently showed extra centromere foci, as seen by expression of
the pWOX2:CENH3:GFP centromeric reporter construct, thus suggesting the formation of
diploid, triploid, and tetraploid male spores [36]. Results indicate defects in the formation
of cell plates between tetrads at telophase II, which suggests that these are recombinant,
SDR-type unreduced gametes [36]. Perhaps this approach could be used in orchid breeding
programs as well.
6. Recombination, Heat Shock and Polyploidy
The relation between heat stress (36–38 ◦C for 24 h) and reduced meiotic recom-
bination has been explored in A. thaliana by immunostaining and transcriptional anal-
yses [44]. Apparently, assembly of the synaptonemal complex is disturbed, as seen by
changes in the distribution and abundance of the chromosome axis protein ASYNAPTIC1
(ASY1, At1g67370) (see Table 2) and lateral element/transverse filament protein ZYP1A
(At1g22260) (see Table 2) and by the absence of bivalents at metaphase I. The expression
of recombinase RAD51 (At5g20850) is also reduced, which suggests impaired processing
of double strand breaks usually implemented by the conserved type-II topoisomerase
SPO11 (At3g13170) [44]. Abnormal tetrads were observed, including what appeared to be
dyads [44]. Thus, taken together, heat stress appears to be valuable for the formation of
unreduced-like gametes, probably nearly FDR-type ones. A potential caveat is that ZYP1A
is also involved in the formation of class I interfering crossovers [58], and in its absence,
crossover formation is not prevented but rather promoted, as seen by an increase of up to
50% in levels of the crossover makers HEI10 and MLH1 [59].
Plants 2022, 11, x FOR PEER REVIEW 12 of 18 
 
dyads [44]. Thus, taken together, heat stress appears to be valuable for the formation of 
unreduced-like gametes, probably nearly FDR-type ones. A potential caveat is that ZYP1A 
Plants 2022, 11, 1421 is also involved in the formation of class I interfering crossovers [58], and in its ab1s2eonfc1e8, 
crossover formation is not prevented but rather promoted, as seen by an increase of up to 
50% in levels of the crossover makers HEI10 and MLH1 [59]. 
Heeaatts strteressss( 3(344◦ C°Cv vs.s.2 121◦ C°C, f, ofroru pupto too noenwe weeeke)kh)a hsabse beneesnh oshwonwtno stoh osrhtoenrtemne mioseiisosinis 
Ainra Abirdaobpidsiospfsriso mfro2m1. 121h.1a ht  2a1t 2◦1C °tCo t1o8 1.18.h1 aht a3t4 34◦C °C[6 [06]0.]I. nInggeennioiouuslsylyc coommbbininininggt timimee--lalappssee 
aannaallyysseess ooff mmiiccrroottuubbuullee oorrggaanniizzaattiioonn ((TTaaggRRFFPP--TTUUAA55)),, bbrreeaakkaaggee oofft thheen nuuccleleaarre ennvveeloloppee 
((NNEEBB)),,l oloccaalliizzaattiioonno offC CDDKKAA;1;1--ssttrreessssg grraannuulleess( (CCDDKKAA;;11--mmVVeennuuss)) aanndd aasssseemmbbllyy ooff tthheeS SCC 
iinn cchhrroommoossoommeess ((AASSYY11--RRFFPP aanndd ZZYYPP11bb--GGFFPP)) ccoonncclluuddeedd tthhaatt dduurriinngg zzyyggootteennee,, AASSYY11 iiss 
uunneexxppeeccteteddlylyd depeplelteetdeda nadndth tehleo alodaidnginogf oZfY ZPY1Pis1a ibs oarbteodrtaebdr uabprtulyp. tTlyh.e Tahbeo ratbioonrtiisopno isss pibolys-
dsuibelyt oduaec ttiov aatcitoivnadtiuorni ndgurpinagch pyatcehnyeteonfea onf aew nelywdlyi sdciosvcoerveedrepdl apnlatncte clel lcl ycyclcelec chheecckkppooinintt 
ccoonnttrroolllleeddb byyt htehek inkiansaesAe TAATXAIAXITAE LTAENLAGNIEGCITEACSTIAASMIAU MTAUTTEADT(EADT M(A; ATMt3;g 4A8t139g04)8[16900].) 
I[n60th].e Isne htheeast-es threeasst-esdtrmesesieodc ymteesi,ohcoymteso,l ohgosmfaoillotgos pfaaiirl ptor oppaeirrl yp,raonpderclhyr, oamndos cohmroembroisdogmese 
abnrdidfgraegs manedn tfsraagremoebnstesr avreed o, pbsoesrsvibeldy, cpaoussseidblbyy canuosne-dh obmy onloong-ohuosmreocloomgobuins arteicoonm[6b0in].aTtihoen 
v[a6l0u].e Tohfet hviaslutyep oef othf iws otyrkpei noft hweocrokn itne xtht eo fcoonrcthexidt obfr eoercdhinidg bisretehdaitntgh eise txhparte tshseio enxpofretshseisoen 
coofn sthtreuscet sccoonusltdrubcetsa tctoeumldp tebde bayttAemgrpotbeadct ebryiu mA-gmroebdaicateteridumtr-amnsefdoiramteadt iotrnainnsfPohramlaaetnioopns iisn,  
aPshiasldaeonnoepsinis,T aasiw isa ndo[5n4e] ifno rTtahiewaanna [l5y4si]s foofr mtheei oasnisaliynsrise sopfo mnseeiotosisa lilnt yrpesepsoonf seex ptoe raimll etynptaels 
coofn edxiptieornims. ental conditions. 
7. Polyploidization and Recombination
7. Polyploidization and Recombination 
In Phalaenopsis orchids, species and primary F1 hybrids are usually diploid, whereas
elite vIna rPiehtaileasenaorpesiste otrrachpildosid, sp[6e1c]i;ese axnamd pplreims aarrye FP1h haylaberniodpss aisreS uosguoalYlyu kdiidpilaonid‘,V w3h’ e(rseeaes 
Feilgituer vea2rAie,tBie)s,  Parhea ltaeetnraoppsloisidT a[6i 1L]i;n exRaemdpAlensg aerle‘ VPh3a1l’aeannodpsPish Saloagenoo YpusiksiBdriaonth ‘eVr3I′ r(esneee F‘Figeunrge 
F2oAn,gB’)., APlhlahlaaevneop2nsis= T4axi =Li7n6 Rcehdro Amnogseolm ‘Ve3s1[′6 a2n].dS Puhchalapeanroepnstiasl Bvraortiheteire sIraernees ‘eFxeunagll Fyocnrogs’.s Aedll 
whiatvheo 2thne r= v4axr i=e t7ie6s cahnrdomspoescoiemsetso [i6n2tr]o. gSruecshs tpraairtesnstuacl hvaarsieqtuieicsk agrero swexthu,aelalys ecorofscsleodn awl riteh-  
portohdeur cvtiaornie,tfireasg arnandc sep, aecnidesr etosi sintatnrocgerteosds itsreaaitsse s[u62c]h. aTsh uqus,icekn hgarnocwetdhs, eexausea lorfe ccolomnbailn raetpioron-
mdiugchtitocno,n fsrtaigturtaenacev, aalnudab rleeshisotratniccue lttou rdailsaesasseet .[62]. Thus, enhanced sexual recombination 
might constitute a valuable horticultural asset. 
 
FFiigguurree2 2. . FFlloowweerr ssppiikkee ((AA)) aanndd ininddiivviidduuaall flfloowweerr ((BB)) inine eliltietet etetrtraapploloididh hyybbrrididP Phhaalalaeennooppssisis SSooggoo 
YYuukkididiaiann‘ V‘V33’ ′( 2(2nn= =4 4xx= = 7766)),, tthhee ssttaannddaarrdd whhiittee hhyybbrriidd moosstt ssoolldd iinnT Taaiwiwaann. .T Thhisise exxttrreemmeelylys statabblele 
mmeeiioottiicc hhyybbrriidd wwaassd deerriviveedd ffrroommP Phhaalalaeennooppssiiss DDoorriiss,, aanno oldldi ninteterrssppeeccifiifcich hyybbrridids syynntthheessizizeedd ffrroomm 
ccrroosssiinnggP Phhaalalaeennooppssiissa ammaabbiilliiss,,P Phhaalalaeennooppssiiss rrimimeessttaaddiiaannaa aannddP Phhaalalaeennooppssiiss aapphhrrooddiittee.. TThhee ssppiikkeess ccaarrryy 
mmuultltipiplelefl folowweerrssa alllle evveennlylys sppaacceedd,,a annddt thheefl floowweerrsst thheemsseellvveessa arrees syymmeettrricicaal,l,l laarrggee,,fl falatt,,a annddl loonngg 
lilviveedd..S Sccaalleeb baarr:: 22..55 ccmm.. PPhhoottooss ttaakkeenn bbyy FF.C.C..C Chheenni ninP PininggttuunnggC Coouunntyty, ,2 2002222. . 
Woorrkki ninB Brarasssicicaaa al lolotrtripiploloididss( A(AAACCg geennoommee,,2 2nn= = 33xx= = 2299)) ddeerriivveedd ffrroomt thhees sppeecciieess 
BBrraasssicicaar raappaa( (AAAAg geennoommee, ,2 2nn= = 22xx= = 2200)) aanndd BBrraasssiiccaa nnaappuuss( (AAAACCCCg geennoomee,,2 2nn= = 44xx == 3388)) 
hhaass sshown an increase in crossoverr ffoorrmaattiioonn ((11.7.7 toto 33.4.4 titmimese)s,) p, opsossisbilbyl ycocrorrersepsopnodnidnign tgo 
ttoytpyep Ie inI tienrtfeerrfienrgin cgrocsrsoosvsoervse [r1s9[]1. 9T]h. iTsh wisowrko irnkvionlvoedlv tehde tuhseeu osfe 1o9f9 1s9in9gslien nguleclneuoctildeoet pidoel-
pyomlyomrpohripshmism (SsN(SPNs)P esv) eenvleyn ldyisdtriisbturitbeudt eadcraocsrso cshs rcohmr omsoomsoems aens da nrdevreeavledal etdhath tahtet hine-
icnrceraesaes einin rereccoommbbininaatitoionn isis tthhee hiigheesstt in pollen-receiving plants, att lloonngg cchhrroommoossoomeess 
aanndda attp peerriicceennttrroommeerricicr reeggioionnss[ 1[199].]. 
Additionally, the combination of cytogenetics and SNP markers can assist in the
identification of regulators for aberrant homoeologous recombination in genotypes that
are suspected to be genetically unstable, as in the case of the B. napus double haploid
 population SGDH [63]. Partial homoeology has been suggested to cause meiotic irregu-
larities and defective tetrad formation in orchids such as Aranda ‘Christine’ C80 [14], an
Plants 2022, 11, 1421 13 of 18
old interspecific hybrid known for being genetically unstable [14]. Illegitimate recombina-
tion between homoeologues may cause aneuploidy (see Table 1) because homoeologous
bivalents, multivalents and univalents may not segregate correctly at meiosis I [64]. Addi-
tionally, the uneven exchange can lead to gain or loss of homoeologous segments when
chromatids segregate [63]. In Brassica napus SGDH plants, the formation of multivalents
seems common, as observed by fluorescent in situ hybridization, and duplications and
deletions are widespread, as confirmed using SNP markers. Most of this variation seems
linked to the presence of a region of 10.3 to 23.9 Mbp nested within chromosome 9, named
BnaA9, which shows a strong change in the expression of orthologues for REPLICATION
PROTEIN A 1C (RPA1C; At5g45400) and MMS and UV Sensitive 81 (MUS81; At4g30870) [63]
(see Table 2). The endonuclease MUS81 is an important mediator in the resolution of recom-
bination intermediates such as double Holliday junctions [64], whereas RPA1C is part of
the heterotrimeric RPA complex that mediates activation of DNA damage checkpoints [65].
Thus, although in polyploid hybrids, it might be possible to fingerprint for alleles
that might cause genetic instability, breeders might want to look for a long-term solution.
Cytologically, in allo-haploid, tissue-cultured Brassica napus cv. Tanto, the reduction in copy
number of ARABIDOPSIS MUTS HOMOLOG 4 (MSH4; At4g17380) (see Table 2), by just
one copy, effectively reduced the frequency of events of homoeologous recombination,
possibly constituting an evolutionary mechanism for fine-tuning meiosis [47]. This gene is
part of the main crossover pathway, called the ZMM pathway because it involves genes
ZIP1, MER3, MSH4, MSH5, SHOC1, HEI10, and PTD. Remarkably, many of them (e.g.,
MER3, MSH4 and MSH5) show rapid loss of duplicates following evolutionary events of
WGD in angiosperms [47]. One might envision the use of CRISPR-Cas9 editing to trim the
number of MSH4 duplicates in valuable orchid polyploids to stabilize meiosis [16]. In fact,
in allopolyploids, the ZMM pathway could be targeted to reduce the number of crossovers
to the bare minimum, one per chromosome pair to prevent inter-homoeologue crossover
formation [47].
8. Post-Polyploid Diploidization in Orchids
In the 1960s in Hawaii, allopolyploid Vanda hybrids showed preferential pairing of
chromosomes during meiosis [9]. Then in the 1980s in Malaysia, the diploid species Calanthe
veratrifolia showed evidence of asynchronous segregation of subgroups of chromosomes
during meiosis, involving multiple spindles, perhaps suggesting a polyploid ancestry or
concealed hybridity [66]. In those days, this behavior was called complement fraction-
ation [66] (see Table 1). Currently this phenomenon is better known as post-polyploid
diploidization (PPD) [21] (see Table 1), and it may have driven the evolution of paleo- and
meso-polyploid lineages into diploid genomes following a WGD event. The process is
thought to involve genome downsizing, subgenome-specific fractionation, and modulation
of gene expression [21]. A key feature of genome fractionation during PPD is that one of
the parental subgenomes generally retains significantly more genes as compared with the
other subgenome, especially in the case of dosage-sensitive genes [67] (see Table 1). Such
genes are involved in macromolecular complexes, transcription regulation and responses
to environmental stimuli [23]. The determination of genome dominance (see Table 1) is
suspected to be linked to differences in the density of transposable elements, methylation,
and siRNA expression [23]. For instance, the subgenome “A” of A. suecica shows decreased
levels of CG methylation and transcriptional up-regulation at loci required for proper
chromatid alignment during meiosis, possibly as a mechanism to promote reproductive
stability [22]. Example loci are homologs of STRUCTURAL MAINTENANCE OF CHRO-
MOSOMES 3/TITAN 7 (SMC3/TTN7; At2g27170), SMC1/TTN8 (At3g54670), SMC6B/MIM
(At5g61460) and PDS5B (At1g77600) (see Table 1).
At the cytological level, PPD may manifest in orchid hybrids as chromosomal rear-
rangements such as centric fissions, inversions and Robertsonian translocations between
homologous and non-homologous chromosomes (see Table 1) that lead to speciation and
diversification. Or if expressed alternatively, PPD may transform a polyploid genome into
Plants 2022, 11, 1421 14 of 18
a quasi-diploid one with a lower base chromosome number (x) (e.g., descending disploidy)
(see Table 1) that reverts the number of linkage groups to the same number as the diploid
ancestors [21]. Inversion loops and translocation junctions have been reported at pachytene
in Doritaenopsis Fuchsia Princess ‘KHM648’ (2x = 38) [1]. However, chromosomal bridges
have been observed at anaphase I in chromosomes of Dtps. Sweet Strawberry ‘Wei’ (4x = 76)
and Dtps. Ben Yu Star ‘Red Dragon’ (4x = 76) [34], both suggesting the existence of PPD
in commercial hybrids of Phalaenopsis. Widespread variation in chromosome number in
Paphiopedilum section Barbata is suspected to be caused by centric fissions (2n = 28, 30, 32,
33, 34, 35, 36, 37, 38, 40, 41, and 42) [20].
Although not entirely obvious, commercial hybrids may offer the opportunity to study
orchid PPD in real time and in a controlled setting to facilitate understanding the molecular
mechanisms that are believed to mediate PPD and genome fractionation, such as chromatin
accessibility and histone modifications [67].
The experimental hurdle is that the Orchidaceae family (Figure 2) comprises more than
28,000 species and 736 genera and that the patterns of karyotype (see Table 1) evolution
are not well understood [21]. What is clear is that the sequenced orchid genomes that are
currently available (Phalaenopsis equestris, Dendrobium catenatum, Dendrobium officinale and
Apostaceae shenzhenica) appear to have high chromosome numbers (2x = 38, 2x = 38, 2x = 38,
and 2x = 68), which suggests paleo-polyploid origins [21,68]. Additionally, a huge disparity
in orchid chromosome numbers, ranging from 12 in Erycina pusilla to 240 in Epidendrum
cinnabarinum, indicates that many WGD events followed by diploidization may have taken
place during evolution across different clades [21,69,70].
In contrast, by comparing the karyotypes of species in Phalaenopsis versus interspecific
hybrids, one may deduce, in a short time, a few trends for PPD. For instance (1) during
the selection of sexually fertile progenies in harlequin and novelty cultivars (P. Chian Xen
Magpie, P. Chian Xen Piano ‘CX339’), there is a strong fractionation bias against large
chromosomes from sections Polychilos, Esmeraldae and Parishianae; (2) in specific triploids
such as P. Golden Sands ‘Canary’, P. Taipei Gold ‘STM’, P. Queen Beer ‘Mantefon’, P.
Joy Spring Canary ‘Taipei’, P. Sogo Relax ‘Sogo F-987’ and P. Liu’s Berry ‘SW’, irregular
pairing of chromosomes is common, presumably due to chromosomal rearrangements;
and 3) the production of unreduced gametes has certainly led to the formation of valuable
tetraploids such as P. Taipei Gold ‘Gold Star’ and P. Paifang’s Queen ‘Brother’ [71]. Finally,
the implementation of genomic in situ hybridization may facilitate the identification of
parental genomes for the introgression of key horticultural traits [71], and the identification
of alleles for OSD1 and TAM might accelerate the development of new elite polyploids [24].
9. Induction of Polyploidy on Meiocytes
A novel polyploid individual may form via different pathways and depending on the
pathway and the level of heterozygosity, a newly formed polyploid will perform better,
as shown by gains in growth, fertility, and horticultural quality [13,19]. This is important
because horticulturally speaking, the current understanding of inter- and intrageneric
polyploidization, and in vitro propagation in Phalaenopsis, Epidendrum, Lycaste, Cymbidium,
and Dendrobium has been obtained from somatic chromosome doubling with colchicine
in solid and liquid medium (0.01–0.005%) [25,61], oryzalin in liquid medium (57 µM) [13],
and amiprophos-methyl (APM) in liquid medium (2.5 µM) [72] , followed by flow cytom-
etry analysis and selection of progenies [72], which may show somaclonal variation [25].
However, this path does not reproduce what usually happens in nature because mitotic
non-disjunction of sister chromatids in meristem tissues, zygotes or embryos is rare and
unfortunately restricts the number of alleles fixed per locus in auto- and allotetraploids [19].
In contrast, meiotic chromosome doubling is known to result in larger genetic variability,
fitness, and heterozygosity than somatic (mitotic) doubling [73].
In cultured Cymbidium, unreduced gametes form at a rate of 0.15% in the cultivar
‘Xiaoxiang’ to 4.03% in cultivar ‘47–17’, and the formation is believed to be the highest in
interspecific hybrids [3]. However, in Begonia, the formation of 2n gametes can be boosted
Plants 2022, 11, 1421 15 of 18
by treating pollen mother cells with trifluralin (10, 100 and 1000 µM in 5% DMSO for
24 h) and nitrous oxide (N2O) at 6 bar (600 kPa) for 48 h [73]. Additionally, in Populus
canescens, the injection of colchicine (0.5% v/v) during pachytene leads to unreduced
gamete production at an astonishing rate of 30%, with the germination rate not significantly
affected (22% vs. 23% in natural unreduced gametes) [74].
Work in Caenorhabditis elegans suggests that during meiosis, colchicine affects the
formation of the SC and disrupts the structure of the nuclear envelope [75]. Colchicine is
a dinitro-sulfonamide herbicide and worm killer that binds selectively to α-tubulin and
inhibits polymerization of microtubules. However, it is also a hydrophobic compound that
may rupture membranes similar to detergents [75]. Oryzalin is a dinitro-aniline herbicide
that has higher affinity to plant tubulins [13]. Trifluralin and N2O are believed to disrupt
meiotic cytokinesis [73]. Trifluralin is a plant-specific dinitro-toluidine herbicide that also
targets tubulin [76]. Trifluralin and colchicine may be applied directly to meiotic flower
buds of P. Sogo Yukidian ‘V3’ (see Figure 1A,B) in lanolin paste (0.09–0.13% and 0.05–0.1%)
to induce the formation of 2n gametes [77].
10. Conclusions
Meiotic polyploidization is an important tool for successful orchid breeding, and it may
allow for the development of new and exciting orchid hybrids, as in Phalaenopsis. However,
as an evolutionary process, it is complex and may involve genome fractionation, dysploidy,
and homoeologous recombination, as seen in organisms such as A. thaliana, A suecica, and B.
napus. Thus, its study in orchids may also allow for better understanding the mechanisms
that shape genome evolution and that may be harnessed during horticultural selection. The
integration of genomics and genome-editing tools may allow for the functional validation of
gene function during orchid meiosis and the induction of valuable traits such as unreduced
gamete formation.
Author Contributions: P.B.-V.; writing—original draft preparation, F.-C.C.; writing—review and
editing. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Vicerrectoría de Investigación, University of Costa Rica grant
numbers C0244 and C2060. And the APC was also funded by Vicerrectoría de Investigación.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors thank their respective institutions for their continuous support.
Conflicts of Interest: The authors declare no conflict of interest.
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