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Polyploidization in Orchids: From Cellular Changes to Breeding Applications

Polyploidy occurs naturally in plants through cell division errors or can artificially be induced by antimitotic agents and has ecological effects on species adaptation, evolution, and development. In agriculture, polyploidy provides economically improved cultivars. Furthermore, the artificial induction of polyploids increases the frequency; thus, it accelerates obtaining polyploid plants used in breeding programs. This is the reason for its use in developing many crops of economic interest, as is the case of orchids in the flower market. Polyploidy in ornamental plants is mainly associated with flowers of larger size, fragrance, and more intense coloring when compared to naturally diploid plants. Currently, orchids represent the largest flower market worldwide; thus, breeding programs aim to obtain flowers with the larger size, durability, intense colors, and resistance to pathogens. Furthermore, orchid hybridization with polyploidy induction has been used to produce improved hybrid cultivars. Thus, the objective of this review was to compile information regarding the natural occurrence, importance, and methods of induction of polyploidy in orchids. The study also summarizes the significance of polyploids and techniques associated with artificially inducing polyploidy in different orchids of commercial relevance.


Introduction
Polyploidy is defined as the increase in chromosome number, generating organisms with more than two complete sets of chromosomes. Polyploidy is one of the essential phenomena in plants and is responsible for species adaptation, diversification, evolution, and development [1]. It is estimated that about 70% of angiosperms have experienced polyploidy during their evolutionary history [2]. The highest frequencies of duplication of genetic material were mainly observed in domesticated plants instead of wild plants. The close relationship between domestication and polyploidy is due to the random selection of polyploid plants for their greater vigor; thus, polyploid species could be more successful and promising for domestication than wild ones [3]. Throughout evolution, the angiosperm genome experienced at least one chromosome duplication event [4], thus allowing original diploid individuals to generate other plants with different ploidy levels.
Polyploid organisms can be classified according to their origin into autopolyploid and allopolyploid [5]. An autopolyploid organism increases its basic number of chromosomes and is formed by the duplication of its genome [5,6]. An allopolyploid is formed from hybridization between different species and is characterized by having more than two basic sets of different chromosomes [6]. It is believed that the majority of flowering plants are allopolyploids (about 75%) [7,8]. Chromosomal and cytological studies of Cymbidium species have demonstrated a predominance of 2n = 40 chromosomes [38,39], with variations observed in Cym. serratum (2n = 41, 43, 60, and 80). From the first Cymbidium polyploids reported in the early 20th century and through the biological and artificial techniques, it has been possible to develop a set of polyploid cultivars of Cymbidium [43]. Cymbidium cultivars are diploids, triploids, and tetraploids with reported differences in chromosomal morphology [40]. The polyploids have been reported in 75.8% of Cym. hybridum cultivars [44], demonstrating an association between the intentional or nonintentional selection of polyploids instead of diploids for superior characteristics.
The high number of commercial tetraploids cultivars demonstrates the importance of polyploidy in the development of superior cultivars of Phalaenopsis. In addition, natural tetraploid species, such as Phal. amabilis and Phal. rimestandiana (both 2n = 4x = 76) and Phal. aphrodite subsp. formosana, are conventionally used in breeding for the production of tetraploid hybrids [6,20,47]. These tetraploid species are used as parental to obtain different groups of Phalaenopsis cultivars with different desired colors for commercial purposes [36,48,49].
In the genus Oncidium, the basic number of chromosomes is believed to be x = 7, but unlike other genera, this has a large chromosomal variation among species, with the majority presenting polyploidy, such as tetraploids, hexaploids, and octoploids, numbers of chromosomes [55].

Endopolyploidy
Endopolyploidy, commonly generated by endoreduplication, has been reported in different genera of orchids (Table 1). Tissue type, stage of development (early or late), and differences between varieties within the same species are the main factors that can influence the frequency and intensity of cellular endoreduplication [9,[56][57][58]. In addition, abiotic environmental factors such as light and nutrients also affect the endoreduplication in plants [9]. For example, Lee et al. [59] reported that temperature influenced the occurrence of endopolyploidy in the cells of Phalaenopsis aphrodite and Oncidium varicosum orchids.
Other factors, such as phytoregulators, also influence the presence of different levels of endopolyploidy in plant tissues [9]. Lim and Loh [60] observed that sexual embryos of Vanda "Miss Joaquin" in the presence of 1-Naphthaleneacetic Acid (NAA) had higher levels of endopolyploidy in their cells compared to embryos in the presence of Gibberellic Acid (GA 3 ), showing that synthetic auxins, such as NAA, are important induction factors that generate variations in ploidy levels in orchid cells.
Several studies have reported endopolyploidy in orchids with a wide variety of cells, tissues, and organs with natural polyploidization events (Table 1). The main types of plant material used for the analysis and identification of endopolyploidy in orchids were leaf tissues (46% of the works), followed by parts of the flowers and roots. Other types of plant tissues were also reported, such as seeds (9%) and ovarian tissue (9%). Plant tissues from in vitro cultivation have also been used to determine ploidy variations in orchid cells. Protocorms (27%) and protocorm-like bodies (PLBs) (27%) are the most used tissue types to analyze the endopolyploidy, followed by embryos (sexual and somatic) and calluses with 23% and 9%, respectively.
The endopolyploidy observed in orchid cells of different types of tissues and organs can be used for the induction and regeneration of complete polyploid plants from endopolyploidy cells, thorugh the use of plant tissue culture techniques [56], and that can be usefull as a biotechnological tool for orchid polyploid cultivar development. Chen et al. [56] developed a technique for the genus Phalaenopsis that consists of successive cycles of horizontal sectioning of protocorms and PLBs, thus inducing the natural endopolyploidy cells in these organs to new PLBs formation, which formed solid polyploid plants after regeneration [56,67,68].

Occurrence of Unreduced Gametes
In orchids, more than one million pollen grains are grouped into a cohesive mass called pollinia [77]. Cytological pollen studies in orchids have shown the formation of unreduced gametes, which are more frequent in cultivars resulting from interspecific and intergeneric hybridization [78]. In some orchid genera like cultivars of Cymbidium, Zeng et al. [79] observed that the frequency of unreduced (2n) gametes ranged from 0.15% to 4.03%, depending on the genotype. After seven different crosses between these cultivars, they observed two tetraploids and three triploid hybrids with good in vitro regeneration behavior and high survival during acclimatization. Thus, progenitor cultivars with a higher frequency of unreduced gametes could be used to induce polyploidy in Cymbidium breeding programs without inhibiting the mitotic spindle and with no carcinogenic risk to animal cells, commonly associated with the manipulation and treatment of plant tissues and organs with antimitotic agents, such as colchicine.
The main mechanisms of natural polyploidization in Phalaenopsis are hybridization and endopolyploidy. However, after analyzing the chromosomes of 60 Phalaenopsis cultivars, Lee et al. [36] suggested that, in addition to endopolyploidy, the formation of unreduced gametes could also be responsible at least in part for the expressive frequency and number of polyploid genotypes.

Artificial Induction of Polyploidy in Orchids
Polyploidy is artificially induced by applying antimitotic agents such as colchicine, oryzalin, trifluralin, propyzamide, and amiprofos-methyl (APM) on tissues, organs, or entire plants [83]. These chemicals are used in vitro to interfere during cell division, generating chromosome duplication in plant cells [84]. Antimitotic agents are grouped according to the phase of the cell cycle that they affect. Some agents can affect the end of the S phase or middle of the M phase (late-stage). Other agents act before the S phase, being the most significant group used for the artificial induction of polyploids [84]. The substance most widely used for polyploidy induction in plants is colchicine, an alkaloid extracted from the seeds and bulbs of Colchicum autumnale plants [84,85]. Before colchicine, Randolph [86] induced artificial polyploidy through high-temperature treatment in early-stage embryos of maize, generating tetraploids. Similarly, Blakeslee and Avery [87] obtained somatic polyploidization using high-and low-temperature heat treatments, but these techniques were not efficient for the induction of polyploids. Blakeslee and Avery [87] and Eigsti [88] conducted the first tests using ex vitro colchicine to plant-inducing polyploidy.
Murashige and Nakano [89] were the first to report spontaneous polyploidy in tobacco callus under in vitro conditions in response to the increase of explant subcultures. They recommended in vitro plant growth as an efficient tool to artificially induce polyploidy [90].
Currently, there are a large number of protocols for in vitro chromosome duplication in many plant species, including orchids. Figure 1 summarizes the main types of explants used and the workflow aimed at obtaining artificial autopolyploid plants in orchids through chemical antimitotics. The efficiency in generating these types of polyploids depends on the type, concentration, and exposure time to the antimitotic agent, explant type and age, in vitro induction protocol, and direct or indirect methods for confirming chromosomal duplication [84]. Among the various benefits are that polyploidy causes increased vigor, allowing more remarkable adaptation to extreme climatic conditions [91]; an overall increase in organs size due to multiple copies of genes, resulting in a phenomenon known as the gigas effect, is also observed [90,91].
Studies at the Laboratory of Plant Physiology and Tissue Culture (CCA/UFSCar, Araras, Brazil) revealed that in vitro autopolyploid plantlets of the Cattleya hybrid induced by colchicine showed distinct morphology. These plants were more compact, with wider and thicker leaves ( Figure 1A,B), than those that were not polyploid ( Figure 1C,D). Other interesting characteristics associated with polyploid organisms include the buffering genome, heterosis, increased heterozygosity, restoration of hybrid fertility, reduced fertility in autopolyploids, and seedless fruits [89,90]. In addition, flowering in polyploid organisms results in improved ornamental features, such as the larger size and intensity of pigments [92] and longer durability [90]. These characteristics associated with polyploidy are desirable and valuable in orchid breeding programs [93]. Among the various benefits of polyploidy for orchid cultivation, the restoration of fertility of hybrids and changes in the morphological and anatomical characteristics, such as increased leaf thickness and length, increased stomata, and the increased size and texture of flowers, besides influencing the flowering periods, are the most significant ones [94,95].

Cattleya Genus
The induced polyploidy in Cattleya can be used for obtaining the compact size of plants, increased flower longevity, a greater number of flowerings throughout the year, flowers with higher firmness (substance), and greater resistance to transport. These are the biggest challenges for the expansion of Cattleya cultivation and marketing [18].
In two studies, polyploidy was induced in Cattleya, where the PLBs and seedlings were used as explants for in vitro cultivation. Colchicine was used in one study at concentrations of 0.05-0.2%, and the exposure time ranged from two to four days. Another study compared the use of two polyploidy-inducing agents, viz., colchicine (0-12.5 mM) and oryzalin (0-50 µM) (Table 2). Unfortunately, despite numerous hybrids used in the Cattleya flower market, only C. intermedia and C. tigrina have been reported in the literature.
used and the workflow aimed at obtaining artificial autopolyploid plants in orchids through chemical antimitotics. The efficiency in generating these types of polyploids depends on the type, concentration, and exposure time to the antimitotic agent, explant type and age, in vitro induction protocol, and direct or indirect methods for confirming chromosomal duplication [84]. Among the various benefits are that polyploidy causes increased vigor, allowing more remarkable adaptation to extreme climatic conditions [91]; an overall increase in organs size due to multiple copies of genes, resulting in a phenomenon known as the gigas effect, is also observed [90,91].  For C.a intermedia, the best treatments for polyploid induction using colchicine were 0.05% (for clone 114-75% of tetraploids) and 0.1% (for clone 121-40% of tetraploids), both treated for eight days, showing a strong genotype-dependent response [96].

Cymbidium Genus
The first use of antimitotics and induction of polyploidy in the genus Cymbidium was reported by Menninger [98], Wimber, and Van Cott [99] and Kim et al. [100]. From 2009 to 2021, there were nine studies on polyploidy induction using antimitotic agents in Cymbidium, of which eight were performed with hybrid cultivars (Table 3). Similar to Cattleya, in Cymbidium, most polyploid induction studies were performed under in vitro conditions, and the PLBs were the primary type of explants (55.6%). Other explants included were protocorms, rhizomes, seedlings, and young shoots (Table 3). The rates of obtaining polyploid plants ranged from 11.1% to 60%, and colchicine was used as an antimitotic in 89% of the studies and oryzalin in only two studies. The highest regeneration rates were obtained with colchicine in 0.03-0.05% concentrations and drug exposure times ranging from 4 to 7 days (Table 3). The use of oryzalin in two Cymbidium hybrids made it possible to obtain tetraploids at concentrations of 5-10 mg L −1 . However, colchicine was more efficient than oryzalin in PLB survival and polyploid frequency [101]. Another fact reported by these authors was the strong genotype-dependent response, and up to 60% of polyploids were reported in Cym. Show Girls, while, in Cym. Mystery Island, maximally 16.7% of the polyploids were obtained. About 75% and 80% of the polyploids were obtained at concentrations of 0.2% of colchicine exposed for two days and 0.05% of colchicine for three days, respectively, using the PLBs as explants [108,109] (Table 4). In addition, three studies used oryzalin, propyzamide, and AMP for polyplodization (Table 4). In addition to oryzalin [110], propyzamide at 100 µM for two days [111] and amiprofos-methyl (AMP) at concentrations of 10 mg L −1 for 12 to 48 h [112] showed good efficiency in obtaining Dendrobium polyploids.  Zhang and Gao [120] PLBs: Protocorm-like bodies.

Phalaenopsis Genus
Griesbach [20] was one of the pioneer used in vitro tools for artificial induction of chromosomal polyploidization in Phalaenopsis. Protocorms of Phal. Equestris, Phal. Fasciata, and Phal. "Betty Hausermann" were exposed to 50 mg L −1 of colchicine for ten days, resulting in 46% with polyploid seedlings [20]. Griesbach [121] used a similar technique with a colchicine treatment to restore the fertility of the triploid hybrid Phal. Golden Sands "Canary" and obtained 50% of hexaploid plants (fertile), which were successfully used as parental plants to develop new cultivars with a greater intensity of colors, sizes, and shapes, such as the pentaploid (2n = 5x = 95) Phal. Meadowlark [121].
Twelve polyploid induction studies were reported for the genus Phalaenopsis, and colchicine was used in 80% of these studies in concentrations ranging from 0.5 to 5000 mg L −1 . The colchicine exposure time lasted from 3 to 10 days. Oryzalin was applied only in one study (Table 5).  The main explants chosen for the in vitro induction of polyploids are protocorms [20,30,121,122]. On the other hand, the explants chosen for the ex vitro induction are seedlings and flowers (bud flowers and pollinated flowers) [123][124][125] (Table 5). Furthermore, it was observed that, unlike other genera, in Phalaenopsis, 50% of the studies in the literature used organs or entire plants exposed to colchicine under ex vitro conditions to obtain polyploid plants.
Interestingly, several studies were performed on Phal. amabilis (64%) during 2013-2021 (Table 5). Despite the reduced commercial importance of this species compared to the hybrids, it has been extensively studied under in vitro culture conditions and for different purposes, possibly serving as an in vitro regeneration model among the many Phalaenopsis genotypes. Among these studies, we would like to highlight the treatment of nitrous oxide to pollen grains of Phal.s amabilis (2n = 2x = 38), which was used to obtain seeds and seedlings from the treated pollen grains. In which the treatment for 24 h resulted in up to 35.6% triploid and 6.7% tetraploid plants, which was more efficient than the treatment for 48 h. Furthermore, Azmi et al. [123] obtained up to 100% tetraploid plants of Phal. amabilis using colchicine (0 to 2000 mg L −1 ) soaked in wet cotton covered with aluminum foil and applied to the ovaries and stigma three days after self-pollination.

Induction of Polyploidy in Oncidium, Vanda, and Others
There have been a few reports on the artificial induction of polyploidy in the genus Oncidium. Unemoto et al. [130] observed that an increased exposure time of protocorms of Onc. flexuosum to colchicine resulted in the increased death of explants, and the surviving seedlings demonstrated morphological alterations with a reduction of shoots and roots. However, the authors did not analyze the ploidy level of the regenerated plants. Therefore, it is difficult to tell if these changes were due to the colchicine phytotoxicity or polyploidization of the regenerated plants. Similarly, Cui et al. [131] also observed morphological changes such as smaller and more robust plants, thick leaves, and longer stomata lengths associated with polyploids, compared to the untreated diploid plants of nonidentified/specified Oncidium obtained from thin cell layers of PLBs treated with different concentrations and exposure times of colchicine.
Nakasone [132] was the first to induce polyploidy in Vanda "Miss Joaquín" using young shoots treated with different concentrations of colchicine (0.5% and 1.5%) for 2 and 6 days of exposure (Table 6). More recently, Tuwo and Indrianto [133] obtained polyploid plants from protocorms treated with colchicine (0.5% for 6 h) for the hybrid of V.a limbiata Blume X V. tricolor Lindl. var. suavis. The regenerated polyploid plants presented a smaller number and width of leaves, smaller number and length of roots, and a greater stomatic size and lower stomatal index ( Table 6).  There have been two studies on the genus Rhynchostylis, correlated with Vanda, in which colchicine was applied to PLBs in one study [134]. The herbicide propyzamide was applied to seeds of two genotypes of this genus to induce polyploidy [111].
In addition to these, studies with the genera Calanthe, Epidendrum, Odontioda, and Paphiopedilum were also reported ( Table 6).

Conclusions
The high frequency of endopolyploidy, together with the presence of polysomatic organs and tissues, were observed in different orchid genera. In vitro regeneration pathways, such as PLBs induction and regeneration from tissues with a high frequency of endoreduplication, can be used to obtain polyploid plants without antimitotic treatment. Additionally, the development of unreduced gametes was reported in some species of Orchidaceae, which is a natural mechanism of polyploidization. These genotypes were used as parents in breeding programs. The use of antimitotic agents is an efficient technique for the artificial production of polyploid plants, which increases the number of genotypes with useful ornamental characteristics in the world flower industry. The genera Cymbidium, Dendrobium, and Phalaenopsis, with the most significant impact on the world's floriculture, have the highest number of published studies and reports on obtaining polyploid plants. In vitro cultivation, using protocorms and PLBs as explants and colchicine as an antimitotic agent has most widely been used for the artificial induction of polyploids in orchids.

Further Prospects
Although colchicine is widely used to increase the frequency of polyploids in orchids, most studies have evaluated the effectiveness of its concentration and exposure. Furthermore, few studies have been focused on assessing the conditions of its application on the explants. The different exposure times, treatment temperatures, and joint applications of products that increase the absorption of colchicine by tissues or reduce its toxicity needs to be better understood. Most studies have reported a pronounced effect of this reagent on the survival of treated explants and different symptoms associated with phytotoxicity on tissues, with biochemical changes and plantlet development after treatment. However, practically no studies have been reported to alleviate the phytotoxic effects of this reagent on explant development, which are limited by successive washing with distilled or deionized water. Another relevant fact of using colchicine, besides its high cost, is toxicity to humans and animals, resulting in short-, medium-, and long-term effects [138]. Thus, the generation of more natural polyploid plants by promoting the formation of new PLBs from polysomatic tissues or by increase studies with unreduced gametes are exciting strategies with lower risks than colchicine induction.
The correlation between polyploidy and genetic improvement is remarkable. In some genera, like Phalaenopsis [36], polyploid commercial hybrids are predominately used in floriculture.
Although most Phalaenopsis hybrid cultivars are protected in terms of commercialization, a strategy that could be further explored by breeding companies would be the use of triploid and pentaploid cultivars, which also have limitations in terms of sexual reproduction.
Another relevant fact is the lack of studies that contemplate haploid and doublehaploid plant technology in orchids, a technology currently used for large crops with diverse breeding and genetic applications.