Scope of whole genome duplication in forages: a plant breeder perspective

Polyploidy is a condition of having more than two sets of chromosomes. Polyploid plants may be artificially obtained through chemical, physical and biological methods (2n gametes). It is considered an interesting approach due to increased genescope and expression, thus resulting in phenotypic changes, such as yield and product quality. Nonetheless, breeding new cultivars through induced polyploidy should overcome deleterious effects partly contributed by genome and epigenome instability after polyploidization. Furthermore, shortening the time required from early chromosome set doubling to the final selection of high yielding superior polyploids is a must. Despite these hurdles, plant breeders have successfully obtained polyploid bred-germplasm in broad range of forages from optimized application methods, concentration and time mainly using colchicine. These experimental polyploids proved to be a valuable tool for understanding gene expression that is driven by dosage dependent gene expression, altered gene regulation and epigenetic changes. Isozymes and DNA-based markers aided on the identification of rare alleles for particular loci when compared with diploids, which may explain their heterozygosity, phenotypic plasticity and adaptability to diverse environments. It has also been observed that experimentally induced polyploid germplasm could enhance fresh herbage yield and quality, e.g. leaf protein content, leaf total soluble solids, water soluble carbohydrates and sucrose content. Offspring of experimentally obtained hybrids underwent selection for several generations to improve their performance and stability.


Introduction
Polyploidy is a condition of having more than two basic sets of chromosomes. It occurs among various life forms [1]. Polyploidy is the widely distributed phenomenon in wild and cultivated crops of forest, legume and grass forage species [2]. Whole genome duplications also occurred at very high rate in forages, e.g. 75% of the speciation underwent genome duplications in group of 1200 species [3].
The remarkable ecological success of the polyploidy prompts the breeders to develop new polyploid germplasm and with increased economic value [2]. These polyploids could also help to understand the evolution of various plant species with economic relevance. The polyploids are also used as bridging species to transfer resistant genes from their parental species, as genes related to biotic and abiotic stresses [4,5]. In contrast to natural polyploidy that have evolved as products of natural selection [6], induced polyploid (polysomic polyploids, and true or segmental allopolyploids) have been mainly developed from after the anti-tubulin effect mainly of the colchicine -(S)-N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo[a]heptalen-7-yl) acetamide; and also of the oryzalin -3,5-dinitro-N 4 ,N 4 -dipropylsulfanilamide; trifluralinα,α,α-trifluoro-2,6-dinitro-N,N,-dipropyl-p-toluidine; and amiprophos methyl-O-methyl chromosome set doubling. Colchicine solution may be applied to the immature buds, resulting in the production of unreduced reproductive cells and, subsequently, produce polyploidy seed in different rates. It is applied to the pedicel of growing immature bud or the inflorescence stalk may be dipped in the growth media solution with a low concentration of colchicine (0.01 to 0.1%), depending upon the forage species (Table 1). Cotton swab dipped in colchicine solution of 0.2% for 10h and consecutive 2 days successfully induced a polysomic tetraploid (12-40%) in guar accessions [15]. Colchicine can also be applied to the callus; i.e., cell culture of meristematic tissues to produce chimeric plants, or seed may be germinated in colchicine solution [16]. Generally, colchicine (0. 1-1%) has been applied to seeds for 24-48h depending upon the forage species to target cells undergoing mitosis (Table 1). Growth arrest is the first symptoms of successful application of colchicine (Fig. 1).
Affected cells usually resume division after a lag period of colchicine application. A comparison of euploid series (2×, 4×, 6× and 8×) in Arabidopsis thaliana showed that induced polyploidy had slower growth, had increased cell size, and lower number of cells per leaf blade. Polyploid cells had lower lignin and cellulose but higher pectin and hemicelluloses in the stem [17]. Frequency of the polyploidy induction can increase with the increase of the colchicine concentration (Table 1). A high colchicine concentration has been, however, toxic to cell and plant tissues. A low colchicine concentration (0.1%) treatment with relative long duration (48 h) has been effective in increasing the yield of polyploid cells in shoot tips in vitro [18] (Table 1). For instance, sorghum seed were subjected to 0.2% concentration of colchicine for 48 h and 72 h, yielding 3.3% and 2.3% polysomic tetraploid plants, respectively [19]. A direct application of colchicine by dipping the apical meristem of seedlings was shown to produce chimeric tissues where induced polyploidy was limited to a particular layer [20].
Polyploid plants can be produced through somatic cell fusion [21]. This method has been used to fuse the protoplast of the same species (polysomic polyploidy) or of two species (amphiploidy or disomic polyploidy) in electric shock chamber without reduction division and it has been successful in creating amphidiploid forage species [22].
In vitro induction of polyploidy was successfully achieved by culturing the nodal segments or callus in tissue culture media supplemented with a low concentration of colchicine. This method has been shown to induce high frequency of polyploidy in various species (Table 1). In comparison with the doubling agents, oryzalin was found to show higher frequency of the induced polyploidy over colchicine [23], but it inhibited the callus growth and seedling regeneration. Liquid medium was more effective in inducing the polyploidy when compared with semi solid media [7,23]. A concentration of 5 µm oryzalin was found highly effective over colchicine and trifluralin in inducing chromosome set doubling of calli obtained from interspecific hybrid plants of elephant grass × pearl millet [24]. In vitro treatment of Trifolium pretense yielded 55.5% higher number of polysomic tetraploids and 1.9 times fewer chimeric individuals when compared with seed imbibition in colchicine solution [25,26]. Colchicine, amiprophosmethyl and oryzalin produced similar frequency of polysomic tetraploid (31-47%) and chimeric plants (14-22%) in T. pratense [25]. Colchicine (0.015% in tissue culture media) was more effective in inducing polysomic tetraploidy (16%) in Trifolium polymorphism species when compared with oryzalin [27].
The demand for pure polyploids and due to different ploidy levels obtained after chromosome set doubling the screening, selection and periodic evaluation of the ploidy level stability of the natural and induced polyploids are needed [28]. Nowadays, the main used method for these purposes is the flow cytometry [8,29] that increases the efficiency of the DNA ploidy level determination and nuclear genome size measurement. From flow cytometry, large numbers of individuals are quickly evaluated in only one day in a quantitative, thus being rapid, reliable and reproducible [11]. This powerful screening tool for ploidy level has been very useful in forage breeding programs. However, the chromosome counting, which is performed through microscopic observation of the metaphase cells, has been also applied to confirm the chromosome number [8,28,29], and, consequently, the ploidy level and detect eventual aneuploids. For example, chromosome counting was only method used to identify polysomic tetraploid and aneuploid plantlets regenerated through direct and indirect somatic embryogenesis of Lilium cernuum Komar and Lilium distichum Nakai, respectively, after colchicine treatment [30]. In relation to flow cytometry (nuclear ploidy level), chromosome number was most adequate to characterize the karyotype and, thus, identify polyploid and few aneuploid (hyperploid and hypoploid) cultivars of Phalaenopsis [31].
Besides these direct tools, indirect analyses have been accomplished. The effects of induced polyploidy on forage species were an increase in the stomatal area and decrease in the stomatal frequency. These changes in the leaf anatomy were due to increase in cell volume promoted by increase in nuclear DNA content [32].

Impact of Polyploidy on Forage Yield and Contributing Traits
There are numerous positive effects of induced polyploidy when compared with diploidy; i.e., larger leaves, herbage yield and slow decay of heterosis [10,33] (Table 2), increase in plant height, persistence, faster re-growth after grazing and increased branching of various forage species were noted in the former [34] ( Table 2). Potential benefits of induced polyploidy of various forage species are noted in Table 2. Disomic tetraploid intersubspecific hybrids (maize × teosinte) had 14% biomass yield increase over their diploid counterpart [16]. Other advantage of tetraploid hybrids included greater leaf area, leaf essential oil contents, and protein contents [16]. Polysomic tetraploid of Lolium species had slow decay of heterosis and has the potential to give better seed yield over several generations without significant decrease in their forage yield and thus their cultivation could be done with cheaper seed that could overcome the drawback of expensive F1 seed [35]. However, chromosome doubling reduced the persistence in the induced 4× ryegrass [35]. Research has shown that neo polyploids have increased production of secondary metabolites, thus enhancing forage quality, succulence and forage intake [36]. Induced polyploids had often poor seed setting and higher pollen sterility [33]. Polysomic tetraploid (4×) were compared with diploids (2×) plants in Vicia villosa roth and induced polysomic tetraploid plants of the species were dwarf with greater leaf area, stomatal diameter, higher chlorophyll contents, but the polysomic tetraploid delayed and lesser flowering than diploid counterparts [37]. Reconstituted polysomic tetraploid (4×) of Festuca pratensis had 26% dry matter and 100% seed yield advantage over standard check cultivars [25]. For some forage species; i.e., as Dactylis polygama and Lolium perenne, polysomic tetraploids did not differ in biomass yield from diploids but they were superior in terms of forage intake [38] and other forage quality parameters [39]. A patent of triploid (3× superior over 4× and 2×) maize has been awarded to produce high molasses, rum and fodder in low sterile genotype [106,107]. Table 2. Impact of induced polyploidy on forage traits in various species

Genetics of Polyploidy
Polysomic polyploid species differ from disomic polyploid (or amphidiploid) species due to their inheritance. The amphidiploid species show peculiar disomic inheritance for locus behaves similar to diploids. Polysomic tetraploid species show quadrivalent (multivalent) pairing, which requires chromosome homology andmay end up in double reductions during meiosis II [40]. Polysomic polyploids may have four copies of alleles (Fig. 2), where two extra copies of alleles were considered as fuel for evolution, because these alleles may undergo the process of sub-functionalization or neofunctionalization [41]. Sub-functionalization and neofunctionalization of alleles may lead to the homoeologous chromosome that may enhance preferential pairing of fully homologous chromosome leading to suppression of quadrivalent pairing. There is a challenge for forage breeder to identify various tetrasomic genotypes {i.e., AAAA (quadriplex), AAAa (triplex), AAaa (duplex), Aaaa (simplex) and aaaa (nulliplex)} and confirm polysomic tetraploidy. Isozyme analysis in ryegrass using two markers loci; i.e., the aspartate aminotransferase and the NAD-specific aromatic alcohol dehydrogenase, showed two alleles per locus and could be used to distinguish heterozygous or homozygous genotypes due to co-dominance of marker alleles [42]. Thus, these isozymes in this study were only able to uncover two alleles per locus in the polyploid ryegrass. Isozyme analysis showed allelic diversity due to polymorphism in enzymatic structure which may be used to differentiate various tetrasomic genotypes in various polysomic tetraploid alfalfa populations [43].
Both enzymes showed diversity in their maker allele size and were successfully used for the identification of tetrasomic genotypes in alfalfa [44]. Tetraploid alfalfa contained three alleles a', a and b for pgm-1. Polysomic tetraploid genotypes contained rare allele a' that was absent in diploid, but was carried in various tetrasomic combinations. Genotype a'a'aa (duplex) was the most frequent. Functional diversity in isozyme alleles at single locus explains the phenotypic plasticity, high level diversity, and more heterozygosity that may provide an adaptability advantage over diploids [44]. Isozyme allelic diversity such as EST-B and POX-b2 was related with fresh leaf weight in alfalfa [45].
DNA-based molecular makers such as amplified fragment length polymorphism (AFLP) were applied to confirm tetrasomic inheritance in tetraploid forage legume species (Lotus corniculatus), which show suppressed quadrivalent pairing. AFLP successfully confirmed the tetrasomic inheritance prevailing in the species, providing evidence of polysomic tetraploid classification of this species [46]. Similarly, tetrasomic inheritance was also confirmed in the other forage species such as alfalfa, the Paspalum using DNA-based molecular markers systems [47,48,49]. Mercedes4.11 as tetra-allelic (ABCD) and Magali2 as tri-allelic (ABCC) [48].
Polysomic tetraploids were difficult as breeding material and shows low decay of heterozygosity, a high degree of inbreeding depression and disomic -tetrasomic inheritance of alleles at single locus [16,50]. The use of DNA-based molecular markers may help to identify various tetrasomic genotypes, to select appropriate parental breeding lines. MAS may help to exploit maximum heterozygosity at single loci to improve the performance of tetraploid genotypes.

"OMICS" Analysis of Polyploidy
Metabolic comparison between forage plants with distinct ploidy level was accomplished by [51].
In comparison to diploid alfalfa, authors evidenced that polysomic tetraploid and polysomic octoploid protoplasts showed the respective nuclear genome size (i.e., confirming 4× and 8×), and, consequently, higher amount of the ribulose-1,5-bisphosphate carboxylase / oxygenase and chlorophyll. For non-forage species, the evaluations about transcriptome and physiology effects after induced chromosome set doubling have been also performed. In addition to morphologic changes, the L. distichum polysomic tetraploids showed higher stomatal conductance than diploid ones, while L. cernuum polysomic tetraploids presented lower stomatal conductance [30]. An increase in chlorophyll content was also detected in polyploids, which resulted in darker green leaves [52]. The "gigas effect" about the morphological and physiological traits of the polysomic tetraploids Raphunus sativus L. was confirmed after comparing to the diploids, and also differences in the endogenous phytohormone levels and flowering genes expression carried out to delay in the flowering and bolting in the polysomic tetraploids [53]. Rao et al. [4] reported that the induced polysomic tetraploidy in Lycium ruthenicum resulted plants that exhibited increase in abscisic acid content in relation to the diploid counterparts due to doubled gene copy and upregulated of the 9-cis-epoxycarotenoid dioxygenase 1 and 9-cis-epoxycarotenoid dioxygenase 2 genes involved with this hormone biosynthesis. The increase in abscisic acid in the polysomic tetraploids also upregulated the ABRE-binding factor 5-like gene and genes that codified osmotic proteins, increasing the drought resistance in relation to the diploids. Induced polyploidy resulted in epigenetic outcomes in alfalfa (Medicago sativa), changing the expression of 189 genes [54]. Brachiaria genus possesses species with diferent basic chromosomes number (× = 6 to × = 9) with polyploid origin. The occurence, mapping and distribution of the gypsy retrotranposons in the karyotype of Brachiaria decumbens, Brachiaria brizantha, Brachiaria ruziziensis and Brachiaria humidicola evidenced that, besides the chromosome number variation, mobile elements also contributed to the karyotype evolution [55]. In addition, genetic variation was showed in polyploid Paspalum species from SSR markers developed for this genus.
As the forage are also affected by abiotic and biotic stresses, interfering in the several traits, the "omics "differences, mainly the transcriptome and metabolome, should be investigated and compared between the ancestor diploids and its induced polyploids. The future approaches involving induced polyploid forage may also include analyses to show the interference of the nuclear gene copy increase in organellar genes expression, both mitochondrial and plastidial, like explored by [56] Oberprieler et al. in diploid, tetraploid, and hexaploid species of Leucanthemum Mill.

Restoration of Fertility
Induced polyploidy has been used as tool to restore the fertility of interspecific hybrids with different ploidy levels or distinct genomes with similar levels, mainly homoploids and anorthoploids. Fertility of allotriploid hybrids between the Pennisetum purpureum and Pennisetum glaucum was restored by germinating the seed in [57] medium supplemented with colchicine.
Induced polyploidy in homoploid interspecific (Zea mays × Zea mays ssp. mexicana) hybrids resulted in higher seed yield than that of diploid crosses when subjected to selection for the regular bivalent formation in maize [16]. Caryopsis and cell size of octoploid Panicum virgatum L. increased and the anatomy of the leaf tissues was changed when compared with diploid individuals, while fertility of the induced plants was decreased significantly [58]. Allotriploid hybrids of Miscanthus × giganteus were sterile but had higher biomass potential and persistence when compared with diploids. Poly-disomic hexaploidy was induced to restore fertility in these allotriploid hybrids and reduce the cost of hybrid seed production [23]. Induced disomic tetraploidy in maize × teosinte hybrids followed by selection for quadrivalent and bivalent formation in induced tetraploid also led to the decrease in hybrid vigor decay, which was investigated throughout various generations [16].

Stability of The Induced Polyploidy
Evolutive research findings suggest that many diploid plant species originated as polyploids and reversed to diploid (Mendelian genetics) forms due to a gradual loss of chromosome during synapsis between homologous (polysomic polyploidy) or homoeologous (amphipolyploidy or disomic polyploidy) chromosomes. These species failed to develop a genetic mechanism to enhance bivalent pairing and to suppress univalent or multivalent pairing of the homologous or homoeologous chromosomes. The failure of regular bivalent pairing and disjunction resulted in a larger frequency of aneuploid gametes that increased the gametophytic sterility.
The chromosome number stability counting in various generation after induced tetraploid (C1-C2) indicated an increase in the aneuploid form in C2 (55%) when compared with C1 (2.5%) generation [64]. In Brachiaria brizantha, failures in spindle fibers formation and absence of metaphase plate were noted in the induced polyploids, as well as micro nuclei were observed in subsequent generation showing abnormalities in meiotic divisions [59]. Homoploid Pennisetum purpureum × Pennisetum glaucum showed alteration in karyotypes, as chromosomal rearrangement and loss of DNA sequences, thus evidencing that interspecific hybrids show intergenomic conflicts which may lead to the loss of the genomic DNA [60], including genes and DNA sequences of the centromere and telomere. Contrary to these findings, induced polysomic tetraploids of Lolium multiflorum showed stability in chromosome number in subsequent generations [61]. Selection for large pollen (2×) production in red clover (Trifolium pratense) increased this trait 4 to 8.9% from C1 to C4, respectively, thus increasing polysomic tetraploid production in subsequent generations [62].
Selection for genotypes with a frequent capacity for quadrivalent pairing in induced tetraploid of maize resulted in a significant reduction in aneuploids and associated pollen sterility [63]. As a result of the selection for quadrivalent formation, the univalent and trivalent formation significantly decreased with corresponding increase in the bivalent and quadrivalent pairing ( Table 3).The selection process led to an increase in the seed yield, whereas herbage yield was affected in 4 th generation of self-pollinated tetraploids (Table 3). Polysomic tetraploid recovery increased over various generations of Lolium perenne L. of selection; i.e., C1 had 25% tetraploid, while self-pollination (4×) led to the recovery of 43% tetraploid [64].

Stress Tolerance of The Induced Polyploidy
The benefits of the induced polyploidy, including polysomic and disomic polyploidy, have been often correlated with increased abiotic and biotic stress tolerance [65,66] (Table 4). Higher tolerance to stress may directly result from induced polysomic polyploidy, which can increase the allelic copies for particular loci, thus increases gene expression and regulatory mechanisms for stress response [1]. Tetraploid species have been considered physiologically efficient due to improved gas exchange characteristics; i.e., carbon exchange rates, water use efficiency, transpiration and stomatal conductance [67] (Table 4). Natural polysomic tetraploid population of Rhodes grass (Chloris gayana L.) retained higher genetic diversity than the diploid form, which could help express diversified mechanisms for adaptation to salinity stress [68]. In contrast, polysomic tetraploid Lolium perenne expressed lower persistence than its diploid form due to lower root biomass and profuse tillering [69]. However, there were no differences between the diploid and tetraploid versions of Lolium perenne cv. 'Alto' in terms of water deficit and invertebrate tolerance [69]. In another study, induced polysomic tetraploid of Lolium perenne had higher competitive ability but lower tolerance to water deficit [70]. Different species of genus Cenchrus differ in their ploidy levels that include diploids, tetraploids and hexaploids. Tetraploid and hexaploid species had better gas exchange traits and morphological traits such as leaf rolling, wilting and plant height than diploid species [71].
Moreover, tetraploid and hexaploid species had better drought recovery than diploid species [71]. Tetraploid and diploid versions of Cenchrus species also differ in the antioxidant system. The tetraploid species had more efficient and expressive antioxidant system (higher values for Δ 1 -pyrroline-5-carboxylate synthetase) to scavenge the reactive oxygen species under water stress which may be translated in higher tolerance to water stress [71,104]. Polysomic tetraploid black locust (Robinia pseudoacacia L.) showed superiority over diploid due to higher defense related enzymatic activity and maintenance of photosynthesis rate under salinity stress [72]. Disomic polyploidy can indirectly improve stress tolerance as the amphidiploids combining a commercially important but susceptible diploid species with a highly resistant species, followed by a doubling of the chromosome number. Triticale is a human-made species originating from the hybridization between tetraploid self-pollinated species wheat (Triticum durum), and diploid outcrossing species rye (Secale cereale), with the aim of combining the nutritional characteristics of wheat with drought and salinity stress tolerance, plus rust resistance of rye [73]. Aluminum tolerance of disomic hexaploid and octoploid triticale showed variable contribution of its parental species and could not be fully expressed in rye synthetic cultivars [74]. Chromosome set doubling in maize × teosinte hybrids improved biomass yield, tillering, and resistance to heat stress [16]. However, spotted stalk borer (Chillo partellus Swinhoe) resistance in the F1 interspecific maize × teosinte hybrids was not noticed because of the effect of the susceptible maize parent [75].

Factor affecting The Success of Induced Polyploidy
The data reviewed above showed some successful examples of about the experimentally obtained polyploids. However, forage breeders considered some decades ago induced polyploidy as an unconventional technique with high expectation and low realization [76]. According to earlier research, it may take many years from the first chromosome set doubling events to the final development of a superior new cultivar [76]. Lolium is a nice example showing that induced polyploidy brought commercial success of polysomic tetraploids of this species [77]. This is probably the best for a forage species for which this methodology has worked, but it has taken well over 50 years from the early doubling events to the point where polysomic tetraploid cultivars are now common place [61]. Tetraploid cultivars of Lolium multiflorum may have larger seed size, leaf size and length, plant height, seed head length when compared with its 2× species [78]. Seed mass of 4× accessions of Lolium species was 3.8 mg (120 seeds) in comparison to 2.4 mg (120 seeds) of the 2× cultivars [79]. The seed mass has been shown to affect seedling vigor and seedling morphological trait [79]. At cellular level, 4× cultivars of Lolium had faster cell elongation rate which increase the cell length, which results in the increase in the size of leaf tissue [80]. 4× tissues had 20 folds larger mesophyll and epidermal cells than in diploid cultivars [80]. DNA contents of 4× Lolium accessions were 11.45 pg 2c -1 while 2× accessions had 5.6 pg 2c -1 [81]. Tetraploid cultivars of Lolium perenne had higher digestibility and water-soluble carbohydrates [82]. These 4× cultivars of Lolium species also offer several other advantages; i.e., act as cheaper source for cultivation than F1 hybrid seed, better tolerance to abiotic stress condition [35] (Table 4). Higher drought tolerance of 4× cultivar of Lolium multiflorum was due to increased production of antioxidants such as phenolics and enzymes which scavenge the reactive oxygen species under stress [83].  [86]. Higher cattle preference for 4× cultivars was due to high leaf proportion and leaf soluble carbohydrates [86]. A study showed higher water-soluble carbohydrate and crown rust susceptibility for 4× cultivars in Lolium [87]. Recently a 4× late maturing cultivar "FL-Red" has been released for cultivation with improved resistance to crown rust and high sustainable yield through selection [88]. Seed mixture of 4× Lolium with Trifolium repens also had better dry matter yield and also with other species. Induced tetraploidy had similar effect on water-soluble carbohydrates of 4× Lolium cultivars as that for recurrent selection for higher water-soluble carbohydrates in 2× diploid cultivars [89]. Water-soluble content could be increased either by intensifying the favorable alleles through recurrent selection or increasing the copy number of alleles in 4× bred germplasm [89].
Slow progress of induced polyploidy after early induction of the chromosome set doubling event of chromosome is the major hindrance of low success rate of polyploidy breeding. The mixoploid condition (2×, 3× and 4×) in freshly developed material and subsequent generation reduced the performance, stability and fertility of induced polyploids [90], which was regarded as a major hindrance for the commercialization of polyploidy species. However, selection of pure and stable polyploidy genotypes in advanced generation can improve the performance of the induced polyploid species. It was observed that selection for several generations could lead to the increased frequency of polysomic tetraploid plants in Lolium multiflorum [90]. Lolium species probably showed better results of the selection and thus was finally commercialized as polysomic tetraploid species [61].
The stabilization of the chromosome number in induced polyploidy require time and may depend on accurately identifying stable offspring regarding chromosome number. Tools such as flow cytometry may be exploited to screen various hybrid-derived offspring with stable chromosome number. Molecular techniques which may accurately determines within genome and epigenome changes such as epigenetic interactions, down and up regulation of key genes related to yield and quality may help to improve the performance of the neo-polyploids.

Conclusion
Polyploidy is widespread phenomenon among grass and legume forages. Research has shown that polyploid species have greater biomass yield, persistence, and regrowth after grazing, and better tolerance to abiotic stresses than diploid species. Methods for induced polyploidy have been developed to increase biomass yield and improve foliage quality. The stability of induced polyploidy may be improved by selection for balanced chromosome pairing and disjunction.
Induced polyploidy has also been used to increase fertility of interspecific hybrids between two cross-incompatible species. Research results suggested that this polyploidy approach should be exploited in forage breeding without any negative consequences. However, the breeding of new cultivars using this approach needs to shorten the lengthy selection cycles from early chromosome doubling stabilize of neo-polyploidy, and address both the poor fertility and loss of vigor in following generations of neo-polyploidy.

Conflicts of interest
The authors declare no conflicts of interest.

Funding statement
This research did not receive any specific funding.