Induced Polyploidy: A Tool for Forage Species Improvement

Polyploidy means having more than two basic sets of chromosomes. Polyploid plants may be artificially obtained through chemical, physical and biological (2n gametes) methods. This approach allows an increased gene scope and expression, thus resulting in phenotypic changes such as yield and product quality. Nonetheless, breeding new cultivars through induced polyploidy should overcome deleterious effects that are 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 after optimizing methods, concentration and time, particularly when using colchicine. These experimental polyploids are a valuable tool for understanding gene expression, which seems to be driven by dosage dependent gene expression, altered gene regulation and epigenetic changes. Isozymes and DNA-based markers facilitated the identification of rare alleles for particular loci when compared with diploids, and also explained their heterozygosity, phenotypic plasticity and adaptability to diverse environments. 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 should undergo selection for several generations to improve their performance and stability.

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 a 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, enlarged cell size, and decreased the number of cells per leaf blade. Polyploid cells had lower lignin and cellulose but higher pectin and hemicelluloses in the stem [17]. Frequency of polyploidy induction can increase by rising 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 example, 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 produced chimeric tissues where induced polyploidy was limited to a particular layer [20].
Polyploid plants can ensue from somatic cell fusion [21]. This method has been used to fuse protoplast of the same species (polysomic polyploidy) or from two species (amphiploidy or disomic polyploidy) in an electric shock chamber without reduction division, thus obtaining 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 induces high polyploidy frequency in various species (Table 1). Although other chromosome doubling agent such as, oryzalin had a higher frequency of induced polyploidy than colchicine [23], it inhibited callus growth and seedling regeneration. Liquid medium was more effective in inducing polyploidy than semi solid media [7,23]. A concentration of 5 µm oryzalin was more effective than colchicine and trifluralin in inducing chromosome doubling of calli obtained from interspecific hybrid plants of elephant grass × pearl millet [24]. In vitro treatment of Trifolium pratense yielded 55.5% more polysomic tetraploids and 1.9 times fewer chimeric individuals than imbibition of seed in a 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 than oryzalin in inducing polysomic tetraploidy (16%) in Trifolium polymorphum [27].
The demand for pure polyploids with different ploidy levels obtained after chromosome doubling needs screening, selection and periodic evaluation of the ploidy level stability of the natural and induced [28]. Nowadays, the main method used for this purpose is flow cytometry [8,29], which increases the efficiency of DNA ploidy level determination and nuclear genome size measurement.
With flow cytometry large numbers of individuals are quickly evaluated in only one day using a quantitative, rapid, reliable and reproducible method [11]. This powerful screening tool for ploidy level has been very useful in forage breeding programs. However, chromosome counting, which is performed through microscopic observation of the metaphase cells, has been applied to confirm the chromosome number [8,28,29], and to detect eventual aneuploids. Besides these direct tools, indirect analyses have also been accomplished. The effects of induced polyploidy on forage species were noted as an increase in the stomatal area and a decrease in the stomatal frequency. These changes in leaf anatomy arose after expanding cell volume due to increasing nuclear DNA content [30].  teosinte seedlings after treatment of seeds for 24 hours (Niazi, I.A.K., permission granted).

Impact of Polyploidy on Forage Yield and Contributing Traits
There are numerous positive effects of induced polyploidy when compared with diploidy in various forage species; i.e., larger leaves, herbage yield and slow decay of heterosis [10,31] (Table 2), increased plant height, persistence, faster re-growth after grazing and augmented branching were noted in the former than in the latter [32] ( Table 2). Potential benefits of induced polyploidy 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]. 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 [36] and other forage quality characteristics [37]. 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 [38,39].

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 and may end up in double reduction during meiosis II [40]. Polysomic polyploids may have four copies of alleles (Fig. 2), of which the two extra copies of alleles may be useful for evolution because these alleles may undergo the process of sub-functionalization or neofunctionalization In the former, redundant alleles may mutate and be subsequently selected during evolution to achieve subfunctions in metabolic pathways but retaining the original ancestral functions, while redundant alleles may mutate and be subsequently selected during evolution to achieve new function of gene related to metabolic pathway in the latter [41]. Sub-functionalization and neofunctionalization of alleles may lead to homoeologous chromosomes that may enhance preferential pairing of fully homologous chromosome, thereby leading to suppression of quadrivalent pairing.
There is always a challenge for a 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 aspartate aminotransferase and NAD-specific aromatic alcohol dehydrogenase showed two alleles per locus, and was used to distinguish heterozygous or homozygous genotypes due to co-dominance of these alleles [42]. Hence, these isozymes were only able to uncover two alleles per locus in the polyploid ryegrass. Isozyme analysis showed allelic diversity due to polymorphism in enzymatic structure that may differentiate various tetrasomic genotypes in various polysomic tetraploid alfalfa (Medicago sativa L.) populations [43]. Esterase enzyme characterized alfalfa synthetic populations into tri-allelic (b1b2b3) and di-allelic (b3b3b3b2, b3b3b1b1) at locus EST-B [43]. Phosphoglucomutase (PGM) is controlled by two loci (Pgm-1 and

Pgm-2)
while Isocitrate dehydrogenase is controlled by a single locus (IDH). Each locus shows tetrasomic inheritance in polysomic tetraploid alfalfa [44]. Both enzymes show diversity in their maker allele size and are successfully used for identification of tetrasomic genotypes in alfalfa [44].
Tetraploid alfalfa contained three alleles (a', a and b) for pgm-1. Polysomic tetraploid genotypes had the rare allele a' that was absent in diploids, but available 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 of tetraploids than in diploids, which may provide an adaptability advantage in the former [44]. For example, isozyme allelic diversity in EST-B and POX-b2 was related with fresh leaf weight in alfalfa [45].
Polysomic tetraploids are difficult to handle as breeding material and show 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 identify various tetrasomic genotypes, could further used to select appropriate parental breeding lines. MAS may assist exploiting maximum heterozygosity at single loci to improve the performance of tetraploid genotypes.
The genome of polyploid species is more complex than that of diploid species. Therefore, identification of single nucleotide polymorphism (SNP)-based markers resulting from next generation sequencing brings challenges. For example, the depth of sequencing and review of the panels are several-fold higher in polyploid species than in diploid species. Distinguishing between homologues or homeologues SNP alleles in sub-genomes of amphidiploids is a complex and difficult task. Understanding homeologues relationships between various SNP alleles of a sub-genome may facilitate understanding the intergenomic interactions affecting quantitative traits.

"OMICS" Analysis of Polyploidy
Transcriptomic and proteomics were utilized to understand the mechanisms underlying the vegetative advantage of polyploidy in a forage species such as alfalfa [51]. Comparison of 2× and 4× bilateral sexual polyploidy of Medicago sativa showed 341 gene differentially expressed between 2× and 4× versions of this species. These genes were regarded as polyploidization sensitive.
Metabolic comparison between forage plants with distinct ploidy level was accomplished [52]. In comparison to diploid alfalfa, authors evidenced that polysomic tetraploid (4×) and polysomic octoploid (8×) protoplasts showed the respective nuclear genome size, and, consequently, higher amount of the ribulose-1,5-bisphosphate carboxylase / oxygenase and chlorophyll. It seems that polyploid species had high gene and protein expression for photosynthesis. 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 those remaining diploids, 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 [53]. The "gigas effect" on morphological and physiological traits of polysomic tetraploids in root vegetable radish (Raphunus sativus L.) was confirmed after comparing it to its diploids forms. There were also differences in endogenous phytohormone levels and flowering genes expression carried out to delay flowering and bolting in polysomic tetraploids [54].
Rao et al. [4] reported that induced polysomic tetraploid plants in Lycium ruthenicum -used for curing blindness in camels, exhibited more abscisic acid content that its 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 their drought tolerance vis-à-vis that of diploids. Induced polyploidy resulted in epigenetic outcomes in alfalfa, thus changing the expression of 189 genes [55]. Brachiaria genus possesses various polyploid species with different basic chromosome number (× = 6 to × = 9).
The occurence, mapping and distribution of gypsy retrotransposons in the karyotype of Brachiaria decumbens, B. brizantha, B. ruziziensis and B. humidicola evidenced that, besides the chromosome number variation, mobile elements also contributed to karyotype evolution [56]. In addition, genetic variation was showed in polyploid Paspalum species from SSR markers developed for this genus.
Forages are also affected by abiotic and biotic stresses, which calls for investigating their "omics" differences, mainly in the transcriptome and metabolome of both the ancestor diploids and their induced polyploids. Further research on induced polyploid forages may also include the analysis regarding the interference of the nuclear gene copy increase in organellar genes expression, both in the mitochondrial and plastids, as was done by Oberprieler et al. [57] in diploid, tetraploid, and hexaploid Leucanthemum spp. 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 P. glaucum was restored by germinating the seed in medium supplemented with colchicine [58]. Induced polyploidy in homoploid interspecific (Zea mays × Z. 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 [59]. Allotriploid hybrids of Miscanthus × giganteus were sterile but had higher biomass potential and persistence than diploids. Poly-disomic hexaploidy was induced to restore fertility in these allotriploid hybrids and to reduce costs for 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 decrease in hybrid vigor decay, which was investigated throughout various generations [16].

Stability of Induced Polyploidy
Research findings suggest that many diploid plant species originated as polyploids and reversed to diploid forms due to a gradual chromosome loss during synapsis between homologous (polysomic polyploidy) or homoeologous (amphipolyploidy or disomic polyploidy) chromosomes [41]. 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 [41].
Chromosome number counting was done in various generation following induced polyploidy.
After induced tetraploid (C1-C2), an increase in the aneuploid form in C2 (55%) was observed when compared with C1 (2.5%) generation [60]. 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 [61]. Homoploid Pennisetum purpureum × Pennisetum glaucum showed alteration in karyotypes, as chromosomal rearrangement and loss of DNA sequences, thus evidencing that interspecific hybrids showing intergenomic conflicts could lose genomic DNA [62], 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 [63]. Selection for large pollen (2n) production in red clover (Trifolium pratense) increased this trait from 4% in C1 to 8.9% in C4, thus increasing polysomic tetraploid production in subsequent generations [64]. 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 [65]. As a result of selection for quadrivalent formation, univalent and trivalent formation significantly decreased with a corresponding increase in bivalent and quadrivalent pairing (Table 3). Selection led to an increase in 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 selection in Lolium perenne L.

Stress Tolerance After Induced Polyploidy
The benefits of induced polyploidy, including polysomic and disomic polyploidy, have been often correlated with increased abiotic and biotic stress tolerance [66,67] (Table 4). Higher tolerance to stress may directly result from induced polysomic polyploidy, which can increase the allelic copies for particular loci, thus increasing 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 [68] (Table 4). A natural polysomic tetraploid population of Rhodes grass (Chloris gayana L.) retained higher genetic diversity than its diploid form, which led to diversifying adaptation mechanisms to salinity stress [69]. In contrast, polysomic tetraploid Lolium perenne had lower persistence than its diploid form due to reduced root biomass and profuse tillering [70]. However, there were no differences between the diploid and tetraploid versions of Lolium perenne cv. 'Alto' regarding water deficit tolerance and host plant resistance to invertebrates [70]. In another study, induced polysomic tetraploid of Lolium perenne resulted in high competitive ability but low tolerance to water deficit [71]. Different species of the grass genus Cenchrus differ in their ploidy; i.e., diploids, tetraploids and hexaploids. Tetraploid and hexaploid species had better gas exchange and morphological traits such as leaf rolling, wilting and plant height than diploid species [72]. Moreover, tetraploid and hexaploid species had better drought recovery than diploid species [72]. which may be translated in higher tolerance to water stress [72,73]. 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 selfpollinated species wheat (Triticum durum), and diploid outcrossing species rye (Secale cereale), with the aim of combining the nutritional characteristics from wheat with drought and salinity stress tolerance, plus rust resistance from rye [74]. 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 [75]. 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 F1 interspecific maize × teosinte hybrids was not noticed because of the effect of the susceptible maize parent [76].

Species: An Example of Commercial 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 [77]. According to earlier research, it may take many years from the first chromosome set doubling events to the development of a superior new cultivar [77]. Lolium is a nice example showing that induced polyploidy brought commercial success of polysomic tetraploids of this species [78]. This is probably the best polyploidization result for a forage species, but it has taken well over 50 years from the early doubling events to the release of tetraploid cultivars [61]. Tetraploid cultivars of Lolium multiflorum may have larger seed size, leaf size and length, plant height, seed head length than its 2× species [79]. Seed mass of 4× accessions of Lolium species was 3.8 mg (120 seeds) versus 2.4 mg (120 seeds) in 2× cultivars [80].
Seed mass affects seedling vigor and morphological traits in early growth [80]. At cellular level, 4× cultivars of Lolium had faster cell elongation rate which increase the cell length, which increased leaf size [81]. Tetraploid tissues had 20-fold larger mesophyll and epidermal cells than in tissues of diploid cultivars [81]. DNA content of 4× Lolium accessions were 11.45 pg 2c -1 , while 2× accessions had 5.6 pg 2c -1 [82]. Tetraploid cultivars of Lolium perenne had higher digestibility and water-soluble carbohydrates [83]. These 4× cultivars of Lolium species also offer several other advantages, e.g. being cheaper for cultivation than F1 hybrid seed, or having tolerance to abiotic stresses [35] (Table 4).
Drought tolerance noted in tetraploid cultivars of Lolium multiflorum was due to increased production of antioxidants such as phenolics and enzymes that scavenge the reactive oxygen species under stress [84]. Tetraploid cultivars of Lolium perenne had an advantage over diploid in late heading cultivars for traits such as herbage mass (kg DM ha -1 ) under low stocking rate, while showing superior sward height, bulk density (kg m -2 ), leaf proportion under both low and high stocking rate [85]. Moreover, intermediate heading tetraploid Lolium cultivars had higher organic matter digestibility, leaf crude protein (%) [86]. Likewise, cows fed on tetraploid cultivars had higher milk yield (kg day -1 ), solid corrected milk yield (kg day -1 ) under both high and low stocking rate, while late heading tetraploid Lolium cultivars were superior under low stocking rates for same traits [85]. Fat, protein and lactose yield (kg day -1 ) were higher in animals fed on tetraploid Lolium cultivars under both high and low stock conditions [86].
In a top cross mating design, tetraploid open pollinated cultivars (female) were crossed with newly generated induced tetraploid plants (male testers), and several uniform hybrid offspring were established, out of which one family had better dry matter yield and water-soluble carbohydrate but lower ground cover than the check for three years [33]. In a trial over 3 years, late heading tetraploid cultivars of Lolium perenne such as Ba13798 and Aberbite had higher total dry matter yield (kg ha -1 ), dry matter digestibility, water soluble carbohydrate but lower crude protein and ground cover than late heating diploid cultivars [33]. The 4× Lolium cultivar 'Green gold' had a dry matter yield advantage of 7% per harvest and superior quality such as water-soluble carbohydrates over the diploid 'cultivar AbderDart' [86]. However, diploid cultivars had higher silage ability than the tetraploids [86]. Livestock got higher dry matter feed (1380 kg ha -1 ) in, and spent more time (10%) grazing tetraploid than diploid cultivars (895 kg ha -1 ) [87]. This cattle preference for tetraploid cultivars arose due to both high leaf proportion and leaf soluble carbohydrates [87]. A study showed high water-soluble carbohydrate and crown rust susceptibility in Lolium tetraploid cultivars [88].
Recently, the late maturing cultivar 'FL-Red' was released due to its host plant improved resistance to crown rust and high sustainable yield [88]. Seed mixture of tetraploid Lolium with Trifolium repens and other species also led to high dry matter yield. Induced tetraploidy had similar effect on watersoluble carbohydrates in tetraploid Lolium cultivars as that noticed after recurrent selection for high water-soluble carbohydrates in 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 tetraploid bred germplasm [90], thus suggesting additive gene action for this characteristic.

Factors Affecting Commercialization of Induced-Polyploid Cultivars
Several factors such as polyploid usefulness, polyploidy maintenance in subsequent generations, or artificial polyploids fertility may ensure success and allow polyploidy moving from being just experimental to commercialization. The economic value of artificial forage polyploids was noted as a result of their high biomass as well as increased forage quality and digestibility. Testing of across agro-ecological zones forage yield and quality of induced polyploid may be necessary to accurately judge their performance and stability after several generations of self-pollination.
Concentration of spindle inhibiting agents may also need to be carefully selected to reduce the mutagenic effects over the genome [91]. Evolution of recessive alleles may pose negative impact over the performance of induced polyploid. Spindle inhibiting agents inducing less deleterious effects over the genome should be found and used [92]. DNA markers may be further used to determine allele frequency changes across the genome, and genotypes with low linkage drag should be selected to develop true-to-type polyploids [91]. Colchicine applications may also induce epigenetic changes, which may not be heritable. Hence, performance of induced polyploids may be investigated over several generations and across testing sites to assess the economic value of synthetic polyploid [93].
Slow progress of induced polyploidy after early induction of the chromosome set doubling event is the major hindrance for the low success rate in polyploid breeding. The mixoploid condition (2×, 3× and 4×) in freshly developed material and subsequent generations reduced the performance, stability and fertility of induced polyploids [94], which was regarded as a major difficulty for commercializing synthetic polyploid forages. A recent study demonstrated that tetraploid Medicago truncatula had higher forage yield (106%) than its diploid form, but derived lines across generations had highly variable fertility and stability [95]. However, selection of pure and stable polyploid genotypes in advanced generations may improve the performance of the induced polyploids.
Selection for several generations could lead to increased frequency of tetraploid plants in Lolium multiflorum [94]. As a result of tetraploid Lolium spp. having interesting results, it was not suprising beig the first to be commercialized as new forage cultivars [63]. interactions, or down-and up-regulation of key genes for yield and quality may assist on improving induced polyploid performance.

Conclusion
Polyploidy is a widespread phenomenon among forages. Research has shown that polyploid species have greater biomass yield, persistence, regrowth after grazing, and better tolerance to abiotic stresses than diploid species. Genetically polyploid organism carried multiple copies of alleles which may be helpful to increase allelic diversity and provide several evolutionary and adaptability

Conflicts of interest
The authors declare no conflicts of interest.

Funding statement
This review of research and other articles did not receive any grant funding.