A new method for selecting cytochimeras by the maximum number of nucleoli per cell in Allium wakegi Araki and A. fistulosum L.

Summary A new method for determining ploidy levels in cytochimeral plants was developed by examining the maximum number of nucleoli per cell. Colchicine-treated plants of Allium wakegi Araki and A. fistulosum L., which have different ploidy levels in the first (LI), the second (LII), and the third (LIII) germ layer, were used together with untreated 2-2-2 plants of the same species. Nucleoli in guard cells for LI and in mesophyll cells for LII were stained in a 50% AgNO₃ aqueous solution at 55° C for three hours under dark, humid conditions. In both species the ploidy levels of LI determined by the maximum number of nucleoli per guard cell accorded well with those determined by guard cell length. In A. fistulosum the ploidy levels of LII determined by the maximum number of nucleoli per mesophyll cell clearly agreed with those determined by pollen size. This method provided more precise and efficient identification for LI and LII ploidy than the conventional methods of using guard cell length for LI and pollen size for LII.     

Bilateral polyploidization inDactylis glomerata L. subsp.lusitanica: occurrence,morphological and genetic characteristics of first polyploids

Summary InDactylis glomerata L. subsp.lusitanica, triploid and tetraploid plants were obtained by bilateral sexual polyploidization in crosses between diploid parents known to produce 2n gametes. The polyploid and diploid progeny were compared for allozyme diversity (allele number and heterozygosity), phenological (pollen fertility, inflorescence emergence date), cellular (stomatic cell size) and morphological characters (vegetative biomass, seed weight, total seed number per plant, panicle number and seed number per panicle length) in four contrasting growing conditions. Stomatal cell size and the total number of alleles were significantly lower in diploids than in polyploids which sometimes recovered more than two distinct alleles at a locus via 2n gametes possessing heterozygous loci. Pollen fertility was similar in diploids and tetraploids, but significantly lower in the triploids. Seed weight was significantly higher in polyploids, although the average number of seeds per panicle length and the total number of seeds was higher in the diploid sibs. No phenological differences occurred among ploidy levels whereas such differences were observed inDactylis in all the natural areas of sympatry between diploids and their related polyploids. Our results provide evidence for an instantaneous effect of polyploidization on certain traits and suggest that natural selection would act subsequently on several other traits to allow the establishment of new polyploids.     

Polyploidy and sexual polyploidization in the genus Vaccinium

Genus Vaccinium,consisting of blueberries, cranberries, lingonberries and many related wild species, includes diploid, tetraploid, and hexaploid species. Most evidence indicates that the tetraploid species are autotetraploids, with non-preferential bivalent chromosome pairing. Although homoploid interspecific crosses usually produce numerous fertile hybrids if the parents are from the same section of the genus, inter sectional crosses at the diploid level normally produce no seedlings, weak seedlings, or seedlings that are very low in fertility. There is a strong but not complete triploid block within Vaccinium. Even with insections, tetraploid × diploid (and the reciprocal) crosses normally give only a few tetraploid and a few triploid hybrids. Hexaploid × diploid crosses within sections are very hard to make, and the few hybrids that have been obtained are pentaploid. The frequency of 2n gametes varies,both among genotypes within species and among species. Vaccinium pollen is normally shed in tetrads, and the frequency of large pollen grains shed in dyads can be used to estimate 2n gamete frequency. Cultivated blueberries occur at both the tetraploid and the hexaploid levels, and there are important genetic resources in the diploids. Unreduced gamete production has permitted transfer of genetic material from the diploid to the tetraploid level and from the diploids and tetraploids to the hexaploid level via triploid hybrids. Intersectional crosses can occasionally produce tetraploid Vaccinium hybrids that appear to behave as amphidiploids and have medium to high fertility. CommercialVaccinium cultivars are normally propagated by cuttings. Intersectional hybridization, chromosome doubling, and asexual propagation could permit the production of novel hybrid combinations with value as ornamentals or in fruit production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Discovery of 2n gametes in tetraploid oat Avena vaviloviana

Summary Sexual polyploidization via the action of 2n gametes (gametes with the sporophytic chromosome number) has been identified as the most important evolutionary mode of polyploidization among plant genera. This study was conducted to determine whether 2n gametes are present in the tetraploid level of the genus Avena (2n=4×=28) Twenty tetraploid Avena lines, representing four species and one interspecific hybrid, were screened for pollen grain size in order to differentiate between n and 2n pollen. Avena vaviloviana (Malz.) Mordv. line PI 412767 was observed to contain large pollen grains at a 1.0% frequency. Cytogenetic analyses of pollen mother cells of PI 412767 revealed cells with double the normal chromosome number (i.e., 56 chromosomes at metaphase I and anaphase I). The mode of chromosome doubling was found to be failure of cell wall formation during the last mitotic division that preceded meiosis. The resulting binucleate cells underwent normal meiotic divisions and formed pollen grains with 28 chromosomes. Based on the formation and function of 2n gametes, three models involving diploid and tetraploid oat lines are proposed to describe possible evolutionary pathways for hexaploid oats. If stable synthetic hexaploid oat lines could be developed by utilizing 2n gametes from diploid and tetraploid oat species through bilateral sexual polyploidization, the resulting hexaploids could be used in breeding programs for transferring genes from diploids and tetraploids to cultivated hexaploids.     

Production of 2n pollen of Asiatic hybrid lilies by nitrous oxide treatment

Tetraploid varieties of lilies have superior agronomic traits such as large flowers and resistance to physiological disorders. In the present study, we attempted to induce 2n pollen of Asiatic hybrid lilies by arresting the meiotic process with nitrous oxide (N₂O) gas. To determine which meiotic stage is optimal for induction of 2n pollen, plants with attached buds at different meiotic stages were treated with N₂O for 24 h in a pressure-tolerant cylinder. A few 2n pollen grains were induced using plants with anthers in prophase I, whereas mixed pollen grains of differing size were produced using plants undergoing meiotic metaphase predominantly in anthers. Although normal lily pollen grains are elliptical, nitrous oxide exposure induced giant pollen grains that appeared spherical. Flow cytometry analysis showed that the giant pollen grains were diploid. When mixed pollen that included normal and giant pollen was crossed to tetraploid cultivars, the resulting seedlings were tetraploid and aneuploid, indicating that the giant pollen grains were diploids that could generate tetraploid seedlings through fusion to diploid eggs supplied from a tetraploid female parent. Thus, treatment with N₂O is useful for the production of 2n lily pollen and may provide a new approach for tetraploid lily breeding.     

Crossability relationships of andean potato varieties of three ploidy levels

Summary The formation of triploids in 4x×2x crosses was studied in primitive potato varieties from the Andes of Peru and Bolivia. Triploid frequencies were as high as 80–100% in some crosses, although the actual number of seeds was low. The triploid block varied according to the tetraploid parent, and was never entirely absent. The likelihood of triploid formation was studied in 4x×2x crosses where pollen from both tetraploids and diploids of known genotype was used. Even though the diploids were chosen for their non-production of 2n gametes, as detected in pollen grain size, 66% of the hybrids were tetraploid. The amount and probability of gene-flow between the tetraploid and diploid gene-pools, either directly through 2n gametes or through triploid bridges was examined. Most of the 20 cultivars of S. x chaucha produced seed in crosses with tetraploids and diploids, although the fertility of the latter crosses was four times higher. The female fertility of some of the triploids thus allows them to exchange genes with the tetraploids and diploids, although gene-flow occurs more rapidly through direct transfer from diploids to tetraploids via 2n gametes.     

A tetraploid hybrid plant from 4x × 2x crosses in Vitis and its origin

To study the origin of unreduced (2n) gametes in diploid Vitis cultivars, we surveyed the occurrence of tetraploid hybrid seedlings from 40 interploid crosses with five tetraploid and seven diploid cultivars. A total of 250 seedlings from the interploid crosses were established through embryo culture and by seed sowing. In 20 2x × 4x crosses, no tetraploid hybrid seedlings were derived from 8,902pollinations. In 20 4x × 2x crosses, two tetraploid hybrid seedlings were obtained from 8,057 pollinations. Investigation of isozyme genotypes of the two tetraploid seedlings using three variable enzyme systems indicated that one of the two seedlings resulted from the union of a diploid egg with a 2n male gamete and that failure of second meiotic division resulted in the formation of the 2n male gamete. The percentage of giant pollen grains in `Muscat Bailey A', a pollen parent of the tetraploid seedling, was relatively high(about 5.9%) and 10.9% of the giant pollen grains germinated on agar medium. These results suggested that 2n pollen of diploid cultivars are useful for breeding tetraploid grape. This revised version was published online in August 2006 with corrections to the Cover Date.     

RAPD analysis of sporting and chimerism in chrysanthemum

Summary The potential of colchicine and the microtubule depolymerizing herbicides trifluralin, oryzalin, and amiprophosmethyl (APM) for in vitro chromosome doubling during B. napus microspore culture was studied. Colchicine was administered during the first 6, 12 or 24 h of culture with 8 different concentrations up to 3 mM, and herbicides at 6 different concentrations up to 30 M for 12 h.Treatments with moderate concentrations of colchicine (3–100 M) produced a small increase in embryo production, while concentrations above 300 M were toxic. Colchicine treatment for 12 h resulted in higher embryo production than treatment for 6 and 24 h. Duration of treatment and concentration of colchicine both had a significant effect on the chromosome doubling. The highest diploidization rates (94% diploid regenerants) were seen after 24 h treatment with 1 mM colchicine.All three herbicides were similar to colchicine in terms of their effect on embryo formation and chromosome doubling comparable to the one of colchicine, but at concentrations approximately 100 times lower. APM was less toxic than trifluralin and oryzalin, but no significant difference in chromosome doubling efficiency was detected between the compounds. The 12 h treatment resulted in a maximum of approximately 65% diploid regenerants with all three herbicides, but APM may have an advantage because of its less toxic effects. Prolonged treatment with APM (20–24 h) may produce 95–100% diploid regenerants.Abbreviations APM amiprophos methyl - DMSO dimethyl sulfoxide     

Parthenogenetic monohaploids (2n=x=12) from Solanum tuberosum L. and S. verrucosum Schlechtd. and the production of homozygous potato diploids

Summary Dihaploid and dihaploid derived clones of Solanum tuberosum and diploid genotypes of S. verrucosum produced 85 viable monohaploids by female parthenogenesis. All were induced using diploid S. phureja clones, homozygous for embryo spot, as pollinator. Frequency of S. tuberosum monohaploids per 100 berries was rather constant in three successive years (14, 17 and 17 respectively). No male and female fertility was found in flowering monohaploids.Colchicine-induced chromosome doubling yielded homozygous s. tuberosum diploids with low pollen quality but good seed fertility.Two diploid self-incompatible species (S. multidissectum and S. berthaultii) produced no monohaploids. The presence of genes for female parthenogenesis in some dihaploids is discussed.     

Generation and characterisation of colchicine-induced autotetraploid Lavandula angustifolia

Lavandula angustifolia (lavender) is a small woody perennial grown for essential oil, which is steam distilled from flowers. To potentially improve size of flowers and oil yield we produced and characterised autotetraploid plants. L. angustifolia seed germinated in the presence of the mitotic spindle inhibitor colchicine at concentrations of 125 mg l⁻¹ or less resulted in plants carrying sports with larger flowers. Propagation of two sports gave rise to putative polyploid cultivars C3/2 and C6/24. Direct chromosome counts in root tip cells of seedlings from four common cultivars of L. angustifolia and the seed lot from which C3/2 and C6/24 were derived was 50 whereas C3/2 and C6/24 had greater than 90 chromosomes indicating they were autotetraploid. Ploidy level assessed by flow cytometry (FCM) of nuclei showed that 12 cultivars of L. angustifolia had similar nuclear DNA content whereas C3/2 and C6/24 had double the amount of DNA confirming autotetraploidy. The genome size (1C-value) of a diploid L. angustifolia cultivar was estimated by FCM to be 0.90 (±0.07) pg. Morphological characteristics were measured in autotetraploid and control plants. Autotetraploids had thicker peduncles, larger flowers and larger seeds than diploids. Scanning electron microscopy revealed peltate glandular trichomes were larger in the tetraploids relative to diploids. Both tetraploid and diploid cultivars had complex non-glandular trichomes on leaves and sepals and two different types of capitate glandular trichomes were identified on leaves. Autotetraploid lavenders represent useful germplasm both for commercial oil production and future breeding.     

Aneuploids of perennial ryegrass (Lolium perenne)

Summary Aneuploid plants of perennial ryegrass (Lolium perenne L.) with 2n=15 to 30 chromosomes were obtained by crossing a near-triploid (2n=3x+1=22) with a diploid or on open-pollination with diploids and tetraploids. Aneuploids occurred with a frequency of 83% in near triploid × diploid progeny and 92% on open-pollination with diploid and tetraploid plants. Aneuploid plants with 15 to 18 chromosomes resembled diploids in morphology and those with 19 to 30 chromosomes were akin to tetraploids. Meiotic studies suggested that most aneuploid plants resulted from transmission of aneuploid egg cells (n=8 to 23). Aneuploid plants with 2n=27 to 30 chromosomes in the progeny of 22×14 cross originated from unreduced egg cells. Plants with 19 to 21 chromosomes were recovered only by immature seed culture. Aneuploid plants with 26 to 30 chromosomes and triploids (2n=21) had higher pollen fertility and bigger seeds than plants with 15 to 22 chromosomes.     

Improved techniques for the induction and isolation of polyploids in the genus Fragaria

Summary The sensitivity of Fragaria seedlings to colchicine is dependent on the plant organ that is treated. Complete immersion of seedlings in a 1.5% colchicine solution results in total lethality, whereas the survival rates were more than 75% even at concentrations of 3.0% when only shoot apices were treated. High proportions of polyploids were isolated by treating shoot apices of seedlings with a 2.0% colchicine solution for 24–28 h. The dropper method is preferred to the tube method as it involves a minimum of manipulation and requires simple equipment. A differential response to colchicine was observed within and between different diploid species, diploid and tetraploid hybrids.     

Reduced fertility in triploids of <Emphasis Type="Italic">Acacia auriculiformis</Emphasis> and its hybrid with <Emphasis Type="Italic">A. mangium</Emphasis>

Polyploid breeding offers the possibility of increased variability in the search for improved growth, site adaptation and disease resistance in tropical acacias. A key focus of breeding in Vietnam has been the production and testing of vigorous triploid clones which are expected to have the added advantage of being sterile. Triploids obtained by manual crosses between diploid and tetraploid trees or by bulk screening of seedlings derived from open-pollinated seedlots were verified using flow cytometry. Thirteen clones are under field testing for growth rate, tree form and fertility. Six of these are now reproductively mature and flowered as prolifically as diploids. However no pollen germinated, either on agar or on their own stigmas under controlled pollination. Only one clone (X01—F₁ hybrid of tetraploid A. mangium and diploid A. auriculiformis) produced open pollinated pods on 0.05% of hermaphrodite flowers and these contained an average of 1.3 filled seeds per pod, about one-fifth the number observed in diploids. Less than 25% of germinated progeny from this triploid clone survived at 3 months after sowing and survivors were severely stunted with arrested growth. Ploidy and genotype analysis revealed them to be predominantly aneuploids, ranging from hyperdiploid to hypotetraploid with 95% being selfs. We are confident that we can select triploid clones which will be effectively infertile if deployed in plantations and may be of particular value in situations where the normal prolific natural regeneration of tropical acacias is highly undesirable.     

The effect of colchicine treatment on Solanum acaule and S. bulbocastanum; A complete analysis of ploidy chimeras in S. bulbocastanum

Summary Seeds of tetraploid Solanum acaule (2n=48) and diploid S. bulbocastanum (2n=24) were germinated in petri-dishes on filter paper soaked in 0.3% colchicine. An additional treatment with 0.3% colchicine was applied one month after sowing at four successive days in the axils of the cotyledons of the seedlings. S. acaule appeared much more sensitive to colchicine (14 surviving seedlings from 500 seeds) than S. bulbocastanum (109 surviving seedlings from 450 seeds). Six S. acaule plants with 2n=96 chromosomes were obtained against 38 S. bulbocastanum plants with 2n=48 chromosomes.The ploidy level in each of the three germ layers L₁, L₂, L₃ was determined in 113 plants of S. bulbocastanum and the following results were obtained. Four of the eight possible ploidy types were detected, viz 2x-2x-2x (72 plants), 4x-2x-2x (3 plants), 2x-4x-4x (9 plants) and 4x-4x-4x (29 plants). Doubling the number of chromosomes resulted in a highly significant increase of the number of chloroplasts in the guard cells of stomata and a greatly significant decrease in the proportion of trimerous pollen, male fertility and leaf index. The variability for all characters studied, except for leaf index, was clearly lowest in the 2x-2x-2x group. All plants with a 2x-L₂ were highly male fertile and self-incompatible, also in the three bud stages tested. Male fertility of the plants with 4x-L₂ varied greatly: 12 plants had more than 90% stainability, 5 plants must be considered male sterile. All non-sterile plants with 4x-L₂ were found to be self-compatible, pointing to a gametophytic system of incompatibility in S. bulbocastanum.     

Male fertility and occurrence of 2n gametes in East African Highland bananas (Musa spp.)

The quantity and quality of pollen produced by a genotype are important attributes to be considered in the selection of male parents for breeding. The objective of this research was to determine the quantity and quality of pollen in 13 selected East African Highland bananas (EAHB) in relation to six diploids commonly used as male parents. Pollen quantity was scored on a scale of 0–6 while the variation of pollen stainability along the rachis was determined using acetocarmine glycerol jelly. Actual male fertility was determined by using pollen from the EAHB varieties on M. acuminata spp. burmannicoides, ‘Calcutta 4’, as the female. The diameter of 200 viable pollen grains was measured under a microscope with the aid of a graduated eyepiece. Significant differences were obtained for mean pollen stainability and pollen diameter. The pollen stainability for diploids was higher than that for the EAHB. Node numbers contributed significantly to variation of pollen stainability in EAHB (P ≤ 0.01) and ‘Pisang lilin’ (P ≤ 0.001). ‘Pisang lilin’ and the EAHB varieties had about 100 nodes as opposed to about 200 in other diploids. The EAHB varieties produced less pollen than most of the diploids. The actual male fertility of EAHB varieties was low (1 seed/bunch), compared to that of ‘Calcutta 4’ (223.3 seeds/bunch) and ‘Pisang lilin’ (13.7 seeds/bunch). Although the production of 2n pollen in EAHB varieties was low (0–7%), the potential exists for using them in new breeding approaches.     

Artificially induced polyploidization in Humulus lupulus L. and its effect on morphological and chemical traits

Chemically induced polyploids were obtained by the colchicine treatment of shoot tips of Humulus lupulus L. ‘Sybilla’. Flow cytometry revealed that most of the treatments resulted in the production of tetraploids. The highest number of tetraploids was obtained when explants were immersed in 0.05% colchicine for 48 h. A field experiment was conducted to compare diploid and tetraploid plants and assess the effect of genome polyploidization on the morphological and chemical characteristics. Tetraploids showed significant differences in relation to diploids. They had thinner and shorter shoots. The influence of chromosome doubling was also reflected in the length, width and area of leaves. The length of female flowers in the tetraploids was significantly shorter than that observed in diploids. Tetraploids produced a diverse number of lupuline glands that were almost twice as large as those observed in diploids. The most distinct effect of genome polyploidization was a significant increase in the weight of cones and spindles. Contents of major chemical constituents of hop cones was little affected by ploidy level. Total essential oils were significantly lower than those in diploids. However there was a significant increase in the proportion of humulene, caryophyllene and farnesene, oils desired by the brewing industry.     

In vitro polyploidisation of <Emphasis Type="Italic">Helleborus</Emphasis> species

Helleborus species are members of the family of the Ranunculaceae. These popular perennials are all diploids (2n = 2x = 32). This study investigates polyploidy induction by different antimitotic agents. Colchicine, oryzalin and trifluralin were tested in vitro on shoots of Helleborus niger, H. orientalis and H. × nigercors. Furthermore the effect of the antimitotic agents on the viability and the multiplication rate of cultured plantlets were analyzed. Flow cytometry demonstrated that polyploidisation was genotype dependent: using H. niger, tetraploids were obtained using either oryzalin (3 μM) or trifluralin (3 or 10 μM), whereas for H. × nigercors only trifluralin (3 or 10 μM) induced polyploidisation. For H. orientalis neither treatment was effective to produce tetraploids or mixoploids. For these three species, colchicine (100 μM) was ineffective. The polyploidisation events in H. niger and H. × nigercors were confirmed by chromosome counts of mounted nuclei derived from root tips (2n = 4x = 64).     

Bilateral sexual polyploidization in potatoes

Summary Bilateral sexual tetraploidization was achieved by intercrossing diploid Phureja × haploid Tuberosum hybrids. Both parents contribute gametes with the somatic number of chromosomes (diplogynoids and diplandroids). More than 100 tetraploid progeny appeared in nine 2x–2x families. The tetraploids are generally more vigorous and significantly outyield their diploid full-sibs. The superiority of the tetraploids is interpreted on the basis of the mode of diplandroid (2n pollen) formation by first meiotic division restitution (FDR). Heterozygosis, epistasis and genetic diversity, if present in the parental diploid genotype, are largely maintained in the 2n gametes formed by FDR. and syngamy brings about more opportunities for heterotic responses in conjunction with polyploidization. Differences in tuber yield among tetraploid clones within families were found to be statistically significant (P <0.05). Thus, unlike somatic chromosome doubling, which can merely increase chromosome numbers, or somatic hybridization, which could in addition bring about heterosis, sexual polyploidization is also associated with genetic variability. The degree of inbreeding and the genotypic structure in the population of newly arisen tetraploids is discussed. The relationship among asexual reproduction, preservation of favorable nonadditive genetic effects by FDR. restoration of sexuality, and polyploidization is explored. The possible evolutionary significance of these factors is emphasized.     

Nematode resistance in bananas: screening results on some wild and cultivated accessions of Musa spp.

Bananas cultivated for export all belong to Cavendish cultivars and are all recognized as very susceptible to nematodes, particularly to the burrowing nematode Radopholus similis and the lesion nematode Pratylenchus coffeae. Even if there have been many changes in the management of banana nematodes in large commercial banana plantations, chemical control still remains most often the last resort method to manage the nematodes, although the number of registered products is definitely declining. Therefore, nematode control though genetic improvement is gaining new interest worldwide. In this study, 55 banana accessions mostly diploids from the Musa acuminata genome group (AA) but including some triploid accessions (AAA), some diploids of the Musa balbisiana genome group (BB) and some interspecific hybrids (AAB, AB) were evaluated for resistance to four nematode species R. similis, P. coffeae, Meloidogyne incognita and M. arenaria. These experiments were conducted in a growth chamber under controlled conditions. All banana accessions were susceptible to nematode species, although many different levels of susceptibility were detected. This study confirmed the good resistance status to R. similis of some cultivars from the Pisang jari buaya and Pisang batuau subgroups and the partial resistance of 17 diploid accessions significantly different from the susceptible reference cv. Grande Naine. This study also showed that 12 diploid accessions exhibited a partial resistance to P. coffeae, including some usual or potential genitors belonging to the wild diploids subspecies burmannica (cvs. Long Tavoy 1 and 2) and burmannicoides (cv. Calcutta 4). No source of resistance to Meloidogyne spp. was found. These screening results, combining for the first time four nematode species, are discussed within the scope of banana breeding in order to produce parental diploid lines with single or combined nematode resistances and further develop triploids that can substitute existing susceptible commercial cultivars.     

Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicine in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses

Summary We cultured colchicine-treated hybrid ovules in vitro to produce fertile amphidiploids of C. persicum (2n=2x=48. referred to as AA) × C. purpurascens (2n=2x=34, referred to as BB). Seedlings and mature plants were obtained from the ovules without colchicine and those exposed to 50 mg/l colchicine for 5, 10 and 15 days, whereas they were not obtained from the ovules exposed to 50 mg/l colchicine for 20 days and 500 mg/l for 5, 10, 15 and 20 days. Although 8 mature hybrids derived from the ovules without colchicine produced a few fertile pollen grains, they failed to produce viable seeds by self-fertilization. The hybrids had 41 somatic chromosomes. Four and 3 mature plants were derived from ovules exposed to 50 mg/l colchicine for 10 and 15 days, respectively. One each among 4 and 3 mature plants showed a high frequency of pollen grain fertility, produced several seeds by self-fertilization, and had 82 somatic chromosomes which is twice the number of hybrid chromosomes (2n=41, AB). These findings indicated that these plants are amphidiploids (2n=82, AABB) between C. persicum and C. purpurascens. Three and 2 viable seeds were derived by the conventional crosses of diploid C. persicum × the amphidiploid and the amphidiploid × C. purpurascens, respectively. Flowering plants that developed from the seeds of diploid C. persicum × the amphidiploid were barely fertile and had 65 somatic chromosomes (2n=65, AAB), whereas those that developed from the seeds of the amphidiploid × C. purpurascens were barely fertile and had 58 somatic chromosomes (2n=58, ABB). The somatic chromosomes indicated that these plants are probably sesquidiploids between the amphidiploid and either C. persicum or C. purpurascens. The interspecific cross-breeding of cyclamen using the amphidiploids and the sesquidiploids is discussed.