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.     

波斯小麦×节节麦杂种F_1直接形成双二倍体的细胞遗传学研究

波斯小麦(Triticum carthlicum,AABB)与节节麦(Aegilops tauschii,DD)杂交,杂种 F_1出现可育性。不同组合的自交结实率21.25—39.72%。细胞学研究表明,可育性是由于杂种 F_1通过两种途径形成了未减数配子:第一,一部分花粉母细胞第一次分裂消失,只发生第二次分裂。即21条染色体集中到赤道面上之后,姊妹染色单体均等分离并移向     

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.     

A statistical mixture model for estimating the proportion of unreduced pollen grains in perennial ryegrass (Lolium perenne L.) via the size of pollen grains

Summary The size of pollen grains is commonly used to indicate the ploidy level of pollen grains. In this paper observations of the diameter of pollen grains are evaluated from one diploid accession of perennial ryegrass (Lolium perenne L.), which was expected to produce diploid (unreduced) pollen grains in addition to haploid pollen grains. The considerable overlap of the diameter distributions of haploid and diploid pollen grains severely hampers the accurate estimation of the proportion of diploid pollen grains. To overcome this problem we develop in this paper a statistical normal mixture model and we describe a method to test for the production of diploid pollen grains from a diploid parent, and to estimate the proportion of diploid pollen grains.     

Matromorphy in Brassica oleracea L. VII. Research on products of microsporogenesis and gametogenesis from prickle pollinated plants

Summary A research has been carried out on the occurrence of 2n gametes in Brassica oleracea and on their possible way of origination. After microsporogenesis it appeared that unreduced PMC's, dyads and triads occurred. Giant pollen grains with two (diploid) and four (haploid) generative nuclei, respectively, were found, resulting from a deviating cytokinesis and karyokinesis. Diploid heterozygous matromorphic embryos may develop from such unreduced gametes formed after macrosporogenesis.     

Meiosis in triploid Solanum tuberosum L.

Summary Meiosis in triploid Solanum tuberosum was studied in plants that originated from crosses between tetraploids and diploids, the latter being mainly dihaploids.Association of chromosomes in first metaphase was regular, being as expected for autotriploid material. In first anaphase lagging chromosomes occurred that mostly divided precociously into their chromatids, the amount of lagging chromosomes being more or less proportional to the average number of univalents at M I. The A I distribution of chromosomes over the poles fitted a normal distribution.In the second meiotic division also lagging chromosomes were observed. During this division disturbances occurred resulting in cells with more or less the somatic chromosome number. The origin of this aberration is discussed, together with the relation between meiotic behaviour and pollen stainability.     

Formation of unreduced gametes is impeded by homologous chromosome pairing in tetraploid <Emphasis Type="Italic">Triticum turgidum</Emphasis> × <Emphasis Type="Italic">Aegilops tauschii</Emphasis> hybrids

It is believed that unreduced gametes with somatic chromosome numbers play a predominant role in natural polyploidization. Allohexaploid bread wheat originated from spontaneous hybridization of Triticum turgidum L. with Aegilops tauschii Coss. Unreduced gametes originating via meiotic restitution, including first-division restitution (FDR) and single-division meiosis (SDM), are well documented in triploid F₁ hybrids of T. turgidum with diploid Ae. tauschii (genomic constitution ABD, usually with 21 univalents in meiotic metaphase I). In this study, two T. turgidum lines known to carry genes for meiotic restitution were crossed to tetraploid Ae. tauschii. The resulting F₁ hybrids (genomes ABDD), had seven pairs of homologous chromosomes and regularly formed 14 univalents and seven bivalents at metaphase I. Neither FDR nor SDM were observed. The distribution of chromosome numbers among progeny obtained by self pollination and a backcross to T. turgidum showed the absence of unreduced gametes. These results suggest that high homologous pairing interfered with meiotic restitution and the formation of unreduced gametes. This may be related to asynchronous movement during meiosis between paired and unpaired chromosomes or to uneven distribution of chromosomes in anaphases, resulting in nonviable gametes.     

Identification and cytological analysis of 2n-pollen producers in Lotus tennis Wald. et Kit.

Two meiotic mutants of L. tennis (2n = 2x = 12) producing unreduced pollen are described. When crossed to male sterile L. corniculatus (2n = 4x = 24) plants, all progeny plants were morphologically similar to L. corniculatus, had 2n = 24 chromosomes, and in the cross, were fully compatible with L. corniculatus, indicating that the male parent plants were 2n-pollen producers. One of them also had ‘giant’ pollen grains. In metaphase II of both genotypes, there were parallel and tripolar spindles leading to dyad and triad formation, the latter being found most frequently. Since both the above-mentioned mechanisms result in first-division restitution-type microspores, the genotypes examined could be useful in breeding Lotus.     

Male fertility and cytology of triploid hybrids between tetraploidSolanum commersonii (2n=4x=48, 2EBN) and Phureja-Tuberosum haploid hybrids (2n=2x=24, 2EBN)

Summary Solanum commersonii Dun. is a diploid (2n=2x=24, 1EBN) wild species of potential value for potato breeding. It is a reproductively isolated species and cannot be crossed with Tuberosum haploids (2n=2x=24, 2EBN) or other diploid 2EBNSolanum species. In order to overcome the EBN barriers, triploid hybrids were produced between Phureja-Tuberosum haploid hybrids, which form 2n pollen grains by parallel spindles, and tetraploidS. commersonii. Microsporogenesis analysis of the triploids indicated a trend towards low values of chromosome distribution at Anaphase I; lagging chromosomes were often observed as well. Despite these abnormalities, the percentage of stainable pollen was very high, ranging from 5.0% to 74.3%. A high variation in pollen grain diameter was also evident. Parallel and tripolar orientation of spindles at Metaphase II of microsporogenesis was a common feature of all the triploids analyzed, but dyads and triads were observed at a very low frequency. Therefore, also the frequency of 2n pollen was very low; the different size of stainable pollen appears to represent the ploidy levels which are possible according to the distribution of chromosomes in Anaphase I. The results obtained also suggest thatS. commersonii could have minor genes acting at the end of meiosis in such a way that, despite the presence of parallel/tripolar spindles, dyads/triads are not formed.Contribution no. 124 from the Research Center for Vegetable Breeding.     

Meiotic chromosome behaviour in an allotriploid derived from an amphidiploid × diploid mating in Cucumis

A synthetic amphidiploid species Cucumis hyriviis Chen & Kirkbride (2n = 4x = 38; genome designated as HHCC) has recently been created from an interspecific mating between C. sativus L. (2n = 2x = 14: genome designated as CC) and C. hystrx. Chakr. (2n = 2x = 24; genome designated as HH). This amphidiploid is resistant to root knot nematode, tolerant to low irradiance, and has higher nutritional value than standard processing cucumber cultivars. An allotriploid (2n = 3x = 26; HCC) was derived from a cross between C. sativus L. and C. hytivus Chen & Kirkbride. Diploid meiotic behaviour in C. sativus and C. hystrix involves the development of seven and 12 metaphase bivalents respectively. In the derived allotriploid. univalents. bivalents, and trivalents (at relatively low frequency) were observed at metaphase I indicating that some homeologues from the C and H genomes can synapse. Based on a comparative karyotype analysis of cucumber (i.e. chromosome size and pairing behaviour) and aliotriploid plants, the seven bivalents observed at metaphase I were ascertained to be cucumber homologues, while the 12 univalents were of C. hystrix origin thus confirming the allotriploid karyotypic constitution to be HCC. On average, the frequency of trivalents was 0.24 at diakinesis and 0.22 in 100 meiocytes at metaphase I. indicating the possibility of genetic exchange due to the homoeology between the C and H genomes. After simultaneous cytokinesis, only polyads were observed in pollen mother cells (PMCs) at telophase II, which led to the production of sterile pollen grains. Multi‐polarization of chromosomes was dominant at anaphase II. However. in about 20% PMCs. chromosomes separated to form a 7C + 1‐2H complement, suggesting a possible method for the production of alien addition cucumber‐C hystrix lines through further backcrossing of the allotriploid to diploid cucumber.     

Screening <Emphasis Type="Italic">A. ventricosa</Emphasis> populations for 2n gametes

Sexual polyploidization via the formation of 2n gametes has been acknowledged as the most significant evolutionary mode of polyploidization among plant species. The present study was conducted in order to determine whether 2n gametes are present in the C-genome diploid Avena ventricosa Bal. ex Coss., a species that contributed to the evolution of the cultivated hexaploid species (Avena sativa L). Individual plants belonging to four different Cypriot populations, were screened for pollen grain size variation with the aim to distinguish 2n gametes. Avena ventricosa ARI00-845 was identified to produce large pollen grains at a low percentage (1.21%). Subsequent analysis using flow cytometry confirmed the presence of 2n gametes in the pollen. Cytogenetic analyses of pollen mother cells revealed cells with twice the typical chromosome number at metaphase I (i.e., 28 chromosomes). We postulate that irregularities in cell wall formation preceding meiosis could have contributed to the mode of chromosome doubling.     

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.     

The first record of tetrasomy in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

Tetrasomic plants with two additional small chromosomes were identified, with a frequency about 2.5%, in the trisomic pea line TRUST-R, which normally contains one extra chromosome covering a sporophyte lethal in the regular chromosome set. As compared to trisomics, tetrasomics exhibited an enhanced expression of the traits resulting from extra chromosome addition: slow growth, enlarged bracts, shortened peduncles, wavy leaflets and stipulae. They were almost sterile, their pollen contained a variable proportion of empty grains and some anomalously large, small or deformed grains. In metaphase I, two extra chromosomes did not form a stable bivalent and only in some cases were situated close to each other. In anaphase I, the extra chromosomes migrated independently to either pole or retarded in the equatorial plain, the same was observed for chromatids in anaphase II. This retardation resulted in anomalous cytokinesis, so that triads, dyads and half-divided or non-divided monads appeared. The retarded extra chromosomes may form small extra nuclei either included into one of the microspores or forming a separate miniature cell; in this way tetrasomics may eliminate extra chromosomes. One of the tetrasomics analysed formed an exceptionally high proportion of microspore pentads. In the regluar TRUST-R trisomics, the sole extra chromosome retarded in the equatorial plain in anaphases I and II. The retardation in anaphase II often makes cytokinesis in trisomics (in general more regular than in tetrasomic) to proceed in two steps: at first cell wall formation separates a pollen mother cell into two dyads and then each of them into two microspores.     

Cytological mechanism of 2<Emphasis Type="Italic">n</Emphasis> pollen formation in Chinese jujube (<Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. ‘Linglingzao’)

In order to elucidate the cytological mechanism of 2n pollen formation in Chinese jujube, a cultivar named ‘Linglingzao’ (2n = 2x = 24) which produces relative more 2n pollens naturally was employed for microsporogenesis analysis. Chromosomes paired in 12 bivalents at diakinesis and the first meiotic division was normal, whereas, the second division was characterized by frequent abnormal spindle orientation (parallel and tripolar spindles). Perpendicular, tripolar, and parallel spindles at metaphase II accounted for 72.93, 17.22, and 9.85% respectively. Perpendicular and tripolar spindles led to tetrads and triads formation respectively. Two types of parallel spindles were observed and a significant association correlation (r = 0.84, P < 0.05) between parallel spindle II (fused spindles) and dyads was found which meant only fused spindles can form dyads, while parallel spindle I with a long distance between two sets of chromosomes seemed to lead tetrad formation. At tetrad stage, the observed frequencies of tetrads, triads and dyads were 80.99, 16.40, and 2.61% respectively. By the rule that each tetrad can form four n pollens, each triad can form one 2n pollen and two n pollens, each dyad can form two 2n pollens, the frequency of 2n pollen estimated was 5.71%. Based on the pollen diameter difference between n and 2n pollens, frequency of 2n pollen observed was 6.15%. No significant difference was observed between frequency of 2n pollen estimated and that of observed. The results indicated that, tripolar spindles and fused spindles at metaphase II followed by more triads and few dyads formation at tetrad stage were responsible for the production of 2n pollen in Chinese jujube. 2n gametes observed correspond to first division restitution. Research on the cytological mechanism of unreduced pollen will provide a platform for unreduced pollen induction and polyploidy breeding in Chinese jujube.     

Cytogenetic analysis of translocations in cotton

In order to create cotton translocation lines, 191 translocations were recovered as heterozygotes following combined treatment of seeds by colchicine and γ‐rays, irradiation of seeds by thermal neutrons and c‐irradiation of pollen. Cytogenetic analysis showed that chromosome translocations involving two chromosomes arose more often (180) than those involving three chromosomes (11). The heterozygous translocations were characterized by different formation and frequencies of the multivalents at metaphase I of meiosis. Only 104 translocations were characterized by a multivalent frequency of more than 0.25 per cell. Most of the translocations (156) exhibited a high meiotic index, but 11 were characterized by a decreasing meiotic index and by an increase in the percentage of tetrads with micronuclei. The pollen fertility of translocations differed significantly and varied from high fertility to pollen sterility. The high frequency of abortive pollen grains was typical for about one half of translocations studied. The translocations were made homozygous and as a result, 12 new reciprocal homozygous translocation lines were obtained in cotton.     

The origin of unreduced microspores due to aberrant cytokinesis in the meiocytes of potato and its genetic significance

Summary In 7 aneuhaploids of Solanum tuberosum and one clone of S. phureja the mechanism of unreduced microspore production has been studied. It has been observed that the unreduced microspores are produced not only by nuclear restitution at meiosis but also by aberrant cytokinesis in the meiocyte. The size and the number of nucleoli that are present in young microspores at the sporad stage give a fairly reliable indication of the ploidy level and the type of nuclear restitution that has occurred. These two aspects are helpful to deduce the meiotic events that have probably led to the formation of unreduced microspores in some cases. It has been found that triads and dyads are formed regardless of the type of nuclear division or restitution, indicating that cytokinesis is independent of karyokinesis to some extent. Cytological basis of unreduced microspore formation in diploid forms of potato appears to be more complex than it was supposed earlier.     

Somatic chromosome elimination and meiotic chromosome pairing in the triple hybrid 6x-(Solanum acaule × S. bulbocastanum) × 2×-S. phureja

Summary Two selected hexaploid F₁ clones from the cross Solanum acaule x S. bulbocastanum were intercrossed and the resulting hybrid plants pollinated with the diploid species S. phureja, in order to obtain tetraploid triple hybrids with the same ploidy level as S. tuberosum cultivars (2n=4x=48).Apart from three trihaploids a large population of triple hybrids was obtained, showing chromosome mosaicism in root tip cells (euploid + hypoploid chromosome numbers) and a uniform, mostly hypoploid chromosome number in the pollen mother cells. It is demonstrated that somatic chromosome elimination in the early stages of development is the most probable cause.From detailed pachytene observations as well as from the chromosome associations observed at metaphase I it is evident, that there is normal pairing between the four genomes in the triple hybrids. Although S. bulbocastanum is a quite distinct species with a rigid crossability barrier with S. phureja, the chromosomes of these two species appear to have a high degree of homology. Especially the formation of quadrivalents involving all twelve groups of four homeologous chromosomes, indicated that the four parental genomes (two from S. acaule, one from S. bulbocastanum and one from S. phureja) are not differentiated to the extent of affecting normal pairing and chiasma formation. These results support the view, that the transfer of valuable characters from S. bulbocastanum to S. tuberosum cultivars is feasible even when these characters are polygenically controlled.     

Effect of high temperature on the production of 2n pollen grains in diploid roses and obtaining tetraploids via unilateral polyploidization

To integrate the gene pool of a wild species (primarily diploid) into a cultivated pool (primarily tetraploid), a crossing between a dihaploid cultivated rose and a hybrid of Rosa wichurana allowed to obtain interspecific diploid hybrids that produced 2n pollen grains. A return to a tetraploid level sought by breeders can then be considered using sexual polyploidization, obtained by crossing a tetraploid cultivated rose with these hybrids. Application of a high‐temperature regime led to a small but significant increase in the percentage of 2n pollen grains in these hybrids of up to 4.6%. This result was obtained by applying high temperatures close to 32°C during the day to plants cultivated in a glasshouse during recurrent cycles of bloom. Crosses were made between an unreleased tetraploid hybrid tea rose, as a female, and the diploid hybrid that produces the most 2n pollen grains. Tetraploid (42.1%) and triploid (57.9%) offspring were obtained. The use of these 2n pollen grains of the first division restitution type should facilitate the introgression of complex traits of interest.     

Monohaploids (n=x=12) from autotetraploid Solanum tuberosum (2n=4x=48) through two successive cycles of female parthenogenesis

Summary Viable monohaploids with 12 chromosomes in their somatic cells were obtained from an autotetraploid cultivar of Solanum tuberosum (2n=4x=48) by inducing female parthenogenesis successively in the tetraploid cultivar and in dihaploids from that cultivar. Both dihaploids and monohaploids were induced using the diploid S. phureja clones IvP 35 and IvP 48 as pollinators. The average frequencies of dihaploids and monohaploids in 1973 were 2.0 and 0.14 per berry respectively.Non-homologous chromosome associations (bivalents, trivalents and even quadrivalents) were observed at metaphase I of meiosis in pollen mother cells of the two monohaploids studied.The occurrence of non-homologous associations of chromosomes during meiosis is discussed. In addition the potential significance of monohaploids for basic research andfor breeding of potatoes is considered.