Variation of phenotype,ploidy level,and organogenic potential of in vitro regenerated polyploids of <Emphasis Type="Italic">Pyrus communis</Emphasis>

A wide range of phenotypic variation was observed among neopolyploids obtained from the diploid pear cultivar ‘Fertility’ by in vitro colchicine treatment. The variant plantlets had alterations in leaf characteristics. Neopolyploids had significantly different ratios of leaf length to leaf width compared to the diploid control. Shoot regeneration from leaf explants and rooting ability from in vitro shoots of neopolyploids was examined. Regeneration frequencies of shoots from leaf explants of seven of the nine neopolyploids were significantly decreased compared to the diploid control. The organogenic potential of neopolyploids was highly genotype-dependent for both shoots and roots. Tetraploid clone 4x − 4 failed to regenerate shoots from leaf explants and the pentaploid clone 5x − 2 failed to root from in vitro shoots. The results suggest that polyploidization caused the decrease in or loss of in vitro organogenic potential. Regenerated shoots derived from neopolyploids showed different phenotypes, depending on the ploidy of the donor plant.     

In vitro embryo rescue culture of F<Subscript>1</Subscript> progenies from crosses between diploid and tetraploid grape varieties

The purpose of this study was to understand factors affecting in vitro embryo rescue culture from hybrids between diploid and tetraploid varieties of grape in creation new triploid germplasm resources. The effects of different media, removal ages of immature seeds and reciprocal crosses of parents on the germination and seedling survival of immature seeds from crosses between diploid and tetraploid grape varieties by in vitro embryo rescue culture were investigated. The results indicated that the medium consisting of NN-1969 + IAA 1.75 mg l⁻¹ + GA₃ 0.35 mg l⁻¹ + CH 400 mg l⁻¹ + AC 2.0 g l⁻¹ was better than other media. The optimal removal age of immature seeds for the best development of embryos was 35–45 days after pollination (DAP). The percentage of germination (PG) for immature seeds and the percentage of seedling survival (PSS) for immature seeds for diploid varieties used as female parents were 10.72% and 4.35% higher than when tetraploid varieties were used as female parents respectively. A total of 41 hybrid progenies from eight combinations were obtained, made up of 17 diploid, 9 tetraploid, 14 aneuploid, and 1 triploid progeny as determined by root-tip chromosome identification. The triploid progeny was from Fujiminori (2n = 4x = 76) × Jingxiu (2n = 2x = 38). These results implied that it was feasible to extend the hybridization range of grape and to create new germplasm resources by in vitro embryo rescue based on the conventional hybridization. The NN-1969 medium supplemented with GA₃ and IAA was more propitious to the development of immature seeds sampled at about 45 DAP. It was easier to obtain plants using diploid as female parent, but triploid progeny was only obtained using tetraploid as female parent.     

DNA Methylation in Genomes of Several Annual Herbaceous and Woody Perennial Plants of Varying Ploidy as Detected by MSAP

Polyploidization is known to accompany altered DNA methylation in higher plants, which plays an important role in gene expression regulation and maintaining genome stability. While the characteristics of DNA methylation in different polyploid plants are still to be elucidated; here, status of genomic DNA methylation in a series of diploid, triploid, and tetraploid annual herbaceous plants (watermelon and Salvia) and woody perennials (pear, Poplar, and loquat) were explored by methylation-specific amplified polymorphism analysis. The results indicated that levels of DNA methylation in triploid watermelon and Salvia were lower than their diploid parents. In triploid Poplar and pear, higher levels of DNA methylation were detected, and no significant difference was observed between triploid and tetraploid in all tested materials. Further data analysis suggested that about half of the total detected sites underwent changes of DNA methylation patterns in triploid watermelons and Salvia, as well as an obvious trend towards demethylation. However, the changes of DNA methylation patterns in three triploid woody perennials were only 17.54–33.40%. This implied that the characteristics of DNA methylation are significantly different during the polyploidization of different plant species. Furthermore, the results suggested that the level of DNA methylation was nonlinearly related to the ploidy level, and triploid plants displayed more interesting DNA methylation status. The characteristics and possible functions of DNA methylation in different ploidy series are further discussed.     

Production of tetraploid plants of non apomictic citrus genotypes

Ploidy manipulation in Citrus is a major issue of current breeding programs aiming to develop triploid seedless mandarins to address consumer demands for seedless fruits. The most effective method to obtain triploid hybrids is to pollinate tetraploid non apomictic cultivars with pollen of diploid varieties. Such non apomictic tetraploid lines are not found in the citrus germplasm and need to be created. In this work we describe a new methodology based on in vitro shoot-tip grafting combined with treatment of the micro-grafted shoot-tip with colchicine and oryzalin to achieve chromosome doubling and a dechimerization procedure assisted by flow cytometry. Stable tetraploid plants of Clemenules, Fina and Marisol clementines and Moncada mandarin have been obtained directly from shoot tip grafting combined with colchicine and oryzalin treatments or after dechimerization of mixoploids plants (2x–4x). These stable tetraploid plants have been used in 4x × 2x hybridizations, to recover over 3,250 triploid hybrids in 3 years.     

梨多倍体化对离体叶片不定梢再生能力的影响

以源于二倍体梨品种Fertility(Pyrus communis L.)通过秋水仙碱离体诱变体细胞染色体加倍获得的不同同源多倍体无性系为试材,以离体叶片为外植体,观察研究了不同倍性无性系叶片的不定梢再生能力。结果表明,多倍体的不定梢再生率显著低于二倍体的再生率。不同多倍体无性系的不定梢再生能力也存在显著差异。三倍体无性系3x-3和四倍体无性系4x-4不能诱导产生不定梢。表明器官发生能力下降或植物细胞全能性的丧失与细胞染色体多倍体化有关。     

Genome size evolution and polyploidy in the Daphnia pulex complex (Cladocera: Daphniidae)

Genome size was estimated in 49 clones of the Daphnia pulex complex from temperate and subarctic locations using flow cytometry and microsatellite DNA analyses. Significant genome size differences were found in diploid species belonging to the two genetically distinct groups (the pulicaria and the tenebrosa groups), with clones from the tenebrosa group having genome sizes 22% larger than those in the pulicaria group. Combined flow cytometry and microsatellite DNA analyses revealed that nearly all polyploid clones in the D. pulex complex are triploid and not tetraploid, as was previously suggested. Sequencing analyses of the ND5 gene to position clones in their respective clades within the D. pulex complex have uncovered three triploid clones of Daphnia middendorffiana with a D. pulex maternal parent. This result was unexpected because Daphnia pulicaria has always been identified as the maternal parent of these hybrid polyploid clones. Triploid clones likely owe their origins to interactions between sexual and asexual populations. Further interactions in the tenebrosa group have generated tetraploid clones but these events have been rare.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 68–79.     

Arrangement of spindle apparatus in mitoses of different ploidy

In non-hypotonically treated mitoses from tissue cultures of Microtus agrestis, both the constitutive heterochromatin of the sex chromosomes and the spindle apparatus were stained by the Giemsa C-banding technique. By means of counting the heterochromatic chromosomes, we determined the cell ploidy and studied the number of centrioles and the spindle arrangement of diploid, triploid, tetraploid and octoploid mitoses. Diploid and triploid prophases contained 2 centrioles in most cases, tetraploid prophases 4, binucleate cells with 2 diploid nuclei likewise 4 and binucleate cells with 2 tetraploid nuclei 8 centrioles. Nearly 99% of diploid and triploid metaphases were bipolar. Of the tetraploid metaphases only 45% were bipolar, 29.5% tripolar, 7.5% quadripolar and 18% formed as a parallel mitosis. In all examined binucleate cells that had had an asynchronous DNA synthesis, a multipolar mitosis was found.     

Triploid bridge and role of parthenogenesis in the evolution of autopolyploidy

Autopolyploidization is considered to play an important role in plant evolution. In polyploidization, the polyploid evolves from the original diploid cytotype, in which the triploid state is considered to mediate the process (triploid bridge). Nevertheless, the fitness of triploid individuals seems to be too low to facilitate the polyploidization process (triploid block). The evolutionary condition of autopolyploidy was analyzed using a mathematical model focusing on the role of parthenogenesis in triploid and tetraploid individuals. In addition, offspring were assumed to arise by sexual reproduction by conjugations between haploid, diploid, and triploid gametes produced by diploid, tetraploid, and triploid individuals. According to the analysis, even if triploid block suppresses the fitness of sexually produced triploids, the polyploidization process can proceed when parthenogenesis occurs frequently. If only triploids frequently reproduce parthenogenetically, the evolutionary consequences tend to depend on the fitness of the tetraploid individuals. On the basis of a predetermined parameter set, if tetraploid fitness is relatively low, all three ploidies can coexist. Otherwise, tetraploidization occurs. In this case, triploid parthenogenesis promotes not only triploidization but also tetraploidization. However, if both triploids and tetraploids frequently reproduce parthenogenetically, the ploidy levels with the highest fitness are likely to dominate in the population through direct competition among cytotypes.     

Ploidy doubling by in vitro culture of excised protocorms or protocorm-like bodies in <Emphasis Type="Italic">Phalaenopsis</Emphasis> species

Endopolyploidy was observed in the protocorms of diploid Phalaenopsis aphrodite subsp. formosana with ploidy doubling achieved by in vitro regeneration of excised protocorms, or protocorm-like bodies (PLBs). Thirty-four per cent of the PLBs regenerated from the first cycle of sectioned protocorms were found to be polyploids with ploidy doubled once or twice as determined by flow-cytometry. The frequency of ploidy doubling increased as the sectioning cycles increased and was highest in diploid followed by the triploid and tetraploid. Regeneration of the endopolyploid cells in the tissue of the protocorms or PLBs is proposed as the source of the development of ploidy doubled plantlets. The frequency of ploidy doubling was similar in seven other Phalaenopsis species, although the rate of increase within cycles was genotype specific. In two species, a comparison of five parameters between 5-month-old diploid and tetraploid potted plants showed only the stomata density differed significantly. The flowers of the tetraploid plant were larger and heavier than those of the diploids. This ploidy doubling method is a simple and effective means to produce large number of polyploid Phalaenopsis species plants as well as their hybrids. The method will be beneficial to orchid breeding programs especially for the interspecific hybridization between varieties having different chromosome sizes and ploidy levels.     

The origin and evolution of parthenogenesis in Heteronotia binoei (Gekkonidae)

Within Australia, the gekkonid lizard Heteronotia binoei exists as diploid bisexual and triploid all-female populations. Three bisexual cytotypes and three triploid clones can be described on the basis of the morphology of gross karyotypes. This paper reports the results of a C-banding analysis that revealed both intrapopulation polymorphism and interpopulation polytypic variation within the most widespread bisexual cytotype (A6). A C-band variant that defined a ZW sex chromosome system in populations from the MacDonnell Ranges in central Australia was also identified. Silver staining confirmed that in all populations the nucleolus organising region always occurred distally on chromosome 6. Examination of C-banding and silver-staining patterns of triploid populations provided strong support for a hybrid origin of the parthenogens that involved the central and western A6 populations. It is proposed that the hybridisation of these populations with the other chromosomally distinct bisexual cytotypes (SM6) resulted in the triploid clones. At present, seven clones have been karyotypically defined, and all the chromosomal variants that were present in the triploids can be accounted for by multiple hybridisation events between the bisexual populations. The analysis also provided evidence that rare sterile tetraploid females are the result of insemination of the triploid parthenogens by male H. binoei. This paper is dedicated to the memory of M.J.D. White (1910–1983)     

Evidence for non-disomic inheritance in a Citrus interspecific tetraploid somatic hybrid between C. reticulata and C. limon using SSR markers and cytogenetic analysis

Artificial tetraploid somatic hybrids have been developed for sterile triploid citrus breeding by sexual hybridization between diploid and tetraploid somatic hybrids. The genetic structure of diploid gametes produced by tetraploid genotypes depends on the mode of chromosome association at meiosis. In order to evaluate tetraploid inheritance in a tetraploid interspecific somatic hybrid between mandarin and lemon, we performed segregation studies using cytogenetic and single sequence repeat molecular markers. Cytogenetic analysis of meiosis in the somatic hybrid revealed 11% tetravalents and 76% bivalents. Inheritance of the tetraploid hybrid was analyzed by genotyping the triploid progeny derived from a cross between a diploid pummelo and the tetraploid somatic hybrid, in order to derive genotypes of the meiospores produced by the tetraploid. A likelihood-based approach was used to distinguish between disomic, tetrasomic, and intermediate inheritance models and to estimate the double reduction rate. In agreement with expectations based the cytogenetic data, marker segregation was largely compatible with tetrasomic and inheritance intermediate between disomic and tetrasomic, with some evidence for preferential pairing of homoeologous chromosomes. This has important implications for the design of breeding programs that involve tetraploid hybrids, and underscores the need to consider inheritance models that are intermediate between disomic and tetrasomic.     

Cytological Diversity and Triploid Frequency in a Complex Population of Ranunculus ficaria L.

An extensive mixed population of Ranunculus ficaria polyplotypeswas mapped, sampled, and examined cytologically. The presenceand relative abundance of different polyplotypes was as follows:31 per cent diploid (2n = 16), 40 per cent tetraploid (2n =32), and 29 per cent presumed intermediate triploid (2n = 24).It was demonstrated that there is no simple character for distinguishingall polyplotypes in the field and chromosome counts must beused for this purpose. The action of winter flood water on bulbildissemination and differences in ecological preference of thepolyplotypes is proposed to account for differences in distributionover the population area. There is some indication that triploidseeds may be produced under a situation of excessive numbersof tetraploid plants relative to diploids which reduces diploid/tetraploidpollen competition on diploid stigmas. Diploid plants with 1–8B-chromosomes were found to occur frequently but no consistenteffect of these B's on vigour, fertility, and meiosis was evident.B-chromosomes were entirely absent from triploid and tetraploidkaryotypes.     

Comparison of chemically induced chromosome loss in a diploid, triploid, and tetraploid strain of Saccharomyces cerevisiae.

Triploid and tetraploid strains of Saccharomyces cerevisiae were constructed and the spontaneous loss during mitosis of one, two or three copies of chromosome VII was determined. In one strain, a triploid (VM2) in which expression of the recessive alleles can be observed only after loss of two copies of chromosome VII (3N-2), the spontaneous frequency of chromosome loss was lower than in the diploid D61.M. In another strain, a tetraploid (VM4) that also requires the loss of two copies of chromosome VII for observation (4N-2) of the recessive alleles, the spontaneous frequency was slightly higher than in the diploid D61.M. The spontaneous frequency of other genetic events (that is, mutation, recombination or chromosome breakage) were lower by 2-3 orders of magnitude than in the diploid strain D61.M. Induction of chromosome loss and other genetic events by nocodazole, ethyl acetate, hydroxyurea and ethyl methanesulfonate was determined in D61.M, VM2, and VM4, and the results were compared. Nocodazole and ethyl acetate induced chromosome loss in both the triploid and the tetraploid strains at lower concentrations than required in the diploid. These compounds also induced elevated frequencies of other genetic events in both the triploid and the tetraploid strains but not in the diploid. Hydroxyurea induced elevated frequencies of chromosome loss in the diploid and the tetraploid. Frequencies of chromosome loss in the triploid treated with hydroxyurea, although elevated, are based on observation of very few colonies of the correct phenotype. Ethyl methanesulfonate failed to induce chromosome loss in any of the three strains. Hydroxyurea and ethyl methanesulfonate did, however, induce very high frequencies of other genetic events.     

Diploid Ancestors of Triploid Export Banana Cultivars: Molecular Identification of 2<Emphasis Type="Italic">n</Emphasis> Restitution Gamete Donors and n Gamete Donors

The origin of triploid export banana cultivars was investigated. They all belong to Cavendish and Gros Michel subgroups of triploid clones and have a monospecific Musa acuminata origin. The appearance of these cultivars is thought to be result of hybridization between partially sterile diploid cultivars producing non reduced gametes and fertile diploids producing normal haploid gametes. To trace these diploid ancestors we compared the RFLP patterns, revealed by 36 probe/enzyme combinations, of 176 diploid clones representing the worldwide available variability with that of clones from the Cavendish and Gros Michel subgroups. This lead us to the identification of the common putative diploid ancestor of cultivars from Cavendish and Gros Michel subgroups which contributed to triploid cultivar formation through the production of 2n restitution gametes. For cultivars of Gros Michel subgroup we also propose a normal gamete donor that may have complemented the triploid allele set.     

不同倍性大青杨的光合特性及叶片解剖结构比较

通过对二年生二倍体、三倍体和四倍体大青杨叶片结构和光合特性的研究,探讨不同倍性大青杨生长性状差异的原因。结果表明：不同倍性大青杨的净光合速率（Pn）、光饱和点（LSP）、光补偿点（LCP）等光合指标差异显著。三倍体的最大净光合速率（Pmax）高出二倍体21%。叶片厚度、表皮细胞厚度、栅栏组织厚度、栅栏组织与海绵组织厚度的比值,都是三倍体和四倍体大青杨比二倍体高,且均呈现增加的趋势。其中,四倍体大青杨表皮细胞最厚,上下表皮分别高出二倍体64%和17%。三倍体大青杨的栅栏组织厚度及栅栏组织与海绵组织厚度的比值最大,比值高出二倍体50%。通过叶片光合特性和解剖结构比较证明,三倍体和四倍体大青杨对光的适应性和光合作用能力强于二倍体。     

Ploidy barrier to endosperm development in maize

Maize kernels inheriting the indeterminate gametophyte mutant (ig) on the female side had endosperms that ranged in ploidy level from diploid (2x) to nonaploid (9x). In crosses with diploid males, only kernels of the triploid endosperm class developed normally. Kernels of the tetraploid endosperm class were half-sized but with well-developed embryos that regularly germinated. Kernels of endosperm composition other than triploid or tetraploid were abortive.-Endosperm ploidy level resulting from mating ig/ig x tetraploid Ig similarly was variable. Most endosperms started to degenerate soon after pollination and remained in an arrested state. Hexaploid endosperm was exceptional; it developed normally during the sequence of stages studied and accounted for plump kernels on mature ears. Since such kernels have diploid maternal tissues (pericarp) but triploid embryos, the present finding favors the view that endosperm failure or success in such circumstances is governed by conditions within the endosperm itself.-Whereas tetraploid endosperm consisting of three maternal genomes and one paternal genome is slightly reduced in size but supports viable seed development, that endosperm having two maternal and two paternal chromosome sets was highly defective and conditioned abortion. Thus, development of maize endosperm evidently is affected by the parental source of its sets of chromosomes.     

Plantlet production from the male inflorescence tips of <Emphasis Type="Italic">Musa acuminata</Emphasis> cultivars from South India

Inflorescence apices are suitable explants for the rapid in vitro propagation of Musa spp. However, the diploid and triploid banana cultivars showed different in vitro responses with respect to the hormone combinations in Murashige and Skoog medium. The diploid cultivar (Sannachenkadali, AA) induced a maximum number of multiple shoots in 8.9 μM 6-benzyl adenine (BA) whereas the triploid cultivar (Red banana, AAA) exhibited maximum multiplication in 22.2 μM 6-benzyl adenine. MS medium supplemented with 11.4 μM indole acetic acid and 17.8 μM BA was also suitable for shoot proliferation in triploid cultivar but not in the diploid cultivar. The regenerated shoots were rooted in Murashige and Skoog basal medium within 10–15 days. The rooted plantlets were transferred to vermiculite and maintained at a temperature of 25 ± 2°C for 10 days and then at room temperature (30–32°C) for 2 weeks before transferring to potted soil compost mixture. The plantlets showed 100% survival.     

Inter-embryo competition in the progeny of autotriploid Aloineae (Liliaceae)

In reciprocal crosses between diploid and triploid Aloineae the progeny are largely diploid or diploid plus one or two chromosomes, but in reciprocal crosses between triploids and tetraploids they are tetraploid or nearly so. Thus the triploids contribute circa haploid gametes to the progeny when crossed with diploids but circa diploid gametes when crossed with tetraploids. These results are compared with those of a number of earlier workers. It is concluded that the bias in the frequency of progeny types towards diploidy or tetraploidy, depending on the ploidy level of the plant which is crossed with the triploid, is caused by inter-embryo competition. Those embryos with an endosperm/embryo factor of 1.5, the value found in normal diploid/diploid crosses having triploid endosperms, are selected in preference to those with factors higher or lower than 1.5.Inter-gamete competition also occurs among the euploid and aneuploid gametes produced by the triploids. This is more pronounced on the male side, because the degree of survival of aneuploid pollen from the triploids into the next generation is much lower than that of aneuploid egg nuclei.Non-reduction in the triploids gives rise to occasional pentaploid progeny in crosses with tetraploids, but it is more probable that in diploid/triploid crosses tetraploid progeny are the products of non-reduction in the diploid.     

Ploidy distribution and DNA content variations of <Emphasis Type="Italic">Lonicera caerulea</Emphasis> (Caprifoliaceae) in Japan

Ploidy level and geographical distribution were investigated in Japanese Lonicera caerulea L. Flow cytometric analysis revealed the presence of DNA diploid and DNA tetraploid plants in Japan. Chromosome observation confirmed that diploid and tetraploid plants showed 2n = 2x = 18 and 2n = 4x = 36, respectively. The DNA diploid populations were found only in lowland mires, Betsukai, Bekanbeushi, Kushiro and Kiritappu located in eastern Hokkaido. On the other hand, DNA tetraploid populations were distributed in a wide area of Hokkaido, and mainland of Japan. The habitats of DNA tetraploid plants were lowland to alpine region. The DNA content measurement with flow cytometry revealed significant differences in the relative DNA contents among DNA tetraploid populations. The relative DNA content within DNA tetraploid populations varied 1.157-fold at maximum, and might correlate with altitude indicating that DNA contents were smaller as altitude increases. The wide area of distribution in various environments of DNA tetraploid plants suggested the adaptability of the tetraploid plants. Although diploid and tetraploid populations were found, no triploid was detected, indicating crossing difficulty between diploid and tetraploid as confirmed by crossing experiment.