T-DNA-tagged chromosome 12 in donor Lycopersicon esculentum × L. pennellii is retained in asymmetric somatic hybrids with recipient Solanum lycopersicoides

Summary Asymmetric somatic hybrid plants were recovered after fusing irradiated mesophyll protoplasts of donor Lycopersicon esculentum × L. pennellii (EP) interspecific hybrid with callus-derived protoplasts of recipient Solanum lycopersicoides. EP plant A54 had been previously transformed by an agrobacterium vector, and the T-DNA insert mapped to the L. esculentum chromosome 12. The T-DNA insert conferred kanamycin resistance to EP that was subsequently used to select cell fusion products and recover asymmetric hybrid plants that retained tagged chromosome 12. Doses of 50- and 100-Gy irradiation promoted the elimination of only a few donor chromosomes. At 200 Gy, the regenerated plants had ploidy levels higher than tetraploid. However, the T-DNA tagged chromosome 12 was always retained in the asymmetric hybrid plants tested. Likewise, all plants from the 100-Gy series, with the exception of number 160, were mixoploid in the root-tip cells. Such mixoploid asymmetric somatic hybrids could be stabilized by inducing adventitious shoots on leaf strips cultured on shoot regeneration medium containing kanamycin. The asymmetric hybrid plants did not produce viable seed when self-pollinated or backcrossed to tomato or S. lycopersicoides. Present address: Department of Biology, University College of London, Gower Street, London, UK     

Morphological,cytological and molecular characterization of a novel symmetric somatic hybrid between N. tabacum and N. glauca

Abstract

By using the protoplast fusion technique, we have obtained 44 regenerated plants, phenotypically different and distinct from their parents, among which we have identified a fertile symmetric somatic hybrid, designated as TG-32, between N. tabacum var. Gexin No.1 and N. glauca. The morphology, fertility, chromosome number and nuclear constitution of the somatic hybrid have been studied in detail. Unlike other asymmetric interspecific somatic hybrids, the chromosome number of the symmetric somatic hybrid is 72, equal to the sum of chromosomes of both parents. The TG-32 plant has flowers similar to those of N. tabacum, but with petals similar to those of N. glauca. Interestingly the offspring of TG-32 vary in seed production ability with temperature, and produce more seeds under a relatively low temperature. Two SCAR markers were used to evaluate genetic variability and structure. The hybrid amplified the expected fragment, but the parents showed only one of two markers. This experimental result supports the hypothesis of the co-existence of two parental genomes in the somatic hybrid.     

Production and cytogenetics of intergeneric hybrids between Brassica napus and Orychophragmus violaceus

The intergeneric hybrid between Brassica napus and Orychophragmus violaceus was obtained by means of embryo culture technique with the latter as the pollen parent. The hybrid was morphologically intermediate between its parents, but could produce a lot of seeds when selfed. Somatic separation of the genomes from the two parental species was observed during the mitotic divisions of some of the hybrid cells. Thus, the hybrid became the mixoploid in nature, consisting of haploid and diploid cells of B. napus, and a nuclear — cytoplasmic hybrid, with the cytoplasm of B. napus and the nuclei of O. violaceus, and the hybrid cells. Pollen mother cells with 19, 12 and 6 bivalents, respectively, were produced by the hybrid. From the selfed progeny of the hybrid, mainly two kinds of plants, B. napus and the hybrid, were found. The hybrid plants of the selfed progeny again produced two kinds of plants, B. napus and the hybrid.     

In vitro multiplication of a sterile interspecific hybrid,Brassica fruticulosa x B. campestris

In vitro methods for plant multiplication of a sterile interspecific hybrid between Brassica fruticulosa and B. campestris through either micropropagation or callus regeneration is described. Shoot-tip, single-node and leaf explants, obtained from in vitro-grown hybrids, regenerated on media containing NAA and BA. In vitro application of colchicine induced chromosome doubling in in vitro-regenerated shoots resulting in the production of fertile amphidiploids. Comparative studies on regeneration potential of the hybrid and its parents were also carried out using callus from leaf explants. The explants of B. fruticulosa and the hybrid were capable of shoot and root formation while those of B. campestris failed to form shoots but produced profuse roots. The results demonstrate the efficacy of an in vitro method in producing a large number of hybrid plants and fertile amphidiploids from incompatible crosses that yield very few hybrid seeds/seedlings.Abbreviations BA benzyladenine - CMS cytoplasmic male sterile - AA diploid genome of B. campestris - FF diploid genome of B. fruticulosa - NAA -naphthaleneacetic acid     

Interspecific hybridization of plants and resistance to herbivores: hypotheses,genetics, and variable responses in a diverse herbivore community

We studied the morphology, molecular genetics, and hebivory of two species of willows (Salix sericea and S. eriocephala) and their interspecific hybrids to test four alternative hypotheses concerning the effects of hybridization on plant resistance. Individually marked plants were identified using morphological traits in the field and measurements of stipule and leaf pubescence were made and compared using Canonical Discriminant Function Analysis. DNA was extracted from the leaves of a sample of the marked plants and RAPD-PCR analysis was performed to establish the genetic status of parental and hybrid plants. RAPD band analysis generally verified the genetic status of parental plants. Hybrid plants were usually correctly identified in the field with a few exceptions. However, the hybrid plants were a heterogeneous group of plants made up of most plants that appear to be F₁s and a few plants that appear to be backcrosses to S. sericea. Morphological variables were useful for distinguishing S. sericea from S. eriocephala and hybrids, but were not as dependable in distinguishing between S. eriocephala and hybrids. We compared the densities of 11 herbivore species and the infection by a leaf rust pathogen (Melampsora sp.) on the leaves and stems of two parents and the hybrids in the field. We found support for the Additive hypothesis (3 species), the Dominance hypothesis (2 species) and the Hybrid Susceptibility hypothesis (7 species, 6 herbivores and the Melampsora rust). We found no evidence for the Hybrid Resistance hypothesis. Guild membership was not a good predictor of similar responses of species to hybrid versus parental plants. A Canonical Discriminant Function Analysis showed discrete separation of the taxa based on herbivore densities, illustrating different community structures on hybrid and parental plants. This study demonstrates the diversity of responses of phytophages in response to interspecific hybridization.     

Production and characterization of somatic hybrid plants between leek (Allium ampeloprasum L.) and onion (Allium cepa L.)

 Results are reported on the production and characterization of somatic hybrids between Allium ampeloprasum and A. cepa. Both symmetric and asymmetric protoplast fusions were carried out using a polyethylene-based mass fusion protocol. Asymmetric fusions were performed using gamma ray-treated donor protoplasts of A. cepa and iodoacetamide-treated A. ampeloprasum protoplasts. However, the use of gamma irradiation to eliminate or inactivate the donor DNA of A. cepa proved to be detrimental to the development of fusion calli, and thus it was not possible to obtain hybrids from asymmetric fusions. The symmetric fusions yielded a high number of hybrid calli and regenerated plants. The analysis of the nuclear DNA composition using interspecific variation of rDNA revealed that most of the regenerated plants were hybrids. Flow cytometric analysis of nuclear DNA showed that these hybrid plants contained a lower DNA content than the sum of the DNA amounts of the parental species, suggesting that they were aneuploid. A shortage of chromosomes in the hybrids was confirmed by genomic in situ hybridization. Chromosome counts in metaphase cells of six hybrids revealed that these plants lacked 2–7 leek chromosomes. One hybrid showed also the loss of onion chromosomes. The hybrids had an intermediate phenotype in leaf morphology. The application of these somatic hybrids in breeding is discussed. Received: 7 April 1997 / Accepted: 10 September 1997     

Restriction endonuclease analysis of chloroplast DNA in interspecies somatic Hybrids of Petunia

Summary Restriction endonuclease cleavage pattern analysis of chloroplast DNA (cpDNA) of three different interspecific somatic hybrid plants revealed that the cytoplasms of the hybrids contained only cpDNA of P. parodii. The somatic hybrid plants analysed were those between P. parodii (wild type) + P. hybrida (wild type); P. parodii (wild type)+P. inflata (cytoplasmic albino mutant); P. parodii (wild type) + P. parviflora (nuclear albino mutant). The presence of only P. parodii chloroplasts in the somatic hybrid of P. parodii + P. inflata is possibly due to the stringent selection used for somatic hybrid production. However, in the case of the two other somatic hybrids P. parodii + P. hybrida and P. parodii + P. parviflora it was not possible to determine whether the presence of only P. parodii chloroplasts in these somatic hybrid plants was due to the nature of the selection schemes used or simply occurred by chance. The relevance of such somatic hybrid material for the study of genomic-cytoplasmic interaction is discussed, as well as the use of restriction endonuclease fragment patterns for the analysis of taxonomic and evolutionary inter-relationships in the genus Petunia.     

Interspecific and temporal variation in herbivore responses to hybrid willows

We studied herbivory of two species of willows (Salix sericea and S. eriocephala) and their interspecific hybrids to test alternative hypotheses concerning the effects of hybridization on plant resistance. Individually marked plants were identified using morphological traits in the field and random amplified polymorphic DNA (RAPD) band analysis was used to verify the genetic status of many parental and hybrid plants. The desities of 12 herbivore species on plants in the field were compared between two parents and their F₂-type hybrids. We found about equal support for the additive, dominance, and hybrid susceptibility hypotheses over 4 years. In one year, one species supported the hybrid resistance hypothesis. Guild membership was not a good predictor of similar responses of species to hybrid versus parental plants. There were marked differences in support for particular hypotheses among years for four herbivore species. This study demonstrates the diversity of responses of phytophages in response to interspecific hybridization, and indicates that year-to-year variation in relative resistance of hybrid plants can be important.     

Plant genetic differences influence herbivore community structure: evidence from a hybrid willow system

To determine the influence of plant genetic variation on community structure of insect herbivores, we examined the abundances of 14 herbivore species among six genetic classes of willow: Salix eriocephala, S. sericea, their F₁ and F₂ interspecific hybrids, and backcross hybrids to each parental species. We placed 1-year-old plants, grown from seeds generated from controlled crosses, in a common garden. During the growing season, we censused gall-inducing flies and sawflies, leaf-mining insects, and leaf-folding Lepidoptera to determine the community structure of herbivorous insects on the six genetic classes. Our results provided convincing evidence that the community structure of insect herbivores in this hybrid willow system was shaped by genetic differences among the parental species and the hybrid genetic classes. Using MANOVA, we detected significant differences among genetic classes for both absolute and relative abundance of herbivores. Using canonical discriminant analysis, we found that centroid locations describing community structure of the insect herbivores differed for each genetic class. Moreover, the centroids for the four hybrid classes were located well outside of the range between the centroids for the parental species, suggesting that more than additive genetic effects of the two parental species influenced community formation on hybrid classes. Line-cross analysis suggested that plant genetic factors responsible for structuring the herbivore community involved epistatic effects, as well as additive and dominance effects. We discuss the ramifications of these results in regard to the structure of insect herbivore communities on plants and the implications of our findings for the evolution of interspecific interactions.     

Somatic hybridization between male sterile Nicotiana tabacum and N. glutinosa through protoplast fusion

Summary Protoplasts derived from suspension cultured cells of cytoplasmic male sterile Nicotiana tabacum (N. debneyi cytoplasm) and of fertile N. glutinosa were fused with the aid of polyethylene glycol (PEG). Out of 1,089 colonies developed from PEG-treated protoplasts, 29 restored whole plants.A somatic hybrid plant was selected on the basis of isoelectrofocusing analysis of Fraction I protein in leaves of regenerated plants. A newly created hybrid contained small subunits of both parents but only a N. glutinosa type large subunit.Male sterile character was conserved in a hybrid plant while leaf morphology was intermediate between the parents. By tobacco mosaic virus infection tests, the hybrid's leaves showed resistant symptoms, hypersensitive local lesions, which were due to N. glutinosa nuclear genome expression.Abbreviations PEG Polyethylene glycol - TMV Tobacco mosaic virus     

BREEDING SYSTEM OF A HYBRID BETWEEN A SEXUAL AND AN APOMICTIC SPECIES OF AMELANCHIER,SHADBUSH (ROSACEAE,MALOIDEAE)

Amelanchier bartramiana, which has been shown to be sexual and self-incompatible, and A. laevis, in which apospory and self-compatibility occur, grow in a mixed population in western Maine. Also present there is the putative interspecific hybrid, A. × neglecta. Principal components and canonical discriminant analyses of seven morphological characters from 96 parental and putative hybrid individuals substantiate morphological intermediacy of the hybrids. A Nomarski differential interference microscopy study of megasporogenesis and megagametogenesis in five hybrid plants shows a predominantly aposporous mode of reproduction, and controlled pollinations indicate that seed formation between A. bartramiana and A. laevis is possible and that the hybrids are self-compatible. The hybrids, which are tetraploids (n = ca. 34) like both parents in Maine, produce viable pollen and seed, but there is little evidence for backcrossing with either parent.     

Age dependency of the meiotic process in an F<Subscript>1</Subscript> interspecific hybrid of <Emphasis Type="Italic">Solanum lnnaeum</Emphasis> L. × <Emphasis Type="Italic">Solanum incanum</Emphasis>

A study of the meiotic process in first- and second-year plants of an F₁ interspecific hybrid of Solanum linnaeum L. × Solanum incanum L. has been carried out. Fruits with seeds were obtained during the second year of vegetation. The numbers of univalents and tetravalents, as well as the percentage of basic impairments, decreased as the age of plants increased. The process of meiosis in the parental forms was normal. A conclusion was made regarding the connection between the regularity of meiosis in the F₁ heterozygotes of Solanum lynnaeum L. × Solanum incanum L., on the one hand, and the hybrid nature of the genotype and age and the influence of environmental factors, on the other hand.     

Mitochondrial DNA polymorphism induced by protoplast fusion in Cruciferae

Summary The mitochondrial genomes of five rapeseed somatic hybrid plants, which combine in a first experimentBrassica napus chloroplasts and a cytoplasmic male sterility trait coming fromRaphanus sativus, and in a second experiment chloroplasts of a triazine resistantB. compestris and a cytoplasmic male sterility trait fromR. sativus, were analyzed by restriction endonucleases. Restriction fragment patterns indicate that these genomes differ from each other and from both parents. The presence of new bands in the somatic hybrid mitochondrial DNA restriction patterns is evidence of mitochondrial recombination in somatic hybrid cells. In both parental and somatic hybrid plants large quantitative variations in a mitochondrial plasmid-like DNA have been observed. Our results suggest that the cytoplasmic support for male sterility is located in the chromosomal mitochondrial DNA instead of the plasmid-like DNA.     

Asymmetric somatic hybrid plants between an interspecific Lycopersicon hybrid and Solanum melongena

Summary Asymmetric somatic hybrid plants were obtained by a modified PEG/DMSO fusion procedure between protoplasts derived from suspension cells of an interspecific tomato hybrid, Lycopersicon esculentum x L. pennellii, and mesophyll protoplasts of Solanum melongena, eggplant. The tomato hybrid was previously transformed with Agrobacterium tumefaciens and contained the kanamycin-resistance marker gene. Prior to fusion, the donor protoplasts of the tomato hybrid were gamma irradiated at 9.0 krad. Thus, non-division of irradiated tomato hybrid protoplasts coupled with kanamycin sensitivity of eggplant enabled selection of somatic cell hybrids. Forty-nine calli selected post-fusion regenerated leaf-like structures in the presence of 50 mg/l kanamycin. However, only four of the 49 calli regenerated intact shoots which rooted in the presence of 50 mg/l kanamycin and were later transferred to the greenhouse. Analysis of phosphoglucoisomerase and peroxidase isozymes, and Southern hybridization with a nuclear-specific pea 45 S ribosomal RNA gene confirmed somatic hybrid status. Cytology revealed that the four hybrid plants had chromosome numbers of 45, 60, 42 and 57, respectively; they were all sterile.     

Hybrid swarms: catalysts for multiple evolutionary events in Senecio in the British Isles

Background: Introgressive hybridisation is an evolutionary catalyst producing novel variants able to explore new ecological niches and evolve as new hybrid taxa. However, the role of ‘hybrid swarms’ – highly variable populations produced following interspecific hybridisation – in generating this evolutionary novelty has been poorly studied.

Aims: We examine the alternative origins of tetraploid hybrid derivatives of Senecio vulgaris and S. squalidus, via local polytopic formation or long-distance dispersal from a single perennial hybrid swarm around Cork, Ireland.

Methods: Morphometric, isozyme and chloroplast DNA analysis.

Results: The Cork hybrid swarm and UK hybrid swarms exhibited a broad range of morphological variation and contained individuals similar to the stable tetraploid hybrid derivatives; S. eboracensis and S. vulgaris var. hibernicus. Chloroplast DNA analysis shows that S. eboracensis did not evolve from the Cork hybrid swarm. However, UK S. vulgaris var. hibernicus populations exhibit a broad range of variation for both chloroplast and isozyme markers, but were not distinguishable from Cork material.

Conclusions: Our study confirms that S. eboracensis did not evolve from the Cork hybrid swarm, and while our analyses could not demonstrate this conclusively for S. vulgaris var. hibernicus the ease with which hybrid swarms have been generated in the past makes a polytopic origin for S. vulgaris var. hibernicus the most likely scenario.     

PATTERNS OF MATING IN WILD SUNFLOWER HYBRID ZONES

Theory predicts that homoploid hybrid speciation will be facilitated by selfing, yet most well-documented hybrid species are outcrossers. One possible explanation for this puzzle is that conditions in hybrid populations may favor selfing, even in otherwise outcrossing species. For example, in self-incompatible plants, mixtures of self and interspecific pollen often induce selfing. Here, we examine patterns of mating in three hybrid zones and four “pure” populations of Helianthus annuus and H. petiolaris, wild, self-incompatible sunflower species that are thought to have parented three homoploid hybrid species. Fourteen to 16 maternal families from each pure population and 44–46 maternal families from each hybrid zone were analyzed for seven polymorphic isozyme loci. Maximum-likelihood (ML) methods were used to estimate multilocus outcrossing rates (T_m) and hybridization frequencies for each maternal family, each phenotypic group within each hybrid zone (annuus-like, hybrid, and petiolaris-like), and each population. As predicted for self-incompatible species, all four parental populations have outcrossing rate ML estimates of 1.0. Within the hybrid zones, outcrossing rates were lowest in the H. annuus–like fraction of the population (0.73, 0.72, and 0.74 in the three hybrid zones, respectively), largely intermediate in the H. petiolaris–like group (0.94, 0.90, and 0.94), and highest in the hybrid group (0.97, 0.93, and 0.97). Although outcrossing rates are lower in hybrid zones than in pure populations, it is unlikely that the observed decrease facilitates hybrid speciation because outcrossing rates in the critical hybrid fraction of the population do not differ significantly from 1.0. Dividing the outcrossed pollen pool into intraspecific and interspecific components revealed that maternal plants are largely fertilized by conspecific pollen, confirming an important role for pollen competition as a reproductive barrier. Highly sterile hybrid plants do not appear to discriminate between parental species pollen, but hybrids with higher fertility tend to be fertilized by pollen from the parental group they resemble genetically. Thus, gametic selection leads to substantial assortative mating in these hybrid zones.     

Somatic hybridisation of Daucus carota and D. capillifolius by protoplast fusion

Summary Protoplasts isolated from cultured cells of albino carrot (Daucus carota) and normal green D. capillifolius were fused by polyethylene glycol. Selection of somatic hybrid plants was based on the restoration of photosynthetic function in hybrids. Green plantlets selected from embryo cultures were characterized on the basis of leaf morphology. The interspecific protoplast fusion resulted in green plants with leaves which were intermediate between those of the parents. The somatic hybrids between orange rooted carrot variety and D. capillifolius with long white roots produced long, white and fleshy roots. The cytological analysis of parasexual hybrids revealed that the chromosome number ranged from 34 to 54. The most frequent chromosome number was 2n = 36. Hybrids were also found with 34 and 35 chromosomes. The somatic hybrid showed the same isoenzyme pattern of leaf peroxidase as D. carota.     

The effect of B chromosomes on homoeologous pairing in species hybrids

Six diploid populations of Lolium rigidum were used in crosses with a standard homozygous line of Lolium temulentum in order to analyse the variation in the effect of B chromosomes on homoeologous pairing in the interspecific hybrid. — Large differences in chiasma frequency were found, both between the progeny of the different populations and, in one case, between the progeny of different plants from the same population. That this variation was due to differences in B chromosome genotype was ruled out by the use of appropriate 1B Lolium rigidum parents. It is concluded therefore that the effect of B's on homoeologous pairing in this interspecific hybrid can be modified quite substantially by the A chromosome genotype of the parents.     

Overexpression of NlgCycB Isolated from Interspecific Hybrid of N. langsdorffii?��?N. glauca Alters Root Growth and Root Hair Development

A cDNA clone encoding cyclin B (NlgCycB) was isolated from an interspecific hybrid of N. langsdorffii × N. glauca. The full-length cDNA had an open reading frame of 1,422 bp, with a deduced amino acid sequence of 473 residues. To investigate the role of NlgCycB, transgenic tobacco plants were constructed to overexpress NlgCycB. The transgenic plants showed premature root hair development in the elongation zone of the root tips. They had more and longer root hairs, which contained nuclei of increased size. Flow cytometric analysis revealed that the proportion of cells with 4C DNA content was increased in the transgenic plants. The NlgCycB gene in transgenic plants was coordinately enhanced with the expression of CDKA genes, especially in the root tips. The transgenic plants had greater growth than the wild-type in MS (1/10-strength) medium with reduced salt concentrations. Taken together, NlgCycB may play an important role in premature root hair development in the elongation zone of root tips and can aid in adaptation to low nutrient salt environments.     

Hybrid dwarfness in crosses between wheat (Triticum aestivum L.) and rye (Secale cereale L.): a new look at an old phenomenon

The existence of hybrid dwarfs from intraspecific crosses in wheat (Triticum aestivum) was described 100 years ago, and the genetics underlying hybrid dwarfness are well understood. In this study, we report a dwarf phenotype in interspecific hybrids between wheat and rye (Secale cereale). We identified two rye lines that produce hybrid dwarfs with wheat and have none of the hitherto known hybrid dwarfing genes. Genetic analyses revealed that both rye lines carry a single allelic gene responsible for the dwarf phenotype. This gene was designated Hdw‐R1 (Hybrid dwarf‐R1). Application of gibberellic acid (GA₃) to both intraspecific (wheat–wheat) and interspecific (wheat–rye) hybrids showed that hybrid dwarfness cannot be overcome by treatment with this phytohormone. Histological analysis of shoot apices showed that wheat–rye hybrids with the dwarf phenotype at 21 and 45 days after germination failed to develop further. Shoot apices of dwarf plants did not elongate, did not form new primordia and had a dome‐shaped appearance in the seed. The possible relationship between hybrid dwarfness and the genes responsible for the transition from vegetative to generative growth stage is discussed.