Cytogenetic study of an interspecific cross of Nicotiana debneyi X N. umbratica

Summary Interspecific F₁ hybrids of Nicotiana debneyi Domin (2n=48) and N. umbratica Burbidge (2n=46), both belonging to the section Suaveolentes, showed a high degree of meiotic chromosome pairing. Two of the five F₂ plants obtained exhibited chromosome mosaicism. The first colchiploid generation (C₁) had the expected chromosome number of 2n=94 while C₂ showed 2n=88, a loss of three pairs of chromosomes. This same chromosome number continued in further colchiploid generations, followed up to C₅, except for a few plants in C₃ which showed chromosome mosaicism. The F₁ phenotype was stable through C₁ to C₅ and fertility was normal in colchiploids through all generations in spite of the loss of three pairs of chromosomes and chromosome mosaicism. This stability and fertility apparently reflect the tolerance of the genomes to the genetic adjustment of chromosome complements which is believed to be associated with the originally polyploid nature of the parental species and the chromosome doubling brought about in the amphidiploids.     

Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids

Genomic in situ hybridization (GISH) methods were used to detect different genome components within Brassica amphidiploid species and to identify donor chromatin in hybrids between Brassica napus and Raphanus sativus. In Brassica juncea and Brassica carinata the respective diploid donor genomes could be reliably distinguished by GISH, as could all R-genome chromosomes in the intergeneric hybrids. The A- and C-genome components in B. napus could not be clearly distinguished from one another using GISH, confirming the considerable homoeology between these genomes. GISH methods will be extremely beneficial for monitoring chromatin transfer and introgression in interspecific Brassica hybrids. Received: 20 May 1997 / Accepted: 28 July 1997     

Streptomycin resistant and sensitive somatic hybrids of Nicotiana tabacum + Nicotiana knightiana: correlation of resistance to N. tabacum plastids

Summary Protoplasts of Nicotiana tabacum SRI (streptomycin resistant) and of Nicotiana knightiana (streptomycin sensitive) were fused using polyethylene glycol treatment. From three heterokaryons 500 clones were obtained. From the 43 which were further investigated, 6 resistant, 3 sensitive, and 34 chimeric (consisting of resistant and sensitive sectors) calli were found. From eight clones, a total of 39 plants were regenerated and identified as somatic hybrids. Chloroplast type (N. tabacum = NT or N. knightiana = NK) in the plants was determined on the basis of the species specific EcoRI restriction pattern of the chloroplast DNA. Regenerates contained NT (13 plants) or NK (15 plants) plastids but only the plants with the NT chloroplasts were resistant to streptomycin. This finding and our earlier data on uniparental inheritance points to the chloroplasts as the carriers of the streptomycin resistance factor.     

A study of wing morphology and fluctuating asymmetry in interspecific hybrids between Drosophila buzzatii and D. koepferae

In this work we investigate the effect of interspecific hybridization on wing morphology using geometric morphometrics in the cactophilic sibling species D. buzzatii and D. koepferae. Wing morphology in F₁ hybrids exhibited an important degree of phenotypic plasticity and differs significantly from both parental species. However, the pattern of morphological variation between hybrids and the parental strains varied between wing size and wing shape, across rearing media, sexes, and crosses, suggesting a complex genetic architecture underlying divergence in wing morphology. Even though there was significant fluctuating asymmetry for both, wing size and shape in F₁ hybrids and both parental species, there was no evidence of an increased degree of fluctuating asymmetry in hybrids as compared to parental species. These results are interpreted in terms of developmental stability as a function of a balance between levels of heterozygosity and the disruption of coadaptation as an indirect consequence of genomic divergence.     

Analysis of fertility in somatic hybrids of Nicotiana rustica and N. tabacum and progeny over two sexual generations

Summary Somatic hybrid plants, produced between Nicotiana rustica and N. tabacum by heterokaryon isolation and culture and also by mutant complementation, were examined regarding their ability to set seed. From a total of seventeen independent somatic hybrids, three were found to be partially self-fertile while the others did not set seed. Differences regarding the methods of hybrid selection, parental varieties and chloroplast composition of hybrids did not appear to be significant regarding the ability of plants to set seed. Much variation in fertility was observed in subsequent generations and by recurrent selection of the most fertile, over two generations, it was possible to increase the level of self-fertility in some of the progeny. One R2 derivative possessed approximately a tenfold higher level of self-fertility than it's somatic hybrid parent. The presence of genetic markers from both parents were observed in all progeny indicating their hybrid nature.     

Rare symmetric and asymmetric Nicotiana tabacum (+) N. megalosiphon somatic hybrids recovered by selection for nuclear-encoded resistance genes and in the absence of genome inactivation

Following protoplast fusion between Nicotiana tabacum (dhfr) and N. megalosiphon (nptII) somatic hybrids were selected on the basis of dual resistance to kanamycin and methotrexate. Despite strong selection for parental nuclear-encoded resistances, only nine N. tabacum (+) N. megalosiphon somatic hybrids were obtained. A preferential loss of the parental N. tabacum nuclear and organelle genome was apparent in some plants in spite of the lack of genomic inactivation by the irradiation or chemical treatment of the parental protoplasts. Only six of the nine hybrids recovered possessed both parental profiles of nuclear RFLPs and isoenzymes. The remaining three hybrids were highly asymmetric with two being identical to N. megalosiphon except for minor morphological differences and rearranged or recombined mitochondrial DNAs (mtDNA), while the other one was distinguishable only by the presence of a rearranged or recombined mtDNA, and was therefore possibly a cybrid. Overall, eight somatic hybrids possessed rearranged or recombined mtDNAs and chloroplast inheritance was non-random since eight possessed N. megalosiphon-type chloroplasts and only one had N. tabacum chloroplasts. In contrast, using the same selection approach, numerous morphologically similar symmetric somatic hybrids with nuclear RFLPs and isozymes of both the parental species were recovered from control fusions between N. tabacum and the more closely related N. sylvestris. In spite of the low frequency of recovery of symmetric N. tabacum (+) N. megalosiphon hybrids in this study, one of these hybrids displayed a significant degree of self-fertility allowing for back-crosses to transfer N. megalosiphon disease-resistance traits to N. tabacum. Plant Research Centre Contribution No. 1579     

Random chloroplast segregation and frequent mtDNA rearrangements in fertile somatic hybrids between Nicotiana tabacum L. and N. glutinosa L.

Patterns of organelle inheritance were examined among fertile somatic hybrids between allotetraploid Nicotiana tabacum L. (2n=4x=48) and a diploid wild relative N. glutinosa L. (2n=2x=24). Seventy somatic hybrids resistant to methotrexate and kanamycin were recovered following fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistant N. tabacum and kanamycin-resistant N. glutinosa. Evidence for hybridization of nuclear genomes was obtained by analysis of glutamate oxaloacetate transaminase and peroxidase isoenzymes and by restriction fragment length polymorphism (RFLP) analysis using a heterologous nuclear ribosomal DNA probe. Analysis of chloroplast genomes in a population of 41 hybrids revealed a random segregation of chloroplasts since 25 possessed N. glutinosa chloroplasts and 16 possessed N. tabacum chloroplasts. This contrasts with the markedly non-random segregation of plastids in N. tabacum (+)N. rustica and N. tabacum (+) N. debneyi somatic hybrids which we described previously and which were recovered using the same conditions for fusion and selection. The organization of the mitochondrial DNA (mtDNA) in 40 individuals was examined by RFLP analysis with a heterologous cytochrome B gene. Thirty-eight somatic hybrids possessed mitochondrial genomes which were rearranged with respect to the parental genomes, two carried mtDNA similar to N. tabacum, while none had mtDNA identical to N. glutinosa. The somatic hybrids were self-fertile and fertile in backcrosses with the tobacco parent.Contribution No. 1487 Plant Research Centre     

Dwarfism and male sterility in interspecific hybrids of Epilobium

Summary Reciprocal differences for male sterility, dwarfism and morphological traits have been studied in intra- and interspecific crosses of five Epilobium species. Male sterility occurred in two interspecific hybrids with E. montanum as the male parent while dwarfism has been found to varying degrees in three interspecific crosses with E. watsonni. In contrast to transient differences in plant height and leaf morphology in reciprocal hybrids of the cross between E. hirsutum and E. parviflorum, male sterility and dwarfism persistently occur as reciprocally different traits which may be influenced by determinants of the cytoplasm. The molecular characterization of the plastid DNA of the parental lines and the F₁ hybrids indicate that the plastome of male sterile and dwarf plants is identical to that of the female parents. Furthermore, in spite of these developmental disturbances, the expression of plastid genes coding for polypeptides of thylakoid-membrane complexes is unchanged. Thus, it seems unlikely that the genetic compartement of the plastids is responsible for the expression of the male sterile or the dwarfed phenotype.     

Morphology and cytology of Hordeum chilense X H. bulbosum hybrids

Summary An interspecific hybrid between Hordeum chilense and H. bulbosum was produced. The hybrid resembles the male parent, but some characters from H. chilense are also present. Transgressive inheritance for other characters has also been observed. Neither chromosome instability nor homoeologous pairing was found.     

Elimination and duplication of particular Hordeum vulgare chromosomes in aneuploid interspecific Hordeum hybrids

Summary Seeds formed in crosses Hordeum lechleri (6x) x H. vulgare (2x and 4x), H. arizonicum (6x) x H. v. (2x), H. parodii (6x) x H. v. (2x), and H. tetraploidum (4x) x H. v. (2x) produced plants at high or rather high frequencies through embryo rescue. Giemsa C-banding patterns were used to analyze chromosomal constitutions and chromosomal locations on the methaphase plate. Among 100 plants obtained from H. vulgare (2x) crosses, 32 plants were aneuploid with 2n=29 (1), 28 (3), 27 (13), 26 (5), 25 (4), 24 (4), or 22 (2); 50 were euploid (12 analyzed), and 18 were polyhaploid (5 analyzed). Four plants had two sectors differing in chromosome number. Two of four hybrids with H. vulgare (4x) were euploid and two were aneuploid. Parental genomes were concentrically arranged with that of H. vulgare always found closest to the metaphase centre. Many plants showed a certain level of intraplant variation in chromosome numbers. Except for one H. vulgare (4x) hybrids, this variation was restricted to peripherally located non-H. vulgare genomes. This may reflect a less firm attachment of the chromosomes from these genomes to the spindle. Interplant variation in chromosome numbers was due to the permanent elimination or, far less common, duplication of the centrally located H. vulgare chromosomes in all 34 aneuploids, and in a few also to loss/gain of non-H, vulgare chromosomes. This selective elimination of chromosomes of the centrally located genome contrasts conditions found in diploid interspecific hybrids, which eliminate the peripherally located genome. The difference is attributed to changed genomic ratios. Derivatives of various H. vulgare lines were differently distributed among euploid hybrids, aneuploids, and polyhaploids. Chromosomal constitutions of hypoploid hybrids revealed a preferential elimination of H. vulgare chromosomes 1, 5, 6, and 7, but did not support the idea that H. vulgare chromosomes should be lost in a specific order. H. vulgare SAT-chromosomes 6 and 7 showed nucleolar dominance. Aneuploidy is ascribed to the same chromosome elimination mechanism that produces haploids in cross-combinations with H. vulgare (2x). The findings have implications for the utilization of interspecific Hordeum hybrids.     

Inheritance of morphological and chemical characters in interspecific hybrids between Papaver bracteatum and Papaver pseudo-orientale

Summary The alkaloid profiles and morphological traits of the capsules of Papaver bracteatum, P. pseudo-orientale, and their hybrids were studied. Dominance of the hexaploid parent P. pseudo-orientale was observed for various characters. A genetic model assuming allelic additive effects and polysomic inheritance was elaborated for the control of isothebaine content in the capsules. The distribution of thebaine content in the segregating generations, F₂ and BCF₁ was evidence of the transfer of genes from the diploid parent P. bracteatum in the gametes of the interspecific hybrid and their expression in its progenies. These findings indicate the potential use of inter-specific hybrids between the Oxytona species in the breeding of cultivars for industrial or ornamental purposes.Contribution No. 3066-E, 1990 series from The Agricultural Research Organization, The Volcani Center, Bet Dagan 50 250, Israel     

In vitro pollen germination of species and two interspecific hybrids from the genus Brassica

In vitro pollen germination of five species and two interspecific hybrids from the genus Brassica was tested in four media. Genetically fixed differences in the demands for optimal pollen germination among species were found. The experiments were designed to define optimal content of mineral salts, sugar, and PEG for every investigated species or hybrids. The differences found among species are discussed in relation to the evolutionary trend.     

Inheritance of freezing resistance in interspecific F1 hybrids of Eucalyptus

Summary The inheritance of freezing resistance in interspecific F₁ hybrid families of Eucalyptus encompassing 27 different species combinations and a range of levels of hardiness was examined. Freezing resistance was assessed by determining the temperatures required to cause either 30% (T30), 40% (T40), or 50% (T50) leakage of electrolytes from excised leaf discs subjected to artificial freezing. Highly significant variation in freezing resistance occurred between species; the maximum difference between parents in any specific combination was over 9°C (E. gunnii x E. globulus). Freezing resistance was inherited in a predominantly additive manner in interspecific hybrids, although there was a tendency towards partial dominance toward the more sensitive species in some combinations (e.g., E. nitens x E. Globulus, E. nitens x E. camaldulensis, E. gunnii x E. globulus). The full expression of this genetic variation appeared to increase with hardiness and in some cases appeared to vary with ontogeny. Estimates of individual narrow-sense heritability of freezing resistance for pure E. nitens families were h ² = 0.66±0.44 and 0.46±0.44. Across all species combinations examined, the heritability of F₁ family means estimated from midparent regression was h ² = 0.76±0.06 and h ² = 0.89±0.06 for T40 and T50 values, respectively. The advantage of using selected parents for interspecific hybridization is demonstrated and the implications of these results for breeding for freezing resistance in Eucalyptus are discussed.     

The fate of recombinant chromosomes and genome interaction in Nicotiana asymmetric somatic hybrids and their sexual progeny

Genomic in-situ hybridization (GISH) was used to monitor the behaviour of parental genomes, and the fate of intergenomic chromosome translocations, through meiosis of plants regenerated from asymmetric somatic hybrids between Nicotiana sylvestris and N. plumbaginifolia. Meiotic pairing in the regenerants was exclusively between chromosomes or chromosome segments derived from the same species. Translocation (recombinant) chromosomes contained chromosome segments from both parental species, and were detected at all stages of meiosis. They occasionally paired with respectively homologous segments of N. sylvestris or N. plumbaginifolia chromosomes. Within hybrid nuclei, the meiotic division of N. plumbaginifolia lagged behind that of N. sylvestris. However, normal and recombinant chromosomes were eventually incorporated into dyads and tetrads, and the regenerants were partially pollen fertile. Recombinant chromosomes were transmitted through either male or female gametes, and were detected by GISH in sexual progeny obtained on selfing or backcrossing the regenerants to N. sylvestris. A new recombinant chromosome in one plant of the first backcross generation provided evidence of further chromosome rearrangements occurring at, or following, meiosis in the original regenerants. This study demonstrates the stable incorporation of chromosome segments from one parental genome of an asymmetric somatic hybrid into another, via intergenomic translocation, and reveals their transmission to subsequent sexual progeny.     

Characterization of interspecific hybrids betweenOrchis mascula andO. pallens (Orchidaceae) by enzyme electrophoresis

The European orchidsOrchis mascula, O. pallens and their hybrids have been analysed by enzyme electrophoresis on starch gels. The two species differ in the electrophoretic mobilities of four out of eight enzymes tested. Three enzymes, phosphoglucomutase, phosphoglucoisomerase and malic enzyme exhibit typical heterozygote patterns in the hybrid plants demonstrating the presence of both differing parental alleles. Thus, species identification is easy by the electrophoretic analysis of a low number of enzyme loci, and hybrids are detectable even if morphological characters fail.     

New hybrids of Hordeum parodii with Hordeum vulgare,H. bogdanii,Agropyron caninum and X Triticosecale

Summary Hybrids were produced from crossing Hordeum vulgare, H. bogdanii, Agropyron caninum and × Triticosecale onto H. parodii (6x or 4x). The rates at which hybrids were produced, expressed in terms of plantlet establishment as percent pollinated florets, ranged from 0.47%, (6x H.parodii × 6x × Triticosecale cv. Welsh) to 6.3% (4x H. parodii × 2x H. vulgare cv. Betzes). Based on frequencies of paired configurations at MI, autosyndetic pairing appeared to be promoted by the presence of a Secale cereale genome but suppressed by the genome of H. vulgare.Contribution No. 759 Ottawa Research Station     

Chloroplast DNA assorts randomly in intraspecific somatic hybrids of Nicotiana debneyi

Summary Plants were regenerated following intraspecific fusion of leaf protoplasts from two naturally occurring genotypes of Nicotiana debneyi. The two genotypes differed in the EcoRl fragmentation pattern of chloroplast DNA and in the nuclear-coded phosphoglucomutase (Pgm) isozymes. There was no conscious selection for hybrid genotypes during protoplast culture or plant regeneration. Among 225 plants screened for Pgm, six were identified as nuclear hybrids. Restriction endonuclease and filter hybridisation analysis revealed that the cytoplasms of the hybrids contained one or other but never both parental chloroplast DNAs. The sorting out of chloroplasts was random and complete; the limit of detecting a rare chloroplast-DNA type in a mixture was 0.1%.     

Karyotype analysis of regenerated plants from callus cultures of interspecific hybrids of cultivated barley (Hordeum vulgare L.)

Summary The karyotype of 82 regenerated plants from callus cultures of interspecific hybrids between cultivated barley (Hordeum vulgare L.) and seven polyploid wild barley species was examined by C-banding or Feulgen staining. The karyotypic changes observed in 46 plants included aneuploidy, double haploidy, amphidiploidy, deletions, inversions, extra C-bands, and extra euchromatic segments. Apparently, chromosome 5, 6, and 7 of H. vulgare were more frequently exposed to elimination or structural change than the other chromosomes of this species. Irradiation of calli seemed to enhance the occurrence of karyotypic variants.     

Production and viability of unreduced gametes in triploid interspecific blueberry hybrids

Summary Three triploid (2n=3x=36) blueberry hybrids were obtained by hand-pollinating approximately 7,000 flowers of tetraploid highbush blueberry cultivars (based on Vaccinium corymbosum L.) with pollen from the diploid species V. elliottii Chapm. Meiotic analysis of these triploids revealed trivalents, bivalents and univalents in all metaphase I cells, with lagging chromosomes evident at anaphase I. Pollen of the three triploids was mostly aborted and did not stain with acetocarmine. However, the three triploids did produce from 0.9%–1.3% giant pollen grains that stained with acetocarmine and were present as monads, dyads or triads, rather than the normal tetrads. Pollination of 10,853 flowers of hexaploid V. ashei Reade cultivars with pollen from the triploids produced 266 berries, which averaged fewer than two fully-developed seeds per berry. One triploid clone showed partial female fertility when crossed to hexaploids, self-pollinated, or intercrossed with other triploids. Ploidy levels of the resulting hybrids were determined.Florida Agricultural Experiment Station Journal Series No. 8672