GISH confirmation of somatic hybrids between Solanum melongena and S. torvum: assessment of resistance to both fungal and bacterial wilts

Interspecific somatic hybrids between Solanum melongena L. (2n = 2x = 24) and two accessions of Solanum torvum Sw. (2n = 2x = 24) were produced in view of transferring resistance to two soil-born pathogens, Ralstonia solanacearum and Verticillium dahliae, from the wild species into the cultivated eggplant. All somatic hybrids were phenotypically homogenous and intermediate between the parents. Their hybrid nature was confirmed by analysis of isozymes and RAPDs. They showed reduced pollen viability, and all but one possessed the chloroplasts from either one or the other parent. As S. melongena and S. torvum chromosomes were morphologically indistinguishable, genomic in situ hybridisation (GISH) was applied to recognise the chromosomes from each parent in the hybrids. As expected, the selected tetraploid plants contained one complete set of chromosomes from each fusion partner. On spread preparations, the two parental genomes were not spatially separated at any time of the cell cycle. Translocation or recombinant chromosomes could not be demonstrated in the mitotic metaphase. Tests for resistance performed in vitro by using suspensions of two strains of R. solanacearum (race 1 and 3) and filtrate of culture medium of one strain of V. dahliae, revealed that S. melongena was susceptible, whereas both accessions of S. torvum had high levels of resistance. Except for two hybrid clones, which were found susceptible to race 3, as was S. melongena, all somatic hybrids tested showed good levels of bacterial and fungal resistance, either intermediate or as high as that of the wild parent.     

Cytogenetic analysis of Lycopersicon esculentum (+) Solanum etuberosum somatic hybrids and their androgenetic regenerants

The aim of the study was to characterize genomic relationships among cultivated tomato (Lycopersicon esculentum Mill.) (2n=2x=24) and diploid (2n=2x=24) non-tuberous wild Solanum species (S. etuberosum Lindl.). Using genomic in situ hybridization (GISH) of mitotic and meiotic chromosomes, we analyzed intergeneric somatic hybrids between tomato and S. etuberosum. Of the five somatic hybrids, two plants were amphidiploids (2n=4x=48) mostly forming intragenomic bivalents in their microsporocytes, with a very low frequency of multivalents involving the chromosomes of tomato and S. etuberosum (less than 0.2 per meiocyte). Tomato chromosomes showed preferential elimination during subsequent meiotic divisions of the amphidiploids. Transmission of the parental chromosomes into microspores was also evaluated by GISH analysis of androgenic plants produced by direct embryogenesis from the amphidiploid somatic hybrids. Of the four androgenic regenerants, three were diploids (2n=2x=24 or 2n=2x+1=25) derived from reduced male gametes of the somatic hybrids, and one plant was a hypertetraploid (2n=4x+4=52). GISH revealed that each anther-derived plant had a unique chromosome composition. The prospects for introgression of desirable traits from S. etuberosum into the gene pool of cultivated tomato are discussed. Received: 2 August 2000 / Accepted: 4 December 2000     

A novel intergeneric hybrid in the Triticeae: Triticum aestivum x Psathyrost achys juncea

Summary Two hybrid embryos of intergeneric origin between Triticum aestivum cv Fukuho (2n=6x=42, AABBDD) and Psathyrostachys juncea (2n=2x=14, NN) were successfully rescued. One hybrid plant had the expected chromosome number of 28 (ABDN), whereas the second plant had 35 chromosomes. The average meiotic chromosome pairing in the 35-chromosome hybrid was 21.87 univalents + 6.38 bivalents + 0.11 trivalents + 0.009 quadrivalents, which indicates that two copies of the N genome were present. Chromosome pairing in the 28-chromosome hybrid was low (1.35 bivalents), and pointed out the lack of homology between the wheat genomes and the P. juncea genome. These new hybrids showed some necrosis and chlorosis, which caused severe floral abortion in the plant that had 35 chromosomes. These problems became gradually less severe after 18 months.Contrib. no. 372     

Production and characterization of somatic hybrids between Solanum melongena L. and S. sisymbriifolium Lam.

Summary Protoplasts of 6-azauracil (AU) resistant cell lines of Solanum melongena L. were fused with protoplasts of S. sisymbriifolium Lam. to create somatic hybrids between these sexually-incompatible species. Following fusion, colonies were selected which were capable of growth in medium containing 1mM AU. These colonies were placed on medium containing zeatin which had been shown to stimulate anthocyanin production during shoot organogenesis in tissue explants of S. sisymbriifolium but not in S. melongena. A total of 37 anthocyanin-producing colonies were identified from which 26 hybrid plants were regenerated. The morphological traits intermediate to those of the parents included: flower colour, leaf shape, and trichome density. Cytogenetic analysis revealed that all hybrids were aneuploids but their chromosome numbers were close to the expected number of 48. Isozyme analysis revealed that nuclear genes of both parents were expressed in the hybrids. In addition, isoelectric focussing of the large subunit of ribulose 1,5-bisphosphate carboxylase (Rubisco) provided evidence that each hybrid expressed only the S. sisymbriifolium chloroplast genome. All hybrids regenerated thus far have been sterile.Contribution No. 787 Ottawa Research Station     

Genetic recombination in Sorghum bicolor x S. macrospermum interspecific hybrids

Sorghum has been improved by public and private breeding programs utilizing germplasm mostly from within the species Sorghum bicolor. Until recently, cross-incompatibilities have prevented hybridization of S. bicolor with most other species within the genus Sorghum. Utilizing germplasm homozygous for the iap allele, hybrids were readily produced between S. bicolor (2n = 20; AAB1B1) and S. macrospermum (2n = 40; WWXXYYZZ). These hybrids were intermediate to the parents in chromosome number (2n = 30) and overall morphology. Meiosis in both parents was regular; S. bicolor had 10 bivalents per pollen mother cell (PMC) and S. macrospermum had an average of 19.96 bivalents per PMC. Six hybrids were studied cytologically and meiosis was irregular, with the chromosomes associating primarily as univalents and bivalents. There was an average of 3.54 bivalents per PMC, with a range of 0-8 bivalents, most of which were rods (98%). Using FISH (fluorescent in situ hybridization), moderate levels (2.6 II per PMC) of allosyndetic recombination were observed. Genomic relationships were sufficient to assign S. macrospermum the genomic formula AAB1B1YYZZ (Y and Z remain unknown). Allosyndetic recombination in the interspecific hybrids indicates that introgression through genetic recombination should be possible if viable backcrosses can be recovered.     

Chromosomal interchange inEleutherine plicata Herb

Chromosome behaviour at metaphase I and anaphase I of meiosis inEleutherine plicata Herb. (2n=14) is studied. Cells with chromosome associations comprising an association of four long chromosomes, in addition to five bivalents were observed more frequently than those with seven bivalents. it is concluded that the ring of four is due to a segmental interchange between the two long non-homologous chromosome pairs. The ring of four at anaphase I showed delayed disjunction, bridge formation and irregular separation of chromosomes in a number of cells while the behaviour of the other bivalents was normal.     

Production and characterization of intertribal somatic hybrids of Raphanus sativus and Brassica rapa with dye and medicinal plant Isatis indigotica

Intertribal somatic hybrids of Raphanus sativus (2n = 18, RR) and Brassica rapa spp. chinensis (2n = 20, AA) with the dye and medicinal plant Isatis indigotica (2n = 14, I I) were firstly obtained by polyethylene glycol-induced symmetric fusions of mesophyll protoplasts. One mature hybrid with R. sativus established in field had intermediate morphology but was totally sterile. It had the expected chromosome number (2n = 32, RRI I) and parental chromosomes were distinguished by genomic in situ hybridization (GISH) analysis, and these chromosomes were paired as 16 bivalents in pollen mother cells (PMCs) at diakinesis and mainly segregated equally as 16:16 at anaphase I (A I), but the meiotic disturbance in second division was obvious. Five mature hybrids with B. rapa established in field were morphologically intermediate but showed some differences in phenotypic traits and fertility, two were partially fertile. Cytological and GISH investigations revealed that these hybrids had 2n = 48 with AAIIII complement and their PMCs showed normal pairing of 24 bivalents and mainly equal segregation 24:24, but meiotic abnormalities of lagging chromosomes and micronuclei appeared frequently during second divisions. AFLP analysis showed that all of these hybrids had mainly the DNA banding pattern from the addition of two parents plus some alterations. Some hybrids should be used for the genetic improvement of crops and the dye and medicinal plant.     

Production of asymmetric hybrids between Solanum tuberosum and irradiated S. brevidens

Summary Asymmetric somatic hybrids were obtained by fusion of Solanum tuberosum (PDH40) protoplasts with 300- or 500-Gy irradiated protoplasts of S. brevidens. These radiation doses were sufficient to prevent the growth of the S. brevidens protoplasts. Putative hybrids were selected on the basis of phenotype from regenerated shoots and identified with a S. brevidens-specific probe. From these, 31 asymmetric hybrids were confirmed by morphological characteristics, isoenzyme patterns and RFLP analysis. The morphology of the asymmetric hybrids was intermediate between that of S. tuberosum and symmetric hybrids of both species (obtained without irradiation treatment). Chromosome counts from 17 asymmetric hybrids showed that the chromosome number of the hybrids ranged from 31 to 64. The asymmetric hybrids probably had one or two genome complements (i.e. either 24 or 48 chromosomes) from S. tuberosum and 7–22 chromosomes from S. brevidens. There was no clear correlation between the radiation dose and the degree of elimination of the S. brevidens genome.     

Elimination of Solanum phureja nucleolar chromosomes in S. tuberosum + S. phureja somatic hybrids

Summary The karyotype of the dihaploid SVP1 line of S. tuberosum (2n=2x=24) showed two nucleolar chromosomes with differently sized satellites. The diploid SVP5 line (2n=2x=24) and tetraploid regenerants of S. phureja had larger but similar satellites. Somatic hybrids between the diploid lines of these potato species with genome combinations 4 tub + 2 ph (plants 1–3), 2 tub + 4 ph (plants 4–7) and 4 tub + 4 ph (plant 8) had lost 2 phureja nucleolar chromosomes if 4 phureja genomes were present. One phureja nucleolar chromosome of plants 1–3 and both of plants 5 and 7 had rearranged satellites. Elimination of the two nucleolar chromosomes occurred preferentially, was under genetic control, and probably took place during early callus development. NOR activity resulting in rear-rangements between NORs may have caused the elimination.     

Haploid meiosis and its bearing on the phylogeny of pearl millet,Pennisetum typhoides stapf et hubb.

Meiotic chromosome associations in a spontaneously originated haploid plant of pearl millet have been studied and their phyletic significance discussed. Chromosome pairing could be observed at pachytene and diplotene. Out of a total of 285 PMC's studied at diakinesis-metaphase 1, 43 showed one bivalent and 7 had two bivalents per cell. Both rod- and ring-bivalents were observed. Apart from synapsis accompanied by chiasma formation, close associations of univalent chromosomes were observed. Out of 150 cells without true bivalents, 41 showed 1 s-s association and five, 2 s-s pairs per cell. On the basis of the realization of a maximum of two bivalents per cell, as also of a maximum of 2 s-s pairs, it has been inferred that the chromosome complement ofP. typhoides (n=7) has evolved from a basic set ofn=5 chromosomes. Other available evidence supporting this inference is also discussed.     

Somatic hybrid plants produced by electrofusion between Solanum melongena L. and Solanum torvum Sw

Summary Somatic hybrid plants between eggplant (Solanum melongena) and Solanum torvum have been produced by the electrofusion of mesophyll protoplasts in a movable multi-electrode fusion chamber. Using hair structure as a selection criteria, we identified a total of 19 somatic hybrids, which represented an overall average of 15.3% of the 124 regenerated plants obtained in the two fusion experiments. Several morphological traits were intermediate to those of the parents, including trichome density and structure, height, leaf form and inflorescence. Cytological analyses revealed that the chromosome numbers of the somatic hybrids approximated the expected tetraploid level (2n=4x=48). Fifteen hybrid plants were homogeneous and had relatively stable chromosome numbers (46–48), while four other hybrids had variable chromosome numbers (35–48) and exhibited greater morphological variation. The hybridity of these 19 somatic hybrid plants was confirmed by analyses of phosphoglucomutase (Pgm) and esterase zymograms.     

Introgression of chromosomes of Festuca arundinacea var. glaucescens into Lolium multiflorum revealed by genomic in situ hybridisation (GISH)

An F₁ hybrid (n=4x=28) between the tetraploid species Festuca arundinacea var. glaucescens (GGG′G′) and a synthetic tetraploid Lolium multiflorum (LmLmLmLm) was backcrossed to diploid L. multiflorum to produce triploid (2n=3x=21) BC₁ hybrids (LmLmG). At metaphase I of meiosis the triploids had a preponderance of ring bivalents and univalents with some linear and frying-pan trivalents. Genomic in situ hybridisation (GISH) differentiated the Festuca chromosomes from Lolium and revealed that the bivalents were exclusively between Lolium homologues, while the univalents were Festuca. Despite the limited amount of homoeologous chiasmata pairing in the triploids, some recombinant chromosomes were recovered in the second backcross when the hybrids were further crossed to diploid L. multiflorum. The progeny from the second backcross was predominantly diploid. Genotypes with recombinant chromosomes and chromosome additions involving an extra Festuca chromosome were identified using GISH. Changes in plant phenotype were related to the presence of Festuca chromatin. Received: 20 September 2000 / Accepted: 05 January 2001     

Development of monosomic alien addition lines and introgression of genes from Oryza australiensis Domin. to cultivated rice O. sativa L.

Oryza australiensis, a diploid wild relative of cultivated rice, is an important source of resistance to brown planthopper (BPH) and bacterial blight (BB). Interspecific hybrids between three breeding lines of O. sativa (2n=24, AA) and four accessions of O. australiensis (2n=24, EE) were obtained through embryo rescue. The crossability ranged from 0.25% to 0.90%. The mean frequency of bivalents at diakinesis/metaphase I in F₁ hybrids (AE) was 2.29 to 4.85 with a range of 0–8 bivalents. F₁ hybrids were completely male sterile. We did not obtain any BC₁ progenies even after pollinating 20,234 spikelets of AE hybrids with O. sativa pollen. We crossed the artificially induced autotetraploid of an elite breeding line (IR31917-45-3-2) with O. australiensis (Acc. 100882) and, following embryo rescue, produced six F₁ hybrid plants (AAE). These triploid hybrids were backcrossed to O. sativa. The chromosome number of 16 BC₁ plants varied from 28 to 31, and all were male sterile. BC₂ plants had 24–28 chromosomes. Eight monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. australiensis were selected from the BC₂ F₂ progenies. The MAALs resembled the primary trisomies of O. sativa in morphology, and on the basis of this morphological similarity the MAALs were designated as MAAL-1, -4, -5, -7, -9, -10, -11, and -12. The identity of the alien chromosome was verified at the pachytene stage of meiosis. The alien chromosomes paired with the homoeologous pairs to form trivalents at a frequency of 13.2% to 24.0% at diakinesis and 7.5% to 18.5% at metaphase I. The female transmission rates of alien chromosomes varied from 4.2% to 37.2%, whereas three of the eight MAALs transmitted the alien chromosome through the male gametes. BC₂ progenies consisting of disomic and aneuploid plants were examined for the presence of O. australiensis traits. Alien introgression was detected for morphological traits, such as long awns, earliness, and Amp-3 and Est-2 allozymes. Of the 600 BC₂ F₄ progenies 4 were resistant to BPH and 1 to race 6 of BB. F₃ segregation data suggest that earliness is a recessive trait and that BPH resistance is monogenic recessive in two of the four lines but controlled by a dominant gene in the other two lines.     

Indeterminate meiotic restitution (IMR): a novel type of meiotic nuclear restitution mechanism detected in interspecific lily hybrids by GISH

A detailed analysis of microsporogenesis was carried out in three diploid lily cultivars (2n=2x=24) and three diploid interspecific hybrids (2n=2x=24) using DNA in situ hybridisation methods (GISH and FISH). In cvs. Gelria (Lilium longiflorum; L genome), Connecticut King and Mont Blanc (both Asiatic hybrids; Agenome) meiosis was regular and only haploid gametes were formed while the three interspecific hybrids between L. longiflorum×Asiatic hybrid (LA) showed a variable frequency of meiotic nuclear restitution and stainable 2n-pollen formation ranging from 3% to 30%. An analysis of meiotic chromosome behaviour of the LA hybrids through GISH and FISH revealed that: (1) the parental chromosomes could be clearly discriminated into univalents, half-bivalents and bivalents in the PMCs; (2) in some of the PMCs the entire complement was present either as univalents or half-bivalents which had the potential to divide equationally (following centromere division) during the first division leading to first division restitution (FDR) gametes; (3) more frequently, however, in one and the same PMC the univalents and half-bivalents divided equationally whereas the bivalents disjoined reductionally at the same time giving rise to 2n-gametes that could vary from the well-known FDR or SDR 2n-gametes. We indicate this novel type of restitution mechanism as Indeterminate Meiotic Restitution (IMR). In order to confirm the occurrence of IMR gametes, the chromosome constitutions of eight triploid BC₁ progenies derived from backcrossing the 2n-gamete producing the LAhybrids to the Asiatic hybrid parents were analysed through in situ hybridisation. The results indicated that there were seven BC₁ plants in which FDR 2n-gametes, with or without homoeologous recombinations, were functional, whereas in one case the 2n-gamete resulting from IMR was functional. In the latter, there was evidence for the occurrence of genetic recombination through homoeologous crossing-over as well as through the assortment of homoeologous chromosomes. A singular feature of the IMR 2n-gamete was that although it transmitted a euploid number of 24 chromosomes to the BC₁ progeny, the number of chromosomes transmitted from the two parental species was dissimilar: 9 L-genome chromosomes and 15 A-genome chromosomes instead of 12 of each. Received: 15 May 2000 / Accepted: 4 December 2000     

Somatic hybridization between potato and Nicotiana plumbaginifolia

Summary Electrofusion was carried out between mesophyll protoplasts from the transformed diploid S. tuberosum clone 413 (2n=2x=24) which contains various genetic markers (hormone autotrophy, opine synthesis, kanamycin resistance, -glucuronidase activity) and mesophyll protoplasts of a diploid wild-type clone of N. plumbaginifolia (2n=2x=20). Hybrid calli were obtained after continuous culture on selection medium containing kanamycin. Parental chromosome numbers, determined at 2 months after fusion, revealed hybrid-specific differences between the individual calli. On the basis of these differences three categories of hybrids were distinguished. Category I hybrids contained between 8 and 24 potato chromosomes and more than 20 N. plumbaginifolia chromosomes; category II hybrids had between 1 and 20 N. plumbaginifolia chromosomes and more than 24 potato chromosomes; category III hybrids contained diploid or subdiploid numbers of chromosomes from both parents. The hybrids were evenly distributed over the three categories. After a 1-year culture of 24 representative hybrid callus lines on selection medium the karyotype of 10 hybrids remained stable, whereas 8 hybrids showed polyploidization of the genome of one parent, together with no or minor changes of the chromosome numbers of the other parent. Six hybrids showed slight changes in the hybrid karyotype. The elimination of chromosomes of a particular parent was not correlated to their metaphase location. The processes of spontaneous biparental chromosome elimination leading to the production of asymmetric hybrids of different categories are discussed.     

Production and characterization of arboreous and fertile <Emphasis Type="Italic">Solanum melongena</Emphasis> + <Emphasis Type="Italic">Solanum marginatum </Emphasis>somatic hybrid plants

In crop plants the shift from being annuals to perennials may allow future agricultural systems requiring less energy inputs. The practicability of this was tested for Solanum melongena. Leaf protoplasts of S. melongena (2n = 2x = 24) and one of the related arborescent species Solanum marginatum (2n = 2x = 24) were electrofused and fertile somatic hybrids with arborescent habit regenerated. The magnetic cell sorter (MACS) technique was used for the selection of heterokaryons. The hybrid nature of 18 regenerated plants was assessed on the banding patterns generated by inter-simple sequence repeat PCR. When taken to maturity in the greenhouse, hybrids grew more vigorously compared to the parental species. Their morphological traits were intermediate between those of S. melongena and S. marginatum. Hybrids flowered and produced an average of 85% stainable viable pollen and fertile fruits. The somatic hybrids were maintained in the greenhouse for more than 3 years and continued to produce flowers developing into two types of fruits with plentiful seeds. Fruits were either striated green containing non-germinable seeds or yellow with fully germinable seeds. Their S₁ progenies showed common features with S₀ hybrids, including fertility and arborescent habit. Cytologically, somatic hybrids exhibited the expected chromosome number of 2n = 4x = 48, while chromosome pairing during microsporogenesis was associated with a low frequency of intergenomic pairing. It is concluded that an arborescent perennial species has been obtained by somatic hybridization. The usefulness of this species per se or in eggplant breeding will depend not only on the transmission of the arborescent habit to cultivated eggplant varieties, but also on the variability that should be created from backcrossing the S. melongena + S. marginatum hybrids to S. melongena.     

Genome relationships between Hordeum procerum (6x) and H. lechleri (6x)

Investigations on the meiotic behaviour of chromosomes in interspecific hybrids (2n=6x=42) between Hordeum lechleri (6x) and H. procerum (6x) and in their component haploids have been utilized to assess the nature of pairing and the extent of genome homology between the two species. In the F₁ hybrids an average of 25 (60%) chromosomes associated at metaphase I, mostly as bivalents. A majority (60%) of the pollen mother cells (PMCs) in H. procerum haploids (2n=3x=21) displayed 21 univalents and even in the remainder, a maximum of two rod bivalents were formed resulting in an average of 0.52 bivalents per cell. In haploids of H. lechleri (2n=3x=21) however, 30% of chromosomes pair. The sum of the chromosomal associations in the component haploids represents only 17% of the complement, far below the observed frequency (60%) in the hybrids. Thus, the pairing displayed in hybrids between H. lechleri and H. procerum was mostly allosyndetic and suggestive of two genomes being common in these species.In haploid H. procerum 1/3 of the PMCs displayed a tripolar organisation of chromosomes leading to triad and hexad formation after divisions I and II respectively. The significance of hexad formation in the trihaploid H. procerum and a possible suppression of homoeologous pairing in H. procerum haploids are discussed.     

The genomic relationship between cultivated sorghum [Sorghum bicolor (L.) Moench] and Johnsongrass [S. halepense (L.) Pers.]: a re-evaluation

Summary The genomic relationship between cultivated sorghum [Sorghum bicolar (L.) Moench, race bicolor, De Wet, 2n=20] and Johnsongrass [S. halepense (L.) Pers., 2n=40] has been a subject of extensive studies. Nevertheless, there is no general consensus concerning the ploidy level and the number of genomes present in the two species. This research tested the validity of four major genomic models that have been proposed previously for the two species by studying chromosome behaviors in the parental species, 30-chromosome hybrids [sorghum, (2n=20) x Johnsongrass, (2n=40)], 40-chromosome hybrids [sorghum, (2n=40) x Johnsongrass, (2n=40)] and 60-chromosome amphiploids. Chromosome pairings of amphiploids are reported for the first time. Chromosomes of cultivated sorghums paired exclusively as 10 bivalents, whereas Johnsongrass had a maximum configuration of 5 ring quadrivalents with occasional hexavalents and octovalents. In contrast, 40-chromosome cultivated sorghum had up to 9 ring quadrivalents and 1 hexavalent. Pairing in the 30-chromosome hybrids showed a maximum of 10 trivalents, and that in the 40-chromosome hybrids exhibited 8 quadrivalents, 5 of which were rings, together with a few hexavalents. Amphiploid plants showed up to 3 ring hexavalents, 1 chain hexavalent and a chain of 12 chromosomes. The data suggest that cultivated sorghum is a tetraploid species with the genomic formula AAB1B1, and Johnsongrass is a segmental auto-allo-octoploid, AAAA B1B1B2B2. The model is further substantiated by chromosome pairing in amphiploid plants whose proposed genomic formula is AAAAAA B1B1B1B1 B2B2.Contribution no. 87-391-J from the Kansas Agriculatural Experiment Station     

Characterization of chromosome instability in interspecific somatic hybrids obtained by X-ray fusion between potato (Solanum tuberosum L.) and S. brevidens Phil

Summary Asymmetric somatic hybrids between Solanum tuberosum L. and S. brevidens Phil. have been obtained via the fusion of protoplasts from potato leaves and from cell suspension culture of S. brevidens. The wild Solanum species served as donor after irradiation of its protoplasts with a lethal X-ray dose (200 Gy). Selection of the putative hybrids was based on the kanamycin-resistance marker gene previously introduced into the genome of Solanum brevidens by Agrobacterium-mediated gene transfer. Thirteen out of the 45 selected clones exhibited reduced morphogenic potential. The morphological abnormalities of the regenerated plantlets were gradually eliminated during the extended in vitro culture period. Cytological investigations revealed that the number of chromosomes in the cultured S. brevidens cells used as protoplast source ranged between 28–40 instead of the basic 2n=24 value. There was a high degree of aneuploidy in all of the investigated hybrid clones, and at least 12 extra chromosomes were observed in addition to the potato chromosomes (2n=48). Interand intraclonal variation and segregation during vegetative propagation indicated the genetic instability of the hybrids, which can be ascribed to the pre-existing and X-ray irradiation-induced chromosomal abnormalities in the donor S. brevidens cells. The detection of centromeric chromosome fragments and long, poly-constrictional chromosomes in cytological preparations as well as non-parental bands in Southern hybridizations with restriction fragment length polymorphism (RFLP) markers revealed extensive chromosome rearrangements in most of the regenerated clones. On the basis of the limited number of RFLP probes used, preferential loss of S. brevidens specific markers with a non-random elimination pattern could be detected in hybrid regenerants.     

Interspecific hybrids of Antheraea roylei and A. pernyi — a cytogenetic reassessment

Summary A rare case of interspecific hybridization between the Indian oak feeding silkworm Antheraea roylei (n=31) and Chinese oak feeding silkworm A. pernyi (n=49) yielding fertile and vigorous offspring is reported. The F₁ and the backcross (A. roylei X A. pernyi X A. pernyi male individuals of the above cross and the F₂₃ and F₃₂ male offspring derived from an earlier cross between another race of A. roylei (n=30) and A. pernyi (n=49) were cytogenetically analysed in order to study their chromosome dynamics. The F₁ hybrids showed 18 trivalents and 13 bivalents in the first meiotic prophase and metaphase. The backcross individuals possessed either 9 trivalents and 31 bivalents or 49 bivalents, in Metaphase I cells. The F₂₃ and F₃₂ individuals were karyotypically alike and exhibited 49 bivalents. The following conclusions were drawn from the above observations: (a) in spite of allopatry and karyotypic divergence in number, a high degree of homology exists between the chromosomal complements of the two species; (b) A. pernyi possibly evolved from A. roylei, during the course of which 18 chromosomes of the latter underwent fission to give rise to the 36 chromosomes of the former. This is demonstrated by trivalent formation and pairing affinities in F₁ hybrids; (c) selection has favoured the elimination of large A. roylei chromosomes which participated in trivalent formation in successive generations of inbred hybrids to establish a stable Karyotype like that of A. pernyi.