<Emphasis Type="Italic">In vitro</Emphasis> root induction in weedy <Emphasis Type="Italic">amaranthus</Emphasis> species to obtain mitotic chromosomes

Summary Mitotic chromosome analysis has proven to be an important tool in monitoring the potential for genetic exchange among related plant species. One major obstacle to using mitotic chromosome analysis in any species is obtaining large numbers of clear, well-spread metaphase chromosomes necessary to perform cytological techniques such as chromosome banding and fluorescent in situ hybridization. The ability to obtain good chromosome spreads is in part determined by the number and morphology of the roots, which contain the metaphase tissue. Many Amaranthus species produce very thin, delicate roots. The technique used in the process described herein provides for much more substantial roots, allowing for higher probability of obtaining well-spread metaphase chromosomes. Seeds were planted in a soilless mixture, and then cuttings and leaves were taken from the plants. The cuttings were sterilized and placed in Murashige and Skoog (MS) media, while leaf tissue was analyzed by flow cytometry, both pre-and post-propagation, to obtain DNA contents. No changes in DNA content were observed. The in vitro procedure produced significantly larger roots than were produced in soilless mix. Furthermore, all of the in vitro roots observed had 32 chromosomes of normal morphology. In vitro root propagation allowed large numbers of roots to be obtained from a single plant, thereby resulting in increased probability of obtaining cells with metaphase chromosomes that reflected the original plants' chromosome numbers and therefore may be used for molecular cytogenetic analysis.     

Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora

Cytological preparations for the fluorescent in situ hybridization (FISH) technique require cytoplasm-free metaphases, with well-spread chromosomes, for the localization of DNA sequences and chromosome mapping. We tested various procedures for FISH analysis of Passiflora cacaoensis, P. gardneri and hybrid F? progeny of P. gardneri x P. gibertii. Two treatments with four enzymes and three incubation times were compared. The material was treated with 1.0 M HCl before enzymatic digestion. The following criteria were used to determine the quality of the metaphases: a) lack or presence of cytoplasm; b) well-spread chromosomes or with overlap; c) complete or incomplete chromosome number (2n). The enzyme Pectinex(?) SP ULTRA gave the best performance, with the shortest incubation time. The best results were observed after 30 min of incubation; more than 70% of the metaphases did not have large amounts of cytoplasm or overlapping chromosomes, and about 75% maintained the chromosome number. FISH was carried out using a 45S rDNA probe (pTa71) labeled with biotin and detected with fluorescein isothiocyanate. Sites with strong staining and without nonspecific signals were observed. Our methodological adaptations allowed the preparation of metaphase slides of high quality for the FISH technique, with less time required for the preparation of samples.     

Constitutive heterochromatin distribution in monocentric and polycentric chromosomes

Monocentric chromosomes of Vicia faba (2n=12) and polycentric chromosomes of Luzula purpurea (2n=6) and L. multiflora (2n=36) were studied by the C-banding technique. C-positive regions exhibited a restricted distribution in V. faba nuclei and were located near the centromeres of the chromosomes. Each chromosome had both a characteristic number and distribution of C-positive regions permitting homologue identification. L. purpurea and L. multiflora C-bands were much more numerous than those of V. faba and were widely distributed throughout both nuclei and chromosomes. Three distinct constitutive heterochromatin distribution patterns were present in L. purpurea metaphase chromosomes permitting homologous chromosome identification. One of three C-band distribution patterns was also evident in L. multiflora chromosomes.     

Chromosome preparations from hepatocytes and bone marrow of Chinese hamsters: A direct method

This report describes a method for the direct preparation of chromosomes from the hepatocytes and bone marrow of the same Chinese hamster (Cricetulus griseus). The technique is a modification of that described by Becker et al. (J. natl. Ca. Inst. 46: 1261–69, 1971) for rat hepatocytes, with the following significant differences: (1) a less extensive partial hepatectomy is employed to initiate hepatocyte regeneration, (2) the use of a larger initial dose of colchicine (4 mg/K) 46–48 hours after hepatectomy instead of 1 mg/K 24 hours after hepatectomy, (3) the use of 0.075 M KCl as hypotonic solution instead of fetal calf serum diluted 1 : 7 with distilled water and (4) flame or blaze drying of chromosome preparations instead of air drying. The combination of the above modifications gave abundant, clear and well-spread chromosomes.     

Identification of Haynaldia villosa chromosomes added to wheat using a sequential C-banding and genomic in situ hybridization technique

Genomic in situ hybridization (GISH) offers a convenient and effective method for cytological detection, but can not determine the identity of the chromosomes involved. We integrated C-banding with GISH to identify Haynaldia villosa chromosomes in a wheat background. All chromosomes of H. villosa showed C-bands, either in telomeric regions or in both telomeric and centromeric regions, which allowed unequivocal identification of each H. villosa chromosome. The seven pairs of H. villosa chromosomes were differentiated as 1–7 according to their characteristic C-bands. Using a sequential C-banding and GISH technique, we have analyzed somatic cells of F₃ plants from the amphiploid Triticum aestivum-H. villosa x Yangmai 158 hybrids. Three plants (94009/5-4,94009/5-8 and 94009/5-9) were shown to contain H. villosa chromosome(s). 94009/5-4 (2n = 45) had three H. villosa chromosomes (2, 3 and 4); 94009/5-8 (2n = 45) possessed one chromosome 4 and a pair of chromosome 5, and 94009/5-9 (2n = 43) was found to have one chromosome 6 of H. villosa. The combination of GISH with C-banding described here provides a direct comparison of the cytological and molecular landmarks. Such a technique is particularly useful for identifying and localizing alien chromatin and DNA sequences in plants.     

短柄草染色体酶解法制片技术

以六倍体短柄草为研究材料,对短柄草的染色体制片方法进行了优化,建立了一种改进的短柄草染色体酶解制片方法。试验结果表明,以45%醋酸固定液固定根尖、酶解时间2h可以获得最佳的根尖染色体制片。此方法不仅可以得到分散良好的有丝分裂中期分裂相,而且还缩短了酶解的时间,提高了制片的效率。     

Karyological observations on Turbellaria Proseriata: karyometric analysis ofMonocelis fusca,M. lineata (Monocelididae) andParotoplana macrostyla (Otoplanidae)

A karyometric analysis of the chromosome set of the marine turbellariansMonocelis fusca, M. lineata andParotoplana macrostyla has been carried out. The karyotype of the twoMonocelis species investigated (2n=6) is formed by three pairs of small and similarly sized chromosomes: InM. fusca, chromosome 1 is metacentric, chromosome 2 acrocentric and chromosome 3 is subtelocentric.M. lineata also presents one pair of metacentric chromosomes (chromosome 2), while chromosomes 1 and 3 are submetacentric.P. macrostyla (2n=12) reveals two pairs of large metacentric and four pairs of small chromosomes, three of which are metacentric, whereas the last is subtelocentric.     

Chromosomes of five artiodactyl mammals

Somatic chromosomes of six specimens belonging to the following five species of artiodactyls (Artiodactyla: Mammalia) are described: A female nilgai (Boselaphus tragocamelus), 2n=46; male baresingha (Rucervus duvauceli), two specimens, 2n=56; a female Himalayan tahr (Hemitragus jemlahicus), 2n=48; a female Kirk's dik-dik (Rhynchotragus kirki), 2n=46; and a male sambar (Cervus unicolor), 2n=58. In the baresingha and the sambar, one or more acrocentric chromosomes carried satellites on their long arms. ³H-thymidine radioautographs of cultured cells of the Himalayan tahr showed a long acrocentric chromosome to be late-replicating, suggesting that it is an X chromosome.     

Karyotaxonomical analysis in the genusAngelica (Umbelliferae)

Morphometric karyotype characters were studied in 25Angelica spp. (Umbelliferae, Apioideae) and in one species of the related genusTommasinia. For three species the chromosome numbers are new. In our study the majority of the species investigated are diploids with 2n = 22, some are tetraploids with 2n = 44 (for these tetraploids also diploid cytotypes are reported in the literature). Among the diploid species,A. miqueliana has a distinct karyotype consisting of submetacentric and acrocentric chromosomes only, the remaining diploids with 2n = 22 as well as tetraploids with 2n = 44 have rather symmetrical karyotypes, consisting of metacentric and submetacentric chromosomes. The very different chromosome number 2n = 28 has been found inA. gmelinii. Its karyotype includes two distinct groups of chromosomes: 8 pairs of rather large metacentrics and submetacentrics and 6 pairs of very short and asymmetrical chromosomes. Chromosome numbers and structures appear to be useful in the taxonomy of some intrageneric taxa inAngelica.     

Cytological studies on six cyprinid fishes

The chromosome complements of six cyprinid fishes were studied, using the routine air-drying Giemsa staining technique. The diploid chromosome number recorded is 2n = 50 (8m+18sm+14st+10t) with NF = 90 in Aspidoparia morar, 2n = 50 (8m+12sm+12st+18t) with NF = 82 in Crossocheilus latius latius, 2n = 50 (6m+12sm–16st+16t) with NF = 90 in Labeo pangusia, 2n = 70 (16m+6sm+16st–32t) with NF = 108 in Perilampus atpar, 2n = 48 (4m+6st+38t) with NF = 58 in Puntius chrysopterus and 2n = 50 (2m+2sm+4st+42t) with NF = 58 in P. tetrarupagus. Sex chromosomes are not identifiable in any of these species. A pair of marker chromosomes has been observed in all species excepting A. morar.     

End-to-end chromosome attachments in mitotic interphase and their possible significance to meiotic chromosome pairing

Cytological studies on telophase and early prophase in roottip cells of several plant species (Allium cepa, 2n=16; four Crepis species, including Crepis capillaris, 2n=6; Callitriche hermaphroditica, 2n=6; Nigella arvensis, 2n=12; Secale cereale, 2n=14) revealed that chromosome ends are attached two by two forming chains of chromosomes (interphase associations). In these chains homologous chromosomes are presumably located adjacent to each other. In Crepis capillaris it was observed that the two nucleolar chromosomes form a separate ring one end attached to the ring of the four remaining chromosomes and the other end attached to the nucleolus. It is proposed that these end-to-end attachments have significance for chromosome pairing in meiosis. The adjacent location of homologous chromosomes in the interphase associations would facilitate rapid and regular synapsis.     

Genomic characterisation and the detection of raspberry chromatin in polyploid Rubus

 This paper reports genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) data for chromosomes of raspberry (Rubus idaeus 2n=2x=14), blackberry (Rubus aggregate, subgenus Eubatus. 2n=2–12x=14–84) and their allopolyploid derivatives used in fruit breeding programmes. GISH was used to discriminate labelled chromosomes of raspberry origin from those of blackberry origin in allopolyploid hybrid plants. The raspberry chromosomes were labelled by GISH at their centromeres, and 1 chromosome was also labelled over the short arm. In one allopentaploid plant a chromosome carried a terminal signal. Karyotype analysis indicated that this is a blackberry chromosome carrying a raspberry translocation. GISH analysis of an aneuoctaploid blackberry cv ‘Aurora’ (2n=8x=58) showed that both whole and translocated raspberry chromosomes were present. The basic Rubus genome has one ribosomal DNA (rDNA) locus, and in all but one case all levels of ploidy had the expected multiples of rDNA loci. Interestingly, in the blackberry cv ‘Aurora’, there were only six sites, two less than might be predicted from its aneuoctaploid chromosome number. Our results highlight the potential of GISH and FISH for genomic designation, physical mapping and introgression studies in Rosaceous fruit crops. Received: 20 February 1998 / Accepted: 12 May 1998     

Homologous somatic association in radial metaphases of Crepis species

The chromosome arrangement in radial metaphases has been analyzed in root tips of Crepis capillaris (2n=6), C. taraxicifolia (2n=8) and C. rubra (2n=10) by using two statistical approaches: 1) measuring the distances between both members of each pair of homologues as the number of intervening chromosomes on the equatorial ring, and 2) applying a new statistical approach developed by Lacadena et al. (1977) which permits to analyze homologous somatic association considering as a whole the n chromosome pairs of the complement. The occurrence of somatic association of homologous chromosomes is clearly demonstrated with both approaches. Previous results obtained by other authors in different materials and with different statistical methods are discussed. The karyotypes of C. taraxifolia and C. rubra are described with numerical data.     

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     

Production and cytogenetics of Brassica campestris-alboglabra chromosome addition lines

 Four different Brassica campestris-alboglabra monosomic addition lines (AA+1 chromosome from C, 2n=21) were obtained after consecutive backcrosses between resynthesized B. napus (AACC, 2n=38) and the parental B. campestris (AA, 2n=20) accession. The alien chromosomes of B. alboglabra (CC, 2n=18) in the addition lines were distinguished by random amplified polymorphic DNA (RAPD) marker analysis and morphology of mitotic chromosomes. Four RAPD marker synteny groups were established, which represented the four different alien chromosomes of B. alboglabra in the four addition lines. Three of the four addition lines were identified to harbour chromosomes 4, 8 or 9 of B. alboglabra. Studies on meiotic pairing in the addition lines revealed intergenomic homoeology relationships among specific chromosome arms between the A- and C-genomes. The long arm of B. campestris chromosome 9 was homoeologous with the long arm of B. alboglabra chromosome 4, while its short arm with the short arms of B. alboglabra chromosomes 8 and 9. Such an intergenomic homoeology relationship supports the hypothesis that B. campestris and B. alboglabra share a common ancestor but that chromosomal rearrangements have occurred during the evolution of the two species. Intergenomic introgression was observed in the progenies of the addition lines. The introgression of an entire B. alboglabra marker synteny group into the B. campestris genome implied the possible occurrence of interspecific chromosomal substitution. Received: 30 May 1996 / Accepted: 18 October 1996     

Chromosome markers and alterations in mitotic cells from interspecific Citrus somatic hybrids analysed by fluorochrome staining

Mitotic cells from Rough lemon (Citrus jambhiri Lush.), Ohta ponkan (C. reticulata Blanco) and two somatic hybrid plants obtained from protoplast fusion were analysed by double staining with chromomycin A₃ (CMA) and 4′-6-diamidino-2-phenylindole. Only CMA-positive bands were observed in metaphasic chromosomes. The two parental karyotypes (2n=2x=18) were heteromorphic, yielding some marker chromosomes that could be identified in the somatic hybrids. One of the somatic hybrids had 2n=37 chromosomes, and the possible extra chromosome was distinguishable. The second somatic hybrid was tetraploid (2n=4x=36), with one of the chromosomes bearing a putative structural alteration. Furthermore, aneusomaty and some mitotic abnormalities were also observed in this latter plant. Such irregularities are reported for the first time for citrus somatic hybrids, and their possible causes and implications are discussed. Received: 23 December 1996 / Revision received: 21 May 1997 / Accepted: 16 June 1997     

A cytological study ofPelargonium sect.Eumorpha (Geraniaceae)

The chromosome numbers of seven species ofPelargonium sect.Eumorpha have been determined from material of known wild origin, and karyotypic comparisons have been made. Within the section there is variation in basic chromosome number (x = 4, 8, 9, 11), variation in chromosome size, and two species have polyploid races. The three species with chromosome numbers based on x = 11 have the smallest chromosomes (1.0–1.5 µm); chromosomes are larger (1.0–3.0 µm) in the other species.P. elongatum has the lowest chromosome number in the genus (2n = 8).P. alchemilloides is exceptional in that it has four cytotypes, 2n = 16, 18, 34 and 36, and the form with 2n = 36 has large chromosomes (2.0–5.0 µm). Evidence from a synthesized hybrid suggests thatP. alchemilloides with 2n = 16 may be of polyploid origin. The three species based on x = 11 appear to be more closely related to species from other sections ofPelargonium that have the same basic chromosome number and small chromosome size, rather than to other species of sect.Eumorpha.     

Destruction of specific heterochromatic chromosomes during spermatogenesis in the Comstockiella chromosome system (Coccoidea: Homoptera)

In male coccids with the Comstockiella chromosome system, the set of chromosomes of paternal orgin becomes heterochromatic (H) during early cleavage. Just prior to prophase I of spermatogenesis, some of the H chromosomes are destroyed; the rest are eliminated following meiosis. In this report a Comstockiella sequence is described from Dactylopius opuntiae (2n=10) in which one chromosome pair is about three times longer than the others. During prophase I the number of small H chromosomes present varied from cyst to cyst, but the long H chromosome was present in every cyst. These observations provide the best evidence to date that in the Comstockiella system a particular chromosome may always escape destruction. An analysis of Kitchin's (1975) data about the frequency of prophase I cysts with 1–4 H chromosomes in three species of Parlatoria with 2n = 8 suggested that in these species chromosomes of similar size may have very different probabilities of being destroyed. Evidence that in other species with the Comstockiella system a particular H chromosome is always retained is reviewed, and the possibility that in Ancepaspis tridentata the variation in the length of the H chromosome retained is due to the partial destruction of the longest chromosome is discussed.     

Improvement of high‐resolution fluorescence in situ hybridisation mapping on chromosomes of Brassica oleracea var. capitata

The low resolution of chromosome‐based Fluorescence in situ hybridisation (FISH) mapping is primarily due to the structure of the plant cell wall and cytoplasm and the compactness of regular chromosomes, which represent a significant obstacle to FISH. In order to improve spatial resolution and signal detection sensitivity, we provide a reproducible method to generate high‐quality extended chromosomes that are ~13 times as long as their pachytene counterparts. We demonstrate that proteinase K used in this procedure is crucial for stretching pachytene chromosomes of Brassica oleracea in the context of a modified Carnoy's II fixative (6:1:3, ethanol:chloroform:acetic acid). The quality of super‐stretched chromosomes was assessed in several FISH experiments. FISH signals from both repetitive 5S rDNA and single‐copy ARC1 on super‐stretched chromosomes are brighter than those on other different types of chromosome due to enhanced accessibility to targets on stretched pachytene chromosomes. In conclusion, the resulting extended chromosomes are suitable for FISH mapping for repetitive DNA sequences and the localisation of a single‐copy locus, and FISH performed on super‐stretched chromosomes can achieve significantly higher sensitivity and spatial resolution than other chromosome‐based FISH mapping techniques.     

Different rate of intrapopulation chromosome polymorphism in two species of Black Sea Blennies (Blenniidae,Pisces)

Somatic karyotypes in seven specimens of Blennius sanguinolentus include 22 subtelocentric and 26 acrocentric chromosomes, whereas one male has 2n=47=1M+22ST+24A: polymorphism is evidently a result of centric fusion of two acrocentrics. Blennius tentacularis is characterized by the availability of four karyomorphs out of which three coincide with karyotypes described earlier (Carbone et al., 1987). Karyttype-I consists of a 48 small uni-armed chromosome, but both karyotypes II and III with 2n=48 and 2n=47 respectively include one large acrocentric chromosome, and karyotype-IV has one large submetacentric out of the 47 chromosomes. Karyotypic variability of B. tentacularis is attributed either to polymorphism by 1–3 chromosome rearrangements or to availability of sex-determining mechanism, including the Y-autosome translocation. This diverse series of male karyomorphs may reflect the complicated behavioural structure.