Normal Diploids Research Articles

A Preliminary Ploidy Analysis of Diploid and Triploid Salmonids

The findings indicate that mosaicism does occur in triploids and especially in triploid hybrids. Immature diploid and triploid salmon were obtained from the University of Alaska – National Marine Fisheries Service. These were fixed in 10% buffered formalin and blocks taken from various tissues including kidney, liver, spleen, and gonads. The paraffin blocks were sectioned at 4 μm and stained with hematoxylin and eosin for histologic evaluation and to quantitate nuclear DNA. The slides for DNA evaluation were stained using the Cell Analysis Systems, Inc. (CAS, Elmhurst, IL) kit following Feulgen hydrolysis. The Cell Analysis Systems, Inc. (Model 200) computer-assisted image analyzer was used for DNA quantitation. An attempt was made to evaluate the frequency of mosaicism, ploidy variation in somatic and gonadal tissues of triploids and hybrids as compared with normal diploids. Hepatic ploidy variation, normal in adult animals, was not present in these specimens. However, in gonads there was evidence of increased mosaicism among the triploids. Because of mosaicism in gonadal tissue, sterility of triploids cannot be presumed with certainty at this stage. Further studies are planned to examine this and other questions raised by these results.

Sex determination and polyploid gigantism in the dwarf surfclam (Mulinia lateralis Say).

Mulinia lateralis, the dwarf surfclam, is a suitable model for bivalve genetics because it is hardy and has a short generation time. In this study, gynogenetic and triploid M. lateralis were successfully induced. For gynogenesis, eggs were fertilized with sperm irradiated with ultraviolet light and subsequently treated with cytochalasin B to block the release of the second polar body (PB2). Triploidy was induced by blocking PB2 in normally fertilized eggs. The survival of gynogenetic diploids was very low, only 0.7% to 8 days post-fertilization (PF), compared with 15.2% in the triploid groups and 27.5% in the normal diploid control. Larvae in all groups metamorphosed at 8-10 days PF, and there was no significant post-larval mortality. At sexual maturation (2-3 months PF), all gynogenetic diploids were female, and there was no significant difference (P > 0.05) in sex ratio between diploids and triploids. These results suggested that the dwarf surfclam may have an XX-female, XY-male sex determination with Y-domination. Compared with diploids, triploids had a relative fecundity of 59% for females and 80% for males. Eggs produced by triploid females were 53% larger (P < 0.001) in volume than those from diploid females. In both length and weight measurements at three months PF, the gynogenetic diploids were not significantly (P > 0.33) different from normal diploid females, suggesting that inbreeding depression was minimal in meiosis II gynogens. Triploid clams were significantly larger (P < 0.001) than normal diploids. We hypothesize that the increased body-size in triploids was caused by a polyploid gigantism due to the increased cell volume and a lack of cell-number compensation.

A genetic and metabolic basis for faster growth among triploids induced by blocking meiosis I but not meiosis II in the larviparous European flat oyster, Ostrea edulis L.

This study establishes a genetic and metabolic basis to faster triploid growth in the oyster Ostrea edulis. Triploidy was induced using cytochalasin B, and image analysis of biopsied tissue employed to ensure similar ploidy of all animals within each class. Results indicate that lifetime growth in total dry tissue weight over 15 months was more than 60% faster ( p<0.001) in meiosis I triploids than in diploid siblings or meiosis II triploids, with no difference between meiosis II triploids and their diploid siblings. For six polymorphic enzyme loci, single-locus heterozygosity was consistently greatest in meiosis I triploids ( p<0.001), so that average multiple-locus heterozygosity in meiosis I triploids was 49% higher than in normal diploids, and 55% higher than in meiosis II triploids ( p<0.001). This suggests that faster growth resulted from increased allelic diversity, rather than the increased allelic quantity that results from the addition of one entire set of chromosomes among triploids generally. Results also confirm that the faster growth of meiosis I triploids resulted from reduced energy expenditure, associated with lower concentrations of RNA per unit total tissue protein, which infer reduced rates of whole-body protein turnover. Statistical analyses confirmed that differences in oxygen consumption and growth were associated with both ploidy class and average multiple-locus heterozygosity, indicating that performance in meiosis I triploids is not only improved as a result of reduced reproductive output, but also through the metabolic consequences associated with increased heterozygosity.

In vitro clonal propagation of Capsicum spp.

The effect of various concentrations of benzyladenine (BA 4.4–177.5 μM) or kinetin (4.7–185.9 μM) on shoot proliferation from shoot-tip explants was investigated in C. praetermissum Heiser & Smith and C. annuum L. Maximum number of shoots were obtained on Murashige & Skoog's medium with 66.6 μM BA or 92.9 μM kinetin in C. praetermissum, and 88.8 μM BA or 116.2 μM kinetin in C. annuum after 4 weeks of culture. Combining 1 μM 2, 3, 5-triiodobenzoic acid (TIBA) with low levels of BA or kinetin significantly increased shoot number as compared to using either cytokinin alone. Rooting of regenerated shoots was achieved on MS medium containing 5.7 μM indoleacetic acid. Best rooting (80–100%) was observed in shoots from TIBA plus BA or kinetin media while only 40–50% of shoots from the BA or kinetin treatments were rootable. Plantlets obtained from TIBA plus BA or kinetin were normal diploids while those from BA or kinetin alone revealed distinct chromosomal aberrations in their root tip squashes. Regenerants from TIBA plus BA or kinetin media were successfully established in the soil (86% survival rate), where they flowered and showed normal meiotic behaviour with 100% pollen viability.

The competence of cells for cell division and regeneration in tobacco explants depends on cellular location, cell cycle phase and ploidy level

This work concerns application of flow cytometry (FCM) and confocal laser scanning microscopy (CLSM) to investigate the competence of cells for cell division and regeneration. FCM analysis of freshly-cut thin cell layer (TCL) explants of Nicotiana tabacum, excised from upper internodes of vegetative plants, revealed that one-quarter of the cells had the 2C nuclear DNA content, whereas of the other cells most nuclei had the 4C and some had the 8C DNA content. Cytometric examination using CLSM showed that the 2C nuclei were mainly located in the epidermis and subepidermis, and the 4C nuclei predominantly in the cortical tissue. During culture of the explants, part of the cortical cells went through mitosis from the first day onwards, and formed callus from which predominantly diploid and some tetraploid roots regenerated at low frequency. Most cortical cells were thus in the G 2 phase of the diploid cell cycle. FCM analysis showed that another fraction of the 4C cortical cells was induced to endoreduplicate to 8C cells. These cells thus had previously switched to the G 1 phase of the tetraploid cell cycle. CLSM analysis revealed that subepidermal and epidermal cells, respectively, underwent cell division from the second and third day onwards. Shoot primordia developed from cells of both cell layers together. Most shoot regenerants were normal diploids, some were mixoploids or tetraploids. The combination of FCM and CLSM allowed identification of the cell cycle phase, the ploidy level, the position of the cell, and the cellular development. The results give insight into the involvement of these parameters in the competence for cell division and regeneration at the level of the individual explant cells, and are therefore relevant for cellular and molecular approaches to plant transformation.

Disease resistance gene transfer in grass carp by sperm chromosome fragmentation and gynogenesis

Diploid gynogenesis was induced in Japanese crucian carp (Carassius cuvieri) eggs using UV-irradiated genetically inactive spermatozoa from mirror carp (Cyprinus carpio L.) or blunt snout bream (Megalobrama amblycephala), with or without cold shock. The optimal radiation dosage was 4200 mJ/cm2 and 3600 mJ/cm2 for mirror carp and blunt snout bream sperm, respectively. At this dosage and without cold shock, the yields were (32.4±3.3)% vs. (33.8±1.4)% gynogenetic haploids and (0.7±0.3)% vs. (0.5±0.3)% hybrid diploids, respectively. At the optimal UV dosage but with cold shock (2 min after fertilization, 0–4°C for 40 min), the hatching rates were (27.8±2.1)% and (29.4±3.3)%, respectively. From hatching to feeding, (15.7±3.4)% and (23.6±4.1)% normal gynogenetic diploids were recorded, respectively. Survival of normal gynogenetic diploids was 56% out of the hatched fry when using irradiated spermatozoa of mirror carp, which was lower than that (up to 80%) when using irradiated spermatozoa of blunt snout bream. This indicated that the sperm of blunt snout bream, with distant genetic relation to the maternal Japanese crucian carp, was more effective than that of mirror carp to induce diploid gynogenesis. The nature of the gynogenetic progeny was identified with external appearance, chromosome number and gonad structure. The presence of only females in gynogenetic progeny probably suggested XX genotype in the female Japanese crucian carp. The gynogenetic diploids have potential values such as faster growth and stronger disease resistance than the normal Japanese crucian carp. All gynogenetic progeny possessed 100 chromosomes whereas all J x B crosses were triploid with 124 chromosomes. The formation of the new triploid hybrids in J x B crosses may be useful in aquaculture.

A statistical approach to estimate the frequency of n, 2n and 4n pollen grains in diploid alfalfa

Using pollen grain diameter, a statistical approach has been applied in order to estimate the percentage of n, 2n and 4n pollen grains produced by diploid plants of alfalfa. Pollen grain size distributions of normal diploids, normal tetraploids and jumbo pollen mutants were used as controls for n<x, 2n<2x and 4n<4x pollen grains, respectively. The least squares method was used to fit the observed distributions of the plants under examination with three reference distributions. Six out of 22 plants analyzed were 2n pollen grain producers and one also produced 4n pollen grains. The results obtained indicate that the method can be efficiently applied in breeding programmes aimed at the selection of alfalfa mutants producing unreduced gametes at high frequency.

Foliar regeneration of the endangered Red Vanda, Renanthera imschootiana Rolfe (Orchidaceae)

An in vitro method was developed to regenerate large numbers of phenotypically uniform plants from the basal parts of the leaves of flowering plants of Renanthera imschootiana Rolfe. Differentiation of up to 10 shoot buds free of callus and protocorm-like bodies occurred in 10–12 weeks from the base of a single leaf implanted in Mitra et al. (1976) medium supplemented with 2% sucrose, 2 g l-1 peptone, 44.4 μM benzyladenine (BA) and 10.7 μM naphthaleneacetic acid (NAA). Subculture of the tissues in medium enriched with 10% coconut water and 35 g l-1 ripe banana pulp resulted in the production of highest average number of 40 shoots in 12 weeks. No difference in the regeneration potential was observed among the three young leaves while mature leaves did not respond. All the leaves of the regenerated shoots were easily recultured to increase shoot multiplication. Shoots readily formed roots on transfer to a medium containing 4.4 μM BA, 10.7 μM NAA and 1% activated charcoal. All regenerated plants examined were normal diploids with 2n=38. Foliar meristem culture appears to have great potential for ex situ conservation and propagation of this extremely endangered orchid.

Production and cytological studies of nine-paired barley (Hordeum vulgare)

Several barley plants with 2n = 18 chromosomes (9 II) were developed from crosses between two lines with 2n = 16 chromosomes (8 II). Cytological studies of the 18-chromosome plants showed nine bivalents at metaphase I and normal 9:9 segregation at anaphase I. Morphologically, these plants with 18 chromosomes were qualitatively similar to normal diploids (2n = 14), in spite of a duplication of a small proximal segment of the short arm of chromosome 3. However, the 18-chromosome plants were as vigorous as some normal diploid cultivars and more vigorous than 16-chromosome parental lines. If the duplicated segment carried beneficial gene(s), it might be useful for enhancement of germplasm in barley genetics and breeding. The significance of the establishment of this new 18-chromosome barley in karyotype evolution is discussed.Key words: nine-paired barley, aneuploid, trisomic, duplication, Hordeum.

Application of DNA Fingerprinting to Confirmation of Clone in Ayu

In order to determine the effectiveness of DNA fingerprinting as a tool for confirmation of clonal nature and identity, we applied a DNA fingerprinting method to three genetically different groups of ayu Plecoglossus altivelis: clonal diploid, mitotic gynogenetic diploid (mitotic-G2N) and normal diploid. Genomic DNA extracted from blood was digested by Pst I restriction endonuclease and was hybridized with repetitive sequence in bacteriophage M13 DNA labelled with [α-32P] isotope. DNA fingerprints of P. altivelis were detected over the whole effective fraction range from 0.5-25 Kilobase pairs, and more than 42 fragment patterns were detected. The DNA fingerprints of clonal fish consisted of at least 21 distinguishable bands which showed identical patterns within a clone. Mitotic-G2N consisted of 12-18 bands, and an individual fingerprint of this group hardly shared the bands of the other individuals. A trend of markedly low levels of band sharing in the DNA fingerprint among individuals of mitotic-G2N fish would be useful as a powerful genetic marker for their respective clonal lines. In N-CONT, 9 of the 18-24 distinguishable bands were shared. These results suggest the usefulness of DNA fingerprinting in fisheries genetics as a marker for the assessment of inbreeding rates, and also for the identification of individuals and families.

Production of Clonal Ayu by Chromosome Manipulation and Confirmation by Isozyme Marker and Tissue Grafting

We attempted to produce clonal fish by chromosome manipulation in ayu Plecoglossus altivelis. To induce mitotic gynogenesis diploids (mitotic-G2N as complete homozygous individuals), the eggs were inseminated with UV-irradiated sperm of normal diploid and were treated with a hy-drostatic pressure of 650kg/cm2 at 80 min after insemination and just before the first cell cleavage. The production of mitotic-G2N was confirmed by isozyme markers. The mitotic-G2N pro-duced from the female parents which were heterozygous at Gpi-1, Me-1 and Pgm loci showed segre-gation into two homozygous types at those loci, and they were female in all individuals. To pro-duce clonal fish, the eggs of mitotic-G2N were inseminated with the UV-irradiated sperm of normal diploid. The diploidization was achieved by retaining the second polar body with cold shock. Two families of clonal fish were produced and designated as clone-1 and clone-2. The clonal fish examined were all homozygous types in which their genotypes were the same as their mothers in each of clonal fish. The graft-tissue (a piece of operculum) transplanted into their intraclonal sibling hosts survived and was accepted. On the other hand, the graft-tissue trans-planted into the different clonal hosts was rejected in both clones. These results suggest that in-traclonal siblings possessed completely identical histocompatibility genes.

Conjugation between unimicronuclear doublets and singlets, and several features of the clones originating from hemikarya of Euplotes woodruffi syngen 1 (ciliophora)

Conjugation of a unimicronuclear doublet with two singlets produced three kinds of exconjugants: an exconjugant singlet cell, with a macronucleus and a micronucleus, originating from a synkaryon (diploid); an exconjugant singlet with a micronucleus and a macronucleus from a hemikaryon (haploid); and an exconjugant doublet consisting of two components, one side bearing a micronucleus and a macronucleus derived from a hemikaryon and the other side with those from a synkaryon. Of the two exconjugant singlets, one died in 64% of all cases and both singlets survived and formed clones in 20% of the cases. Using mainly the latter, the length of the immaturity period and fission rates were compared between diploid and haploid clones. Diploid clones were the same as the control clones in the morphology of their nuclear apparatus. Haploid clones showed faintly stained small micronuclei; They sometimes produced amicronucleates. Exconjugants which were considered to be diploids showed an immaturity period of over 40 fissions and fission rates of over 1.43, while those considered to be haploids often produced clones with short immaturity periods, short clonal life, and low fission rates. The F 1 singlets with short immaturity, when crossed to normal diploids, produced F 2 clones with lower viability and shorter immaturity than those produced by crosses between two normal diploids.

Somatic embryogenesis and plant regeneration from callus cultures of Bunium persicum Boiss

Callus was obtained from mericarps of Bunium persicum Boiss. on MS medium supplemented with 2.0 mg/1 2,4-D and 4.0 mg/1 Kn. Small white clumps of compactly packed cells developed on the callus on a medium containing 1.0 mg/1 2,4-D and 0 mg/1 Kn. These cell clumps differentiated numerous globular embryos on the same medium. Embryo maturation and germination was achieved on the basal as well as on 1 mg/1 Kn supplemented medium. All regenerated plants examined were normal diploids with 2n=14.

Ploidy Manipulation and Gynogenesis in Fishes: Cytogenetic and Fisheries Applications

Manipulation of fish chromosomes dates back to the early part of this century. The earliest experiments involved induction of gynogenesis with sperm inactivated by radiation or chemical treatments. Temperature or pressure shocks applied soon after fertilization resulted in the retention of the second polar body and reconstitution of diploidy; triploidy resulted from shocks to fish ova fertilized with normal sperm. More recently, it has been possible to suppress the first mitotic division offish eggs with high-pressure or -temperature treatments applied at the time of first cleavage to produce mitotic gynogenetic diploids and tetraploids. Androgenesis has been successfully induced in fish by irradiation of ova, fertilization of eggs with normal sperm, and suppression of the first mitosis with high-pressure treatments. Gynogenetic diploids have been used for cytogenetic studies of meiotic phenomena and gene mapping. The general finding to date is that the arrangement of genes on chromosomes is highly conserved in fish genomes. Inbreeding in successive generations of gynogenetic diploids is not as rapid as was anticipated, due to extensive gene-centromere crossing-over and chiasmata interference. However, isogenic lines can be produced more rapidly from gynogenetic diploids than from inbred lines of normal diploids, which require many generations of sibmating. Mitotic gynogenetic diploids and androgenetic diploids are completely homozygous and similar to inbred or isogenic lines; they can be used to study genetic versus environmental effects on fish. Triploids are of interest because they are expected to be sterile, to grow faster than diploids as they reach the age of sexual maturity, and to live longer than diploids because of the biological costs associated with reproduction; however, results on the performance of adult triploidy have been equivocal. For some species, adult triploidy outperformed diploids in growth and other production characters, whereas the triploidy of other species did not possess superior performance traits.

Physical mapping of the B-hordein loci on barley chromosome 5 by in situ hybridization

Physical mapping of the B-hordein genes in barley (Hordeum vulgare cv. Betzes) was achieved by in situ hybridization of a biotin-labelled B-hordein cRNA probe to chromosome spreads. Lines trisomic for chromosome 5 showed up to 3 signals on interphase nuclei, while no more than 2 signals were observed in normal diploids. An enzyme pretreatment, which produced broken cells, increased the frequency of signal detection. A clear signal was observed on 23% of interphase nuclei and 0.025% of metaphase spreads. The B-hordein genes mapped to 46% of the distance from the centromere on the short arm of chromosome 5. These results are discussed in relation to the barley genetic map and to previous reports of in situ hybridization of low-copy and unique-copy genes in plants.Key words: in situ hybridization, Hordeum vulgare, B-hordein genes, genetic map.

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl−1) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl−1 BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl−1). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.

The postimplantation development of spontaneous digynic triploid embryos in LT/Sv strain mice.

When spontaneously ovulating LT/Sv female mice are mated with fertile males, between one third and one half of the zygotes analyzed at the first cleavage mitosis are found to be triploid. This is due to the fact that LT/Sv females ovulate both primary and secondary oocytes, all of which are capable of being fertilized. Fertilization of the former group results in the production of digynic triploid conceptuses, while their diploid littermates result from the fertilization of normal secondary oocytes. The present study was therefore carried out in order to investigate the 'spontaneous' level of triploidy in these mice, and to provide insight into the developmental fate of the LT/Sv triploid embryos, as previous studies had indicated that in this species triploids invariably fail to develop beyond the early postimplantation period. This study revealed that when autopsies were carried out on the 7th and 8th days of gestation, it was generally difficult to distinguish between the karyologically normal diploids and the digynic triploid conceptuses when only morphological criteria were used. However, by the 10th day of gestation, the triploid conceptuses could usually be readily distinguished from their diploid littermates by their smaller size and (occasionally) by their disorganized or abnormal morphological appearance.

Cytogenetics of Lolium perenne

A number of diploid inbred lines of Lolium perenne were treated with colchicine at the early seedling stage to induce chromosome doubling. In each inbred line the colchicine-treated undoubled diploids were kept as controls, as well as the normal untreated diploids. Comparisons of vegetative growth and development, involving the three treatments within each line, revealed that colchicine treatment of seedlings has long-lasting effects upon plant growth and development independent of chromosome doubling, and that for certain characteristics the effects of chromosome doubling are confounded with other effects of the treatment used to produce tetraploids. This colchicine induced variation in the diploids is transmitted through the seed generations in at least one of the inbred lines. The variation appears to be non-random and also shows a strong genotypic component. In so far as the effects of chromosome doubling could be determined, they showed the usual gigas response but were again strongly influenced by genotype.

Localization of the human prealbumin gene to 18p11.1-q12.3 by gene dose effect study of Southern blot hybridization.

The human prealbumin gene was tried to map by the analysis of gene dose effects on Southern hybridization bands of various chromosome 18 abnormalities. The32P-labeled probe “pPA1” which contains the prealbumin cDNA hybridized to an 11 kb and a 4.6 kbHind III-fragments from cells each of which has a different chromosome 18 constitution. Autoradiograms (hybridization bands) for pseudodicentric 18 (trisomy of almost whole of a chromosome 18) showed 1.5-fold density compared with those of the normal diploid. Autoradiograms for trisomy for 18q21.3-qter and monosomies for 18p11.2-pter and for 18q21.1-q21.3 were all not different in density from those of the diploid control. Our results provide evidence for regional assignment of this gene to 18p11.1-q12.3, most likely to cen-q12.3.

Spontaneous Neurinoma in an African Lungfish, &lt;italic&gt;Protopterus annectens&lt;/italic&gt;, and DNA Repair Studies on Normal and Neoplastic Tissues&lt;xref ref-type="fn" rid="FN1"&gt;2&lt;/xref&gt;

Neurinomas developed in an African lungfish (Protopterus annectens), living in an aquarium in Western Japan. The 2 tumors, measuring 7.5 X 9.0 X 6.5 and 13 X 4 X 6 cm, were located on the skin. As shown by light microscopy, tumor cells were composed of spindle-shaped cells with huge pleomorphic nuclei, which were arranged in parallel rows or whorls in interlacing connective tissue. Long-term culture of these tumor cells was achieved in vitro at 25 degrees C with use of conditioned medium over a period of more than 4 months. The nuclear DNA contents of erythrocytes (normal diploids, 2C) and tumor cells dispersed from the fixed tumor tissues were measured by 4',6-diamidino-2-phenylindole hydrochloride-DNA microfluorometry by using mouse cerebellar small granule cells (normal diploids, 2C) as a reference. The 2C value of the lungfish was approximately 28-fold greater than that of the mouse. Furthermore, consistent with the nuclear pleomorphism observed by light microscopy, the nuclear DNA contents of tumor cells showed a wide distribution from hypo-2C to hyper-4C. DNA repair synthesis was measured autoradiographically in organ cultures of the tumor, lung, and skin, exposed to chemical carcinogens or UV radiation. Considerable repair was observed in the tumor and skin cells exposed to 1-methyl-1-nitrosourea (CAS: 684-93-5), N-methyl-N'-nitro-N-nitrosoguanidine (CAS: 70-25-7), or 254-nm or sunlamp UV light. Only traces of repair synthesis were detected in lung exposed to 1-methyl-1-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. 4-Hydroxyaminoquinoline 1-oxide (CAS: 4637-56-3) did not induce repair in any of the three tissues. The observed values for repair, relative to the amount of DNA, were similar to those in other fishes.