Co-segregation of nitrate-reductase activity and normal regeneration ability in selfed sibs of Nicotiana plumbaginifolia somatic hybrids,heterozygotes for nitrate-reductase deficiency

Summary The nitrate-reductase (NR) defective cell lines of Nicotiana plumbaginifolia isolated in our laboratory could not be regenerated into plants on the standard medium (Márton et al. 1982 a). The normal regeneration potential, however, was restored in somatic hybrids obtained by fusing the NR^– (green) lines with a pigment deficient (P^–), but NR⁺ line, A28. Somatic hybrid plants were fertile in two combinations (A28 + NA9 and A28 + NX9). As expected, segregation for NR^– and P^– was found after selfing the somatic F1 (SF1) obtained by protoplast fusion, and in the F2. The variable segregation ratios are explained by chromosome abnormalities. Co-segregation of the NR^– phenotype and the altered response to shoot induction on standard medium suggest the involvement of the nitrate-assimilatory pathway in determining shoot regeneration ability.     

Transformation of the Hprt gene with DNA from spermatogenic cells

DNA-mediated transformation of hypoxanthine guanine phosphoribosyl transferase (HPRT)-deficient cells was used to assess the state of the X chromosome Hprt gene in spermatogenic cells. It had been shown previously that DNA from the inactive X chromosome of somatic cells functions poorly or not at all in HPRT transformation, indicating that DNA modification is involved in somatic cell X chromosome inactivation (XCI). In contrast, DNA from mature sperm does function in HPRT transformation suggesting that DNA modification may not be the basis of XCI in mature sperm. In this paper, transformation of HPRT^– mouse and hamster cells has been performed to test the nature of XCI during earlier stages of spermatogenesis. DNA from these developing murine germ cells was shown to be capable of HPRT transformation, extending the observation that XCI in sperm does not appear to involve a DNA modification. We also show here that DNA from mature sperm of marsupials functions in HPRT transformation, a result consistent with a role for sperm XCI in the evolution of somatic X inactivation.     

Evidence for tetrameric structure of mammalian hypoxanthine phosphoribosyltransferase

A fast electrophoretic variant of hypoxanthine phosphoribosyltransferase (HPRT) has been detected in Mus musculus bactrianus, a mouse subspecies from Middle Asia (USSR). The electrophoretic HPRT pattern yielded by hybrids between the somatic cell of LMTK^– (deficient in thymidine kinase) and the splenocytes of a male of M. m. bactrianus was five-banded. The pattern obtained from the germ cells of the ovaries from 14.5-day-old embryos from laboratory CBA mice × M. m. bactrianus crosses was also composed of five bands. The hybrids between the somatic cells of human fibroblasts × LMTK^– cells gave a three-banded electrophoretic HPRT pattern because the asymmetrical heteropolymeric isozymes are probably unstable. Taken together, all the evidence is in favor of a tetrameric structure of mammalian HPRT.     

The Effect of Transposon P{GUS · p53.259H} on the Frequency of Tumor Clones in Drosophila melanogaster wtsP2/+ Heterozygotes

We showed that transposon P{GUS · p53.259H}, mapped to chromosome 3 and carrying a dominant mutation p53 ^259H.GUS, has a positive effect on the frequency of spontaneous and carcinogen-induced tumor mosaic clones warts ^– in Drosopila melanogaster heterozygotes for the tumor suppressor gene warts located in the same chromosome. The transposon effect could be explained either by the arrest of apoptosis in the cells expressing mutant p53 ^259H.GUS gene and containing carcinogen-induced pre-mutations, and/or by genetic instability introduced into chromosome 3 by the P{GUS · p53.259H} transposon itself. The effect of the P{GUS · p53.259H} appeared to be carcinogen-specific. It substantially increased the frequency of tumors induced by supermutagenic platinum complex, oxoplatin, and did not increase the frequencies of tumors induced by polycyclic aromatic hydrocarbons, benzo()pyrene and pyrene. In the spectrum of mutations induced by all carcinogens tested, somatic recombination events prevailed over somatic mutations. Hence, carcinogen-specificity of the P{GUS · p53.259H} effect cannot be explained by preferential induction of somatic mutations or somatic recombination by one of the carcinogens. Organ-specificity of the increased frequency of mosaic warts ^– clones induced by P{GUS · p53.259H} was established.     

Observations on the extent and temporal stability of latitudinal clines for alcohol dehydrogenase allozymes and four chromosome inversions in Drosophila melanogaster

The chromosomes of embryos within thelytokous females of Forda spp. were studied, in samples from galls on Pistacia in the Middle East, and from roots of Poaceae in the Middle East, Europe and North America. The nuclei of oogonial cells, oocytes and early cleavage stages have consistently more chromosomes than the nuclei of dividing cells in the somatic tissues of young embryos from the same mothers. Elimination of the extra germ line chromosomes apparently occurs in late cleavage. In F. marginata Koch the germ line chromosome number varies from 25 to 40 in different populations and the somatic cell number varies from 17 to 20; in F. formicaria the germ line has 21–23 chromosomes and somatic nuclei have 18 or 20. In both species variation occurs between samples from galls on Pistacia as well as between populations on roots of Poaceae. The numbers and relative sizes of the eliminated chromosomes also differ between populations. Comparable phenomena in other insects are briefly discussed.     

Role of clozapine in the occurrence of chromosomal abnormalities in human bone-marrow cells in vivo and in cultured lymphocytes in vitro

Summary Bone-marrow chromosomes were examined from 38 mentally and physically retarded and two psychiatric patients who were being treated with a variety of neuropharmacologic drugs. Twenty of these patients used clozapine (Leponex^®). The clastogenic effects of clozapine in vitro were studied in the lymphocyte cultures of three patients-one free of hematologic disease and two who 6 months earlier had had agranulocytosis attributed to the use of clozapine. The mean frequency of cytogenetic abnormalities in the bonemarrow cells of patients who used clozapine was significantly increased (P> 0.05). The two patients who had had agranulocytosis had a greater frequency of cytogenetic abnormalities in their cultured lymphocytes in vivo and in vitro than the patient free of hematologic disease. A clone with a 13/14 chromosome translocation was detected in one of the patients. As all patients received a number of drugs during the in vivo and in vitro studies no definite conclusions could be drawn regarding the role played by clozapine in the occurrence of chromosomal abnormalities.     

Effect of sucrose on polyploidization in early callus cultures of Solanum tuberosum

Leaf segments of a monohaploid, dihaploid and tetraploid genotype of the potato (Solanum tuberosum; x = 12) were cultured on callus-inducing medium with 10, 20, 30 or 40 gl^–1 sucrose. After 5 and 7 days of culture, metaphases contained the somatic or polyploidized number of mono- or diplochromosomes. The percentages of polyploidized metaphases were inversely correlated with the number of chromosome sets of the genotypes. In monohaploid leaf segments the percentages of polyploidized metaphases and of metaphases with diplochromosomes increased when the sucrose concentration was raised from 10 or 20 to 30 gl^–1 and remained constant or decreased from 30 to 40 gl^–1. Higher concentrations of sucrose but not higher osmolalities of the medium due to mannitol induced endoreduplication in more cells. The frequency of polyploidized metaphases and metaphases with diplochromosomes in dihaploid and tetraploid leaf segments remained constant through increases in sucrose concentrations.     

Comparison of the effects of kanamycin and geneticin on regeneration of papaya from root tissue

Kanamycin and geneticin are commonly used for the selection of neomycin phosphotransferase II (npt II) transformed plants. Since papaya tissue is sensitive to both antibiotics, it is difficult to explore their effects on the regeneration process solely based on using non-transformed tissues. Adventitious roots derived from npt II-transgenic and non-transgenic papaya shoots in vitro were used as explants in this investigation. The effects of kanamycin and geneticin on callus formation, embryogenesis, and conversion of somatic embryos to shoots were compared. Callus growth derived from npt II-transformed root explants was apparently enhanced on kanmycin within 50–200 mg l^–1 or on geneticin within 12.5–50 mg l^–1 as compared to those on antibiotic-free controls. The percentages of npt II-transformed somatic embryo-forming callus were not significantly different (16.3–18.3%) on geneticin less than 6.25 mg l^–1 and only slightly reduced (11.2–15.7%) on geneticin within 12.5–50 mg l^–1, whereas, formation of somatic embryos was strongly suppressed on kanamycin media. Conversion rates of npt II-transformed somatic embryos to shoots were not significantly different among all kanamycin or geneticin treatments. Percentages of the callus derived from non-transformed root explants were greatly reduced on the medium containing more than 25 mg l^–1 kanamycin or geneticin, and no somatic embryos formed from untransformed callus on any kanamycin or geneticin media. Our results indicated that somatic embryogenesis of callus derived from npt II-transformed root explants of papaya was strongly inhibited by kanamycin. Thus, to regenerate npt II-transformed cells from papaya root tissue, we recommend using the lower concentration geneticin (12.5–25 mg l^–1) to avoid the adverse effects of kanamycin on embryogenesis.     

Direct somatic embryogenesis fromBegonia gracilis explants

Direct somatic embryogenesis ofBegonia gracilis was achieved from microcultured laminar segments and petioles on Murashige and Skoog medium with 0.5 mg 1^–1 kinetin and 2% coconut water. Somatic embryos were obtained with greater frequency from petiole explants than from leaf blade sections. Under red light (45 mol m^–2 s^–1), approximately 80% of the petiole explants successfully produced somatic embryos but only 30% of the leaf blade sections responded. However, somatic embryos were significantly more abundant on responding lamina explants (60–70 embryos/leaf section) than on petioles (40–50 embryos/petiole). These trends were similar for explants kept in the dark, but overall production was lower. Somatic embryos were produced more quickly (5 weeks) from petioles than from lamina explants (8 weeks). The somatic embryos germinated to produce plantlets and subsequently shoot cultures with the same appearance as the parental clone.Abbreviations BA benzyladenine - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid - SE somatic embryo     

Influence of plant growth regulators,carbon sources and iron on the cyclic secondary somatic embryogenesis and plant regeneration of transgenic cherry rootstock `Colt' (<Emphasis Type="Italic">Prunus avium</Emphasis> × <Emphasis Type="Italic">P. pseudocerasus</Emphasis>)

The frequency of long-term secondary somatic embryogenesis and shoot meristem development from embryogenic masses of the cherry rootstock `Colt' ( Prunus avium × P. pseudocerasus), differentiated from transgenic roots containing the T-DNA of Agrobacterium rhizogenes, has opened the way for genetic improvement by biotechnological techniques. Whole plants were produced by stimulating shoot meristem development from somatic embryos. The combination of 4 mg l^–1 of kinetin and 2% of maltose under illumination stimulated shoot development and, subsequently, whole plants have been recovered by applying 1.5 mg l^–1 kinetin to the rooting medium. Although numerous treatments have been tested involving both embryogenic masses and whole embryos, normal embryo germination was observed sporadically. Cold treatment was effective in stimulating secondary somatic embryogenesis with embryo development to the cotyledonary stage, but did not promote their germination. Similarly, a higher concentration (44–55 mg l^–1) of chelated iron than that commonly used in tissue culture media (36.7 mg l^–1) produced, after 3 weeks in culture, almost a 50% increase in the number of embryos at the cotyledonary stage per embryogenic mass. Among the cytokinins tested, 1 mg l^–1 of 6-benzylaminopurine and 0.1 mg l^–1 of thidiazuron were effective in inducing secondary somatic embryogenesis; however, each of them expressed highest efficiency with specific medium and environmental conditions. Furthermore, application of 1 mg l ^–1 thidiazuron reverted morphogenic callus to non-morphogenic callus, particularly in medium containing 2% sucrose. Finally, hormone free medium with 2% maltose enhanced maturation of the emb-ryos to the normal cotyledonary stage. This paper has improved knowledge of embryo culture and plant production in this important genotype, opening the way for genetic improvement by biotechnological techniques, mainly with the aim of modifying the growth pattern of the canopy of sweet cherry grafted on it.     

Male and female reproductive toxicity induced by sub-chronic ethanol exposure in CF-1 mice

Since genetic damage induced by ethanol exposure is controversial and incomplete and because germ and somatic cells constitute bioindicators for monitoring reproductive toxicity and genotoxic actions of ethanol consumption, the purpose of the present investigation was to evaluate morphological sperm, oocyte alterations and parental genotoxic effects after sub-chronic ethanol intake in the CF-1 outbred mouse strain. Ethanol 10% was administered to CF-1 adult male (treated males, TM) and female (treated females, TF) mice for 27 days, whereas water was given to controls from both sexes too (CM and CF). Post-treatment micronucleus frequency (MN-PCE/1,000/mouse) and gamete morphology were evaluated. To test whether change of female reproductive status results in maternal genotoxicity, CF-1 females received ethanol 10% (exposed group, periconceptionally treated females (PTF)) or water (control group, pregnant control females (PCF)) in drinking water for 17 days previous and up to 10 days of gestation. TM had a high percentage of abnormal spermatozoa vs CM (p < 0.001) and elevated parthenogenetic activated oocyte frequency appeared in TF vs CF (p < 0.001). Sub-chronic ethanol ingestion induced increased MN frequency in TM and TF (p < 0.01). In PTF, where blood alcohol concentrations were between 19–28 mg/dl, very significantly increased MN frequency was found vs PCF (p < 0.01), whereas MN values were similar to TF. These results show that sub-chronic alcohol ingestion in CF-1 mice produces sperm head dysmorphogenesis and oocyte nuclear anomalies, suggesting that morphological abnormalities in germ cells are probably related to parental genotoxicity after ethanol consumption.     

Cellular genetic study of a somatic instability in a tobacco mutant: in vitro isolation of valine-resistant spontaneous mutants

Summary A chlorophyll-deficient mutant line of tobacco (Nicotiana tabacum), named tl, displays spontaneously on leaves green, white, and twinned green/white somatic variations at high frequencies (10^–3 to 10^–2). The frequency of cell events leading to the somatic variations has been shown to be closely dependent on the stage of differentiation of cells during plant development. The activity of transposable elements is suspected in the tl genotype, and a study of its mutagenic ability was performed by selecting in vitro new mutant cellular types. The cellular marker chosen was the resistance to toxic doses of valine conferred by a permeation deficiency. A reproducible method allowing efficient selection of valine-resistant mutant clones from haploid tobacco mesophyll protoplast-derived cells was used. In 10 out of 12 experiments, the frequency of spontaneous valine-resistant clones obtained with the wild-type control was null for cell populations tested to the 10⁶. On the other hand, spontaneous valine-resistant clones were repeatedly isolated at variable and sometimes high frequencies (greater than 10^–3) from cell populations of the tl type. Valine resistance of plants regenerated from these clones was transmitted to the progeny as a single monogenic mutation. These results indicate an increased mutagenic ability of the tl genotype, as compared to the wild-type line.     

A comparison of the protective action of chicken and quail egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa

Egg yolk-based diluents provide adequate cryoprotection for the sperm of several mammalian species. Traditionally, chicken egg yolk has been used as additive for the freeze preservation of spermatozoa because of its wide availability. Variations in the chemical composition of the egg yolk of different avian species appear to influence the protection afforded during cooling, freezing, and thawing. The aim of the present study was to assess the use of quail egg yolk as a novel additive for the epididymal spermatozoa of a threatened wild ruminant species—the Spanish ibex—and to compare its efficacy with chicken egg yolk. Epididymal spermatozoa were diluted using a Tris–citric acid–glucose medium (TCG) composed of 3.8% Tris (w v⁻¹), 2.2% citric acid (w v⁻¹), 0.6% glucose (w v⁻¹), 5% glycerol (v v⁻¹), and 6% egg yolk (v v⁻¹). Sperm masses from the right epididymes were diluted with TCG-6% chicken egg yolk medium, while those from the left were diluted with TCG-6% quail egg yolk. The thawed spermatozoa preserved with TCG-6% quail egg yolk extender exhibited lower motility (P < 0.001), membrane integrity (P < 0.001), and viability (P < 0.01) than those diluted with the TCG-6% chicken egg yolk extender. The fertility of spermatozoa frozen in TCG-6% chicken egg yolk tended to be higher than in those frozen with TCG-6% quail egg yolk (63.3% vs 36.4%, P = 0.19). These results show that quail egg yolk offers no advantages over chicken egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa.     

High Frequency Somatic Embryogenesis in Cotton

A highly reproducible system for efficient somatic embryogenesis was developed to regenerate plantlets from cotton (Gossypium hirsutum L.) cultivars (Nazilli M-503 and Nazilli 143). Shoot apices, hypocotyls and nodes of 10-d-old seedlings were used as explants. High frequency (100 %) embryogenic calli was initiated from all tested explants on Murashige and Skoog (1962) (MS) media supplemented with 1 g dm^–3 polyvinylpyrrolidone (PVP), 1 mg dm^–3 6-benzylaminopurine (BAP), 0.5 mg dm^–3 kinetin for Nazilli M-503 and 1 g dm^–3 PVP, 2 mg dm^–3 BAP, 0.5 mg dm^–3 kinetin for Nazilli-143. Globular stage somatic embryos were produced 4 months after transfer to hormone-free MS medium supplemented with 1 g dm^–3 PVP. Subsequent subculture of globular embryos every 3 weeks on hormone-free MS medium led to the development of torpedo and cotyledonary stage embryos with the frequency of 75 and 83.2 % from hypocotyl explants of Nazilli M-503 and Nazilli-143, respectively. Afterwards, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination and development into plantlets. The highest germination frequency (42.9 %) for Nazilli M-503 somatic embryos were obtained on hormone-free MS medium after 5 months with hypocotyl explants, whereas germination frequencies of Nazilli-143 embryos from hypocotyl, node and apex explants varied between 22 – 30 %.     

Taenia crassiceps: A secretion-substance of low molecular weight leads to disruption and apoptosis of seminiferous epithelium cells in male mice

The present research was performed to isolate and study the effects of a low molecular weight (<1300 Da) parasite-associated substance, obtained from peritoneal fluids of female mice infected with Taenia crassiceps cysticerci, on seminiferous epithelium cells of male mice testis. The results showed an intense disruption of Sertoli cells and germ cells within the seminiferous tubules of experimental mice, along with the destruction of their gap junction (GJ). Significant generalized apoptosis of germ cells within seminiferous tubules was determined by TUNEL staining (P = 0.0159). In addition, a significant number of infiltrating macrophages were found in the luminal space of these seminiferous tubules (P < 0.0001). Finally, electron microscopy studies revealed structural and morphological abnormalities in the somatic cells (Sertoli and Leydig cells) and in the germ cells, primarily in the round and elongate spermatids.     

Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.)

Summary We report the isolation and regeneration of protoplasts from an embryogenic banana (Musa spp.) cell suspension culture initiated from in vitro proliferating meristems. A high yielding isolation method (up to 6×10⁷ protoplasts.ml^–1 packed cells) is discussed. Optimal regeneration, with more than 50% of the protoplasts showing initial cell division, occurred when high inoculation densities (10⁶ protoplasts.ml^–1) or nurse cultures were applied. Under these conditions, the frequency of microcolony formation was 20–40%. These microcolonies developed directly, without an intervening callus phase, into somatic embryos which later germinated and formed plantlets.     

Radiation protection of human lymphocyte chromosomes in vitro by orientin and vicenin

Orientin (Ot) and Vicenin (Vc), two water-soluble flavonoids isolated from the leaves of Indian holy basil Ocimum sanctum have shown significant protection against radiation lethality and chromosomal aberrations in vivo. In the present study the protective effect of Ot and Vc against radiation induced chromosome damage in cultured human peripheral lymphocytes was determined by micronucleus test. In order to select the most effective drug concentration, fresh whole blood was exposed to 4 Gy of cobalt-60 γ-radiation with or without a 30 min pre-treatment with 6.25, 12.5, 15.0, 17.5 or 20 μM of Ot/Vc. Micronucleus (MN) assay was done by cytochalasin induced cytokinesis block method. Radiation significantly increased the MN frequency (16 times normal). Pre-treatment with either Ot or Vc at all concentrations significantly (P<0.05–0.001) reduced the MN count in a concentration dependent manner, with the optimum effect at 17.5 μM. Therefore, fresh blood samples were incubated with/without 17.5 μM Ot/Vc for 30 min and then exposed to 0.5–4 Gy of γ-radiation. Radiation increased the MN frequency linearly (r²=0.99) with dose. Pre-treatment with Ot or Vc significantly (P<0.01–0.001) reduced the MN counts to 51–67% of RT alone values, giving DMFs of 2.62 (Ot) and 2.48 (Vc). Both the compounds showed significant antioxidant activity in vitro at the above concentrations, which was significantly higher than that of DMSO at equimolar concentrations. Thus, the results demonstrate that both the flavonoids give significant protection to the human lymphocytes against the clastogenic effect of radiation at low, non-toxic concentrations. The radioprotection seems to be associated with their antioxidant activity. The clinical potential of these protectors in cancer therapy needs to be investigated.     

Nonhomologous pairing in oogonia and ganglia of Drosophila melanogaster

A high frequency of chromosome pairing between an X-duplication (Dp) and a compound 4 (–44), each of which lacks its homologue in the genome, has been observed in somatic ganglia cells in female and male larvae as well as in oogonia of adult Drosophila melanogaster. Three Dp's of different sizes were used. Pairing was determined both by inspection and by precise measurement of the distances between chromosomes; the two methods agreed in virtually every case. Nonhomologous pairing frequencies ranged from 23.0 to 62.6%, showing a strong dependency on size. The Dp that was closest in size to –44 produced the greatest frequency of pairing in each of the three tissues. Homologues in the same cells paired with a frequency of 89% or greater. The X and Y in the male ganglia showed a pairing frequency of only 45.7%, thus resembling nonhomologues in their pairing behavior. Genetic studies were also carried out on the three genotypes. Segregation data for the Dp's and –44 established that the same pattern of pairing observed in the mitotic cells was present in the oocytes — the Dp which was closest in size to the –44 again producing the greatest frequency of pairing. In the oocytes the pairing frequencies were much higher than those observed in the mitotic cells.From a dissertation submitted to the University of Tennessee in partial fulfillment of the requirements for the degree of Doctor of Philosophy. This work was jointly sponsored by National Science Foundation Grant No. GZ-1323, and by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.     

Toxic Bacillus pumilus from indoor air, Recycled Paper Pulp,Norway Spruce, Food Poisoning Outbreaks and Clinical Samples

Forty-four B. pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B. cereus and B. licheniformis. The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B. licheniformis, whereas the B. pumilus food poisoning and clinical isolates were lower in toxicity. The type strain also produced inhibitory substances. The toxic substances were insensitive to heat (100 °C, 20 min), to pH 2 or pH 10 and to digestion with pronase. The substances were readily soluble in methanol and chloroform, but less soluble in toluene. Exposure of boar spermatozoa to 1–10 μg ml^–1 (EC₅₀) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction. No change was observed in human peripheral blood lymphocytes exposed to concentrations of B. pumilus extract that affected spermatozoa. The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition.