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  • 1
    Electronic Resource
    Electronic Resource
    Molecular analysis of nuclear genes in somatic bybrids (1992)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 85 (1992), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: This paper reviews recent developments in the use of molecular probes for analyzing the genetic makeup of somatic hybrids. Successful application of somatic hybridization to the interspecific transfer of traits encoded in the nucleus is still having limited success. A major difficulty is hybrid infertility, particularly in hybrids between sexually incompatible species. The formation of asymmetric hybrids is being explored as an approach for improving hybrid fertility. Evaluation of the degree of chromosome elimination and chromosome stability and instability in asymmetric hybrids is difficult when the traditional approaches of chromosome counting and isozyme analysis are used. Two new approaches are resolving this difficulty. The use of species-specific repetitive DNA probes in dot blotting and in situ hybridization to chromosomes is providing quantitative data on chromosome elimination and allows detection of translocations. Use of restriction fragment length polymorphism (RFLP) probes for analysis of hybrids between genetically mapped species makes it possible to account for the presence or absence of individual chromosomes and chromosomes arms. Wider use of such molecular probes should greatly improve our understanding of the genetics of both symmetric and asymmetric somatic hybrids and may lead to new strategies for the effective interspecific transfer of nucleus-encoded traits by protoplast fusion.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1992.tb04739.x
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  • 2
    Electronic Resource
    Electronic Resource
    Protein synthesis and auxin-induced growth: Inhibitor studies (1979)
    Springer
    Planta 145 (1979), S. 437-442 
    Details
    ISSN: 1432-2048
    Keywords: Auxin ; Avena ; Cell elongation ; Protein synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have compared the effects of cycloheximide (CHI) and two other rapid and effective inhibitors of protein synthesis, pactamycin and 2-(4-methyl-2,6-dinitroanilino)-N-methyl proprionamide (MDMP), on protein synthesis, respiration, auxin-induced growth and H+-excreation of Avena sativa L. coleoptiles. All three compounds inhibit protein synthesis without affecting respiration. The effectiveness of the inhibitors against H+-excretion and growth correlates with their ability to inhibit protein synthesis. Both CHI and MDMP inhibit auxin-induced H+-excretion after a latent period of 5–8 min, and inhibit growth after a 8–10-min lag. These results support the idea that continued protein synthesis is required in the initial stages of the growth-promoting action of auxin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00380097
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  • 3
    Electronic Resource
    Electronic Resource
    Separation of tonoplast and plasma membrane potential and resistance in cells of oat coleoptiles (1982)
    Springer
    The journal of membrane biology 66 (1982), S. 15-23 
    Details
    ISSN: 1432-1424
    Keywords: plant cells ; Avena ; vacuolar membrane ; plasmalemma ; membrane potential ; membrane resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Membrane potential and resistance were recorded from parenchymal cells of oat (Avena) coleoptiles, using one and two intracellular electrodes. Membrane potential is largest (−100 mV) in impalements with low input resistance (2–4 MΩ), and is less negative (−50 mV) in penetrations with high input resistance (〉 20 mΩ). The interpretation is that the electrode lodges in the vacuole which is positive to the cytoplasm (but still negative to the external solution), and that measurements of net membrane potential are compromised to varying degrees by leakage shunts introduced across the high resistance vacuolar membrane by the electrode. This conclusion is supported by several additional lines of evidence. (1) It is possible to convert large-R/small-V impalements into small-R/large-V penetrations by passing excess current through the electrode or by briefly ‘ringing’ the capacitance neutralization circuit in the amplifier. The cells usually recover their resistance in a few minutes, with a concomitant decrease in the negativity of the membrane potential. (2) Changes in external [K] affect the measuree potential by an amount that is independent of the input resistance of the impalement. This is consistent with an effect of [K] o on the potential of the plasma membrane and the occurrence of leakage shunts primarily at the tonoplast. (3) Quantitatively, the effects of a change in [K] o on resistance indicate that nearly 90 percent of the input resistance of unshunted cells resides in the tonoplast. (4) The effects of metabolic inhibitors (DNP, CN−) on potential are smaller in large-R than in small-R impalements. This observation suggests there are electrogenic pumps contributing to the membrane potential at both the plasmalemma and tonoplast. Finally, we conclude that with an electrode in the vacuole it is possible to record potentials that are dominated by the contribution of the plasma membrane, provided care is taken to select impalements combining both large, negative potential and low input resistance.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01868478
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  • 4
    Electronic Resource
    Electronic Resource
    Bromodeoxy uridine combined with UV light and gamma irradiation promotes the production of asymmetric somatic hybrid calli (1996)
    Springer
    Plant cell reports 15 (1996), S. 986-990 
    Details
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The degree of gamma- or X-ray-induced donor chromosome elimination in asymmetric somatic hybrids is highly variable. Here the beneficial use of bromodeoxyuridine and UV light as additional chromosome destabilizing agents is described. Protoplasts of Nicotiana tabacum were fused with protoplasts of Nicotiana plumbaginifolia (Np) that carried the kanamycin-resistance and glucuronidase (GUS) genes on separate chromosomes. Prior to fusion, the Np donor protoplasts were pretreated with bromodeoxyuridine and then were inactivated by treatment with iodoacetate ± UV light ± 200 Gy gamma irradiation. Hybrids were selected on medium containing kanamycin. The elimination of Np DNA was assessed by scoring of the fraction of hybrid calli that expressed GUS and by dot-blot analysis using a Np-specific probe. Gamma irradiation alone resulted in elimination of 50% of Np DNA. Pretreatment with bromodeoxyuridine (10 μM) followed by 2.5 to 5 min UV light resulted in the elimination of 35–45% of the donor genome, but incorporation of bromodeoxyuridine (10 μM) followed by 2.5 to 5 min UV light and 200 Gy gamma irradiation resulted in 85 to 90% elimination of Np DNA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00231601
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  • 5
    Electronic Resource
    Electronic Resource
    Protein patterns in the oat coleoptile as influenced by auxin and by protein turnover (1980)
    Springer
    Planta 148 (1980), S. 429-436 
    Details
    ISSN: 1432-2048
    Keywords: Auxin action ; Avena ; Coleoptile ; Protein synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Synthesis of growth-limiting proteins (GLP) is required for continued auxin-induced elongation of oat (Avena sativa L.) coleoptiles. In order to determine whether GLP synthesis is dependent or independent of auxin, a double-labeling ratio technique, coupled with disc-gel electrophoresis, has been used to assess the effect of auxin on the pattern of protein synthesis. Sections were peeled to enhance amino-acid uptake; proteins were labeled with [14C]- or [3H] leucine in the presence or absence of indole-3-acetic acid for 40 min to 6 h, and were separated into soluble, membrane-associated, and wall-associated fractions. Regardless of the conditions used, or the protein fraction examined, no changes in response to auxin were detected in the pattern of protein synthesis. In order to escape detection by this technique an auxin-induced protein would have to comprise less than 0.75% of the total newly synthesized protein. Thus the synthesis of GLP appears to be independent of auxin. The same technique has been used to follow protein turnover. During the chase, proteins are initially degraded at an average rate of 8% h−1, and some protein bands showed as much as 14% h−1 degradation. No protein was detected which had a turnover rate as rapid as the GLP.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02395310
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  • 6
    Electronic Resource
    Electronic Resource
    Rapid response of the plasma-membrane potential in oat coleoptiles to auxin and other weak acids (1983)
    Springer
    Planta 159 (1983), S. 231-237 
    Details
    ISSN: 1432-2048
    Keywords: Acid, weak, uptake ; Auxin, action and specificity ; Avena, IAA and membrane potential ; Coleoptile ; Cytoplasmic pH ; Electrogenic proton transport ; Plasma membrane potential
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have compared the effects of the auxin, indole-3-acetic acid (IAA) with that of other weak acids on the plasma-membrane potential of oat (Avena sativa L.) coleoptile cells. Cells treated with 1 μM IAA at pH 6 depolarize 20–25 mV in 10–12 min, but they then repolarize, until by 20–25 min their potentials are about 25 mV more negative than the initial value. Similar concentrations of benzoic and butyric acids cause the initial depolarization, but not the subsequent hyperpolarization. The hyperpolarization is therefore specific to IAA. All the weak acids, including IAA, evoke a rapid hyperpolarization when their concentrations are raised to 10 mM. This result indicates that at high concentrations, the uptake of undissociated weak acids activates electrogenic proton pumping, most likely by lowering cytoplasmic pH. In contrast, the hyperpolarization observed with concentrations of IAA four orders of magnitude lower appears to be a specific hormonal effect. This specific, auxin-induced hyperpolarization occurs at the same time as the initiation of net proton secretion and supports the hypothesis that auxin initiates extension growth by increasing proton pumping.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00397530
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  • 7
    Electronic Resource
    Electronic Resource
    Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana (1985)
    Springer
    Planta 165 (1985), S. 217-224 
    Details
    ISSN: 1432-2048
    Keywords: Electrical fusion ; Heterokaryon ; Nicotiana (protoplast fusion) ; Protoplast fusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·105 protoplasts (0.5 ml of cells at 106/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00395044
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  • 8
    Electronic Resource
    Electronic Resource
    Protein patterns in the oat coleoptile as influenced by auxin and by protein turnover (1980)
    Springer
    Planta 148 (1980), S. 429-436 
    Details
    ISSN: 1432-2048
    Keywords: Auxin action ; Avena ; Coleoptile ; Protein synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Synthesis of growth-limiting proteins (GLP) is required for continued auxin-induced elongation of oat (Avena sativa L.) coleoptiles. In order to determine whether GLP synthesis is dependent or independent of auxin, a double-labeling ratio technique, coupled with disc-gel electrophoresis, has been used to assess the effect of auxin on the pattern of protein synthesis. Sections were peeled to enhance amino-acid uptake; proteins were labeled with [14C]- or [3H] leucine in the presence or absence of indole-3-acetic acid for 40 min to 6 h, and were separated into soluble, membrane-associated, and wall-associated fractions. Regardless of the conditions used, or the protein fraction examined, no changes in response to auxin were detected in the pattern of protein synthesis. In order to escape detection by this technique an auxin-induced protein would have to comprise less than 0.75% of the total newly synthesized protein. Thus the synthesis of GLP appears to be independent of auxin. The same technique has been used to follow protein turnover. During the chase, proteins are initially degraded at an average rate of 8% h−1, and some protein bands showed as much as 14% h−1 degradation. No protein was detected which had a turnover rate as rapid as the GLP.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00552655
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  • 9
    Electronic Resource
    Electronic Resource
    Bromodeoxyuridine combined with UV light and gamma irradiation promotes the production of asymmetric somatic hybrid calli (1996)
    Springer
    Plant cell reports 15 (1996), S. 986-990 
    Details
    ISSN: 1432-203X
    Keywords: Abbreviations: BrdU, bromodeoxyuridine (5‐bromo‐2'‐deoxyuridine); GUS, glucuronidase; HPT hygromycin B phosphotransferase; KmR, kanamycin resistant, Np, Nicotiana plumbaginifolia; NPT, neomycin phosphotransferase II; Nt, Nicotiana tabacum; UV, ultraviolet
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary. The degree of gamma‐ or X‐ray‐induced donor chromosome elimination in asymmetric somatic hybrids is highly variable. Here the beneficial use of bromodeoxyuridine and UV light as additional chromosome destabilizing agents is described. Protoplasts of Nicotiana tabacum were fused with protoplasts of Nicotiana plumbaginifolia (Np) that carried the kanamycin‐resistance and glucuronidase (GUS) genes on separate chromosomes. Prior to fusion, the Np donor protoplasts were pretreated with bromodeoxyuridine and then were inactivated by treatment with iodoacetate ± UV light ± 200 Gy gamma irradiation. Hybrids were selected on medium containing kanamycin. The elimination of Np DNA was assessed by scoring of the fraction of hybrid calli that expressed GUS and by dot‐blot analysis using a Np‐specific probe. gamma irradiation alone resulted in elimination of 50% of Np DNA. Pretreatment with bromodeoxyuridine (10 μM) followed by 2.5 to 5 min UV light resulted in the elimination of 35–45% of the donor genome, but incorporation of bromodeoxyuridine (10 μM) followed by 2,5 to 5 min UV light and 200 Gy gamma irradiation resulted in 85 to 90% elimination of Np DNA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050162
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  • 10
    Electronic Resource
    Electronic Resource
    Factors affecting protoplast electrofusion efficiency (1987)
    Springer
    Plant cell reports 6 (1987), S. 337-340 
    Details
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The electrofusion efficiency of protoplasts isolated from a carrot (Daucus carota) suspension culture was increased by treatment with 0.1 mg/ml lysolecithin, 2.5% dimethylsulfoxide (DMSO), or 0.5 mM Ca2+. The lysolecithin and DMSO treatments substantially increased protoplast lysis, whereas calcium treatment did not. The enzymes used for protoplast isolation were also found to have a dramatic effect on the efficiency of fusion. A mixture of Cellulysin and Driselase led to a two-fold enhancement of fusion as compared with Driselase alone. The stimulation by Cellulysin appears to be due to enzymatic modification of the cell surface. However, comparison of the time course for wall digestion with the development of susceptibility to electrofusion suggests that the effect of Cellulysin is not simply due to removal of the cell wall. Brief treatment of the cells with pronase or proteinase K also doubled the efficiency of fusion. Taken together, these results indicate that electrofusion efficiency can be enhanced by the method used for protoplast isolation; they also suggest that modification of membrane/cell-surface proteins during protoplast isolation may be particularly important in determining electrofusion efficiencies.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00269554
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