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  • 1
    Electronic Resource
    Electronic Resource
    Transfer of the Brassica tournefortii cytoplasm to B. napus for the production of cytoplasmic male sterile B. napus (1996)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 96 (1996), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The donor-recipient fusion method was used to combine the cytoplasm of Brassica lournefortii with the nucleus of B. napus for the production of cytoplasmic male sterile (CMS) plants. X-ray-irradiated mesophyll protoplasts of B. tournefortii were fused with iodoacetamide (lOA)-inactivated hypocotye protoplasts of B. napus. Selective conditions of IOA concentrations and X-ray doses were determined, which resulted in recovery of fusion products and inhibition of further growth of unfused parental cells. In total, 54 plants were obtained from different fusion experiments, of which 25 were verified as cybrids or partial hybrids. Mitochondrial DNA (mtDNA) analyses using 5 mitochondrial gene probes revealed that 20 of the 25 fusion-derived plants had mtDNA either identical, or with varying degrees of similarity, to B. lournefortii. These plants were classified into four groups on the basis of pollen viability and number. Seven plants were categorised as male sterile since they did not produce pollen or had non-viable pollen. Of the male sterile plants, five had a mtDNA pattern identical to B. tournefortii and a nuclear DNA content corresponding to B. napus. The nuclear-mito-chondrial constitution of these plants thus indicates that the combination of B. tournefortii cytoplasm with the B. napus nucleus results in CMS. Furthermore, mtDNA analysis of the two additional male sterile plants which displayed a rearranged mtDNA, revealed that the only mtDNA similarity shared among all male sterile plants was specific for B. tournefortii atp6 pattern. This indicates that the atp6 region of B. tournefortii may be involved in the expression of CMS.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1996.tb00192.x
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  • 2
    Electronic Resource
    Electronic Resource
    Transformation of Brassica napus by using the aadA gene as selectable marker and inheritance studies of the marker genes (1994)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 92 (1994), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: An Agrobacterium tumefaciens-mediated transformation system for Brassica napus has been improved. We investigated several marker genes for transformation of Brassica napus, and the aadA gene, which confers resistance to streptomycin and spectinomycin, was found to be the most suitable. Forty-three out of 193 putative transformants in the T1 generation were investigated by Southern blot analysis. Transformants containing a range of 1 to 10 integrated T-DNA copies per genome were found. Total DNA from 35 plants showed hybridisation to both the aadA and the nptll marker gene probes, from 5 plants only to one marker gene probe and from 3 plants DNA did not hybridise to any of the gene probes. Furthermore, more complex integration patterns such as direct repeated copies of the T-DNA, both as tandem and inverted copies, were observed. Inheritance of the marker genes in the T2 generation was studied in 37 of the plants. This revealed that 22% of the plants that contained both marker genes, segregated as one single locus (3:1) for both genes, while 46% of the plants gave a segregation pattern corresponding to one T-DNA locus for at least one of the marker genes. Moreover, these inheritance patterns appeared to be more or less independent of the number of genes seen in the Southern blot analysis of the T, generation. In this study we show that the introduced marker genes are inherited by the T; generation in a less predictable way than was earlier reported for B. napus.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1994.tb06652.x
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  • 3
    Electronic Resource
    Electronic Resource
    Electroporation of rapeseed protoplasts – transient and stable transformation (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 88 (1993), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Protoplasts of Brassica napus hypocotyls were transfected using electroporation. Parameters such as discharge potential, protoplast density and buffer constituents were tested to determine the most suitable conditions for gene transfer. To monitor the introduction of DNA into protoplasts a plasmid containing the β-glucuronidase (EC 3.2.1.31), and the neomycin phospotransferase (EC 2.7.1.95) genes was used. By using this construct, expression of a screenable marker gene for transient expression analysis as well as an antibiotic resistance marker gene for selection of stable transformants were obtained. Refined electroporation conditions resulted in a frequency of 0.1% transiently transformed protoplasts. Microcalluses were cultured under selective conditions in a bead-type culture system. Resistant callus, with an absolute transformation frequency of 4.9 × 10−5 and a relative transformation frequency of 0.3% could be achieved. X-ray irradiation of newly electroporated protoplasts did not enhance absolute transformation frequencies. From some of the resistant calluses, transgenic plants could be regenerated which were characterized by molecular analysis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1993.tb01378.x
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  • 4
    Electronic Resource
    Electronic Resource
    Fusion of male-sterile tobacco causes modifications of mtDNA leading to changes in floral morphology and restoration of fertility in cybrid plants (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: Protoplasts from male-sterile Nicotiana tabacum cultivars with cytoplasms of N. suaveolens, N. bigelovii and N. undulata were fused in different pairwise combinations. Cybrid plants were obtained and categorized according to their flower morphologies into groups as parental male-sterile types, recombined biparentals, novel male-steriles and male fertiles. Restriction enzyme analyses and DNA gel blot hybridizations of the mitochondrial DNA (mtDNA) of these cybrid plants revealed that all plants with parental male-sterile morphologies had banding patterns identical to the parental patterns, whereas all cybrids with flower morphologies different from their parents had novel patterns. Several restriction fragments were found to be unique to the mtDNA of male-fertile plants with perfectly restored stamen morphologies when compared to the mtDNA from cybrid plants with incomplete restoration. MtDNA gel blot hybridization analysis of fertile plants revealed that the probe for atpA hybridized to two restriction fragments, one from each parent. Further, fertile plants with complete stamen restoration lacked an expressed sequence in the 3’flanking region of atpA, in contrast to both male-sterile parents.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1992.tb04743.x
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  • 5
    Electronic Resource
    Electronic Resource
    High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae (1984)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 61 (1984), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Protoplasts isolated from 4-day-old hypocotyls of various species of Brassica (Brassica napus, B. campestris and B. oleracea) produced callus with high efficiency in media containing casein hydrolysate and high concentrations of the auxin 2,4-D (4.5 μM). Cell division began after 24 h and 60% of the cells had divided after 48 h. In contrast, protoplasts isolated from stem and mesophyll of plants grown in vitro or in the greenhouse began to divide after a delay of 3–5 days. In these cases 40–50% of the cells had divided after 5 days as compared to 70% for hypocotyl protoplasts. To obtain a high frequency of regeneration, rapidly growing calli were transferred to media having a high cytokinin:auxin ratio as early as possible, usually 3 weeks after protoplast isolation. The average regeneration frequency for calli obtained from mesophyll protoplasts was 50%, while as many as 70% of the calli derived from hypocotyl protoplasts of B. napus regenerated plantlets on a medium containing zeatin (9.1 μM) and IAA (0.6 μM). On the same medium regeneration of Brassica oleracea was obtained. A low percentage of calli (1%) from Brassica campestris formed shoots when cultured on a combination of zeatin (4.6 μM), BA (4.4 μM) and IAA (0.6 μM).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1984.tb06097.x
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  • 6
    Electronic Resource
    Electronic Resource
    Protease activities in non-embryogenic and embryogenic carrot cell strains during callus growth and embryo formation (1984)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 62 (1984), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Embryogenic and non-embryogenic cell strains of Daucus carota L. were examined for their protease activity using a wide range of chromogenic synthetic peptides as substrates. High arginine-specific activity was present in all strains, but no protease activity “specific” for embryogenic or non-embryogenic strains could be detected with the substrates tested. The specific protease activity was 5–10 times higher in the non-embryogenic as compared to the embryogenic strain for most tested substrates, and this difference was not due to release of proteases in the latter. All strains showed a decrease in protease activity when cultured in media without 2,4-dichlorophenoxyacetic acid, but the embryos had high protease activity in comparison with the nondifferentiated cell aggregates. In the latter aggregates, hydrolyzing activity towards three of the substrates (H-D-Phe-Pip-Arg-p-nitroanilide, Suc-Ala-Pro-Phe-p-nitro-anilide and Bz-Phe-Val-Arg-p-nitroanilide) was absent, whereas the embryos were able to hydrolyze them.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1984.tb04603.x
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  • 7
    Electronic Resource
    Electronic Resource
    Somatic Hybridization of Nitrate Reductase Deficient Mutants of Nicotiana tabacum by Protoplast Fusion (1978)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 44 (1978), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Protoplasts were isolated from two mutant cell lines of Nicotiana tabacum L. cv. Gatersleben and fused with the aid of polyethylene glycol. Both mutants lacked nitrate reductase and were thus auxotrophic for reduced nitrogen. The fusion resulted in a high frequency of hybrid cells which were detected by their regained ability to grow in media containing nitrate as sole nitrogen source. Thus, the two mutants were found to complement each other in the hybrids. In control experiments, back mutation and cross-feeding were excluded as possible explanations for the occurrence of cell lines utilizing nitrate. A total of 1061 hybrid lines capable of sustained proliferation were isolated. Some of them were further characterized with respect to nitrate reductase activity, chlorate sensitivity, chromosome number, and shoot formation.The results demonstrate that protoplast fusion can be used for the genetic analysis of cell variants of higher plants and that nitrate reductase-deficient mutants provide efficient selective systems for hybrid cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1978.tb08631.x
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  • 8
    Electronic Resource
    Electronic Resource
    Pretreatment of Cell Suspensions as a Method to Increase the Protoplast Yield of Haplopappus gracilis (1977)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 40 (1977), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: A low yield of free protoplasts was obtained when fast-growing cell suspensions of Haplopappus gracilis were treated with Driselase, an active mixture of cellulase and pectinase. If the cells in the suspension culture were pretreated by increased levels of auxin, reduced sugar concentration, addition of sulphur-containing amino acids, or by the reducing agent mercaptoethanol, protoplasts were released from a higher proportion of the cells. These pretreatments did not adversely effect division of the protoplasts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1977.tb04078.x
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  • 9
    Electronic Resource
    Electronic Resource
    Agglutinating Effects of Concanavalin A on Isolated Protoplasts of Daucus carota (1974)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 31 (1974), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The agglutinating effects of Concanavalin A (Con A) on protoplasts isolated from cell suspensions of Daucus carota were studied. Con A was shown to agglutinate the plant protoplasts in a manner similar to the way some animal cells are agglutinated. The agglutination process is dependent on the Concanavalin A concentration, protoplast density, treatment time, the temperature, and the membrane condition, α-D-Methylgluco-pyranosid completely inhibited Con A induced agglutination. The results are discussed in relation to membrane structure and morphology.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1974.tb03696.x
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  • 10
    Electronic Resource
    Electronic Resource
    Somatic hybridization in Nicotiana: Segregation of organellar traits among hybrid and cybrid plants (1981)
    Springer
    Planta 153 (1981), S. 504-510 
    Details
    ISSN: 1432-2048
    Keywords: Chloroplast ; Cybrid ; Nicotiana ; Protoplast ; Somatic hybrid ; Sterility, male
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts from a nitrate reductase-deficient mutant of Nicotiana tabacum L. were fused with protoplasts from a stamen-less, cytoplasmically malesterile cultivar of tobacco containing the cytoplasm from N. suaveolens Lehm. Plants were regenerated from the fused protoplasts and characterized with respect to stamen development, chromosome number, and chloroplast composition. Of 29 regenerated plants, stamen production was restored in 26 plants and pollen production in 22. One plant was male sterile and two plants have never flowered. Analysis of the electrophoretic mobility of ribulose-1,5-bisphosphate carboxylase (RuBPcase) showed that 19 of the plants contained RuBPcase of the N. suaveolens type, six plants contained enzyme of the N. tabacum type, and four plants contained both types. Analysis of resistance to tentoxin in seedlings from 20 of the plants demonstrated that 14 had N. suaveolens-type chloroplasts, three had N. tabacum type, and three contained both types. Many of the plants which produced stamens and pollen still contained chloroplasts of the N. suaveolens type. Thus, the trait of cytoplasmic male sterility in tobacco is not an expression of the type of chloroplast genetic material.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00385533
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