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  • 1
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The expression of cytosolic and plastid pyruvate kinase (PK; E.C. 2.7.1.40) in various tissues of tobacco (Nicotiana tabacum cv. Petit Havana SR1) was investigated. To facilitate this study, a cDNA clone for cytosolic pyruvate kinase (PKc) was isolated from a tobacco seed cDNA expression library. This cDNA has an open reading frame capable of encoding a 508-amino acid polypeptide with a predicted molecular mass of 55.1 kDa. The deduced amino acid sequence has 76% identity with the sequence of potato PKc. Southern blot analysis shows that N. tabacum contains two copies of the PKc gene, each derived from the single gene found in its progenitors, N. tomentosiformis and N. sylvestris. Northern blots detected a 1.9-kb band in flower, seed, root, stem and leaf tissues of N. tabacum. Immunoblotting using anti-[castor oil seed (COS) PKc]-immunoglobulin G (IgG) detected 58- and 56-kDa polypeptides in all tobacco tissues examined. In developing seeds, mRNA levels were low except for a peak at 8 days post anthesis (dpa), whereas the highest level of the PKc polypeptides was detected 10–16 dpa. The persistence of PKc well after the peak in mRNA levels suggests that PKc is stable in developing seeds. Our previous studies have shown that leucoplast PK (PKp) mRNA could be detected throughout tobacco seed development as well as in somatic tissues. In developing seeds, steady state PKc mRNA levels showed a broad peak of accumulation from 8–20 dpa, reaching maximum levels at 16 dpa. In the present study, polypeptides of 63 and 60 kDa were detected on immunoblots probed with anti-(COS PKp)IgG in developing tobacco seeds 10–20 dpa. The observed pattern of PKp protein accumulation closely correlated with the profile of steady state mRNA levels suggesting that this isoenzyme has a much greater rate of turnover than that of PKc. In contrast to PKc, there was no detectable accumulation of PKp protein at other times during seed development or in somatic tissues even though significant levels of PKp mRNA could be detected. The differences in the protein accumulation and mRNA expression patterns of PKc and PKp suggest that the tissue-specific and developmental expression of these isoenzymes in tobacco may be controlled by independent transcriptional and post-transcriptional mechanisms.
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  • 2
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Company
    Nature biotechnology 5 (1987), S. 1053-1056 
    ISSN: 1546-1696
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: [Auszug] A recombinant cauliflower mosaic virus (Ca-MTII) was constructed by inserting a cDNA clone of Chinese hamster metallothionein II into the open reading frame II of the cloned virus pCa-BB1. Systemically-infected Brassica campestris tissue contained metallothionein at a level of 0.5 percent of the ...
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  • 3
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Alfalfa (Medicago sativa L.) protoplasts derived from cell suspension cultures divided inefficiently in liquid culture. The onset of cell division activity occurred synchronously among the protoplasts; however, many were blocked at cytokinesis and therefore did not complete first division. Very few of the cells that began to divide continued to do so. Immobilization of protoplasts in agarose after 1 to 4 days in liquid culture overcame this inhibition of division. Continuous growth in agarose was restricted and therefore microcolonies were transferred to agar medium to complete callus development. Plating efficiencies of 2–10% were achieved within 30 days of protoplast isolation. The agarose treatment was responsible for a 5- to 30-fold improvement in plating efficiency.
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  • 4
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The CAT gene which codes for the enzyme chloramphenicol acetyltransferase was found to be ineffective as a reporter gene in cells and tissues of Brassica species. High levels of endogenous CAT activity were found to be widespread among this genus and did not appear to be distributed in a tissue- or cell-specific manner. Moreover, the presence of an inhibitor of CAT activity was discovered in Brassica napus and Brassica juncea. This inhibitor appeared to act selectively on bacterial CAT in transgenic plants. These findings provided an explanation for difficulties experienced in the detection of transgenic CAT activity in B. napus.
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  • 5
    ISSN: 1432-203X
    Keywords: Brassica napus ; microspore ; density ; conditioned medium ; feeder ; embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In microspore cultures of Brassica napus L. cv. Topas, embryo yield increases with culture density up to about 40,000 microspores per ml. A much higher density (100,000 per ml) appears inhibitory to embryogenesis. A relatively high culture density (30,000 or 40,000 per ml) for the first 2–4 days of culture is crucial for embryogenesis, after which cultures may be diluted to allow better embryo growth. Medium conditioned by culturing microspores at 30,000 or 40,000 per ml for 1 day improved microspore-embryo yield in low density cultures (3,000 or 4,000 per ml) more than 3-fold. In contrast, media conditioned with microspores from 1–4 days or 0–4 days of culture were inhibitory. Use of feeder cultures resulted in up to 10-fold increase of embryo yield in low density microspore cultures, depending on the method used. Filter papers and other membranes placed on top of feeders greatly inhibited embryogenesis in the feeder layer as well as microspores cultured on the feeder, possibly due to poorer gaseous exchange.
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  • 6
    ISSN: 1432-2048
    Keywords: Key words:Glycine (seed coat) – Seed coat – Parenchyma (thick walled) – Subtilisin-like gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. A seed coat-specific gene, SCS1 (Seed Coat Subtilisin 1), from soybean, Glycine max [L.] Merill, has been identified and studied. The gene belongs to a small family of genes with sequence similarity to the subtilisins, which are serine proteases. Northern blot analysis showed that SCS1 RNA accumulates to maximal levels in seed coats at 12 days post anthesis, preceding the final stages of seed coat differentiation. The SCS1 RNA was not found in other tissues including embryos, seed pods, flowers, stems, roots or leaves. In-situ hybridization studies confirmed the temporal pattern of expression observed by Northern blot analysis and further revealed a restricted pattern of RNA accumulation in thick-walled parenchyma cells of the seed coats. These cells are important in the apoplastic translocation of nutrients en route to the embryo from the vascular tissues. The tissue-specific subtilisin-like gene may be required for regulating the differentiation of the thick-walled parenchyma cells.
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  • 7
    ISSN: 1432-203X
    Keywords: Transgenic ; Acetohydroxyacid synthase ; Sulfonylurea ; Imidazolinone ; Selection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Genes coding for the enzyme acetohydroxyacid synthase, often referred to as acetolactate synthase (AHAS, ALS; EC 4.1.3.18), from wild type Arabidopsis thaliana and a sulfonylurea-resistant mutant line GH50 (csrl-1; Haughn et al. 1988) were introduced in Nicotiana tabacum. Both genes were expressed at high levels with the 35S promoter. The csrl-1 gene conferred high levels of resistance to chlorsulfuron whereas the wild type gene did not. As selectable markers, chimaeric AHAS genes yielded transgenic plants on chlorsulfuron but at much lower efficiencies than with a chimaeric neomycin phosphotransferase gene on kanamycin (Sanders et al. 1987). Shoot differentiation from leaf discs was delayed on chlorsulfuron by 4–6 weeks. This study indicated a role for mutant AHAS genes in the genetic manipulation of herbicide resistance in transgenic plants but as selectable markers for plant cells undergoing differentiation no advantage over other genes was perceived.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell reports 8 (1989), S. 303-306 
    ISSN: 1432-203X
    Keywords: Ti plasmid ; Virulence ; Brassica napus ; Brassica juncea
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Brassica napus and Brassica juncea were infected with a number of Agrobacterium tumefaciens strains. Tumourigenesis was very rapid and extremely efficient on B. juncea with all but one of the strains. Tumourigenesis on B. napus varied widely. It was very efficient with the nopaline strains, was reduced with the succinamopine strain A281 and was very weak with the octopine strains. The latter observation was confirmed with six different B. napus rapeseed cultivars. The selectivity was due to differences in the virulence of Ti plasmids with B. napus, rather than the tumourigenicity of the T-DNA or virulence of the chromosomal genes associated with the strains. An exception was strain LBA4404. The virulence of the octopine strains was increased by coinfection with more virulent disarmed strains and by induction with acetosyringone.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Methods in cell science 12 (1989), S. 139-144 
    ISSN: 1573-0603
    Keywords: microinjection ; genetic transformation ; protoplasts ; microspores ; Brassica napus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary This paper describes a general method suitable for the microinjection ofBrassica napus protoplasts, unicellular microspores, and multicellular microspores. By incorporating components taken from other methods, manual operations frequently involved in the microinjection of plant cells have been simplified and microinjection rates increased. The embedding of cells in agarose provides a simple alternative to the variety of sophisticated immobilization strategies devised for different plant cell types thereby reducing the manipulations often involved in the culture of microinjected cells. Use of an automatic microinjector eliminated the operation of fine control systems on manual injectors; however, precision in sample delivery was reduced. Analyses indicate that transformed tissues can be recovered from microinjected protoplasts and microspores at high frequencies.
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  • 10
    ISSN: 1573-5044
    Keywords: Brassica napus ; β-glucuronidase ; polymerase chain reaction ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Approximately 2,000 embryogenic uninuclear microspores of rapeseed (Brassica napus) cv. Topas were intranuclearly injected with a chimaeric β-glucuronidase (Escherichia coli Uid A) gene. Stable integration had not occurred among 55 plants that were regenerated. Coinjection of the dye Lucifer Yellow and detection of injected DNA by the polymerase chain reaction revealed high frequencies of transfer. However, the amount of DNA injected was less than 20 copies, which may have been insufficient for stable transformation of microspores.
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