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  • Microspore  (4)
  • Transformation  (4)
  • 1
    ISSN: 1432-203X
    Keywords: Brassica campestris ; Microspore ; Derived Embryos ; Acyl Lipid Biosynthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The native lipid composition and the capacity of cell-free extracts to biosynthesize acyl lipids in vitro were determined for the first time using the recently reported microspore-derived (MD) embryo system from the Brassica campestris low erucic acid line BC-2 (Baillie et al. 1992). The total lipid fraction isolated from midcotyledonary stage MD embryos (21 days in culture) was composed primarily of triacylglycerol (76%) with an acyl composition quite similar to that of mature BC-2 seed. When incubated in the presence of glycerol-3-phosphate, 14C 18∶1-CoA, and reducing equivalents, homogenates prepared from 21-day cultured MD embryos were able to biosynthesize glycerolipids via the Kennedy pathway. The maximum in vitro rate of triacylglycerol biosynthesis could more than account for the known rate of lipid accumulation in vivo. The homogenate catalyzed the desaturation of 18∶1 to 18∶2 and to a lesser extent, 18∶3. The newly-synthesized polyunsaturated fatty acids initially accumulated in the polar lipid fraction (primarily phosphatidic acid and phosphatidylcholine) but began to appear in the triacylglycerol fraction after longer incubation periods. As expected for a low erucic acid cultivar, homogenates of MD embryos from the BC-2 line were incapable of biosynthesizing very long chain monounsaturated fatty acyl moieties (20∶1 and 22∶1) from 18∶1-CoA in vitro. Nonetheless, embryo extracts were still capable of incorporating these fatty acyl moieties into triacylglycerols when supplied with 14C 20∶1-CoA or 14C 22∶1-CoA. Collectively, the data suggest that developing BC-2 MD embryos constitute an excellent experimental system for studying pathways for glycerolipid bioassembly and the manipulation of this process in B. campestris.
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  • 2
    ISSN: 1432-203X
    Keywords: Key words Pollen ; Polygalacturonase ; Promoter ; Brassica ; Transformation ; GUS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A 647-bp 5′-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed in Brassica napus pollen was fused to the β-glucuronidase (GUS) marker gene. This fusion construct was introduced into B. napus plants via Agrobacterium tumefaciens transformation. Analysis of the transgenic B. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.
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  • 3
    ISSN: 1432-203X
    Keywords: Brassica campestris ; Microspore ; Embryogenesis ; Haploid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protocol previously developed for B. napus microspore culture was modified to produce embryos from several lines of Brassica campestris. Bud size, genotype, media constituents, and incubation time and temperature were examined. Donor plants were grown in a growth cabinet at a day/night temperature of 10/5°C. Microspores were isolated from buds 2.0 – 2.9 mm in length and cultured in modified Lichter (1982) medium containing 17% sucrose, pH 6.2. After 48 h at 32°C, the incubation medium was replaced with NLN (Lichter 1982) medium containing 10% sucrose. Microspores were cultured at 24°C in darkness and embryos developed after three weeks. More than 1000 plants have thus far been regenerated. Genotypic differences were observed for microspore embryogenesis. The majority of the regenerants were haploid, however colchicine could be effectively used to achieve chromosome doubling.
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  • 4
    ISSN: 1432-203X
    Keywords: Brassica rapa ; Microspore ; Embryogenesis ; Haploid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Isolated microspore culture techniques are being widely used in Brassica breeding programs to generate haploid and doubled haploid plants. A number of factors influence regeneration response in vitro including genotype. In order to assess the effect of genotype on microspore embryogenesis in B. rapa L. var. oleifera, 17 cultivars and breeding lines were evaluated. Embryos developed from all but one genotype when using NLN medium with 17% sucrose, followed by a reduction in sucrose concentration to 10%, 48 h later. The number of embryos /100 buds differed between genotypes, ranging from 0 to 70. Further studies indicated that sucrose concentration and incubation time influenced embryogenesis. Selection studies carried out with an Agriculture and Agri-Food Canada breeding line have resulted in the identification of a highly embryogenic B. rapa line. This line produced thousands of microspore-derived embryos /100 buds and will be useful in mutant selection and gene transfer as well as biochemical and developmental studies.
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  • 5
    ISSN: 1432-203X
    Keywords: Pollen ; Polygalacturonase ; Promoter ; Brassica ; Transformation ; GUS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A 647-bp 5′-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to theβ-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.
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  • 6
    ISSN: 1432-203X
    Keywords: Key words Brassica ; Mustard ; Transgenic ; Transformation ; Regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Shoot organogenesis and plant regeneration were readily achieved from cotyledonary petioles and hypocotyls of Brassica carinata. These explants were used for Agrobacterium-mediated transformation. A construct containing the selectable marker genes, neomycin phosphotransferase II, phosphinothricin acetyl transferase and the reporter gene β-glucuronidase, under the control of a tandem 35S promoter, was used for transformation. Although transformation was achieved with both cotyledonary petioles and hypocotyls, cotyledonary petioles responded best, with 30–50% of the explants producing GUS-positive shoots after selection on 25 mg/l kanamycin. Direct selection on L-phosphinothricin also produced resistant shoots but at a lower frequency (1–2%).
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Theoretical and applied genetics 55 (1979), S. 65-67 
    ISSN: 1432-2242
    Keywords: Brassica campestris ; Embryogenesis ; Haploid ; Microspore ; Temperature
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Culture of Brassica campestris anthers at 35°C for one or three days prior to culture at 25°C significantly stimulated the yield of microspore-derived embryos. More than 100 plants were regenerated from cultured embryos and haploids were identified amongst them. The haploid frequency was greater than 70% if all small-flowered sterile plants were considered to be haploid. The yield of microspore-derived plants in B. campestris is approaching the level where anther culture may be utilized as a practical breeding tool.
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  • 8
    ISSN: 1432-2242
    Keywords: Nicotiana tabacum ; N. Debneyi ; Somatic hybridization ; Transformation ; Organelle inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A simple, yet effective selection system was used to produce fertile somatic hybrids betweenNicotiana tabacum andN. debneyi. This approach utilized transgenic antibiotic-resistantN. tabacum andN. Debneyi as donor plants for mesophyll protoplast fusions. Thirteen somatic hybrid plants were regenerated from calli capable of growth on medium containing both antibiotics. The majority of the hybrids displayed a range of leaf and floral morphologies and growth habits that were intermediate to those of the parental species, and had chromosome numbers varying from amphidiploid (2n = 96) to hypoaneuploid (2n = 60). Isoenzyme and RFLP analysis demonstrated the presence and expression of nuclear genes from both parents in all of the hybrids. Most plants are fully fertile. Thus, these plants differ from the malesterile tobacco ‘cybrids’ and alloplasmic lines produced by transferring theN. debneyi cytoplasm to tobacco. A nonrandom pattern of cytoplasmic segregation in the fusion products occurred with a bias towards the presence ofN. debneyi cp and mtDNA. Evidence for the presence of rearranged or recombinant cp and mtDNA in some of the hybrids was obtained. The somatic hybrids were successfully backcrossed to theN. tabacum parent and are now being tested for immunity to black root rot, a trait specific toN. debneyi, but not existent in theN. tabacum parental line.
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