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  • 1
    Electronic Resource
    Electronic Resource
    Transient marker-gene expression during zygotic in-vitro embryogenesis of Brassica juncea (Indian mustard) following particle bombardment (1996)
    Springer
    Planta 198 (1996), S. 211-220 
    Details
    ISSN: 1432-2048
    Keywords: Brassica ; Embryogenesis ; Firefly luciferase ; Microprojectile bombardment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A method has been established that allows the transfer of genes into single cells of excised globular-stage zygotic Brassica juncea L. embryos. The fate of single, genetically marked cells was followed during in-vitro embryogenesis. A simple and defined embryo culture medium has been designed on which zygotic B. juncea embryos, excised at the globular or at later stages, develop normally into mature, fully grown embryos. The smallest embryos which can be efficiently cultured are 30 μm long (embryo proper without suspensor) and are comprised of less than 20 cells. The embryos grow on the surface of solid medium without embedding and are freely accessible to microprojectile bombardment. Shooting at globular, transition and early heart-shaped embryos using both a particle inflow gun and a micro-targeting particle accelerator resulted in transient expression of genes encoding visible markers. For both particle-acceleration devices the shooting conditions have been optimised based on transient β-glucuronidase (GUS) expression. Bombarding embryos under optimal conditions had no deleterious effects on in-vitro embryogenesis. Multicellular GUS-expressing sectors were obtained, showing that cells can survive receiving a particle and resume normal development. The examination of these sectors has provided new information about the cell division patterns characterising early B. juncea embryogenesis. To be able to follow the development of particular genetically marked sectors, we tried to identify reporter genes that, in contrast to the uidA gene (which encodes GUS), can be non-destructively assayed in embryonic cells. Preliminary data has shown that expression of the firefly luciferase gene (Luc) can be detected in bombarded embryos without affecting their viability.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00206246
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  • 2
    Electronic Resource
    Electronic Resource
    Gene transfer to inflorescence and flower meristems using ballistic micro-targeting (1994)
    Springer
    Sexual plant reproduction 7 (1994), S. 135-143 
    Details
    ISSN: 1432-2145
    Keywords: Flower ; Meristem ; Gene transfer Particle bombardment ; Triticum aestivum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Direct gene transfer to floral meristems could contribute to cell-fate mapping, to the study of flower-specific genes and promoters, and to the production of transgenic gametes via the transformation of sporogenic tissues. Despite the wide potential of its applications, direct gene transfer to floral meristems has not been achieved so far because of the lack of suitable technology. We show in this paper that ballistic micro-targeting is the technique of choice for this purpose, and in this way, we were able to transfer genes efficiently into excised wheat immature spikes. Particle size was adjusted for optimal penetration into the L1 and L2 cell layers of the spikes with limited cell damage. Spikes at different developmental stages were shot either with a plasmid containing two genes involved in anthocyanin biosynthesis or with a plasmid bearing the uidA (β-glucuronidase) gene. The transient expression of these marker genes was observed in the different developmental stages tested and in cells of both the L1 and the L2 layers. The transient expression of the uidA gene was significantly increased when the sucrose concentration in the culture medium was increased from 0.06 to 0.52 M. At the highest concentration, 100% of the targeted spikes expressed the uidA gene, with an average of 69 blue cells per spike. Twelve days after microtargeting, multicellular sectors showing transgene expression and containing up to 17 cells were found in 85% of the shot immature inflorescences. This indicated that targeted cells survived particle bombardment. Sectors were found in primordia of both vegetative and reproductive organs.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00230582
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  • 3
    Electronic Resource
    Electronic Resource
    Deleterious effect of minimal enzymatic treatments on the development of isolated maize embryo sacs in culture (1995)
    Springer
    Sexual plant reproduction 8 (1995), S. 313-317 
    Details
    ISSN: 1432-2145
    Keywords: Embryo sac ; Zea mays ; Enzymatic isolation ; Zygotic embryogenesis ; Microinjection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The long-term viability of isolated embryo sacs was studied in maize. Fertilised embryo sacs were digested in order to remove most of the nucellus cells present on their surfaces and then transferred to culture. Experiments on 161 embryo sacs showed that isolation treatments using even minimal enzymatic digestion affected the further development of the embryo sacs. Few embryo sacs survived in culture and those produced only abnormal embryos; they produced no plants. We concluded that embryo sacs isolated through enzymatic digestion may offer limited prospects for long-term studies where normal embryogenic development is required. Alternative strategies are discussed for maize.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00229389
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  • 4
    Electronic Resource
    Electronic Resource
    Transient expression of visible marker genes in meristem cells of wheat embryos after ballistic micro-targeting (1993)
    Springer
    Planta 192 (1993), S. 84-91 
    Details
    ISSN: 1432-2048
    Keywords: Direct gene transfer ; Microprojectile bombardment ; Micro-targeting ; Meristem ; Triticum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Seven days after anthesis, the shoot apical meristem of immature embryos of wheat (Triticum aestivum L.) is not yet covered by the coleoptile or leaf primordia and provides an optimal target for ballistic micro-targeting. Gold particles 1.2 μm in diameter at a concentration of 5·105 particles per μl and propelled by 110-bar nitrogen penetrated up to four cell layers into embryo apical meristems but produced no deleterious effects on germination. The use of diaphragms with internal diameters of 100 or 200 μm restricted bombardment to meristem cells or also included surrounding tissues, respectively. The results of transient-expression experiments indicated successful delivery of foreign DNA into meristem cells. Cells of the central zone of the meristem or pro-meristem transiently expressed foreign genes driven by the Cauliflower mosaic virus (CaMV) 35S and rice actin1-D constitutive promoters. Partial plasmolysis before bombardment and slow recovery of normal turgor pressure increased transient-expression frequencies. Meristem cells transiently expressed foreign genes at frequencies 10-fold less than surrounding tissues under identical conditions. Transgenic sectors were observed in both coleoptiles and leaf primordia.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00198696
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  • 5
    Electronic Resource
    Electronic Resource
    Nuclear DNA amounts in the egg and zygote of maize (Zea Mays L.) (1995)
    Springer
    Planta 197 (1995), S. 641-645 
    Details
    ISSN: 1432-2048
    Keywords: Fertilization (in vivo) ; Karyogamy ; Zea embryogenesis ; Zygotic DNA synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The nuclear DNA content of isolated eggs and zygotes of maize was estimated using 4′,6-diamidino-2-phenylindole (DAPI) staining and microspectrofluorometry. The data indicate that egg nuclei contain the 1C level of DNA (basic haploid amount) at the time of karyogamy, and that, by inference, the sperm nuclei are also at 1C. Fertilization occurred in most ovules by 24–28 h post-pollination (hpp), and DNA synthesis was well underway by 27–31 hpp. By 30–34 hpp, 80% of the zygotes were at the 3C DNA level or above, and many were undergoing mitosis. This study provides information that is pertinent to experiments on the microinjection of exogenous DNA into isolated zygotes of maize, and it will serve as a comparative base for future determinations of the DNA content of zygotes produced and cultured in vitro.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00191572
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