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  • 1955-1959  (8)
  • 1930-1934
  • 1890-1899
  • gene expression  (4)
  • protoplasts  (4)
  • 1
    ISSN: 1573-5060
    Keywords: Oryza sativa L. cv. Taipei 309 ; rice ; protoplasts ; direct DNA uptake ; kanamycin-resistant transgenic plants ; field trial ; glasshouse trial ; neomycin phosphotransferase II (npt II) gene ; gene expression and inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The phenotypes of seed progeny (R2 generation) of Oryza sativa L. cv. Taipei 309, which carried the neomycin phosphotransferase II (npt II) gene, were compared with those of non-transformed, protoplast-derived plants of the same generation and non-transformed, seed-derived plants under field and glasshouse conditions. Under both conditions the transgenic plants were generally smaller, took longer to flower and had reduced fertility. Significant differences were observed between individuals within the group of transgenic plants. The npt II gene was present in most of the transgenic plants, but NPT II activity was only detected in a minority of individuals.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-5060
    Keywords: bleomycin ; direct gene transfer ; expression ; irradiation ; petunia ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The production of transgenic plants by means of direct gene transfer to protoplasts is now a widely-used technique. The biological mechanisms underlying the transformation are still poorly understood, but many investigations have attempted to shed light on some components of this process. Varying the experimental conditions has in some cases led to better transformation rates, but further improvements of the protocols are possible. Such improvements will require a better understanding of how the alien DNA enters the cells, becomes integrated into the chromosomes and is treated as a part of the plant genome. Irradiation with sublethal doses of X-rays or UV-light has been shown to increase the transformation frequency, while certain drugs have been shown to act in a similar manner. The effects of these and other factors are discussed.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-5060
    Keywords: Agrobacterium ; Brassica napus ; CaMV 35S promoter ; mas promoter ; gene expression ; risk assessment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Gene fusions between the β-glucuronidase (GUS) reporter gene and the promoters of the cauliflower mosaic virus 35S RNA transcript (CaMV 35S) and the mannopine synthase (mas) genes were introduced into rapeseed varieties via Agrobacterium-mediated transformation. Fluorometric assay of β-glucuronidase activity indicated different expression patterns for the two promoters. In seedlings, the CaMV 35S promoter had maximum activity in the primary roots, while the mas promoter was most active in the cotyledons. Etiolated seedlings cultured in the dark showed reduced activity of the mas promoter. Before vernalization at the rosette stage, both promoters were more active in older plant parts than in younger ones. At this stage the highest activity was recorded in cotyledons. After the plants had bolted reduced promoter function was detected in the upper parts of the transformed plants. Both promoters were found to be functional in the majority of the studied organs of transgenic rapeseed plants, but the promoter activity varied considerably between the organs at different developmental stages. The ability of pollen to transfer the introduced genes to other varieties and related species (e.g. Brassica napus and Diplotaxus muralis) by cross-pollination was studied in greenhouse experiments, and field trials were carried out to estimate the distance for biologically-relevant gene dispersal. In artificial crossing, the introduced marker gene was transferable into other varieties of Brassica napus. In field trials, at a distance of 1 metre from the source of transgenic plants, the frequency of an outcrossing event was relatively high (10-3). Resistant individuals were found at 16 and 32 metres from the transgenic pollen donors, but the frequency of an outcrossing event dropped to 10-5.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1573-5060
    Keywords: barley ; electroporation ; PEG-mediated DNA uptake ; promoter analysis ; protoplasts ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary In some cereal species that are still recalcitrant to stable transformation and regeneration, transient expression in isolated protoplasts is a useful tool for the study of gene expression and regulation. We have successfully applied these techniques to barley protoplasts derived from developing endosperm, aleurone, leaves and roots in order to characterize functionally cis-acting motives in two gene promoters, corresponding to trypsin inhibitor BTI-CMe and to sucrose synthase Ss1. Gene specificity is maintained in transient expression assays with protoplasts isolated from these different barley tissues and the pattern of expression parallels the mRNA levels observed for the corresponding genes in the same tissues.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1573-5060
    Keywords: protoplasts ; protoclonal variation ; somatic hybridization ; top-fruit trees ; woody ornamentals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Until recently, temperate fruit trees and woody ornamentals have been regarded as recalcitrant to biotechnological breeding approaches based on protoplasts. This however should no longer be the case, as procedures are now available, not only for the regeneration of complete plants from protoplasts of various tissues of such species, but also for the exploitation of protoplast technology for their genetic manipulation. This paper will examine the recent advances and state of the art in this domain, with particular attention to the use of protoplast technology as a novel tool in the breeding of rosaceous top-fruit tree species and woody ornamentals. Problems and their solutions within the context of regenerating plants from isolated protoplasts of stone (Prunus spp.), pome (Pyrus spp., Malus spp.) and small (Rubus spp.) fruits, and of several shrubby ornamental genotypes (Lonicera spp., Weigela spp., Forsythia spp., Cotoneaster spp.) will be addressed. Interspecific (Prunus spinosa + Prunus cerasifera) and intergeneric (Forsythia spp. + Syringa spp.) somatic hybridization within this group of species, as well as the use of protoplasts for host/pathogen interaction studies (Pyrus/Erwinia amylovora) will also be discussed.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1573-5060
    Keywords: carotenoids ; ethylene ; gene expression ; Lycopersicon esculentum Mill. ; polygalacturonase ; pectinesterase ; phytoene synthase ; ACC oxidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The common cultivated tomato (Lycopersicon esculentum Mill.) provides a major focus for improvement of crop quality through genetic engineering. Identification of ripening-related cDNAs has enabled the modification of specific aspects of ripening by manipulating gene expression in transgenic plants. By utilizing ‘antisense RNA’ to modify expression of ripening genes, we have inhibited the production of the cell wall-metabolising enzymes polygalacturonase and pectinesterase and created transgenic plants that contain, effectively, single, targeted mutations affecting these genes. Furthermore, this approach has been used with previously unidentified cDNA clones to enable both functional identification and manipulation of genes involved in ethylene production (ACC oxidase) and carotenoid biosynthesis (phytoene synthase). The use of antisense RNA targeted to specific genes to alter ripening phenotypes and improve commercial utility of fruit by affecting shelf-life, processing characteristics and nutritional content is discussed. We have used the extreme ripening-impaired mutant, ripening inhibitor (rin) to identify additional genes implicated in the ripening process. This approach has resulted in the cloning of several novel ripening-related mRNAs which are now being studied by antisense experiments. This may enable identification and manipulation of additional genes involved in processes such as softening, flavour and aroma generation and susceptibility to pathogens.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1573-5060
    Keywords: microprotoplast fusion ; partial genome transfer ; monosomic additions ; kanamycin resistance ; β-glucuronidase ; gene expression ; potato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Various aspects of a microprotoplast fusion technique and the strategies followed for intergeneric partial genome transfer (one or a few chromosomes) and alien genes from sexually-incongruent donor species to recipient species are described. The essential requirements of the microprotoplast fusion technique are the induction of micronuclei at high frequencies, as well as the isolation and enrichment of sub-diploid microprotoplasts in donor species, efficient fusion of the donor microprotoplasts with normal recipient protoplasts and stable regeneration of plants from fusion products. The results on the production of microprotoplast hybrid plants between the transformed donor lines of Solanum tuberosum and Nicotiana Plumbaginifolia carrying various genetic markers, and a recipient line of Lycopersicon peruvianum or Nicotiana tabacum, and on the transfer and expression of alien genes (kanamycin resistance, β-glucuronidase) are presented. The data obtained on microprotoplast hybrid plants between S. tuberosum and L. peruvianum showed that many of the hybrids contained one potato chromosome carrying nptII and GUS, and 24 or 48 L. peruvianum chromosomes (monosomic additions), and that they were male-and female-fertile. Various applications of chromosome transfer by this technique, especially for economically-important traits (e.g. disease or stress resistance) from sexually-incompatible wild species, for construction of chromosome-specific DNA libraries through microdissection and microcloning of chromosomes, or by flow-sorting of chromosomes for genome analysis, are discussed.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1573-5060
    Keywords: Vicia narbonensis ; gene transfer ; gene expression ; seeds ; 2S albumin ; methionine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Epicotyl explants were co-cultivated with Agrobacterium tumefaciens EHA101 to transfer a chimeric 2S albumin gene construct carried in the binary Ti plasmid vectors pGSGLUC1 or pGA472 into the grain legume Vicia narbonensis. This gene encoding the sulphur-rich Brazil nut albumin was under the control of either the CaMV 35S promoter which permits gene expression in all organs, or the Vicia faba legumin B4 promoter which elicits seed-specific gene expression. After callus formation and selection for kanamycin resistance, somatic embryos were induced which, in the case of transformation with the vector pGSGLUC1, were screened for GUS activity. Embryos that produced GUS were in addition analysed for 2S albumin formation. Selected transgenic embryos were cloned by multiple shoot regeneration. Rooted and fertile plants were obtained by grafting transgenic shoots on the appropriate seedlings. R1 and R2 generations were raised and analysed for GUS as well as 2S albumin gene expression. Expression of the 35S promoter/2S albumin gene fusion took place in all organs of the transgenic plants including the cotyledons of seeds, whereas seed-specific gene expression was found in transformants with the legumin promoter/2S albumin gene fusion. The 2S albumin accumulated in the 2S protein fraction of transgenic seeds and its primary translation product was processed into the 9 and 3 kDa polypeptide chains. The foreign protein was localised in the protein bodies of the grain legume. Analysis of the R2 plants indicated Mendelian inheritance of the 2S albumin gene. In homozygous V. narbonensis plants the amounts of 2S albumin were twice that present in the corresponding heterozygous plants. Whereas only low level formation of the foreign protein was achieved if the gene was under the control of the 35S promoter, approximately 3.0% of the soluble seed protein was 2S albumin if seed-specific gene expression was directed by the legumin B4 promoter. Some of these transformants exhibited a three-fold increase in the methionine content of the salt-soluble protein fraction extracted from seeds.
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