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  • 1990-1994  (53)
  • 1955-1959  (12)
  • 1920-1924
  • gene transfer
  • genetic engineering
  • somatic hybridization
  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell reports 13 (1994), S. 145-148 
    ISSN: 1432-203X
    Keywords: muskmelon ; gene transfer ; Agrobacterium ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cotyledon explants of muskmelon (Cucumis melo L., cv. Amarillo Oro) seedlings were co-cultivated with disarmed Agrobacterium tumefaciens strain LBA4404 that contained the binary vector plasmid pBI121.1. The T-DNA region of this binary vector contains the Nopaline synthase/neomycin phosphotransferase II (NPTII) chimeric gene for kanamycin resistance and the Cauliflower Mosaic Virus 35S/β-glucuronidase (GUS) chimeric gene. After infection, the cotyledon pieces were placed in induction medium containing 100 mg/l kanamycin. Putative transformed shoots were obtained, followed by the development of morphologically normal plantlets. The transgenic nature of regenerants was demonstrated by polymerase chain reaction, Southern blot analysis, plant growth on medium selective for the transgene (NPTII) and expression of the co-transformed GUS gene. Factors affecting the transformation procedure are discussed.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-203X
    Keywords: gene transfer ; selection for phosphinothricin resistance ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts isolated from embryogenic suspension cultures of wheat (Triticum aestivum cv. Hartog) were electroporated in the presence of plasmid pEmuGN and/or pEmuPAT, which contained the reporter gene gus and selectable marker gene bar, respectively. Under optimised electroporation conditions, up to 0.9% of viable protoplasts displayed gus activity two days after electroporation. To select for phosphinothricin (PPT) resistant colonies, electroporated protoplasts were incubated for six weeks in a medium containing 10 μg/ml PPT. The cells surviving the selection were maintained as individual colonies on solid medium or as suspension cultures. More than 60% of these colonies exhibited tolerance to 40 μg/ml PPT when tested 10 months after initial selection. To date, 57 green plants have been regenerated from these colonies and 24 have been transferred to soil. Southern blot analyses of colonies and plants, using the bar gene sequence as the probe, confirmed transformation of the cells. Positive PAT assays of both regenerated colonies and plants indicated the presence of the bar gene product. These results provide a basis for the establishment of routine procedures for transformation of wheat by direct gene transfer into protoplasts.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-5028
    Keywords: fertile transgenic barley ; gene transfer ; Hordeum vulgare ; neomycin phosphotransferase II ; particle bombardment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T0), and four of the 90 T0 spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T1). The four transgenic shoots of Toivo (the T0 plant) produced 25 plantlets as T1 progeny. Altogether fifteen of these T1 plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T2 progeny.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 291-301 
    ISSN: 1573-5044
    Keywords: clesteroviruses ; cytopathology ; fleek disease ; gene transfer ; tissue culture ; virus purification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Agrobacterium rhizogenes-mediated transformation was applied toVitis spp. andNicotiana spp. infected by different grapevine phloem-limited viruses (grapevine fleck virus, grapevine virus A, grapevine virus B) to obtain root cultures for virus purification. All plant species were successfully transformed, and several clones were established in liquid culture. Transformed grapevine roots contained as much virus as non transformed roots and more than leaves, as assessed by ELISA and thin sectioning. Likewise, transformed roots ofNicotiana benthamiana Domin. contained in average more GVA than leaves, especially those at the base and the top of the plant, whereas withNicotiana occidentalis wheel., GVB was apparently less concentrated than in leaves.Nicotiana root grew faster than those ofVitis. All viruses multiplied and persisted in root cultures, which were successfully used for purification. Virus yields were the same (GFkV and GVB) or higher (GVB) than those reported in the literature. Grapevine roots may prove useful for culturing and purifying other non-mechanically transmissible grapevine viruses.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1573-5028
    Keywords: anti-bacterial protein ; genetic engineering ; precursor processing ; synthetic gene ; thionin ; transgenic
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Hordothionins (HTHs) are small anti-bacterial proteins present in barley endosperm which are processed from larger precursor proteins, consisting of an amino-terminal signal peptide (SP), the mature highly basic HTH and a carboxy-terminal acidic peptide (AP). Different HTH precursor proteins were expressed in tobacco to study the effects of the pre-sequences (SP) and pro-sequences (AP) on expression, processing, sorting and biological activity and hence the feasibility of engineering bacterial disease resistance into crops which lack these proteins. Maximum HTH expression levels of approximately 0.7% (11 μmol/kg) of total soluble protein in young tobacco leaves were obtained using a semi-synthetic gene construct encoding a complete chimaeric HTH precursor protein. Tenfold lower HTH expression levels (maximum 1.3 μmol/kg) were obtained using synthetic gene constructs without the AP-coding sequence and no expression was found in plants containing synthetic HTH gene constructs without SP-and AP-coding sequences. In both cases where expression was found, the precursors were apparently correctly processed, although the HTH produced in plants containing a construct without AP sequence appeared to be slightly modified. No effect on plant phenotype was observed. Localization studies indicated that the HTH was in identical fractions of plants expressing the two different precursors, albeit at a different ratio, and was not secreted into the intercellular spaces of leaves or culture medium by protoplasts. Our results indicated that the AP is not involved in sorting and suggested that it might facilitate transport through membranes. The in vitro toxicity of HTH isolated from transgenic tobacco plants expressing the two different precursor proteins for the bacterial plant pathogen Clavibacter michiganensis subsp. michiganensis appeared similar to that of the HTH purified from barley endosperm.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1573-5028
    Keywords: Antibody ; coordinated expression ; genetic engineering ; protein assembly ; root ; secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To explore the feasibility of employing antibodies to obtain disease resistance against plant root pathogens, we have studied the expression of genes encoding antibodies in roots of transgenic plants. A model monoclonal antibody was used that binds to a fungal cutinase. Heavy and light chain cDNAs were amplified by PCR, fused to a signal sequence for secretion and cloned behind CaMV 35S and TR2′ promoters in a single T-DNA. The chimeric genes were cloned both in tandem and in a divergent orientation. The roots of tobacco plants transformed with these constructs produced antibodies that were able to bind antigen in an ELISA. Immunoblotting showed assembly to a full-size antibody. In addition, a F(ab′)2-like fragment was observed, which is probably formed by proteolytic processing. Both antibody species were properly targeted to the apoplast, but the full-size antibody was partially retained by the wall of suspension cells. The construct with divergent promoters showed a better performance than the construct with promoters in tandem. It directed the accumulation of functional antibodies to a maximum of 1.1% of total soluble protein, with half of the plants having levels higher than 0.35%. The high efficiency of this construct probably results from coordinated and balanced expression of light and heavy chain genes, as evidenced by RNA blot hybridization.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    World journal of microbiology and biotechnology 10 (1994), S. 243-248 
    ISSN: 1573-0972
    Keywords: Biotechnology ; development ; genetic engineering ; sub-Saharan Africa ; sustainable
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Whether development is defined by the long-standing economic parameter of per capita gross national product (GNP) or by the newly introduced Human Development Index (HDI), which is not based exclusively on per capita GNP, the countries of sub-Saharan Africa rank at or near the bottom of the developing world. Agriculture and agro-based processing are the mainstays of the economies of the majority of these countries. Because of this, and also because many of the diseases endemic in these countries are communicable, the application of modern biotechnology (including genetic engineering, tissue culture and monoclonal antibody technology) and related biotechnologies could play an important part in creating sustainable development in the region. There is, therefore, an urgent need to train more of the region's indigenous citizens, and to equip more laboratories, in modern biotechnology. It is suggested that, in order to accelerate the harnessing of the fruits of biotechnology, more countries in the region should affiliate with the International Centre for Genetic Engineering and Biotechnology (ICGEB). It is further suggested that a regional equivalent of the ICGEB be built and the services of non-governmental biotechnology organizations used.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 147-153 
    ISSN: 0006-3592
    Keywords: aqueous two-phase systems ; β-galactosidase ; T4 lysozyme ; partitioning ; charge modifications ; genetic engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have examined the effect of genetically engineered charge modifications on the partitioning behavior of proteins in dextran/polyethylene glycol two-phase systems containing potassium phosphate. By genetically altering a protein's charge, the role of charge on partitioning can be assessed directly without the need to modify the phase system. The charge modifications used are of two types: Charged tails of polyaspartic acid fused to β-galactosidase and charge-change point mutations of T4 lysozyme which replace positive lysine residues with negative glutamic acids. The partition coefficient Kp for these proteins was related to measured interfacial potential differences Δφ using the simple thermodynamic model, In Kp = In Ko + (F/RT)Zp δφ. The protein net charge Zp was determined using the Henderson-Hasselbalch relationship with modifications based on experimentally determined titration and isoelectric point data. It was found that when the electropartitioning term Zp δφ was varied by changing the pH, the partitioning of T4 lysozyme was quantitatively described by the thermodynamic model. The β-galactosidase fusions displayed qualitative agreement, and although less than predicted, the partitioning increased more than two orders of magnitude for the pH range examined. Changes in the partitioning of lysozyme due to the various mutations agreed qualitatively with the thermodynamic model, but with a smaller than expected dependence on the estimated charge differences. The β-galactosidase fusions, on the other hand, did not display a consistent charge based trend, which is likely due either to the enzyme's large size and complexity or to nonelectrostatic contributions from the tails. The lack of quantitative fit with the model described above suggests that the assumptions made in developing this model are oversimplified. © 1994 John Wiley & Sons, Inc.
    Additional Material: 6 Ill.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Transgenic research 3 (1994), S. 109-115 
    ISSN: 1573-9368
    Keywords: gene transfer ; metabolic engineering ; overexpression ; secondary metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Plants interact with their environment by producing a diverse array of secondary metabolites. Many of these compounds are valued for their medicinal, industrial or agricultural properties. Other secondary products are toxic or otherwise undesirable and can reduce the commercial value of crops. Gene transfer technology offers new opportunities to modify directly plant secondary product synthesis through metabolic engineering. This article reviews some of the strategies which have been used to increase or decrease the synthesis of specific plant metabolites, as well as methods for expanding the biosynthetic capabilities of individual species.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Transgenic research 3 (1994), S. 13-19 
    ISSN: 1573-9368
    Keywords: gene transfer ; electroporation ; stable transformation ; protoplasts ; transgenic trees ; Populus tremula x P. alba
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts from leaflets ofin vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying thenptII gene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene,als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 105 to 104 protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 μM paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS ofArabidopsis and 25 μM phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated. Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.
    Type of Medium: Electronic Resource
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  • 11
    ISSN: 1573-9368
    Keywords: Solanum tuberosum ; genetic modification ; transformation ; gene transfer ; genetic isolation ; risk assessment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Information on the extent of transgene dispersal by pollen to adjacent potato plots and to related weed species is an important requisite for risk assessment; a procedure followed before novel transgenic plants are evaluated under field conditions. The purpose of the investigation was to determine the frequency of cross-pollination between potato (Solanum tuberosum) plants at different distances, using a kanamycin resistnace transgene (nptII) as a selectable marker. All potato plants were from the variety Désirée. Non-transgenic potato plants, used as potential recipients of transgene-containing pollen, were planted in 12 sub-plots, at distances of 0–20 m from the nearest transgenic potato plants. Seeds harvested from the non-transgenic plants were screened for resistance to kanamycin, and molecular methods were used to confirm that resistant progeny contained thenptII gene. Where transgenic and non-transgenic potato plants were in alternate rows (leaves touching), 24% of seedlings from the non-transgenic parent plants were kanamycin-resistant. Comparable seedlings from plants at up to 3 m distance had a resistance frequency of 2%, at 10 m the frequency was 0.017% and at 20 m no resistant progeny were observed. Plants of the weed speciesS. dulcamara andS. nigrum were also planted close to the transgenic potatoes to test for evidence of hybridization, and no kanamycin-resistant seedlings were observed among progeny fromS. dulcamara andS. nigrum. This investigation provided evidence that the extent of gene dispersal from transgenic potatoes to non-transgenic potatoes falls markedly with increasing distance, and is negligible at 10 m. There was, also, no evidence of transgene movement from potato toS. dulcamara andS. nigrum under field conditions. These data will be valuable in defining genetic isolation procedures for the early field evaluation and the use of novel transgenic potato genotypes.
    Type of Medium: Electronic Resource
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  • 12
    Electronic Resource
    Electronic Resource
    Springer
    Transgenic research 3 (1994), S. 263-278 
    ISSN: 1573-9368
    Keywords: Brassica napus ; oilseed rape ; transgenic plants ; interspecific hybridization ; gene transfer ; risk assessment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Before novel transgenic plant genotypes are grown outside containment facilities and evaluated under field conditions, it is necessary to complete a risk assessment to consider the possible consequences of that release. An important aspect of risk assessment is to consider the likelihood and consequences of the transgene being transferred by cross-pollination to related species, including other crops, weeds and ruderal populations. The purpose of this report is to review the literature to assess the ease with whichBrassica napus can hybridize with related species. The evidence for hybridization is considered at three levels: a) by open pollination, b) by hand pollination and c) by the use ofin vitro ovule and embryo rescue techniques; and also examines the fertility and vigour of the F1, F2 and backcross generations. Four species are reported to hybridize withB. napus by open pollination:B. rapa andB. juncea using fully fertile parents; andB. adpressa andR. raphanistrum using a male-sterileB. napus parent. Seventeen species are reported to form hybrids (including the four species above) withB. napus when pollination is carried out manually. At least 12 of these species were unable to form F2 progeny, and eight were unable to produce progeny when the F1 was backcrossed to one of the parental species. Many factors will influence the success of hybridization under field conditions, including: distance between the parents, synchrony of flowering, method of pollen spread, specific parental genotypes used, direction of the cross and the environmental conditions. Even where there is a possibility of hybridization betweenB. napus and a related species growing in the vicinity of a release, poor vigour and high sterility in the hybrids will generally mean that hybrids and their progeny will not survive in either an agricultural or natural habitat.
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  • 13
    ISSN: 1573-9368
    Keywords: gene transfer ; polyamines ; S-adenosylmethionine decarboxylase ; tobacco ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key regulatory enzyme in the polyamine biosynthetic pathway. Numerous studies have shown that the enzyme activity and polyamine levels are generally correlated with cellular growth in plants, animals and bacteria. In order to gain more insight into the role of polyamines in plants, human SAMDC cDNA under control of the 35S promoter of cauliflower mosaic virus, along with a neomycin phosphotransferase gene, was transferred to tobacco (Nicotiana tabacum cv. Xanthi) viaAgrobacterium tumefaciens. Transgenic plants showed the presence of human SAMDC mRNA and a 2-4-fold increase in SAMDC activity. In the transformed tissues, putrescine levels were significantly reduced, while spermidine content was 2–3 times higher than the control tissues. Cellular spermine content was either increased or remained unchanged. Excised leaf segments from transformed plants frequently produced shoots even on callus inducing medium.
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  • 14
    Electronic Resource
    Electronic Resource
    Springer
    Transgenic research 3 (1994), S. 401-405 
    ISSN: 1573-9368
    Keywords: gene transfer ; transgenic ; swine ; growth hormone ; PEPCK-bGH
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transgenic pigs were created that harboured a phosphoenol pyruvate carboxykinase-bovine growth hormone construct (PEPCK-bGH). Four founder animals and two transgenic offspring from one line were evaluated between 61/2 and 12 months of age. There was no evidence of severe hepatic or renal lesions in these pigs, which characterised transgenic PEPCK-bGH mice previously described. While glomerular and tubular lesions in kidney sections were not identified in the transgenic pigs, mesangial cell proliferation was observed in two transgenic offspring from a single line. Additionally, glomerular size was significantly increased in four of four puberal transgenic swine when compared to age- and sex-matched controls (28.30±4.1 vs. 14.2±2.7×105 μm3; representing 3 transgenic lines,p〈0.05). Surprisingly, no mature adipocytes were observed in subcutaneous sections obtained in transgenic GH pigs. Histological evaluation of these transgenic pigs further illustrates the requirement for precise control of growth-related genes and their protein products.
    Type of Medium: Electronic Resource
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  • 15
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 56 (1994), S. 168-170 
    ISSN: 0730-2312
    Keywords: abzyme ; bacteriostatic agent ; bioactive material ; biomimetic structure ; biosensor ; blood coagulation ; ceramic ; coating ; combinatorial synthesis ; drug delivery ; encapsulation ; enzyme ; extracorporeal device ; genetic engineering ; implant ; in vitro enolution ; LB film ; liqid ; liposome ; molecular imprinting ; molecular machine ; nanostructure ; orthopedics ; porey ; engineering ; sensor ; silk ; S-layer ; surface ; smart material ; synthetic chemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Materials that mimic or extend the properties of natural molecules are being developed for medical appoications. Recent breakthroughs in genetic engineering, polymer synthesis, molecular self-assmbly and related areas are greatly expanding the variety of structures available for use in physiological settings.
    Type of Medium: Electronic Resource
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  • 16
    Electronic Resource
    Electronic Resource
    Chichester : Wiley-Blackwell
    Journal of Chemical Technology AND Biotechnology 61 (1994), S. 273-281 
    ISSN: 0268-2575
    Keywords: fed-batch culture ; pH-stat ; recombinant Escherichia coli ; genetic engineering ; fuzzy control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: In the cultivation of genetically engineered Escherichia coli it is very important to control the substrate concentration at an appropriate level in order to avoid the accumulation of acetate, thereby elevating the expression level of plasmid-encoded protein. In this paper, a pH-stat mode of fuzzy control was considered for the overexpression of β-galactosidase in the fed-batch cultivation of recombinant E. coli. In the simple pH-stat fuzzy control, the response of pH change in the culture broth to the feeding rate of glucose was used to estimate the glucose consumption rate. In the modified pH-stat fuzzy control, the glucose consumption rate was accurately estimated by using pH change and the change in the carbon dioxide content of the exhaust gas. With this control strategy, the cell density could be increased to 72 g DCW dm-3, which was twofold higher than that attained in the cultivation with the simple pH-stat fuzzy control. The bulk β-galactosidase concentration was increased to 4150 U cm-3, which was threefold higher than when the simple pH-stat control was used.
    Additional Material: 6 Ill.
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  • 17
    Electronic Resource
    Electronic Resource
    Springer
    Cellular and molecular life sciences 49 (1993), S. 225-234 
    ISSN: 1420-9071
    Keywords: Ecological theory ; environmental safety ; exotic species ; genetic engineering ; introduced species ; recombinant DNA ; risk analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The exotic or non-indigenous species model for deliberately introduced genetically engineered organisms (GEOs) has often been misunderstood or misrepresented. Yet proper comparisons of ecologically competent GEOs to the patterns of adaptation of introduced species have been highly useful among scientists in attempting to determine how to apply biological theory to specific GEO risk issues, and in attempting to define the probabilities and scale of ecological risks with GEOs. In truth, the model predicts that most projects may be environmentallysafe, but a significant minority may be very risky. The model includes a history of institutional follies that also should remind workers of the danger of oversimplifying biological issues, and warn against repeating the sorts of professional misjudgments that have too often been made in introducing organisms to new settings. We once expected that the non-indigenous species model would be refined by more analysis of species eruptions, ecological genetics, and the biology of select GEOs themselves, as outlined. But there has been political resistance to the effective regulation of GEOs, and a bureaucratic tendency to focus research agendas on narrow data collection. Thus there has been too little promotion by responsible agencies of studies to provide the broad conceptual base for truly science-based regulation. In its presently unrefined state, the non-indigenous species comparison would overestimate the risks of GEOs if it were (mis) applied to genetically disrupted, ecologically crippled GEOs, but in some cases of wild-type organisms with novel engineered traits, it could greatly underestimate the risks. Further analysis is urgently needed.
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  • 18
    ISSN: 1573-6857
    Keywords: plant ; genetic engineering ; nutritive value ; agrobacterium ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract This review describes work aimed at the improvement of the nutritive value of grain and forage legumes using gene transfer techniques. Two traits which are amenable to manipulation by genetic engineering have been identified. These are plant protein quality and lignin content. In order to increase the quality of protein provided by the legume grains peas and lupins, we are attempting to introduce into these species chimeric genes encoding a sunflower seed protein rich in the sulphur-containing amino acids methionine and cysteine. These genes are designed to be expressed only in developing seeds of transgenic host plants. Chimeric genes incorporating a similar protein-coding region, but different transcriptional controls, are being introduced into the forage legumes lucerne and subterranean clover. In this case the genes are highly expressed in the leaves of transformed plants, and modifications have been made to the sunflower seed protein-coding sequences in order to increase the stability of the resultant protein in leaf tissue. Another approach to increasing plant nutritive value is represented by attempts to reduce the content of indigestible lignin in lucerne.
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  • 19
    Electronic Resource
    Electronic Resource
    Springer
    European journal of plant pathology 99 (1993), S. 259-268 
    ISSN: 1573-8469
    Keywords: antibacterial ; antimicrobial ; genetic engineering ; thionin ; toxicity assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Purothionins (PTHs) and hordothionins (HTHs) were purified by cation-exchange chromatography from petroleum-ether extracts of wheat and barley flour respectively. The HTHs could be separated into two fractions, HTH-1 and HTH-2. Radial diffusion assays and micro-plate broth dilution assays with a number of plant pathogenic bacteria showed that these proteins were toxic forClavibacter michiganensis subsp.michiganensis, the causal agent of bacterial canker on tomato,C. m. subsp.sepedonicus, the causal agent of ring rot on potato, andXanthomonas campestris pv.vesicatoria, the causal agent of a spot disease on tomato and pepper. Only minor differences in toxicity between PTHs and HTHs, and between HTH-1 and HTH-2, were detected. Minor differences in toxicity of these thionins were also detected for different strains of these bacteria. The use of these plant proteins for engineering bacterial disease resistance into solanaceous crops will be discussed.
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  • 20
    Electronic Resource
    Electronic Resource
    Springer
    Methods in cell science 15 (1993), S. 69-71 
    ISSN: 1573-0603
    Keywords: protoplast fusion ; gene transfer ; alkaline phosphatase ; expression cloning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A method is described that facilitates performing a large number of protoplast fusions to mammalian cells simultaneously and successfully. This method makes it possible to circumvent typical hurdles to the use of transient expression in mammalian cells, facilitating expression cloning of DNA enoding the newly detected gene product of interest. The original in vitro assay used to define the new activity is of interest, adapted to microtiter plates, combined with protoplast fusion, extends the reach of expression cloning to such cases as products of a rare message, or activities involving a multisubunit or unstable protein or both.
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  • 21
    ISSN: 1573-5028
    Keywords: antifreeze proteins ; gene transfer ; preproprotein processing ; α-helix stability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression of fish antifreeze protein (AFP) genes in plants is a possible means of increasing their frost resistance and freeze tolerance. Initial work involved transfer into tobacco of an AFP gene from winter flounder which codes for the alanine-rich, α-helical Type I AFP. Plants were transformed with a gene construct in which the preproAFP cDNA was inserted between the cauliflower mosaic virus 19S RNA promoter and the nopaline synthetase polyadenylation site. Although transgenic plants produced AFP mRNA, no AFP was detected on western blots. Re-evaluation of AFP expression in these transgenic plants showed that AFP accumulated to detectable levels only after exposure of the plant to cold. Extracts of plants incubated at 4°C for 24 h contained a protein which co-migrated with winter flounder proAFP and was cross-reactive to Type I AFP antisera. Two other minor protein bands of slightly higher apparent M r also cross-reacted with the antisera and are thought to represent processing intermediates. The proAFP was unique to the transgenic plants and was absent in extracts taken prior to cold exposure. AFP levels increased over the first 48 h of cold incubation then remained stable. Since the α-helix content of Type I AFP has been shown to decrease markedly at warmer temperatures, we postulate that Type I AFP stability in transgenic plants is dependent on its secondary structure.
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  • 22
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 33 (1993), S. 247-250 
    ISSN: 1573-5044
    Keywords: gene transfer ; GUS gene-marker ; particle acceleration ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Construction and operation of an airgun device for transient gene-expression studies in monocots is described. Compressed air in a cylinder of an airgun was used as the source of propulsion for DNA-coated gold or tungsten particles. Under a partial vacuum of 700 mm Hg, velocity of the macrocarrier was measured at 520 m sec−1 and 432 m sec−1 at atmospheric pressure. Optimum distance from the stopping plate to different target cells during bombardment ranged from 4 to 7 cm. Mean transformation efficiency of the GUS-gene marker was estimated at 350 transformants per 65 mg fresh weight of the maize cultured cells. Up to 200 GUS transformed cells were detected per 100 mg of embryogenic rice callus. Use of gold flakes or tungsten powder as microcarriers resulted in similar transformation rates. No transformation was observed in any cells when DNA constructs contained prokaryotic translation initiation sequences for the GUS gene. Based on transient GUS assays, further modification of the airgun device will likely be necessary to obtain high stable transformation rates.
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  • 23
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    Euphytica 73 (1993), S. 109-114 
    ISSN: 1573-5060
    Keywords: wild-germplasm ; interspecific-hybridization ; gene pools ; introgression ; gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Wild species which are crossable to cultivated pea, lentil, and chickpea have been collected and are maintained in major germplasm collections throughout the world. Wild species of Vicia crossable to the cultivated faba bean have not been found. The primary, secondary, and tertiary gene pools of the cool season food legumes represent potential genetic diversity that may eventually be exploited in cultivated types to overcome biotic and abiotic stresses. Technical difficulties in obtaining hybrids beyond those within the primary gene pool is a major obstacle. Reproductive isolation, embryo breakdown, hybrid sterility, and limited genetic recombination are major barriers to greater use of wild germplasm. Conventional crossing has been successful in producing interspecific hybrids in Lens, Cicer and Pisum and those hybrids are being evaluated for desired recombinants. In vitro culture of hybrid embryos has been successful in overcoming barriers to wider crosses in Lens. The successful transfer of genes from wide sources to cultivated types can be assisted by repeated backcrossing and selection designed to leave behind undesired traits while transferring genes of interest. Molecular marker assisted selection may become a valuable tool in the future use of wild species. In general, too little is known about the possible genetic variation available in wild species that could be valuable in developing resistance to biotic and abiotic stresses. Current efforts on the use of wide hybridization in the cool season food legumes are reviewed and discussed.
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  • 24
    ISSN: 1573-5060
    Keywords: gene cloning ; gene transfer ; virus and insect resistance ; genome analysis ; DNA figerprinting
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The potential of plant gene technology encompasses a multitude of different techniques ranging from the isolation of useful genes, their characterization and in vitro manipulation to the reintroduction of the modified constructs into target plants, where they are expressed at a rate that alters the phenotype of the plants. Genome analysis, on the other hand, aims at characterizing the genome architecture and function(s). Plant gene technology has catalyzed progress in plant breeding, as will be exemplified by a few examples, but has not yet been applied to food legume improvement on a large scale. Genome analysis, however, has a series of practical implications, as is illustrated by the successful introduction of DNA fingerprint and PCR fingerprint techniques to chickpea (Cicer arietinum L.) breeding and Ascochyta rabiei pathotyping. The present overview addresses both areas of plant molecular biology to illustrate their potential for food legume breeding.
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  • 25
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    Euphytica 74 (1993), S. 165-185 
    ISSN: 1573-5060
    Keywords: transgenic legumes ; genetic engineering ; particle bombardment ; Agrobacterium ; direct DNA transfer ; crop legumes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The absence of variety-independent gene transfer methods for major agronomic species has, until now, limited the usefulness of recombinant DNA techniques to crop improvement programs. Until recently, only Solanaceous crops could be used to study fundamental and applied problems in plant sciences. During the past five years rapid advances in cell biology, in combination with the development of novel gene transfer methodology allowed utilization of the tools of plant molecular biology in conventional breeding programs. Cereal and leguminous species were considered to be recalcitrant to genetic manipulation. As a result of the development of direct DNA transfer methodology into organized tissue, we are now in a position to introduce any foreign gene into almost all of the major cereals and legumes. This can be achieved efficiently, often in a variety-independent fashion. The object of this review is to provide a comprehensive account of the state of the art in gene transfer for the cultivated leguminous crops. Important oilseed and feed species primarily in industrialized countries, as well as minor but equally important species for sustaining growth populations in developing countries will be examined. Advantages of the various gene transfer methods that were shown to be useful for specific crops, as well as limitations and problems associated with each crop and gene transfer method will be discussed. Data from field trials of transgenic legumes, where available, will be presented.
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  • 26
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    Euphytica 70 (1993), S. 53-59 
    ISSN: 1573-5060
    Keywords: Brassica napus resynthesis ; DNA fingerprinting ; simple repetitive sequences ; somatic hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Brassica napus plants, artificially synthesized through somatic hybridization of B. oleracea and B. campestris protoplasts, were analyzed by oligonucleotide fingerprinting. While the fingerprint patterns of the different hybrid plants looked very much alike, they did not simply represent a combination of the parental patterns. Instead, the absence of parental bands as well as the presence of new bands suggest that elimination and/or rearrangements occurred during or after the fusion of the two genomes. The fingerprints of individual F1 progeny plants of selfed hybrids did not detect major changes. Thus, once formed, the artificially resynthesized amphidiploid B. napus genome appears to be stable. Taken together, our experiments demonstrate the usefulness of oligonucleotide fingerprinting for the characterization of artificial hybrids in the genus Brassica.
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  • 27
    ISSN: 1573-9368
    Keywords: rice ; small cell groups ; electrophoresis ; gene transfer ; stable integration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A new method has been developed to introduce foreign DNA into rice cells. Gene delivery occurred when an electrophoretic drive with cycles of intervallic electric field was applied to a mixture containing partially digested small cell groups (SCGs) and plasmid DNAs. Gene transfer efficiency was evaluated by the detection of β-glucuronidase (GUS) activity resulting from expression of a chimaeric plasmid DNA. The optimal combination of treatment conditions (3 V/cm, 30 s pulse and 30 min electrophoretic run) produced a frequency of up to 8.2% of blue cells in transformed microcalluses 40 days after culture of treated SCGs without selection for kanamycin resistance. Southern hybridization showed that the foreign gene had integrated into the chromosomal DNA. These results demonstrate that pulsed electrophoretic drive is applicable to the transfer of foreign genes into plant cells.
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  • 28
    ISSN: 1573-6830
    Keywords: herpesvirus vector ; gene transfer ; neuron-specific enolase (NSE) promoter ; lacZ, stereotactic delivery ; rat CNS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary 1. Herpesvirus infection with genetically engineered vectors is a way to deliver foreign gene products to various cell populations in culture andin vivo. Selective neuronal gene expression can be achieved using the neuron-specific enolase (NSE) promoter regulating expression of a transgene placed in and delivered by a herpesvirus vector. 2. We sought to determine the anatomical specificity and efficiency of herpesvirus-mediated gene transfer into the rat brain following placement of virus particles carrying a transgene (lacZ) under control of the NSE promoter. The virus utilized was thymidine kinase (TK) deficient and therefore replication deficient in the brain. 3. Infusion of 106 plaque-forming units of virus into the striatum caused a limited number of striatal neurons to express thelacZ transgene mRNA and protein product 7 days postinfection. In addition, small numbers of neurons expressing the transgene mRNA and protein were found ipsilateral to the viral injection in the frontal cortex, substantia nigra pars compacta, and thalamus. Neurons at these anatomic loci project directly to the striatal injection site. No other cells within the brains of injected animals expressed thelacZ gene. 4. While this herpesvirus NSE vector was capable of introducing novel functional genetic information into postmitotic neurons within defined neuroanatomic constraints, the numbers of neurons expressing detectable levels ofβ-galactosidase was minimal. The calculated efficiency of delivery and transgene expression at 7 days postinfection was 1 transgenic neuron per 104 virus particles infused. 5. We conclude that NSE probably is not an optimal promoter for use in gene delivery to CNS neurons in herpesvirus vectors and that the efficacy of gene delivery using other neuron-specific promoters placed at various sites in the herpes viral genome needs to be explored.
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  • 29
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    Transgenic research 2 (1993), S. 125-133 
    ISSN: 1573-9368
    Keywords: chicken embryo ; gene transfer ; retroviral vector ; cloned DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The application of transgenic technology to domestic poultry offers an alternative means to conventional practice for improvement of this highly productive agricultural species. The hen's reproductive system has unique characteristics which have imposed limitations on the use of established methods for artificial gene transfer. In this article, we review the various strategies that have been adopted to overcome the problem. Target sites for gene insertion include the fertilized ovum, the blastodermal embryo in the unincubated egg, and the primordial germ cells. Notable success in obtaining somatic and germline transformation has been achieved with the use of retroviral vectors to infect the blastodermal embryo. Current attempts to introduce DNA directly into the genome, without resort to pathogen-derived vectors, are discussed.
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  • 30
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    European journal of plant pathology 98 (1992), S. 1-12 
    ISSN: 1573-8469
    Keywords: genetic engineering ; resistance genes ; transgenic virus resistance ; viral genes ; virus interactions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Knowledge of the nucleotide sequences in the genomic nucleic acid of several potato viruses has enabled the open reading frames to be identified. These open reading frames are expressed by a variety of strategies, to produce proteins with functions in virus nucleic acid replication, virus particle production, cell-to-cell transport of virus and virus transmission by vectors. The activity of such proteins depends on their interactions with other viral or non-viral materials. Several other biological properties of plant viruses can also be related to individual viral gene products. For example, in plants co-infected with a specific pair of unrelated viruses, one virus can benefit from an ability to use the gene product of the second virus in replication, cell-to-cell transport or transmission by vectors. Similarly, different host resistance genes are targeted against viral replicase, movement protein or coat protein. Thus it is becoming possible to relate gene-for-gene (or more accurately, viral gene domain-host gene) interactions to events at the molecular level. Genetically engineered resistance to plant viruses likewise can be targeted against individual viral genes, and probably also against viral regulatory sequences. Such transgenic resistance seems likely to be as durable as conventional host resistance but durability should be improved by producing plants with combinations of resistances of different kinds, either conventional or genetically engineered, or both.
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  • 31
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    European journal of plant pathology 98 (1992), S. 29-36 
    ISSN: 1573-8469
    Keywords: genetic engineering ; resistance genes ; transgenic virus resistance ; viral genes ; PVX ; PVY ; PLRV
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract To engineer resistance against potato virus X (PVX), the viral coat protein (CP) gene has been introduced into two potato cultivars. Stable expression of the gene in transgenic clones throughout the growing season has been obtained and resulted in considerably increased virus resistance. With varying frequencies depending on the original cultivar used, true-to-type PVX resistant transgenic clones have been obtained. Since deviant light sprout characteristics were invariably associated with aberrations in plant phenotype, they can be used in procedures to early screen for deviations. Furthermore, it has been possible to unequivocally discriminate between the original untransformed and independent transgenic cultivars. Although no relation has been found between the presence, if any, of the CP of potato virus Y (PVY) or potato leafroll virus (PLRV) in CP gene transgenic potato, appreciable levels of resistance to these viruses has been obtained. This suggests that the mechanism by which a viral CP gene in the potato genome evokes resistance, differs amongst various viruses.
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  • 32
    ISSN: 1573-5028
    Keywords: Brazil nut ; 2S albumin gene ; gene transfer ; Phaseolus vulgaris ; particle bombardment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Bean (Phaseolus vulgaris L.) mature embryos were transformed using biolistic methods with a plasmid containing 2S albumin and β-glucuronidase structural sequences, both under the control of the 35S CaMV promoter. We have shown that chimaeric tissues could be obtained and that both structural sequences were expressed to similar levels.
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  • 33
    ISSN: 1573-5028
    Keywords: genetic engineering ; phage P1 ; recombinase ; luciferase ; selectable markers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Cre-lox site-specific recombination system of bacteriophage P1 was used to excise a firefly luciferase (luc) gene which had previously been incorporated into the tobacco genome. The excision event was due to site-specific DNA recombination between two lox sequences flanking the luc gene and was catalyzed by the Cre recombinase introduced by cross-fertilization. Recombination resulted in the fusion of a promoter with a distally located hygromycin phosphotransferase (hpt) coding sequence and the excision event was monitored as a phenotypic change from expression of luc to expression of hpt. The efficiency of recombination was estimated from the exchange of gene activity and confirmed by molecular analysis. The relevance to potential applications of site-specific deletion-fusion events for chromosome engineering are discussed.
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  • 34
    ISSN: 1573-5044
    Keywords: nitrate reductase deficiency ; potato ; somatic hybridization ; tomato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts of two ‘leaky’ nitrate reductase deficient and thus nitrate auxotrophic (NAR) mutants of tomato and their wild types, were fused with protoplasts of monoploid potato. In all four combinations hybrid calli grew more vigorously than parental calli and this somatic hybrid vigour as such provided a useful enrichment for somatic hybrids. Selection against nitrate auxotrophy further increased the efficiency of the enrichment, particularly if a molybdenum cofactor mutation was used as the basis for the selection. It is concluded that the nitrate auxotrophy of these NAR mutants is sufficiently expressed at the level of the cell, to allow its use in somatic hybridization experiments with potato.
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  • 35
    ISSN: 1573-5044
    Keywords: Agrobacterium transformation ; Cucumis sativus ; gene transfer ; neomycin phosphotransferase ; tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cucumber (Cucumis sativus L.) petiole and leaf segments of two pickling genotypes were transformed with Agrobacterium tumefaciens strain LBA 4404, an octopine Ti-plasmid deletion mutant that is avirulent (disarmed plasmid), but to which were added T-DNA inserts on binary plasmids (pBIN 19, ca. 10 kb, and pCGN 783, ca. 25 kb). Expression of neomycin phosphotransferase (NPT II) encoding resistance to the aminoglycoside kanamycin was used as a selectable marker. Factors which influenced the frequency of callus development on medium containing kanamycin (75 mg l-1) were explant size, bacterial concentration and length of exposure, cocultivation period, and presence of acetosyringone. The optimal procedure involved exposing segments of petiole (4–6 mm) or leaf (0.5 cm2) segments to a bacterial suspension (108 cells ml-1) containing 20 μM acetosyringone for 5 min, followed by a 48 h cocultivation period on a tobacco feeder layer. Explants were placed on MS medium containing 500 mg l-1 carbenicillin, 75 mg l-1 kanamycin, and NAA/BA (5.0/2.5 μM) or 2,4-d/BA (5.0/5.0 μM) and subcultured twice, each after a 2–3 week period, onto fresh media. The overall frequency of transformed callus was 20–50%; the frequency of plantlet regeneration from transformed callus was 8–15%. Twenty-one out of 23 individual plants recovered from two genotypes of pickling cucumber were NPT II positive (transformation frequency of 9%). Copy number of the NPT II gene insert (35S-NPT II-3′ fragment, ca. 2.2 kb) in three transformed plants was estimated at ten per haploid genome, indicative of multiple insertions within the cucumber genome. Multimers of the gene (visible as 4.4 and 6.6 kb fragments in Southern analysis) were detected in one plant, suggestive of tandem duplications or repeats. Progeny from a cross between this transformed plant and a nontransformed control showed segregation for the NPT II gene in dot-blot assays; at least 24 plants out of 32 were kanamycin positive. Copy number in the progeny was variable, and ranged from none to ten.
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  • 36
    ISSN: 1573-5060
    Keywords: Brassica napus ; fertility ; interspecific hybridization ; self-incompatibility ; somatic hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Brassica napus is a natural allotetraploid derived from the diploid species B. rapa L. (syn. campestris L.) and B. oleracea L. Somatic hybrids synthesized from highly heterozygous lines of these two diploid species were evaluated for fertility. The hybrids were obtained from two fusion experiments which differed in the B. rapa full-sibling parent used as the source of protoplasts. Both B. rapa siblings were lelf-incompatible (SI) yet contained different S-alleles; the B. oleracea species parent was self-compatible (SC). Eight tetraploid hybrids examined had very high female and male fertility; eight hybrids with higher ploidy had low fertility. Hybrids derived from one B. rapa sibling were self-incompatible, whereas those derived from the other B. rapa sibling were fully self-compatible. These data suggest that the different S-alleles of each B. rapa sibling displayed varying penetrance relative to the SC of the B. oleracea parent when combined in B. napus.
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  • 37
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    Sociological forum 6 (1991), S. 551-565 
    ISSN: 1573-7861
    Keywords: agriculture ; capitalism ; development ; world economy ; Third World ; genetic engineering
    Source: Springer Online Journal Archives 1860-2000
    Topics: Sociology
    Notes: Abstract Frederick Buttel was one of the pioneers in studying the social impacts of biotechnology, claiming originally that it will involve profound changes in social structure. Recently Buttel turned around his argument proposing that, rather than revolutionary, biotechnology is more a “substitutionist” technological form to be applied to declining sectors of the economy than an “epoch-making” technology. This paper provides both external and internal critiques of Buttel's new position based on the concept of the “third technological revolution,” looking at the impact of new technologies as a global and interrelated phenomenon, and not on an individual case-by-case basis. The concluding section suggests the necessity of bringing into the analysis those living in the Third World: 60% of this population lives from agriculture and will be affected by the deployment of agricultural biotechnologies, whether through “substitutionism” or through totally new products.
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  • 38
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    European journal of epidemiology 7 (1991), S. 207-212 
    ISSN: 1573-7284
    Keywords: Rickettsia ; Intracellular parasite ; genetic engineering ; molecular biology ; rickettsial genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Our understanding of the biology of the rickettsiae, organisms that are the archetype of the obligate intracytoplasmic bacterial parasites, remains muddy and fragmentary. For example although we all appreciate that the rickettsiae can exploit their unique environment, the host cell cytoplasm, but are unable to grow axenically, the basis of this fact is still one of microbiology's central mysteries. It is unfortunate, but true, that because of the inherent difficulty of working within this system, progress on the answers to such questions will be slow and laborious. However, with the application of molecular biological methods, that is, the powerful modern approaches of genetics and biochemistry, the rickettsiology community has the realistic prospect that this field is far from being at a stand-still and that significant increases in our comprehension of the fundamental problems of rickettsial biology are occurring and will continue to occur at ever accelerating rates. Some examples, both in terms of scientific conclusions and technical approaches, of the progress made in recent years and expectations for the near future will be presented.
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  • 39
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    Neurological sciences 12 (1991), S. 257-268 
    ISSN: 1590-3478
    Keywords: Human muscle cultures ; clones ; nerve-muscle cocultures ; cell lines ; heterokaryons ; myoblast transfer ; gene transfer ; Myo D ; cybrid clones ; hereditary human myopathies
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Sommario L'Autore illustra l'utilità e i limiti delle colture di muscolo umano nello studio delle miopatie umane ereditarie. In particolare nelle miopatie dovute a difetti di enzimi-specifici muscolari, l'utilizzo di tecniche di cocoltura muscolo-nervo con avanzato grado di maturazione muscolare permette di delucidare i meccanismi molecolari patogenetici di tali malattie. L'uso di avanzate tecniche di biologia molecolare e cellulare, quali linee permanenti, trapianto di mioblasti, trasferimento genico e di mitocondri, oltre che fornire utili informazioni a livello molecolare potranno trovare applicazione, in futuro, persino nella terapia di molte miopatie ereditarie.
    Notes: Abstract In this article I illustrated the use of regenerating human muscle cultures for studying the hereditary human myopathies. Although some of the data are still controversial, they do point up the great potential of this “in vitro system”. For hereditary myopathies due to developmentally regulated proteins that are expressed only at a more advanced stage of muscle differentiation, the use of highly differentiated nerve-muscle cocultures might contribute significantly to a better understanding of their developmental pathogenesis. More advanced techniques (permanent human muscle cell lines, heterokaryons, myoblast transfer, gene transfer, myogenic conversion of human non-muscle cells, cybrid clones) may provide a great deal of information at molecular level and may also have practical applications in the diagnosis or even in the treatment of hereditary human myopathies.
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  • 40
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    Euphytica 55 (1991), S. 157-169 
    ISSN: 1573-5060
    Keywords: gene transfer ; genetic manipulation ; chimaeric genes ; legumes ; transformation ; somatic hybridisation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The merits and limitations of somatic cell techniques involving Agrobacterium-mediated transformation, direct gene transfer and protoplast fusion, are discussed in relation to the genetic improvement of forage and grain legumes. Whilst progress with legumes is limited compared to that with plants of other families such as the Solanaceae, the fact that many legumes are readily amenable to tissue culture now permits somatic cell techniques to be targetted to these species. Future development of the subject will necessitate close collaboration between molecular biologists and plant breeders to enable novel plants generated by in vitro technologies to be incorporated into conventional breeding programmes.
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  • 41
    ISSN: 1573-4919
    Keywords: human growth hormone ; rat pituitary tumor cells ; tissue-specificity ; gene transfer ; DNase 1 footprinting
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary Placental chorionic somatomammotropin (hCS-A or B) and growth hormone variant (hGH-V) are members of the human growth hormone family, and are related by structure and function to pituitary growth hormone (hGH-N). However, while the hGH-N gene is expressed specifically in the anterior pituitary, hGH-V and hCS are produced in the placenta. Hybrid hGH-N, hGH-V and hCS-A genes containing 5′-flanking sequences, including the endogenous promoter, are preferentially expressed in rat pituitary tumor (GC) cells, after gene transfer. Since interaction with a pituitary-specific protein (Pit 1) is required for efficient hGH-N as well as rat growth hormone (rGH) gene expression in GC cells, binding of pituitary proteins to the hGH-V and hCS-A promoter sequences was investigated. Rat Pit 1 binds at two locations on the hGH-N gene, a distal (−140/−107) and proximal site (−97/−66), in a similar manner to that observed with the rGH gene. By contrast, efficient Pit 1 binding was seen only to the distal site of the hGH-V gene and the proximal site of the hCS-A gene. Although binding of a protein to the distal hCS-A sequences was observed, the site of interaction was truncated (−140/−116), not pituitary-specific, and was more consistent with the binding of Sp1. These data indicate that rat Pit 1 binds to the placental hGH-V and hCS-A genes and correlates with their promoter activity in GC cells after gene transfer. However, the data also indicate that rat Pit 1 binds to human and rat pituitary growth hormone in a similar manner (two sites of interaction) and that the pattern of binding is distinct from the placental members of the hGH gene family. These data indicate that human Pit 1, unlike the rat equivalent, might distinguish these genes functionally (tissue-specifically) as well as structurally.
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  • 42
    ISSN: 1573-5028
    Keywords: storage protein ; genetic engineering ; transgenic plants ; zeins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chimeric genes composed of the β-phaseolin promoter, an α-zein coding sequence and its modified versions containing lysine codons, and a β-zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation. α-Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population. At 25 days after pollination the 19 kDa α-zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants. The transgenic plant with the highest level of zein gene expression had an α-zein content that was approximately 0.003% of the total seed protein. The amount of α-zein in other transgenic plants varied between 1 × 10−4% and 1 × 10−5% of the total seed protein. The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the α-zein protein. Polysomes translating α-zein mRNA isolated from tobacco seeds contained fewer ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis. In vivo labelling and immunoprecipitation indicated that newly synthesized α-zein was degraded in tobacco seeds with a half-life of less than 1 hour.
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  • 43
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    Journal of agricultural and environmental ethics 4 (1991), S. 101-107 
    ISSN: 1573-322X
    Keywords: agricultural bioethics ; bovine somatotropin ; ownership of germ plasm ; genetic engineering ; animal rights ; intellectual property rights
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Philosophy
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  • 44
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 38 (1991), S. 296-303 
    ISSN: 0006-3592
    Keywords: ethanol ; genetic engineering ; Escherichia coli ; lignocellulose ; xylose ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The conversion of xylose to ethanol by recombinant Escherichia coli has been investigated in pH-controlled batch fermentations. Chemical and environmental parameters were varied to determine tolerance and to define optimal conditions. Relatively high concentrations of ethanol (56 g/L) were produced from xylose with excellent efficiencies. Volumetric productivities of up to 1.4 g ethanol/L h were obtained. Productivities, yields, and final ethanol concentrations achieved from xylose with recombinant E. coli exceeded the reported values with other organisms. In addition to xylose, all other sugar constituents of biomass (glucose, mannose, arabinose, and galactose) were efficiently converted to ethanol by recombinant E. coli. Unusually low inocula equivalent to 0.033 mg of dry cell weight/L were adequate for batch fermentations. The addition of small amounts of calcium, magnesium, and ferrous ions stimulated fermentation. The inhibitory effects of toxic compounds (salts, furfural, and acetate) which are present in hemicellulose hydrolysates were also examined.
    Additional Material: 5 Ill.
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  • 45
    ISSN: 1573-0778
    Keywords: genetic engineering ; Namalwa cells ; perfusion culture ; scaling-up ; serum-free medium ; stable production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We investigated the basic technology of cell culture conditions for production of useful substances such as cytokines, and related proteins produced by Namalwa cells. Namalwa cells (Klein, 1972), human B lymphoblastoid cells, were used for large scale production of alpha-interferon (Klein, 1979). Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 × 107 cells/ml in suspension mode by the use of a perfusion culture system, Biofermenter™, containing a cone-type cell-sedimentation column as cell separator (Sato, 1983). Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells (Miyaji, 1990a,b,c). Some of these were produced in large quantities by use of a gene amplification method with dhfr (Miyaji, 1990c), even though the Namalwa KJM-1 cells contained endogenous dhfr genes. For stable production of the target protein, Namalwa KJM-1 cells are very useful host cells, because they have no effective endogenous protease activity in the conditioned medium. Using Biofermenter with micro-silicone fibers and a dialysis system, the specific productivity of the target proteins was not depressed at a high cell density.
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  • 46
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 261-267 
    ISSN: 0730-2312
    Keywords: tissue transplantation ; catecholamines ; dopamine ; L-DOPA ; genetic engineering ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In animal models, grafts derived from several different tissues, principally fetal substantia nigra and adrenal medulla from young adults, have been found to be effective in alleviating some of the manifestations of lesions of the substantia nigra. It has been suggested that these grafts function by diffusely secreting dopamine, by exerting trophic effects on the host brain, or by producing a new innervation of the host corpus striatum. Evidence for each of these modes of action is briefly reviewed. Several brain tissue transplantation techniques have been described. Each of these techniques has significant limitations in animal models. The significance of these limitations for human application is described, and possibilities for improving the efficacy of brain tissue transplantation in animal models and for human application are discussed.
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  • 47
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 268-272 
    ISSN: 0730-2312
    Keywords: hematopoiesis ; stem cell ; perfusion ; hematopoietic growth factor ; genetic engineering ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The successful ex vivo reconstruction of human bone marrow is an extraordinarily important basic scientific and clinical goal. Fundamentally, the system is the paradigm of a complex interactive tissue, in which the proliferation and regulated differentiation of one parenchymal cell type (the hematopoietic stem cell) is governed by the surrounding stromal cells. Understanding and reproducing the molecular interactions between bone marrow stromal cells and stem cells in tissue culture models is therefore the critical step in successful bone marrow tissue culture. Clinically, successful reconstruction of human bone marrow would permit the controlled production of mature blood cells for transfusion therapy, and immature bone marrow stem cells for bone marrow transplantation. In approaching the bone marrow culture system, we recognize the critical role that hematopoietic growth factors (HGFs) play in hematopoiesis. Since stromal cells in traditional human bone marrow cultures produce little HGFs, we have begun by asking whether local supplementation of hematopoietic growth factors via genetically engineered stromal cells might augment hematopoiesis in liquid cultures. The results indicate that locally produced GM-CSF and IL-3 do augment hematopoiesis for several weeks in culture. In combination with geometric and dynamic approaches to reconstructing physiological bone marrow microenvironments, we believe that this approach has promise for reconstructing human bone marrow ex vivo, thereby permitting its application to a variety of basic and clinical problems.
    Additional Material: 1 Ill.
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  • 48
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 353-358 
    ISSN: 0730-2312
    Keywords: bone marrow ; peripheral blood ; gene transfer ; IL-2 ; neo gene ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Human T-lymphocytes are long lived, easily accessible, mature, and capable of proliferation. They are theoretically a suitable target for retroviral mediated gene transfer. To test this hypothesis, normal human T-cells obtained from bone marrow and peripheral blood were stimulated with phytohemagglutinin (PHA) and infected 24 h later with the retroviral vector N2 which carries the bacterial neo gene. T-lymphocytes were propagated in culture for up to 14 weeks with interleukin-2 (IL-2). Analysis by whole cell RNA dot/blot using a single stranded RNA probe demonstrated persistent expression of the neo gene. Preliminary functional studies revealed that both helper and suppressor functions were preserved in the infected cells in culture. These results demonstrate that normal T-cells are capable of long-term expression of genes introduced by retroviral mediated gene transfer and are potential target cells for somatic gene therapy.
    Additional Material: 3 Ill.
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  • 49
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    Theoretical medicine and bioethics 11 (1990), S. 103-110 
    ISSN: 1573-1200
    Keywords: genetic engineering ; human embryos ; medical ethics ; medical technology ; in vitro fertilization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Philosophy
    Notes: Abstract In this paper I will discuss three areas in which advances in human reproductive technology could occur, their uses and abuses, and their effects on society. First is the potential to drastically increase the success rate and availability of in vitro fertilization and embryo freezing. Second is the ability to perform biopsies on embryos prior to the onset of pregnancy. Finally, I will consider the adding or altering of genes in embryos, commonly referred to as “genetic engineering”. As new reproductive technologies pass from experimental models into the potential for medical utilization, I believe that it will be important for lawmakers everywhere to avoid the impulse to outlaw procedures that a society believes to be ‘unnatural’ at a first glance. Rather, I would hope that they can respond thoughtfully with legislation that serves two purposes — to protect the rights of couples to overcome infertility or to reduce the risk of genetic disease in their children-to-be, and more importantly, to protect children-to-be from the abuses that could result from some of the practices that I will discuss.
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  • 50
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    Hydrobiologia 204-205 (1990), S. 151-159 
    ISSN: 1573-5117
    Keywords: Enteromorpha ; genetic engineering ; micro spectrophotometry ; parasexual fusion ; seaweeds ; Ulvaria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Enteromorpha linza and Ulvaria oxysperma in North Carolina reproduce exclusively by asexual zoospores. Calcofluor white staining indicated that newly released zoospores lack significant cellulose cell wall material, making them suitable for treatment as protoplasts in a parasexual fusion process using high pH-Ca2+, PEG and centrifugation. Presumptive fusion products were identified by their larger size, twin chloroplasts and eyespots, and presence of fluorescence labelled and unlabelled portions. Parasexual fusion and karyogamy were confirmed by elevated levels of nuclear DNA in fusion cell germlings. In addition, aceto-orcein staining of fusion cell products revealed a diploid chromosome complement of 2N = 20 in Enteromorpha linza. Fusion cells were isolated by killing the more numerous adjacent unfused zoospores with 2-3 min exposure to blue light (410–490 nm). Unexposed fusion cells could be readily distinguished and recovered by micropipette at the 10-day stage.
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  • 51
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    Plant molecular biology 14 (1990), S. 501-511 
    ISSN: 1573-5028
    Keywords: chloroplast SOD ; paraquat ; stress ; genetic engineering
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The petunia nuclear gene which encodes the chloroplast isozyme of superoxide dismutase, SOD-1, has been fused with an efficient rbcS promoter fragment and 3′ flanking region and introduced into tobacco and tomato cells. Transformed plants carrying this chimeric gene have up to 50-fold the levels of SOD-1 which occur in wild-type plants. However, tobacco plants with 30-to 50-fold the normal SOD-1 activity do not exhibit resistance to the light-activated herbicide paraquat. Similarly, tomato plants with 2-to 4-fold increases in SOD-1 do not exhibit tolerance to photoinhibitory conditions known to increase superoxide levels (high light, low temperatures and low CO2 concentrations). Our data indicate that increasing the chloroplastic SOD level in a plant cell is not sufficient to reduce the toxicity of superoxide.
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  • 52
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    Plant molecular biology 15 (1990), S. 527-538 
    ISSN: 1573-5028
    Keywords: β-glucuronidase ; CaMV 35S promoter ; genetic engineering ; immunohistochemistry ; Oryza sativa ; transgenic rice
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The cauliflower mosaic virus promoter is commonly used to drive transcription of chimeric genes in transgenic plants, including the cereals. To determine the tissue and cell types of cereal plants that the promoter functions in, transgenic rice plants containing a CaMV 35S promoter/GUS chimeric gene were analyzed for GUS activity. Insertion of a 35S/GUS chimeric gene at low copy number into chromosomal DNA of plants regenerated from electroporated protoplasts was confirmed by gel blot hybridization analysis of uncut and endonuclease-digested DNA. Quantitative measurement showed that GUS activity was some tenfold higher in rice leaves than in tobacco leaves [8] whereas activities obtained for rice roots were similar to those reported for tobacco roots. Histochemical localization of GUS activity confirmed that the CaMV 35S promoter functions in cells of the leaf epidermis, mesophyll and vascular bundle. It is also active in the cortex and vascular cylinder of the root, but only marginally active in the root epidermis. The generally similar distribution and levels of GUS activity obtained in differentiated tissue of stably transformed rice plants indicates the value of the CaMV 35S promoter as a positive control for studies in gene activity in transgenic monocots and dicots.
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  • 53
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    Journal of agricultural and environmental ethics 3 (1990), S. 114-146 
    ISSN: 1573-322X
    Keywords: genetic engineering ; herbicide resistance ; herbicide tolerant crops ; agricultural ethics ; morality ; values ; justice ; sustainable agriculture ; alternative agriculture ; socio-economic effects ; weeds ; biotechnology ; agribusiness ; family farms
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Philosophy
    Notes: Abstract Should we continue to support publicly funded research on genetically engineered herbicide resistant crops? In Part One, I discussed the difference between science and ethics, presented a brief history of weed control, and explained three moral principles undergirding my environmentalist perspective. I then argued that unqualified endorsement (E) of the research is unjustified, as is unqualified opposition (O). In Part Two, I argue against qualified endorsement (QE), and for qualified opposition (QO).
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  • 54
    ISSN: 1573-5060
    Keywords: Oryza sativa ; Indica-type rice ; genetic engineering ; vitamin A endosperm ; insect resistance ; virus resistance ; fungus resistance ; essential amino acids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Indica-type rice provides the staple food for two billion people in Third World countries. Several problems involved in the stable and sustained production of high quality food cannot be solved by traditional breeding. Methods have been established for gene transfer to Indica rice breeding lines to study possible contributions from genetic engineering. Experiments are in progress on the development of transgenic resistance towards Yellow Stem Borer, resistance towards Rice Tungro Virus, accumulation of provitamin A in the endosperm, increase of essential amino acids in the endosperm such as lysine, cysteine and methionine and resistance towards fungal pests such as Rice Blast and Sheath Blight. Transgenic clones from Indica rice breeding lines have been recovered from several of the approaches mentioned, some of which have been regenerated to plants.
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  • 55
    ISSN: 1573-5060
    Keywords: Solanum tuberosum ; somatic hybridization ; regeneration ; asymmetric fusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary This paper reviews investigations into the application of protoplast fusion to the genetic and agronomic improvement of potato. Fusion studies involving Solanum tuberosum are reviewed under the categories of: fusion with wild relatives, dihaploid fusion and asymmetric strategies. The selection and characterisation of putative somatic hybrid material is identified as a critical stage in the process and certain specific aspects of this technology are identified. Future prospects for the wider uptake and integration of these techniques into breeding programmes are also discussed.
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  • 56
    ISSN: 1573-5060
    Keywords: Lactuca sativa ; Lactuca virosa ; Lactuca tatarica ; Lactuca perennis ; Iettuce ; sexual hybridization ; embryo rescue ; somatic hybridization ; protoplast fusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Various genes for disease resistance identified in wild Lactuca are difficult, even impossible to exploit in lettuce breeding, due to sexual incompatibility between L. sativa and wild Lactuca sp. We adapted two cellular biology techniques to overcome these interspecific barriers: in vitro embryo rescue and protoplast fusion. In vitro rescue of immature embryos was used successfully for sexual hybridization between L. sativa and L. virosa. Vigorous hybrid plants were produced between L. sativa and seven accessions of L. virosa. Protoplast fusion permitted the regeneration of somatic hybrids between L. sativa and either L. tatarica or L. perennis. Hybrids between L. sativa and L. tatarica were backcrossed to L. sativa.
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  • 57
    ISSN: 1573-5060
    Keywords: Linum usitatissimum ; linseed ; mutation breeding ; somaclonal variation ; fatty acids ; genetic engineering
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary In the early 1980s the phenomenon of somaclonal variation induced by cell culture was exploited to produce genetic variation in linseed. The linseed variety Andro, derived from the widely grown Canadian variety McGregor, was selected in saline culture and was released for production in Canada. ‘Andro’ possesses traits very different from its parent, such as increased seedling vigour and tolerance to heat stress. Additional stable somaclonal variation in characters such as yield, days to maturity, seed weight and oil content were subsequently induced in ‘McGregor’. However, despite extensive screening of the somaclonal variants, no significant variation in the fatty acid profile was found. Chemical mutagenesis using ethyl methanesulphonate was, however, succesful in modifying the fatty acid profile of McGregor. Initial screening of M2 seed by the thiobarbituric acid colourimetric procedure was followed by gas chromatography to select half-seeds with atypical fatty acid profiles. Two independent, partially dominant genes were identified that were responsible for reducing the linolenic acid (18 : 3) from 50% to 2% while increasing linoleic acid (18 : 2) to 70%. A single, partially dominant gene, inherited independently of the linolenic acid genes, increased palmitic acid (16 : 0) from 7% to 30% and palmitoleic acid (16 : 1) from trace amounts to 4%. Agrobacterium-mediated transformation of linseed has also been successful. Herbicide tolerance genes for glyphosate, sulfonylurea and phosphinothricin have been incorporated into Canadian varieties. Commercially useful levels of tolerance to sulfonylurea herbicides have been achieved with no adverse agronomic affect. It is expected that a transgenic variety containing this resistance will be registered for commercial production in Canada in 1994. Standard breeding techniques, the application of antisense technology and the overexpression of fatty acid synthesis genes are being used to further modify the fatty acid profile of linseed, as well as for the transfer of abiotic stress-related genes identified in bromegrass.
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  • 58
    ISSN: 1573-5060
    Keywords: genetic engineering ; introgression ; molecular markers ; potatoes ; resistances ; Solanum ; technology mansfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Potato genetic improvement has been facilitated using new knowledge of potato reproductive biology and new techniques. Many wild diploid species as well as landrace cultivars have been used in breeding at the diploid level, a strategy which is supported by 1) 2n gametes and 2) haploids from tetraploid cultivars. Different categories of wild species which have been under-utilized are now being exploited further in more systematic enhancement programmes using semi-conventional and biotechnological methods. Molecular maps of the potato genome are used actively to achieve marker-assisted introgression and improved selection among the germplasm collections to facilitate the use of valuable wild genetic resources. As an alternative method to incorporate a high level of fesistance, genetic engineering has been employed to facilitate the initial breeding process using various gene constructs for controlling major biotic stresses in the world.
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  • 59
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    Euphytica 85 (1955), S. 217-233 
    ISSN: 1573-5060
    Keywords: crop improvement ; alien gene transfer ; progeny analysis ; somatic hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary In recent years, the rapid development of somatic cell genetics has made possible the transfer of alien genes over wide taxonomic distances by somatic hybridization. In this review, the potential of somatic hybridization in the breeding of crops within the Brassicaceae, Fabaceae, Poaceae and Solanaceae is discussed. It is evident from these studies that many hybrids, either symmetric or asymmetric, which are fertile have the potential to be used as a bridge between the alien species and the crop. Progeny analysis of some hybrid combinations also reveals intergenomic translocations which may lead to the introgression of the alien genes. Furthermore, fusion techniques enable the resynthesis of allopolyploid crops to increase their genetic variability and to restore ploidy level and heterozygosity after breeding at reduced ploidy level in polyploid crops.
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  • 60
    ISSN: 1573-5060
    Keywords: gene transfer ; Hordeum vulgare ; neomycin phosphotransferase II ; particle bombardment ; transgenic barley
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for β-glucuronidase (GUS). Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T0). This chimeric plant passed the transferred nptII gene to its T1 progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T0 spikes and 15 T1 offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T2 and T3 plants were produced by isolating embryos from green grains of transgenic T1 and T2 plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T2 offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed. Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticin R or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile.
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  • 61
    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.
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  • 62
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    Euphytica 85 (1955), S. 1-12 
    ISSN: 1573-5060
    Keywords: genetic engineering ; gene targets ; mapping ; markers ; transformation ; QTLs
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
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  • 63
    ISSN: 1573-5060
    Keywords: meristem ; shoot apex ; ballistic microtargeting ; gene transfer ; wheat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The classical approach of gene transfer to a given plant species delivers the foreign gene to transformable cells and then puts the effort into generating plants. This approach is very difficult in many important crop plants, including cereals, and the results of regeneration are very genotype-dependent. In contrast, we use regenerable cells and try to transform them. Shoot apical meristems provide a tissue which regenerates in situ a fertile plant for most given genotypes or species. Transformation of meristem cells may lead to transgenic sectors in chimeras. These sectors may contribute to the gametes and, thus, to transgenic offspring, which then should be homohistonts and not sectorial chimeras like their parents. Our model plant for these studies is wheat. Microtargeting is a ballistic approach which is particularly suitable for the controlled delivery of microprojectiles to meristem cells in situ (Sautter et al., 1991). We summarize in this paper our experience with ballistic microtargeting of transgenes to wheat shoot apical meristem cells in situ.
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  • 64
    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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  • 65
    ISSN: 1573-5060
    Keywords: gene transfer ; crop species ; particle bombardment ; transgenic plants ; cereals ; legumes ; woody plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The limiting component in the creation of transgenic crops has been the lack of effective means to introduce foreign genes into elite germplasm. However, the development of novel direct DNA transfer methodology, by-passing limitations imposed by Agrobacterium-host specificity and cell culture constraints, has allowed the engineering of almost all major crops, including formerly recalcitrant cereals, legumes and woody species. The creation of transgenic rice, wheat, maize, barley, oat, soybean, phaseolus, peanut, poplar, spruce, cotton and others, in an efficient and in some cases, variety-independent fashion, is a significant step towards the routine application of recombinant DNA methodology to the improvement of most important agronomic crops. In this review we will focus on key elements and advantages of particle bombardment technology in order to evaluate its impact on the accelerated commercialization of products based on agricultural biotechnology and its utility in studying basic plant developmental processes and function through transgenesis. Fundamental differences between conventional gene transfer methods, utilizing Agrobacterium vectors or protoplast/suspension cultures, and particle bombardment will be discussed in depth.
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