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  • 1990-1994  (155)
  • 1955-1959  (12)
  • 1890-1899
  • Agrobacterium
  • genetic engineering
  • protoplasts
  • 1
    Electronic Resource
    Electronic Resource
    A reporter gene under the control of tms or aux promoters is differentially expressed in tobacco and barley protoplasts (1994)
    Springer
    Plant cell reports 13 (1994), S. 155-158 
    Details
    ISSN: 1432-203X
    Keywords: Agrobacterium ; auxin biosynthesis genes ; barley and tobacco protoplasts ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Agrobacterium tumefaciens and some Agrobacterium rhizogenes strains possess auxin biosynthesis genes (tms and aux genes respectively), responsible for a de novo auxin biosynthetic pathway in transformed plant cells. A comparison is presented of the potential expression of these genes in a monocotyledonous (barley) and a dicotyledonous plant (tobacco). The promoters of the genes were translationally fused to the β-glucuronidase reporter gene and analysed in transient expression experiments. The tms and aux fusions were highly expressed in tobacco, but not in barley. However, the aux enhancer active in tobacco, conferred low β-glucuronidase expression in barley when fused to a truncated cauliflower mosaic virus 35S promoter. The results are discussed in relation to the differential responses to Agrobacterium infection in monocots and dicots.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00239883
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  • 2
    Electronic Resource
    Electronic Resource
    Stable expression of the GUS reporter gene in chrysanthemum depends on binary plasmid T-DNA (1994)
    Springer
    Plant cell reports 14 (1994), S. 59-64 
    Details
    ISSN: 1432-203X
    Keywords: Agrobacterium ; transformation ; T-DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three chrysanthemum (Dendranthema grandiflora) cultivars were cocultivated with 2 Agrobacterium tumefaciens strains in combination with 4 pBIN19 derived binary plasmids, all carrying the Nosnptll selection gene and 35Sgus(intron) reporter gene. All binary plasmids transferred DNA to chrysanthemum explants but only pMOG410 gave good stable expression of GUS. This plasmid differs from the other plasmids in 2 aspects: 1) It carries a restored nptll gene and 2) the selection gene is positioned at the left border side of the reporter gene. Cocultivation with AGLO(pMOG410) yielded up to 13 GUS positive shoots per 100 explants. The presence of the gus and nptll gene in recovered shoots was confirmed by PCR and Southern blot analysis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233300
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  • 3
    Electronic Resource
    Electronic Resource
    Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro) (1994)
    Springer
    Plant cell reports 13 (1994), S. 145-148 
    Details
    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
    URL: http://dx.doi.org/10.1007/BF00239881
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  • 4
    Electronic Resource
    Electronic Resource
    Isolation and transformation of rice aleurone protoplasts (1994)
    Springer
    Plant cell reports 13 (1994), S. 394-396 
    Details
    ISSN: 1432-203X
    Keywords: Rice ; α-amylase ; protoplasts ; aleurone ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Protoplasts isolated from the aleurone have been used extensively in molecular studies focusing on hormone-mediated regulation of gene expression in barley seed. To extend the use of aleurone protoplasts to other species, we have determined the conditions necessary for the isolation of protoplasts from rice aleurone layers of germinated seed. Many of the common cell wall degrading enzymes used in making protoplasts were tested for their ability to release protoplasts from rice aleurone layers. Cellulysin was found to be the most effective. Transformation of these aleurone protoplasts was accomplished using polyethylene glycol and DNA constructs containing the firefly luciferase reporter gene under the control of two different promoters were tested. Luciferase expression was 24-fold greater when the reporter gene was under the control of the CaMV 35S promoter than when the promoter from the alcohol dehydrogenase 1 gene was used. With the isolation and transformation of aleurone protoplasts from rice, it is now possible to investigate molecular events occurring in this tissue during germination.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00234145
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  • 5
    Electronic Resource
    Electronic Resource
    A reproducible procedure for plant regeneration from seedling hypocotyl protoplasts of Vigna sublobata L. (1994)
    Springer
    Plant cell reports 14 (1994), S. 175-179 
    Details
    ISSN: 1432-203X
    Keywords: Vigna sublobata ; protoplasts ; microcalli ; shoot bud formation ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Viable protoplasts of Vigna sublobata L. were isolated enzymatically from hypocotyls of axenic seedlings. Protoplast yields were dependent upon seedling age, with maximum yields (2.25 ± 0.35 × 106 g fwt−1) from seedlings aged 6 d. Protoplasts regenerated cell walls and underwent sustained divisions when cultured in either agarose-solidified or liquid K8P medium. The plating density affected the division frequency and plating efficiency; the division frequency (68 ±0 6.0%) was maximum at 4.0 × 104 ml−1 while plating efficiency was maximum (1.3 ± 0.1%) at 5.0 × 104 ml−1. Dividing protoplasts developed into microcalli, which produced glossy green compact nodular calli on transfer to 8.0 gl−1 w/v agar-solidified medium containing MS salts, B5 organic components, 30 g l−1 sucrose, NAA (0.2–0.5 mg l−1), zeatin riboside (0.5–2.0 mg l−1) and GA3 (0.5–1.0 mg l−1). These calli, after sub-culture on the same medium, produced shoot buds which underwent elongation following transfer of tissues to 6.0 g l−1 agar-solidified B5 medium containing 30g l−1 sucrose, IBA (0.01 mg l−1) and BAP (1.0 mg l−1). Elongated shoots developed roots after transfer to 8.0g l−1 agar-solidified, hormone-free MS medium with 30 g l−1 sucrose.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233785
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  • 6
    Electronic Resource
    Electronic Resource
    Synergistic enhancement of protoplast growth by oxygenated perfluorocarbon and Pluronic F-68 (1994)
    Springer
    Plant cell reports 13 (1994), S. 251-255 
    Details
    ISSN: 1432-203X
    Keywords: Petunia hybrida ; protoplasts ; oxygen delivery ; perfluorochemicals ; Pluronic F-68 ; surfactant ; cell division
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Cell suspension-derived protoplasts of albino Petunia hybrida were grown for 10 d at the interface between aqueous culture medium (KM8P) and an oxygenated (10 mbar for 15 min) perfluorocarbon liquid, perfluorodecalin. Protoplasts synthesised new cell walls and divided normally at the perfluorodecalin/culture medium interface, with a mean viability after 10 d of 〉 92.0%. The mean plating efficiency of protoplasts was elevated by 37% (P〈0.05) following culture at the perfluorodecalin/medium interface, but was unaltered by perfluorodecalin or oxygen separately. The mean plating efficiency of protoplasts cultured at the interface was further increased to a maximium of 52% above control, in the presence of oxygenated perfluorodecalin and KM8P medium supplemented with the non-ionic, co-polymer surfactant, Pluronic F-68 at 0.01% (w/v). These findings demonstrate the effectiveness of oxygenated perfluorodecalin for promoting protoplast growth, by facilitating oxygen delivery. The finding that Pluronic F-68 further increased the plating efficiency of protoplasts cultured at the perfluorocarbon/aqueous interface suggests that these agents improve growth through separate, but cumulative, mechanisms.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233314
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  • 7
    Electronic Resource
    Electronic Resource
    Effect of plant genotype on the transformation of cultivated alfalfa (Medicago sativa) by Agrobacterium tumefaciens (1994)
    Springer
    Plant cell reports 13 (1994), S. 330-334 
    Details
    ISSN: 1432-203X
    Keywords: Medicago sativa ; Alfalfa ; Transformation ; Agrobacterium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The trait for somatic embryogenesis is being introduced sexually into alfalfa (Medicago sativa) breeding populations to facilitate genetic transformation of this crop. Cocultivation experiments were conducted with an agronomically-improved embryogenic clone from one such population as well as with two other embryogenic clones, one of which was the source of the embryogenic trait in the breeding populations. Transgenic plants were produced from the agronomically-improved clone whereas none were produced from the other two clones. Among the 16 transgenic plants analyzed there was a range in both copy number and number of integration sites for the NPT-II gene; those plants regenerated after a prolonged selection phase in vitro generally had the highest numbers in both respects. There was no evidence of sectoral chimerism of the transgene in a subsample of transgenic plants analyzed by PCR.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00232631
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  • 8
    Electronic Resource
    Electronic Resource
    Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions (1994)
    Springer
    Archives of microbiology 161 (1994), S. 300-309 
    Details
    ISSN: 1432-072X
    Keywords: Agrobacterium ; 16S rDNA ; 16S rDNA-23S rDNA Intergenic spacer ; tmr Gene ; nos Gene ; virA Gene ; virB2 Gene ; Polymerase chain reaction ; Restriction fragment length polymorphism ; Crown gall
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chromosomes and Ti plasmids of 41 Agrobacterium strains, belonging to biovars 1, 2, 3, and Agrobacterium rubi species were characterized by the restriction fragment length polymorphism of PCR-amplified DNAs. Profiles that were obtained by the analysis of the amplified 16S rDNA confirmed the grouping of the strains according to their species. Higher polymorphism was detected in the intergenic spacer between the 16S rDNA and 23S rDNA genes, allowing efficient discrimination of strains. Identification of most strains was possible, and the genetic relatednesses of Agrobacterium strains could be estimated. The analysis of the plasmid Ti encoded regions between the tmr and nos genes, and the virA and virB2 genes, allowed fingerprinting of Ti plasmids. Genomic typing by the rapid PCR-RFLP method is thus shown to be useful for an independant identification of strains and of the conjugative Ti plasmids.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00303584
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  • 9
    Electronic Resource
    Electronic Resource
    Several phenotypic changes in the cell envelope of Agrobacterium tumefaciens chvB mutants are prevented by calcium limitation (1994)
    Springer
    Archives of microbiology 161 (1994), S. 310-315 
    Details
    ISSN: 1432-072X
    Keywords: Agrobacterium ; β-1,2-Glucan ; chvB Mutants—Ca2+
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The chvB gene of Agrobacterium tumefaciens encodes a 235 kDa proteinaceous intermediate involved in the synthesis of β-1,2-glucan. chvB mutants show a pleiotropic phenotype. Besides not to produce cyclic β-1,2-glucan, chvB mutants have been reported to be avirulent, attachment-deficient, and nonmotile. In this study we report additional differences from the parent strain, probably all linked to changes in the cell envelope. This pleiotropic phenotype — except for attachment and virulence — could largely be prevented by growing chvB cells with low levels of calcium. Although a role for β-1,2-glucan in osmoadaptation has been proposed, the mode of action of β-1,2-glucan is not known. We speculate that in A. tumefaciens β-1,2-glucan stabilizes membranes, which would be important especially in hypotonic media containing calcium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00303585
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  • 10
    Electronic Resource
    Electronic Resource
    Expression of biologically active hordothionins in tobacco. Effects of pre- and pro-sequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting (1994)
    Springer
    Plant molecular biology 24 (1994), S. 83-96 
    Details
    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
    URL: http://dx.doi.org/10.1007/BF00040576
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  • 11
    Electronic Resource
    Electronic Resource
    Localization of the VirA domain involved in acetosyringone-mediatedvir gene induction inAgrobacterium tumefaciens (1994)
    Springer
    Plant molecular biology 25 (1994), S. 899-907 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; gene regulation ; sensor protein ; signal transduction ; VirA protein ; acetosyringone
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The VirA protein ofAgrobacterium tumefaciens is thought to be a receptor for plant phenolic compounds such as acetosyringone. Although it is not known whether the interaction between VirA and the phenolics is direct or requires other phenolic-binding proteins, it is shown in this study that the first 280 amino acids of the VirA protein are not essential for the acetosyringone mediatedvir gene induction response. Considering the fact that the cytoplasmic region between the amino acids 283 and 304 is highly conserved between the different VirA proteins, and that deletion of this region abolishes VirA activity, we suggest that the acetosyringone receptor domain is located in this cytoplasmic domain of the VirA protein.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00028884
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  • 12
    Electronic Resource
    Electronic Resource
    The small, versatilepPZP family ofAgrobacterium binary vectors for plant transformation (1994)
    Springer
    Plant molecular biology 25 (1994), S. 989-994 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; binary vectors ; gentamycin resistance ; kanamycin resistance ; tobacco
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The newpPZP Agrobacterium binary vectors are versatile, relatively small, stable and are fully sequenced. The vectors utilize the pTiT37 T-DNA border regions, the pBR322bom site for mobilization fromEscherichia coli toAgrobacterium, and the ColE1 and pVS1 plasmid origins for replication inE. coli and inAgrobacterium, respectively. Bacterial marker genes in the vectors confer resistance to chloramphenicol (pPZP100 series) or spectinomycin (pPZP200 series), allowing their use inAgrobacterium strains with different drug resistance markers. Plant marker genes in the binary vectors confer resistance to kanamycin or to gentamycin, and are adjacent to the left border (LB) of the transferred region. A lacZ α-peptide, with the pUC18 multiple cloning site (MCS), lies between the plant marker gene and the right border (RB). Since the RB is transferred first, drug resistance is obtained only if the passenger gene is present in the transgenic plants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00014672
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  • 13
    Electronic Resource
    Electronic Resource
    Purification and partial characterization of a glycoprotein from pea (Pisum sativum) with receptor activity for rhicadhesin, an attachment protein of Rhizobiaceae (1994)
    Springer
    Plant molecular biology 24 (1994), S. 171-183 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; attachment ; plant receptor ; rhicadhesin ; Rhizobium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Attachment of Rhizobium and Agrobacterium bacteria to cells of their host plants is a two-step process. The first step, direct attachment of bacteria to the plant cell wall, is mediated by the bacterial protein rhicadhesin. A putative plant receptor molecule for rhicadhesin was purified from cell walls of pea roots using a bioassay based on suppression of rhicadhesin activity. This molecule appeared to be sensitive to treatments with pronase or glycosidase. Its isoelectric point is 6.4, and its apparent molecular mass was estimated to be 32 kDa before and 29 kDa after glycosidase treatment, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ultrafiltration. The sequence of the first 29 N-terminal amino acids was determined: A-D-A-D-A-L-Q-D-L-C(?)-V-A-D-Y-A-S-V-I-L-V-N-G-F-A-S-K(Q)-(P/Q)-(L)-(I). No homology with known proteins was found. In the course of this research project the extracellular matrix protein vitronectin was reported to inhibit attachment of A. tumefaciens to carrot cells [29]. A variety of adhesive proteins, including vitronectin, contain a common cell attachment determinant with the sequence R-G-D. Since we could not detect other cell wall components able to suppress rhicadhesin activity, and since an R-G-D containing hexapeptide was also active as a receptor, we speculate that the plant receptor for rhicadhesin is a glycoprotein containing an R-G-D attachment site.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00040583
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  • 14
    Electronic Resource
    Electronic Resource
    Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco (1994)
    Springer
    Plant molecular biology 26 (1994), S. 1701-1710 
    Details
    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
    URL: http://dx.doi.org/10.1007/BF00019485
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  • 15
    Electronic Resource
    Electronic Resource
    A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.) (1994)
    Springer
    Plant cell reports 13 (1994), S. 541-545 
    Details
    ISSN: 1432-203X
    Keywords: Trifoliate orange ; Epicotyl segment ; Agrobacterium ; Transformation ; rolC promoter ; β-glucuronidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.) was developed using epicotyl segments. The segments were infected with Agrobacterium harboring the binary vector pBI121 or pBI101-O12-p1. Both vectors contained the neomycin phosphotransferase II (NPTII) and the β-glucuronidase (GUS) genes. In the plasmid pBI101-O12-p1, the GUS gene was directed to the promoter region of ORF12 (rolC) of the Ri plasmid. On a selection medium containing 100 or 200 μg/ml kanamycin, adventitious shoots were formed from 21.7–44.6% of the segments. Histochemical GUS assay showed that 55.4–87.7% of the shoots expressed the GUS gene. The stable integration of this gene was also confirmed by polymerase chain reaction (PCR) analysis and by Southern blot analysis. When the pBI101-O12-p1 plasmid was used, the GUS activity was found to be located in phloem cells of leaf, stem and root. More than 100 transformed plants were obtained using this method within 2–3 months.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00234507
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  • 16
    Electronic Resource
    Electronic Resource
    Expression and inheritance pattern of two foreign genes in petunia (1994)
    Springer
    Theoretical and applied genetics 88 (1994), S. 433-440 
    Details
    ISSN: 1432-2242
    Keywords: Agrobacterium ; Transformation ; Gene expression ; Petunia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transgenic petunia (Petunia hybrida Vilm.) plants were obtained from Agrobacterium-mediated shoot apex transformation. Studies at the phenotypic as well as molecular level established both the presence of the NPT II (neomycin phosphotransferase II) and GUS (β-glucuronidase) genes and their level of activity. Twenty-nine primary transformed plants showed varying patterns of phenotype expression of both genes. NPT II and GUS expression in 7 primary plants over a 4-month interval showed varying levels of gene expression within and among individual plants. All primary transgenic plants were self-pollinated and backcrossed to establish the inheritance patterns of both genes. Mendelian and non-Mendelian inheritance patterns for both genes were observed. Analysis of the progeny showed poor transmission of the foreign genes through the pollen especially when two or more bands were present in the Southern hybridization. Most plants whose progeny segregated in Mendelian ratios for either the NPT II or GUS gene had just one copy of the gene. In this study where both foreign genes were examined in both self and test crosses, no transgenic plant showed Mendelian patterns of inheritance for both foreign traits.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00223657
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  • 17
    Electronic Resource
    Electronic Resource
    Cybridization in Nicotiana tabacum L. using double inactivation of parental protoplasts and post-fusion selection based on nuclear-encoded and chloroplast-encoded marker genes (1994)
    Springer
    Theoretical and applied genetics 88 (1994), S. 1017-1022 
    Details
    ISSN: 1432-2242
    Keywords: Agrobacterium ; Iodoacetamide Nicotiana protoplast fusion ; Nitrosomethyl urea Rhodamine 6G ; X-irradiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An effective selection system preceded by double inactivation of parental protoplasts was used to transfer Nicotiana suaveolens Leh. cytoplasmic male sterility into a commercial tobacco (N. tabacum L.) breeding line. Mesophyll protoplasts from transformed plants of N. tabacum cultivar WZ2-3-1-1 possessing a neomycin phosphotransferase II gene were used as the nuclear donors, while those isolated from N. suaveolens plants carrying a chloroplast mutation for resistance to spectinomycin, induced using nitrosomethyl urea, were the cytoplasm donors in somatic cybridizations. Prior to fusion, nuclear donor protoplasts were inactivated with iodoacetamide or rhodamine 6G, while those of the cytoplasm donor were inactivated by X-irradiation. The resultant microcalli were cultured on a shoot regeneration medium containing both kanamycin and spectinomycin to select cybrids. Only regenerants that had typical characteristics of the N. tabacum cultivar were selected for transfer to the glasshouse. Four putative cytoplasmic male-sterile (CMS) plants, out of a total of 44 regenerated plants transferred to the glasshouse, were obtained. Intraspecific somatic transfers of the CMS trait between N. tabacum cultivars with distinctlydifferent morphologies using single inactivation and nonselective shoot regeneration medium were demonstrated. The implications of the results for practical tobacco breeding as a means of circumventing lengthy backcrossing procedures are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00220810
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  • 18
    Electronic Resource
    Electronic Resource
    Transformation of protoplasts and intact cells from slowly growing embryogenic callus of wheat (Triticum aestivum L.) (1994)
    Springer
    Theoretical and applied genetics 89 (1994), S. 577-582 
    Details
    ISSN: 1432-2242
    Keywords: Wheat ; Transformation ; Agrobacterium ; Electroporation ; Polyethylene glycol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A procedure for culturing protoplasts from slowly growing embryogenic calli of wheat was developed. The procedure was dependent on the ability to isolate large numbers of culturable protoplasts from slowly growing embryogenic callus. Approximately 68% of the isolated protoplasts divided, and 22% formed colonies; of the latter, 67% continued to proliferate. Plating efficiency was reduced when protoplasts were transformed by polythylene glycol, electroporation, and/or Agrobacterium. Intact cells were also directly transformed by electroporation. Direct electroporation of the Agrobacterium binary vector into intact cells resulted in a significant increase of GUS activity over the control.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00222451
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  • 19
    Electronic Resource
    Electronic Resource
    T-DNA-insert-independent mutations induced in transformed plant cells duringAgrobacterium co-cultivation (1994)
    Springer
    Transgenic research 3 (1994), S. 317-325 
    Details
    ISSN: 1573-9368
    Keywords: Agrobacterium ; mutagenesis ; Nicotiana plumbaginifolia ; nitrate reductase ; ploidy ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transformation frequencies were determined for 1n, 2n, and 4n Nicotiana plumbaginifolia protoplast cultures inAgrobacterium-mediated gene transfer experiments. An unexpected large drop (50%) in plating efficiencies was observed in the non-selected (control) 1n populations after transformation treatment with virulent strains. This effect was not observed in the 2n or 4n cultures or in the 1n cultures when treated with avirulent bacteria. The mortality was disproportionally high and could not be explained by the low (0.1–0.5%) transformation efficiency in the 1n population, indicating mutagenesis of the cell populations independently from the T-DNA insertions. Mutagenesis was also indicated in gene tagging experiments where nitrate reductase-deficient (NR−) mutants were selected from haploidNicotiana plumbaginifolia protoplasts, as well as from leaf disc cultures or protoplasts of diploid plants that were heterozygotic for a mutation either in the NR apoenzyme gene (nia/wt) or one of the molybdenum-containing cofactor genes (cnxA/wt), afterAgrobacterium co-cultivation. The chlorate-resistant isolates were tested for the T-DNA-specific kanamycin resistance trait only after NR-deficiency had been established. Thirty-nine independent NR-deficient mutants were analysed further by Southern blot hybridization. There was no indication of integrated T-DNA sequences in the mutated NR genes, despite the fact that NR-deficient cells were found more frequently in cell populations which became transformed during the treatment than in the populations which did not. These observations suggest that transformation-competent cells undergo mutagenesis during theAgrobacterium gene transfer process not only as a result of stable integration events, but also through accompanying events that do not result in major changes in the mutated loci. The nature of these changes at the molecular level remains to be elucidated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01973592
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  • 20
    Electronic Resource
    Electronic Resource
    Spurious localizations of diX-indigo microcrystals generated by the histochemical GUS assay (1994)
    Springer
    Transgenic research 3 (1994), S. 176-181 
    Details
    ISSN: 1573-9368
    Keywords: Agrobacterium ; β-glucuronidase ; cytoenzymology ; reporter gene ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract For several models expressing theuidA orgus reporter gene with or without a presequence for mitochondrial targeting, we have demonstrated that the compartmentation of β-glucuronidase (E.C. 3.2.1.31) activity was not in agreement within situ localization of the diX-indigo microcrystals generated by the cytoenzymological GUS assay. These crystals were generally associated with the various cytomembranes and lipid inclusions. Experiments with purified β-glucuronidase or withgus-expressing bacteria incubated with 5-bromo-4-chloro-3-indolyl-β-d-glucuronide and maize oil-phosphate buffer emulsion indicated that the intermediate products resulting from the GUS assay actively diffused and crystallized preferentially in association with lipids, sometimes far from the site of enzyme activity. This phenomenon could not be suppressed by the addition of potassium ferricyanide in the incubation medium. These findings are discussed with regard to previously reported biochemical and histochemical data on animal tissues, and focus on the necessity for caution in studies of tissue-specific gene expression using the GUS assay, particularly for lipid-rich plant models.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01973985
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  • 21
    Electronic Resource
    Electronic Resource
    The NocR repressor-activator protein regulates expression of the nocB and nocR genes of Agrobacterium tumefaciens (1994)
    Springer
    Molecular genetics and genomics 244 (1994), S. 367-373 
    Details
    ISSN: 1617-4623
    Keywords: Agrobacterium ; Nopaline ; Repressor Activator ; LysR family
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The NocR protein of Agrobacterium tumefaciens was found to regulate expression of the divergently transcribed nocB and nocR genes of the pTiT37 nopaline catabolism (noc) region. Experiments using the firefly luciferase (luc) gene as reporter demonstrated that NocR represses and activates transcription from the nocB promoter in the absence and presence of nopaline, respectively. NocR also negatively autoregulates its own synthesis irrespective of the presence of nopaline. Regulation of expression of both nocB and nocR is mediated by binding of the NocR protein to the nocR promoter. A 12 by symmetrical sequence, which lies 3 by downstream of the −10 hexamer of the nocR promoter, was confirmed to be essential for binding of the NocR protein. Functional localization of the nocB and nocR promoters verified that they do not overlap at all, and that the interrupted dyad, at which NocR binds, is 137 by upstream of the regulated nocB promoter. The in vivo and in vitro results described here and those published previously suggest that a novel type of regulatory mechanism, which may involve changes in DNA topology, controls gene expression in the noc operon of pTiT37.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00286688
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  • 22
    Electronic Resource
    Electronic Resource
    Agrobacterium vitis nopaline Ti plasmid pTiAB4: relationship to other Ti plasmids and T-DNA structure (1994)
    Springer
    Molecular genetics and genomics 245 (1994), S. 493-505 
    Details
    ISSN: 1617-4623
    Keywords: Agrobacterium ; Bacterial evolution ; Nopaline strains ; Ti plasmids ; Grapevine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Ti plasmid of the Agrobacterium vitis nopaline-type strain AB4 was subcloned and mapped. Several regions of the 157 kb Ti plasmid are similar or identical to parts of the A. vitis octopine/cucumopine (o/c)-type Ti plasmids, and other regions are homologous to the nopaline-type Ti plasmid pTiC58. The T-DNA of pTiAB4 is a chimaeric structure of recent origin: the left part is 99.2% homologous to the left part of the TA-DNA of the o/c-type Ti plasmids, while the right part is 97.1 % homologous to the right part of an unusual nopaline T-DNA recently identified in strain 82.139, a biotype Il strain from wild cherry. The 3′ non-coding regions of the ipt genes from pTiAB4 and pTi82.139 are different from those of other ipt genes and contain a 62 by fragment derived from the coding sequence of an ipt gene of unknown origin. A comparison of different ipt gene sequences indicates that the corresponding 62 by sequence within the coding region of the AB4 ipt gene has been modified during the course of its evolution, apparently by sequence transfer from the 62 by sequence in the 3′ non-coding region. In pTi82.139 the original coding region of the ipt gene has remained largely unmodified. The pTiAB4 6b gene differs from its pTi82.139 counterpart by the lack of a 12 by repeat in the 3′ part of the coding sequence. This leads to the loss of four glutamic acid residues from a series of ten. In spite of these differences, the ipt and 6b genes of pTiAB4 are functional. Our results provide new insight into the evolution of Agrobacterium Ti plasmids and confirm the remarkable plasticity of these genetic elements. Possible implications for the study of bacterial phylogeny are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00302262
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  • 23
    Electronic Resource
    Electronic Resource
    Genetic manipulation in vesicular-arbuscular mycorrhizal fungi (1994)
    Springer
    Plant and soil 159 (1994), S. 171-178 
    Details
    ISSN: 1573-5036
    Keywords: Agrobacterium ; Gigaspora margarita ; Glomus intraradix ; PCR ; mycorrhizae ; rDNA gene ; root organ culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The symbiosis between vesicular-arbuscular mycorrhizal (VAM) fungi and host plants develops after successful interactions between both partners. These interactions probably involve signal molecules produced by the host plant, by the fungi, or by both. So far the biotrophic status of VAM fungi has hampered the understanding of the processes regulating their physiology. However, among different methods for co-cultivating VAM fungi, root organ cultures (ROC) appear to be a useful technique for studying VAM development. This system has been useful in defining the nutritional requirements of VAM fungi in the precolonization stage and in obtaining axenic fungal material in various developmental stages. The work discussed here focuses on the application of Polymerase Chain Reaction (PCR) technology and the potential of promoting hyphal growth in the absence of the plant. These techniques are being used to study VAM fungi in two main areas. The first concerns the determination of the DNA sequences coding for the SSU ribosomal RNA of two VAM fungi. This approach has allowed the design of specific primers for the rapid identification and quantification of VAM fungi. The second area of research concerns the potential use of PCR technology to study selective expression of specific genes during fungal spore development in defined in vitro conditions. The achievement of this future prospect depends on the ability to prepare PCR-based cDNA libraries from small amounts of fungal material after stimulation of hyphal growth with CO2 and plant flavonols.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00000106
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  • 24
    Electronic Resource
    Electronic Resource
    The biosynthetic pathway of the S-alk(en)yl-L-cysteine sulphoxides (flavour precursors) in species of Allium (1994)
    Springer
    Plant cell, tissue and organ culture 38 (1994), S. 181-188 
    Details
    ISSN: 1573-5044
    Keywords: Allium cepa ; biosynthesis ; compartmentation ; γ-glutamyl peptides ; onion ; protoplasts ; S-alkenyl-L-cysteine sulphoxide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Pulse labelling experiments with 35SO4 2- fed for 24h to intact plants (shooted onion sets)of Allium cepa (onion) showed that 〉70% of the label appeared in the S-alkenyl-L-cysteine sulphoxides within 18h, reached a maximum at 48h and thereafter decreased. The amount of label detected in the γ-glutamyl peptide fractions was below 20% of the total label at any time. It is concluded that in intact plants (at the growth stage used) the γ-glutamyl peptides are not the immediate precursors of the S-alkenyl-L-cysteine sulphoxides. The major S-alkenyl-L-cysteine sulphoxide in onion was found to be compartmentalized mainly within the endoplasmatic reticulum.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00033876
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  • 25
    Electronic Resource
    Electronic Resource
    Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains (1994)
    Springer
    Plant cell, tissue and organ culture 38 (1994), S. 257-262 
    Details
    ISSN: 1573-5044
    Keywords: Agrobacterium ; Atropa belladonna ; double transformation ; phytohormone content ; tropane alkaloids ; viviparous leaves
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00033885
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  • 26
    Electronic Resource
    Electronic Resource
    Production and characterization of intergeneric somatic hybrids through protoplast electrofusion between potato (Solanum tuberosum) and Lycopersicon pennellii (1994)
    Springer
    Plant cell, tissue and organ culture 37 (1994), S. 137-144 
    Details
    ISSN: 1573-5044
    Keywords: electrofusion ; L. pennellii ; protoplasts ; S. tuberosum ; salt tolerance ; somatic hybrids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Mesophyll protoplasts of Lycopersicon pennelli Corr., a wild relative of tomato, were electrofused with those from a dihaploid potato clone, cv Nicola, with the objectives of transferring saline tolerance from L. pennellii to cultivated potato. 150 calli were selected from the fusion experiments, finally giving 2 hybrid shoots. Their hybrid nature was verified by examining isoenzyme patterns for esterases (EST), peroxidase (PRX), phosphogluconate dehydrogenase (6-PGD), and glutamate oxaloacetate transaminase (GOT). The hybrid plants had an intermediate morphology, and grew vigorously in vitro. When transplanted to soil, they were less vigorous, due to difficulties in rooting, but were still capable of flowering, and forming short stolons and mishaped tubers, probably resulting from the effects of gene dosage due to the novel association of two genomes from a tuberizing (potato) and a non tuberizing species (L. pennellii). The characteristics of such mishaped tubers provided strong evidence of a hybrid nature for the selected plants. The hybrid plants were highly sterile, producing only 3–7% viable pollen. Tests for salt tolerance showed that the growth of the somatic hybrid plants was reduced by 50% as for L. pennellii, whilst potato did not grow at all under saline conditions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00043607
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  • 27
    Electronic Resource
    Electronic Resource
    Isolation of cells and protoplasts from leaves of in vitro propagated peach (Prunus persica) plants (1994)
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 99-105 
    Details
    ISSN: 1573-5044
    Keywords: cell division ; peach ; protoplasts ; Prunus persica ; tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Yields of 106–108 peach mesophyll cells and protoplasts · gfw-1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4–10 mm in length, were a superior source for protoplast isolation than medium or big expanded leaves, 22–30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml-1. Purification of protoplasts on a sucrose gradient yielded about 107 protoplasts · gfw-1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00048320
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  • 28
    Electronic Resource
    Electronic Resource
    Highly efficlent plant regeneration from mesophyll protoplasts of Indian field cultivars of tomato (Lycopersicon esculentum) (1994)
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 255-258 
    Details
    ISSN: 1573-5044
    Keywords: tomato ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three Indian cultivars ofL. esculentum were assessed for shoot regeneration from protoplast-derived calli. Consistent yields of viable protoplasts (〉9.0×106 g f.wt.-1) were obtained from leaflets of 14 days old cultured shoots. Protoplast viability (88–94%) and planting efficiency (55–70%) were recorded for the three cultivars. Up to 71% of the protoplast-derived tissues regenerated shoots.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00037728
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  • 29
    Electronic Resource
    Electronic Resource
    Expression of human α-interferon cDNA in transgenic rice plants (1994)
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 197-204 
    Details
    ISSN: 1573-5044
    Keywords: interferon ; Lipofectin ; protoplasts ; rice ; transgenic plant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The plasmid pIG3031 containing human α-interferon cDNA and the neomycin phosphotransferase II coding sequence was successfully transferred into rice protoplasts (Indica type rice) by Lipofectinmediated transformation. Assays for NPT II enzyme activity indicated that the transformation frequency was 10%. Transgenic plants were regenerated from transformed calli. Southern blots showed that the human α-interferon cDNA sequence was present in rice DNA. RNA slot blots indicated apparent transcription of human α-interferon cDNA under the control of the plant-active promoter PI'. Extracts of transgenic cell cultures and plants contained apparent interferon activity as measured by resistance of a human amniotic cell line to viral infection in the presence of plant extracts. These studies demonstrate that human α-interferon cDNA may be correctly expressed in rice cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00037720
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  • 30
    Electronic Resource
    Electronic Resource
    Isolation and culture of protoplasts from young sporophytes ofSalvinia natans aseptically obtained by co-culture of female and male gametophytes (1994)
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 237-242 
    Details
    ISSN: 1573-5044
    Keywords: aseptic culture ; female gametophyte ; heterospory ; male gametophyte ; protoplasts ; Salvinia natans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Sporophytes were aseptically obtained by co-culture of female and male gametophytes derived from two types of spores (megaspores and microspores) of the heterosporous fernSalvinia natans All. Protoplasts isolated enzymatically from juvenile leaflets of sporophytes were cultured in a 1/10 Murashige and Skoog's medium containing 2.2 μM naphthalene acetic acid, 2.2 μM 6-benzyl-aminopurine, 0.35 M mannitol, and 0.05 M sucrose. Cell division took place within 6 days of culture, and cell-clusters composed of 9–10 cells were observed after 30 days of culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00037725
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  • 31
    Electronic Resource
    Electronic Resource
    Single-cell cloning of a crown gall from protoplasts regenerated in hormone-free medium. Establishment of pure transformed cell-lines of Catharanthus roseus (1994)
    Springer
    Plant cell, tissue and organ culture 37 (1994), S. 25-30 
    Details
    ISSN: 1573-5044
    Keywords: Catharanthus roseus ; crown gall tumor ; heterokaryons ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts enzymatically isolated from cell line of Catharanthus roseus G. Don crown gall, were cultured at high density (105 P ml-1) in modified B5 liquid medium (Gamborg et al. 1976). In the absence of growth regulators C. roseus protoplasts were able to regenerate a cell-wall, divide and, subsequently, yield very numerous clones in the absence of growth regulators. After two weeks, the cultures were greatly diluted in order to obtain clones of single-cell origin. Most of the clones individually transferred onto solid medium can proliferate indefinitely, without growth regulators. Among analyzed clones, 90% were nopaline positive. Their ajmalicine and serpentine content was compared with that of the parental crown gall line, and was found to be low. The CR10 protoplasts were very easy to grow, they were an interesting model for the development of pure tumorous lines. Moreover, we found that the tumorous protoplasts were useful for cell fusion experiments or for the delicate culture of tree protoplasts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00048113
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  • 32
    Electronic Resource
    Electronic Resource
    Biotechnology and sustainable development in sub-Saharan Africa (1994)
    Springer
    World journal of microbiology and biotechnology 10 (1994), S. 243-248 
    Details
    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
    URL: http://dx.doi.org/10.1007/BF00414855
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  • 33
    Electronic Resource
    Electronic Resource
    Genetically engineered charge modifications to enhance protein separation in aqueous two-phase systems: Electrochemical partitioning (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 147-153 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260440202
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  • 34
    Electronic Resource
    Electronic Resource
    Recovery of transgenic trees after electroporation of poplar protoplasts (1994)
    Springer
    Transgenic research 3 (1994), S. 13-19 
    Details
    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
    URL: http://dx.doi.org/10.1007/BF01976022
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  • 35
    Electronic Resource
    Electronic Resource
    Self-assembling biomolecular materials in mediaine (1994)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 56 (1994), S. 168-170 
    Details
    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
    URL: http://dx.doi.org/10.1002/jcb.240560208
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  • 36
    Electronic Resource
    Electronic Resource
    Efficient fuzzy control strategies for the application of pH-stat to fed-batch cultivation of genetically engineered Escherichia coli (1994)
    Chichester : Wiley-Blackwell
    Journal of Chemical Technology AND Biotechnology 61 (1994), S. 273-281 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jctb.280610316
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  • 37
    Electronic Resource
    Electronic Resource
    The true meaning of ‘exotic species’ as a model for genetically engineered organisms (1993)
    Springer
    Cellular and molecular life sciences 49 (1993), S. 225-234 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01923530
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  • 38
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    Electronic Resource
    Selection of anthocyanin-accumulating potato (Solanum tuberosum L.) cell lines from calli derived from seedlings produced by gamma-irradiated seeds (1993)
    Springer
    Plant cell reports 12 (1993), S. 555-558 
    Details
    ISSN: 1432-203X
    Keywords: Solanum tuberosum ; anthocyanins ; gamma-irradiation ; protoplasts ; protoclones
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Callus cell lines of potato (Solanum tuberosum L. cv. Zarevo) were obtained from seedlings germinated from gamma-irradiated seeds (200 Gy). Some of these cell lines produce red-violet pigments which were identified as acylated anthocyanins. The major anthocyanin was determined to be peonidin 3-O-[6-O-(4-O-E-p-coumaroyl-rhamnosyl)-glucoside]-5-O-glucoside (“peonanin”). Single cell-derived protoclones from non-pigmented protoplasts sometimes also gave rise to pigmented cell clusters thus indicating that the changes in the expression of the anthocyanin pathway can also occur after the stage of initial callus induction.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233059
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  • 39
    Electronic Resource
    Electronic Resource
    Plant regeneration from mesophyll-protoplasts of four different ecotypes and two marker lines from Arabidopsis thaliana using a unique protocol (1993)
    Springer
    Plant cell reports 12 (1993), S. 569-572 
    Details
    ISSN: 1432-203X
    Keywords: Arabidopsis thaliana ; ecotypes and mutant lines ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of four ecotypes (Col C24, Per-1, Bur-0, Landsberg erecta) and two marker lines (M4 and M10) of Ardbidopsis thaliana is described. The different lines showed plating efficiencies between 1.0 and 3.9% using Nitsch medium or this medium supplemented with coconut water. For the differentiation of callus into normal shoots a single shoot regeneration medium was applicable to all ecotypes, but depending on the line other regeneration media showed to be more suitable. The results indicated that the protoplast culture procedure is applicable, with minor modifications, to all tested genotypes but the most suitable shoot regeneration medium should be established for each A. thaliana line.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233062
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  • 40
    Electronic Resource
    Electronic Resource
    Fertile plant regeneration from protoplasts of meadow fescue (Festuca pratensis Huds.) (1993)
    Springer
    Plant cell reports 12 (1993), S. 95-100 
    Details
    ISSN: 1432-203X
    Keywords: forage grasses ; Festuca pratensis ; suspension cultures ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Suspension cultures from mature embryo-derived compact callus were initiated in seven meadow fescue (Festuca pratensis Huds.) cultivars. Four to six months after initiation, embryogenic suspension cultures with a moderate growth rate were established from three of them (cvs. Barmondo, Belimo and Leopard). These suspension cultures showed the capacity, maintained over six months, to regenerate green plants which could be grown to maturity under greenhouse conditions. Morphogenic suspension cultures from single genotypes of three F. pratensis cultivars (cvs. Barmondo, Belimo and Leopard) yielded large numbers of protoplasts, which upon culture in agarose beads using nurse cells formed microcalli with an overall plating efficiency in the range of 10-3 to 10-4. Mature plants were reproducibly regenerated and established in soil, from such protoplasts during a period of six months. The regeneration of fertile plants from protoplasts derived from suspension cultures of meadow fescue and its implications on gene transfer technology for this species are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00241942
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  • 41
    Electronic Resource
    Electronic Resource
    Analysis of genetic stability of plants regenerated from suspension cultures and protoplasts of meadow fescue (Festuca pratensis Huds.) (1993)
    Springer
    Plant cell reports 12 (1993), S. 101-106 
    Details
    ISSN: 1432-203X
    Keywords: Festuca pratensis ; suspension cultures ; protoplasts ; plant regeneration ; somaclonal variation ; genetic fidelity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials. The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00241943
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  • 42
    Electronic Resource
    Electronic Resource
    Effect of 1,2-benzisoxazole-3-acetic acid on plant regeneration from protoplasts of Nicotiana tabacum (1993)
    Springer
    Plant cell reports 12 (1993), S. 121-124 
    Details
    ISSN: 1432-203X
    Keywords: Auxin ; benzisoxazole-3-acetic acid ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Benzisoxazole-3-acetic acid, a new synthetic growth regulator, was administered to protoplast cultures from Nicotiana tabacum and subsequently to the developed microcalluses, to test its activity on plant regeneration from protoplasts in different culture conditions. Such activity, compared to that of naphthalene-acetic acid, proved to be rather low in the stage of cellular division and microcallus formation but particulary high in the stage of shoot induction from microcallus, thus confirming that the activity of this compound is mainly morphogenetic.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00239090
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  • 43
    Electronic Resource
    Electronic Resource
    Transformation of peas (1993)
    Springer
    Plant cell reports 12 (1993), S. 180-183 
    Details
    ISSN: 1432-203X
    Keywords: Peas ; Seed ; Transformation ; Agrobacterium ; Meristems
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The lateral cotyledonary meristems present in germinating seed were inoculated with a non-oncogenic strain of A. tumefaciens carrying a gene conferring kanamycin resistance as a selectable marker and a β-glucuronidase sequence as a reporter gene. Kanamycin resistant plants were derived from the meristems and shown to be transformed on the basis of Southern blots, polymerase chain reaction analysis and tests for β-glucuronidase activity. The plants were fertile and tests of their progeny confirmed the transmission of integrated sequences through a sexual generation. This transformation method has the merit of an unlimited supply of material for inoculation and a relatively short time scale from inoculation to the production of rooted plants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00239102
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  • 44
    Electronic Resource
    Electronic Resource
    Somatic embryogenesis and plant regeneration from hypocotyl protoplasts of sunflower (Helianthus annum L.) (1993)
    Springer
    Plant cell reports 12 (1993), S. 260-263 
    Details
    ISSN: 1432-203X
    Keywords: sunflower ; protoplasts ; somatic embryogenesis ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A sunflower genotype (Helianthus annuus L. cv. Florom-328) able to regenerate plants from in vitro cultures was identified by screening hybrids and inbred lines. Protoplasts of this genotype were isolated from dark grown hypocotyls and were cultured in droplets of agarose-solidified V-KM medium covered by liquid V-KM supplemented with naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). One week later colonies were subjected to 2,4-dichlorophenoxyaceticacid for a one week period. Further culture in V-KM with reduced concentrations of NAA and BAP resulted in the appearence of somatic embryos. Maturation of embryos was achieved by culture on MS medium supplemented with NAA, BAP, gibberellic acid A3 and the ethylene inhibitor AgNO3. Embryos were then transferred onto hormone free MS medium for germination. The frequency of shoot formation in the best case was 9.6 percent of viable colonies (1.3 percent of protoplasts plated). Some of the shoots with roots could be transplanted into soil, others were grafted on hypocotyls of in vivo germinated seedlings. Eighty percent of grafted shoots and over 95 percent of rooted shoots survived. The plants flowered and produced 5 to 10 seeds each. Factors affecting the frequency of embryo formation and plant regeneration are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00237131
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  • 45
    Electronic Resource
    Electronic Resource
    Conditional inhibition of β-glucuronidase expression by antisense gene fragments in petunia protoplasts (1993)
    Springer
    Plant molecular biology 23 (1993), S. 45-55 
    Details
    ISSN: 1573-5028
    Keywords: Key words ; antisense RNA ; β-glucuronidase ; protoplasts ; transient gene expression ; PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Antisense RNA-mediated inhibition of gene expression is a valuable tool to induce mutant phenotypes. We are interested in the application of antisense gene fragments with the aim to improve the efficiency of inhibition and to be able to selectively suppress gene family members in plants. Protoplasts may provide a rapid system to screen the efficiency of antisense gene segments. As a first step, we set up a transient expression system for leaf protoplasts of Petunia hybrida and used as a model system the inhibition of β-glucuronidase (uidA) expression by uidA antisense gene segments. Both GUS enzyme activities and uidA RNA levels were measured. Co-introducing equal amounts of a full-length uidA antisense gene and a uidA sense gene reduced GUS activity by 60–70%. Various uidA antisense fragments also inhibited expression although with different efficiencies and we show that strong antisense fragments can be retrieved from weak antisense gene fragments. A promoter-less antisense gene did not reduce uidA expression indicating that the inhibition is mediated by antisense transcripts. Using quantitative PCR on first-strand cDNA we show that expression of functional antisense genes lead to reduced levels of uidA mRNA. This suggests that the mechanism of antisense RNA inhibition in protoplasts is similar to that in transgenic plants and that the protoplast system in combination with PCR can be used to preselect antisense fragments of any gene.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00021418
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  • 46
    Electronic Resource
    Electronic Resource
    Phenotype and hormonal status of transgenic tobacco plants overexpressing the rolA gene of Agrobacterium rhizogenes T-DNA (1993)
    Springer
    Plant molecular biology 23 (1993), S. 1199-1210 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; growth retardants ; plant hormones ; Ri plasmid ; transgenic tobacco
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The rolA gene of the TL-DNA of Agrobacterium rhizogenes Ri-plasmid plays a major role in establishing the hairy root syndrome in transgenic plants. Transgenic tobacco plants (Nicotiana tabacum L.) expressing constitutively the rolA gene under the transcriptional control of the 35S RNA promoter show pronounced phenotypical alterations. P35S-rolA transgenic tobacco plants are characterized by stunted growth, dark green wrinkled leaves with an altered length-to-width ratio, condensed inflorescences, retarded onset of flowering, a reduced number of flowers and shortened styles. To investigate whether the pleiotropic alterations of growth and development are linked to an altered hormonal status we have compared the immunoreactive content of indole-3-acetic acid, cytokinins, abscisic acid, gibberellin and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) of seedlings and different tissues of P35S-rolA transgenic plants, transgenic plants expressing the rolA gene under control of its own phloem-specific promoter and wild-type plants. Multiple tissue-specific alterations of phytohormone concentrations are the consequence of rolA gene activity. Changes of phytohormonal content can explain part of the rolA-induced phenotypic alterations. Most strikingly, in young and fully developed leaves of rolA and P35S-rolA transgenic clones a 40–60% reduction of immunoreactive gibberellin A1 was found, as compared to wild-type leaves. Treatment of wild-type tobacco plants with inhibitors of gibberellin biosynthesis phenotypic alterations similar to those of rolA transgenic plants. This suggests that the reduction of gibberellic acid content is indirectly but causally involved in rolA-induced alterations of stem elongation and planar leaf blade growth.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00042353
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  • 47
    Electronic Resource
    Electronic Resource
    Agrobacterium-mediated production of transgenic rice plants expressing a chimeric α-amylase promoter/β-glucuronidase gene (1993)
    Springer
    Plant molecular biology 22 (1993), S. 491-506 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; α-amylase promoter ; α-glucuronidase ; Oryza sativa ; potato suspension culture ; transgenic rice
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have successfully transferred and expressed a reporter gene driven by an α-amylase promoter in a japonica type of rice (Oryza sativa L. cv. Tainung 62) using the Agrobacterium-mediated gene transfer system. Immature rice embryos (10–12 days after anthesis) were infected with an Agrobacterium strain carrying a plasmid containing chimeric genes of β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Co-incubation of potato suspension culture (PSC) with the Agrobacterium inoculum significantly improved the transformation efficiency of rice. The uidA and nptII genes, which are under the control of promoters of a rice α-amylase gene (αAmy8) and Agrobacterium nopaline synthase gene (nos), respectively, were both expressed in G418-resistant calli and transgenic plants. Integration of foreign genes into the genomes of transgenic plants was confirmed by Southern blot analysis. Histochemical localization of GUS activity in one transgenic plant (R0) revealed that the rice α-amylase promoter functions in all cell types of the mature leaves, stems, sheaths and roots, but not in the very young leaves. This transgenic plant grew more slowly and produced less seeds than the wild-type plant, but its R1 and R2 progenies grew normally and produced as much seeds as the wild-type plant. Inheritance of foreign genes to the progenies was also confirmed by Southern blot analysis. These data demonstrate successful gene transfer and sexual inheritance of the chimeric genes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00015978
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  • 48
    Electronic Resource
    Electronic Resource
    The GAPDH gene system of the red alga Chondrus crispus: promotor structures, intron/exon organization, genomic complexity and differential expression of genes (1993)
    Springer
    Plant molecular biology 23 (1993), S. 981-994 
    Details
    ISSN: 1573-5028
    Keywords: cis-acting elements ; intron conservation ; intron secondary structure ; pre-mRNA splicing ; CpG suppression ; protoplasts ; transcript levels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Our previous phylogenetic analysis based on cDNA sequences of chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPDH; genes GapA and GapC, respectively) of the red alga Chondrus crispus suggested that rhodophytes and green plants are sister groups with respect to plastids and mitochondria and diverged at about the same time or somewhat later than animals and fungi. Here we characterize the genomic sequences of genes GapC and GapA of C. crispus with respect to promotor structures, intron/exon organization, genomic complexity, G+C content, CpG suppression and their transcript levels in gametophytes and protoplasts, respectively. To our knowledge this is the first report on nuclear protein genes of red algae. The GapC gene is G+C-rich, contains no introns and displays a number of classic sequence motifs within its promotor region, such as TATA, CAAT, GC boxes and several elements resembling the plant-specific G-box palindrome. The GapA gene has a moderate G+C content, a single CAAT box motif in its promotor region and a single intron of 115 bp near its 5′ end. This intron occupies a conserved position corresponding to that of intron 1 in the transit peptide region of chloroplast GAPDH genes (GapA and GapB) of higher plants. It has consensus sequences similar to those of yeast introns and folds into a conspicuous secondary structure of - 61.3 kJ. CpG profiles of genes GapC and GapA and their flanking sequences show no significant CpG depletion suggesting that these genomic sequences are not methylated. Genomic Southern blots hybridized with generic and gene specific probes indicate that both genes are encoded by single loci composed of multiple polymorphic alleles. Northern hybridizations demonstrate that both genes are expressed in gametophytes but not in protoplasts where appreciable amounts of transcripts can only be detected for GapC.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00021813
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  • 49
    Electronic Resource
    Electronic Resource
    Quantification of indole-3-acetic acid in untransformed and Agrobacterium rhizogenes-transformed pea roots using gas chromatography mass spectrometry (1993)
    Springer
    Planta 189 (1993), S. 55-59 
    Details
    ISSN: 1432-2048
    Keywords: Agrobacterium ; Auxin ; Pisum (hairy roots) ; Root (auxin levels) ; Transformation (root)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Root segments of Pisum sativum L. were transformed by several strains of Agrobacterium rhizogenes. The resulting hairy roots, as well as apical segments from untransformed pea roots, were used to initiate root lines cultured in vitro. Levels of free IAA were quantified in the sub-cultured lines by gas-chromatography coupled to mass spectrometry, using selected ion monitoring. For most of the cultured untransformed and transformed root lines the IAA content was very small, compared with levels in untransformed intact primary roots. However, an agropine-type hairy root line (incited by strain 15834) contained significantly higher amounts of IAA. The peculiar phenotype of this root line (abundant production of calli) appears to be associated with an increased IAA level, as opposed to most of the hairy root lines, where the extensive secondary root proliferation associated with the hairy-root disease cannot be merely attributed to a markedly enhanced IAA content.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00201343
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  • 50
    Electronic Resource
    Electronic Resource
    Variation amongst Brassica juncea cultivars for regeneration from hypocotyl explants and optimization of conditions for Agrobacterium-mediated genetic transformation (1993)
    Springer
    Plant cell reports 12 (1993), S. 462-467 
    Details
    ISSN: 1432-203X
    Keywords: Brassica juncea ; hypocotyl ; plant regeneration ; Agrobacterium ; genetic transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Twelve cultivars of Brassica juncea grown in different agroclimatic regions of the world were tested for their ability to regenerate in vitro from hypocotyl explants and, accordingly, were divided into three groups. One group of cultivars regenerated on MS medium supplemented with 2,4-D, BAP and with NAA, BAP combinations; another group regenerated only on MS with 2,4-D, BAP; and the third group showed very low regeneration on both of these combinations. Inclusion of silver nitrate in the medium was essential for high frequency of regeneration. In general, Indian cultivars were more responsive than the cultivars of CIS and Australian origin. Using the media optimal for regeneration and an Agrobacterium-based binary vector carrying hpt and gus-intron genes, conditions for genetic transformation of B. juncea hypocotyl explants were optimized. Transformation frequencies, identified by GUS staining at the initial stages of growth, were lower on MS medium with 2,4-D, BAP than on MS with NAA, BAP. Plants resistant to 20 μg/ml hygromycin were regenerated at a frequency of 11–36% from hypocotyl explants and were shown to be transformed by Southern blotting, GUS staining and progeny analysis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00234713
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  • 51
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    Electronic Resource
    Improvement of protoplast culture protocols for Beta vulgaris L. (sugar beet) (1993)
    Springer
    Plant cell reports 12 (1993), S. 339-342 
    Details
    ISSN: 1432-203X
    Keywords: Alginate embedding ; Beta vulgaris ; feeders ; protoplasts ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The effects of NaCl, feeder cells and the embedding of protoplasts in calcium alginate have been investigated in an attempt to improve culture conditions of recalcitrant sugar beet (Beta vulgaris L.) mesophyll protoplasts. While the use of NaCl in all instances proved detrimental to protoplast development, the other two treatments had clear beneficial effects. Minimum plating densities, necessary to sustain cell division, could be reduced to 〈5% (〈4000 protoplasts / ml) of the control levels and plating efficiencies could be significantly enhanced by approx. 10 fold. Plants could still be regenerated from soft calli derived from mesophyll protoplasts cultured under the modified conditions at a frequency of 20–30 %. In particular, the use of alginate is considered of potentially great importance for the further application of beet protoplasts for other aims e.g. asymmetric hybridization.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00237431
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  • 52
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    Electronic Resource
    Genetic transformation of Brassica nigra by agrobacterium based vector and direct plasmid uptake (1993)
    Springer
    Plant cell reports 12 (1993), S. 418-421 
    Details
    ISSN: 1432-203X
    Keywords: Brassica nigra ; Transformation ; Agrobacterium ; Direct gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Genetic transformation systems have been established for Brassica nigra (cv. IC 257) by using an Agrobacterium binary vector as well as by direct DNA uptake of a plasmid vector. Both the type of vectors carried nptII gene and gus gene. For Agrobacterium mediated transformation, hypocotyl tissue explants were used, and up to 33% of the explants produced calli on selection medium. All of these expressed B-glucuronidase gene on histochemical staining. Protoplasts isolated from hypocotyl tissues of seedlings could be transformed with a plasmid vector by FEG mediated uptake of vector DNA. A number of fertile kanamycin resistant plants were obtained using both the methods, and their transformed nature was confirmed by Southern blot analysis and histochemical staining for GUS. Backcrossed and selfed progenies of these transformed plants showed the presence of npt and gus genes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00234704
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  • 53
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    Electronic Resource
    NewAgrobacterium helper plasmids for gene transfer to plants (1993)
    Springer
    Transgenic research 2 (1993), S. 208-218 
    Details
    ISSN: 1573-9368
    Keywords: Agrobacterium ; plant transformation ; helper plasmids ; host range ; Ti plasmid ; T-DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We describe the construction of new helper Ti plasmids forAgrobacterium-mediated plant transformation. These plasmids are derived from three differentAgrobacterium tumefaciens Ti plasmids, the octopine plasmid pTiB6, the nopaline plasmid pTiC58, and the L,L-succinamopine plasmid pTiBo542. The T-DNA regions of these plasmids were deleted using site-directed mutagenesis to yield replicons carrying thevir genes that will complement binary vectorsin trans. Data are included that demonstrate strain utility. The advantages ofAgrobacterium strains harbouring these ‘disamed’ Ti plasmids for plant transformation viaAgrobacterium are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01977351
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  • 54
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    Electronic Resource
    Localization of target cells and improvement ofAgrobacterium-mediated transformation efficiency by direct acetosyringone pretreatment of carrot root discs (1993)
    Springer
    Protoplasma 174 (1993), S. 10-18 
    Details
    ISSN: 1615-6102
    Keywords: Acetosyringone ; Agrobacterium ; Carrot ; Cell cycle ; GUS assay ; Indole acetic acid levels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Localization of target cells forAgrobacterium-mediated transformation in the carrot root disc model has been achieved after inoculation with a disarmedA. tumefaciens strain harbouring a GUS-intron construct. The first GUS positive cells could be detected on both sides of the discs 48 h after inoculation. The transformed cells were always more numerous on the apical side, mainly localized in the intrafascicular cambium and in the immature phloem strands. The kinetics of free endogenous IAA levels on both sides after wounding have been determined, indicating that rapid IAA accumulation on the apical side was not simply due to polar migration from the basal side. Attempts to optimize transformation efficiency were made by pretreating the discs with various concentrations of acetosyringone (AS) and/or naphthalene acetic acid (NAA). Surprisingly, while 25 μM AS applied to bacteria prior to the inoculations was ineffective, the same AS concentration applied as a pretreatment to the discs strongly increased the number of transformed cells in the target tissues and decreased the lag time for the appearance of the first GUS positive cells. NAA pretreatment on the basal side enhanced the AS effect. AS pretreatment was found both to advance the reentry of competent cells with a potential for cell division into the S phase of the cell cycle and to stimulate bacterial attachment to the cell walls. The relationship between transformation efficiency and DNA synthesis in the host cells is discussed. AS treatment of plant tissues prior to inoculation is proposed as a means of increasing the transformation rates.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01404037
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  • 55
    Electronic Resource
    Electronic Resource
    Analysis of the toxicity of purothionins and hordothionins for plant pathogenic bacteria (1993)
    Springer
    European journal of plant pathology 99 (1993), S. 259-268 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01974307
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    Rapid divergence of Agrobacterium vitis octopine-cucumopine Ti plasmids from a recent common ancestor (1993)
    Springer
    Molecular genetics and genomics 240 (1993), S. 49-57 
    Details
    ISSN: 1617-4623
    Keywords: Agrobacterium ; Microbial evolution ; RFLP analysis ; Ti plasmids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The octopine/cucumopine (o/c) Ti plasmids of the grapevine-associated Agrobacterium vitis strains constitute a family of related DNA molecules. Restriction maps were established of two limited-host-range o/c Ti plasmids, pTiAg57 and pTiAB3, and of the wide-host-range o/c Ti plasmid pTiHml. Together with the previously obtained map of the wide-host-range o/c Ti plasmid pTiTm4, about 1000 kb were mapped with a resolution of 0.2 kb, allowing a detailed comparison of the various structures. One region of the o/c Ti plasmids is highly conserved and differs mainly by the presence or absence of relatively small DNA fragments (0.9–2.7 kb); the other region has been modified more extensively and carries large sequences specific for each Ti plasmid type. The sequence similarity within large conserved regions shows that these plasmids have diverged recently and that their evolution was driven by large-scale genetic events rather than single nucleotide changes. These results have important implications for studies on bacterial evolution.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00276883
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  • 57
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    Nopaline causes a conformational change in the NocR regulatory protein-nocR promoter complex of Agrobacterium tumefaciens Ti plasmid pTiT37 (1993)
    Springer
    Molecular genetics and genomics 241 (1993), S. 65-72 
    Details
    ISSN: 1617-4623
    Keywords: Agrobacterium ; Nopaline ; Gene regulation ; Protein-DNA interaction ; DNA topology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The nocR gene of Agrobacterium tumefaciens Ti plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37. We have cloned and sequenced nocR, which encodes a DNA-binding protein. The deduced amino acid sequence is similar to those of members of the LysR family of prokaryotic activator proteins. Gel retardation experiments demonstrated that the NocR protein binds to the nocR promoter in both the presence and absence of nopaline. The increased mobility of the complex and alterations in the DNase I footprints revealed a nopaline-induced conformational change in the NocR-DNA complex. Sequence analysis of the NocR binding site indicated the presence immediately downstream of the −10 sequence of the nocR promoter of a 12 by putative operator overlapping a consensus gyrase recognition sequence and an 18 by long alternating purine-pyrimidine sequence. These results suggest that nopaline-induced alterations in the NocR protein-nocR promoter complex might control gene expression in the noc operon.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00280202
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  • 58
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    Investigations of boron uptake at the cellular level (1993)
    Springer
    Plant and soil 155-156 (1993), S. 143-146 
    Details
    ISSN: 1573-5036
    Keywords: barley ; cell wall ; Hordeum vulgare ; pollen selection ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The nature of expression of the tolerance of barley to high levels of B at the cellular level was investigated with a view to identifying ways by which this level of expression might be exploited in a breeding programme. Using protoplasts derived from leaf tissue, it was found that genetic differences between B tolerant and intolerant barleys were not expressed in the absence of cell walls. Barley genotypes differing in their tolerance to B were subjected to high levels of B in the growth medium from pollen formation onwards. The genetic distribution of segregating populations in the next generation was not changed for tolerance to high B. Results also suggested that genetic tolerance to B is expressed by pollen in vitro.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00025004
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  • 59
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    Plant regeneration from protoplasts isolated from cell suspension cultures of the wild tomato, Lycopersicon chilense Dun. (1993)
    Springer
    Plant cell, tissue and organ culture 32 (1993), S. 311-317 
    Details
    ISSN: 1573-5044
    Keywords: cell suspension ; Lycopersicon chilense ; plant regeneration ; protoplasts ; wild tomato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protocol has been established for rapid, high frequency plant regeneration from protoplasts of the wild tomato species Lycopersicon chilense Dun. Cell suspension cultures were obtained from calli initiated from seedling stem explants. Protoplasts were isolated from cell suspensions by an overnight one-step enzyme digestion, purified by washing in salts solution and cultured in liquid medium. Dilution of liquid medium every 3 days, with medium containing low levels of growth regulators and sucrose, was critical for sustained colony formation. Up to 70% of protoplast-derived calli regenerated shoots when cultured on agar-solidified medium with Murashige & Skoog (1962) salts and vitamins, 2.0 mg l-1 zeatin and 0.1 mg l-1 indole acetic acid for 21 days, followed by transfer to the same medium lacking indole acetic acid.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00042294
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  • 60
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    A study of some factors affectingAgrobacterium transformation and plant regeneration ofDendranthema grandiflora Tzvelev (syn.Chrysanthemum morifolium Ramat.) (1993)
    Springer
    Plant cell, tissue and organ culture 33 (1993), S. 171-180 
    Details
    ISSN: 1573-5044
    Keywords: Agrobacterium ; Dendranthema grandiflora ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In an attempt to develop a system for producing transformed plants from explants ofDendranthema grandiflora, the susceptibility of the cultivar Super White to various wild-type strains ofAgrobacterium tumefaciens andA. rhizogenes was investigated. Tumour formation was not a reliable indicator of the ability of a related disarmed strain to mediate transformation. Following inoculation of explants with disarmedAgrobacterium strains, a number of shoots developed on selective media. However, none of these shoots were transformed. By co-cultivating stem internode explants with a mixed inoculum of wild-type and disarmed strains, it was possible to obtain a callus stably transformed withAgrobacterium carrying a disarmed T-DNA. Histological analysis of explants revealed that shoot regeneration initially occurred from the cells of the epidermis and subsequently from the cortex. However, the cells which were susceptible to T-DNA transfer were confined to the vascular tissue.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01983231
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  • 61
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    Culturing embryogenic protoplasts of ‘Hamlin’ sweet orange in calcium alginate beads (1993)
    Springer
    Plant cell, tissue and organ culture 34 (1993), S. 19-25 
    Details
    ISSN: 1573-5044
    Keywords: Ca-alginate ; citrus ; plating efficiency ; protoplasts ; somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts were enzymatically isolated from log phase embryogenic sweet orange [Citrus sinensis (L.) Osbeck cv. Hamlin] suspension cultures, embedded in 3-mm diameter Ca-alginate beads, and cultured in a growth cabinet (15–20 μ mol m-2 s-1, 4-h photoperiod, 27°C). Plating efficiency exceeded 90% for Ca-alginate embedded protoplasts vs. 30% for protoplasts cultured in a liquid medium. Embryoids formed from protoplasts were recovered after 20 days by dissolving the Ca-alginate matrix with a calcium sequestrant. Embryoids readily formed shoots that were rooted on MS+0.01 μM NAA+5% sucrose. Potential applications are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00048459
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  • 62
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    The biotechnology of crop legumes (1993)
    Springer
    Euphytica 74 (1993), S. 165-185 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00040399
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  • 63
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    Regeneration of whole plants from Gracilaria asiatica Chang et Xia protoplasts (Gracilariaceae, Rhodophyta) (1993)
    Springer
    Hydrobiologia 260-261 (1993), S. 429-436 
    Details
    ISSN: 1573-5117
    Keywords: Gracilaria ; protoplasts ; callus-like ; regeneration ; plant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A large number of viable protoplasts were produced by enzymatic digestion of Gracilaria asiatica vegetative tissue. The protoplasts underwent initial division after 5–7 d in culture and developed into callus-like cell-masses. Many filaments grew from the periphery of these cell-masses and disappeared after about one month in culture. Simultaneously, the central part of the callus-like cell-masses thickened and its color deepened. The first buds appeared from the center of the cell-masses and developed into whole plants after three months in aerated culture. Many new buds formed around the first plant and more than 20 plants grew per callus-like cell-masses in less than four months. Filaments taken from callus-like cell-masses developed into young plants after about 20 d of culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00049052
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  • 64
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    Microtubule organization in the apical cell of Sphacelaria (Phaeophyceae) and its related protoplast (1993)
    Springer
    Hydrobiologia 260-261 (1993), S. 167-172 
    Details
    ISSN: 1573-5117
    Keywords: Phaeophyceae ; protoplasts ; apical cells ; immunofluorescence ; microtubules ; cell cycle ; polarity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The growth of the filamentous brown alga Sphacelaria depends on a large, strongly polarized, apical cell. The protoplast derived from this cell can be distinguished in a heterogeneous suspension by cytological markers, so it is possible to study development of the cytoskeleton during protoplast isolation and the first steps of regeneration. In the initial cell, microtubules show an asymmetric distribution along the axis; they are mainly located at the distal part around the physodes. After protoplast isolation, this polarity initially seems to be maintained; subsequently, the microtubules radiate from the two centrioles and spread out to the plasmalemma. This experimental model is suitable for investigating the development of the polarity of the initial cell, and the sequence of the first morphogenetic events leading to protoplast regeneration.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00049016
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  • 65
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    Protoplasts of Gelidium robustum (Rhodophyta) (1993)
    Springer
    Hydrobiologia 260-261 (1993), S. 421-427 
    Details
    ISSN: 1573-5117
    Keywords: protoplasts ; Gelidium robustum ; agarophyte ; Rhodophyta
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Viable protoplasts were isolated from apices of the agarophyte Gelidium robustum (Gardn.) Hollenb. & Abb. using a combination of commercial cell-wall degrading enzymes and extracellular wall-degrading enzymes isolated from a marine bacterium. The protoplasts were approximately 8–15 µm in diameter, liberated mainly from the surface cell layers and from cells at the distal ends of medullary filaments. The bacterial enzyme alone was not sufficient to liberate significant numbers of protoplasts. Maximum yield was 9 × 105 protoplasts/g tissue (wet wt.). Optimum osmolality occurred between 1750–1950 mOs kg−1; yield and viability were severely diminished at osmolalities less than 1350 mOs kg−1. Viability, as determined by flurorescein diacetate staining and Evans Blue exclusion 1 hr after removal from the enzyme solution, was approximately 80–95%. Roughly 80% of the cells did not show Calcofluor fluorescence, while 40% stained positively for the presence of sulfated polysaccharides. Cell wall regeneration was observed with inconsistent reproducibility, and no cell division was observed when the protoplasts were placed in culture medium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00049051
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    Genetic engineering of grain and pasture legumes for improved nutritive value (1993)
    Springer
    Genetica 90 (1993), S. 181-200 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01435039
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  • 67
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    Effect of digitonin on membrane-bound and chitosomal chitin synthetase activity in protoplasts from yeast cells ofCandida albicans (1993)
    Springer
    Antonie van Leeuwenhoek 64 (1993), S. 67-74 
    Details
    ISSN: 1572-9699
    Keywords: Candida albicans ; chitin synthetase ; digitonin ; proenzyme activation ; protoplasts ; solubilization ; zymogen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) fromC. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen,in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processesin vivo andin vitro or, alternatively, that both enzyme activities (activein vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml−1 of digitonin and 64 µg ml−1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 µg ml−1 of protease, whereas activity strongly decreases in the presence of 64 µg ml−1 of trypsin. Digitonin does not produce zymogen activationper se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00870923
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    An efficient protoplast-to-plant system for the hybrid ornamental shrub,Weigela ×florida cv. Bristol Ruby (Caprifoliaceae) (1993)
    Springer
    Plant cell, tissue and organ culture 33 (1993), S. 315-320 
    Details
    ISSN: 1573-5044
    Keywords: protoplasts ; plant regeneration ; woody ornamentals ; Weigela
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Strategies were developed for the successful isolation of large numbers of highly viable protoplasts from the leaves, stems and roots of axenic plants of the hybrid ornamental shrubWeigela ×florida cv Bristol Ruby. Protoplasts, of all sources, were cultured on different media, leading to the establishment of sustained divisions, and coupled with the production of multi-celled (〉50 cells) colonies. However, those colonies derived from mesophyll protoplasts only were capable of a further proliferation to the callus stage. Upon transfer to a regeneration medium consisting of MS salts and organics plus a range of concentrations of NAA and BAP, such calli underwent caulogenesis, with optimum responses for a medium with 1.0 mg l−1 NAA and 1.0 mg l−1 BAP. The protoplast-derived shoots thus obtained were multiplied on MS medium with 0.1 mg l−1 IBA, 0.5 mg l−1 BAP and 0.1 mg l−1 GA3. Individual shoots were subsequently rooted on a half-strength MS medium plus 3.0 mg l−1 IBA, and complete protoplast-derived plants were finally transferred to the glasshouse for acclimatization.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02319017
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  • 69
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    Efficient plant regeneration from protoplasts of Arachis paraguariensis Chod. et Hassl. using a nurse culture method (1993)
    Springer
    Plant cell, tissue and organ culture 34 (1993), S. 83-90 
    Details
    ISSN: 1573-5044
    Keywords: Arachis species ; nurse culture ; plant regeneration ; protoplasts ; tissue culture ; wild peanut
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An efficient protocol has been developed for protoplast culture and plant regeneration from wild peanut (A. paraguariensis) using a nurse culture method. Protoplasts were isolated from suspension cultures initiated from leaf-derived callus, imbedded in agarose blocks and co-cultured with nurse cells of the same species. Up to 10% of the protoplasts divided and formed compact callus colonies. The protoplast plating efficiency was correlated with both the length of the nurse cell co-cultivation period and the protoplast plating density. The optimal nurse culture duration was 14 d. The optimal plating density was 2×104 protoplasts/ml plating medium. Multiple shoots (up to 10 shoots per colony) were readily regenerated from protoplast-derived callus after transfer of callus to semi-solid modified MS medium containing 0.5 mg l-1 NAA and 1 mg l-1 BA. Plantlets with normal leaflets were obtained by rooting shoots on porous rootcubes saturated with modified MS medium containing 1 mg l-1 NAA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00048467
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  • 70
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    Agrobacterium-mediated genetic transformation of oilseed Brassica campestris: Transformation frequency is strongly influenced by the mode of shoot regeneration (1992)
    Springer
    Plant cell reports 11 (1992), S. 506-513 
    Details
    ISSN: 1432-203X
    Keywords: Genetic transformation ; Oilseed Brassica campestris ; Agrobacterium ; Shoot differentiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Protocols were developed for efficient shoot regeneration from hypocotyl and cotyledon explants of oilseed Brassica campestris (brown sarson) cv. ‘Pusa Kalyani’. These were used for genetic transformation by an Agrobacterium based binary vector carrying neomycin phosphotransferase (npt) gene and β-glucuronidase (gus)-intron gene for plant cell specific expression. Transformed plants were recovered from hypocotyl explants at a frequency of 7–13%. Addition of silver nitrate markedly enhanced shoot regeneration in hypocotyl explants under non-selection conditions and was found to be an absolute requirement under selection conditions. Cotyledon explants, inspite of being more regenerative, proved to be highly refractory to transformation. Only two chimeric transformed shoots were obtained from more than 10,000 cotyledons treated with Agrobacterium. In hypocotyl explants, shoot regeneration occurred from the vascular parenchyma both with and without the intervention of callus phase. Only the shoot buds differentiating from callus tissue were positive for GUS activity. In cotyledons, shoot buds originated only directly from the vascular parenchyma, generally at a distance of about 450–625 μ from the cut surface. Such shoots were negative for GUS activity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00236266
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    Auxin autonomy in cultured tobacco teratoma tissues transformed by an auxin-mutant strain of Agrobacterium tumefaciens (1992)
    Springer
    Planta 188 (1992), S. 123-128 
    Details
    ISSN: 1432-2048
    Keywords: Agrobacterium ; Auxin (tumor tissue) ; Compensation (tms genes) ; Nicotiana (cell transformation) ; tms genes ; Tumor morphology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have studied the mechanism of auxin autonomy in tobacco (Nicotiana tabacum L.) crowngall tissues transformed by the auxin-mutant (tms −) A66 strain of Agrobacterium tumefaciens. Normally, tms − tobacco tumor tissues require the formation of shoots to exhibit auxin-independent growth in culture. We have isolated from tms − tobacco cells several stable variants that are fully hormone-independent and grow rapidly as friable, unorganized tissues, thus mimicking the growth and morphology of tms + tobacco cells that produce high levels of auxin. However, none of the variants contained the high levels of auxin found in tms + tumor cells. The variants could be divided into two classes with respect to their response to applied auxin. The first class was highly sensitive to applied auxin: low concentrations (1 μM) of α-naphthaleneacetic acid (NAA) severely inhibited growth and markedly stimulated the accumulation of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The second class of variants showed a low sensitivity to applied auxin: growth was promoted by concentrations of NAA up to 10 μM, and growth inhibition and high ACC levels were observed only at high NAA concentrations (100 μM). Unorganized variants with low auxin sensitivity were also isolated from a variant line with high auxin sensitivity. The isolation of tumor cells that exhibited the growth phenotype of tms + cells while retaining the low auxin content and low auxin sensitivity of tms − cells indicates that full hormone autonomy, characteristic of wild-type crown-gall tumors, can be achieved by a mechanism that is independent of changes in the auxin physiology of the cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00198948
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    Auxin autonomy in cultured tobacco teratoma tissues transformed by an auxin-mutant strain ofAgrobacterium tumefaciens (1992)
    Springer
    Planta 188 (1992), S. 123-128 
    Details
    ISSN: 1432-2048
    Keywords: Agrobacterium ; Auxin (tumor tissue) ; Compensation (tms genes) ; Nicotiana (cell transformation) ; tms genes ; Tumor morphology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have studied the mechanism of auxin autonomy in tobacco (Nicotiana tabacum L.) crowngall tissues transformed by the auxin-mutant (tms −) A66 strain ofAgrobacterium tumefaciens. Normally,tms − tobacco tumor tissues require the formation of shoots to exhibit auxin-independent growth in culture. We have isolated fromtms − tobacco cells several stable variants that are fully hormone-independent and grow rapidly as friable, unorganized tissues, thus mimicking the growth and morphology oftms + tobacco cells that produce high levels of auxin. However, none of the variants contained the high levels of auxin found intms + tumor cells. The variants could be divided into two classes with respect to their response to applied auxin. The first class was highly sensitive to applied auxin: low concentrations (1 μM) of α-naphthaleneacetic acid (NAA) severely inhibited growth and markedly stimulated the accumulation of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The second class of variants showed a low sensitivity to applied auxin: growth was promoted by concentrations of NAA up to 10 μM, and growth inhibition and high ACC levels were observed only at high NAA concentrations (100 μM). Unorganized variants with low auxin sensitivity were also isolated from a variant line with high auxin sensitivity. The isolation of tumor cells that exhibited the growth phenotype oftms + cells while retaining the low auxin content and low auxin sensitivity oftms − cells indicates that full hormone autonomy, characteristic of wild-type crown-gall tumors, can be achieved by a mechanism that is independent of changes in the auxin physiology of the cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01160721
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    Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana (1992)
    Springer
    Planta 188 (1992), S. 439-456 
    Details
    ISSN: 1432-2048
    Keywords: Agrobacterium ; Arabidopsis ; Cotyledon ; Root explant (in vitro culture) ; Transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The insertion of foreign DNA in plants occurs through a complex interaction between Agrobacteria and host plant cells. The marker gene β-glucuronidase of Escherichia coli and cytological methods were used to characterize competent cells for Agrobacterium-mediated transformation, to study early cellular events of transformation, and to identify the potential host-cell barriers that limit transformation in Arabidopsis thaliana L. Heynh. In cotyledon and leaf explants, competent cells were mesophyll cells that were dedifferentiating, a process induced by wounding and-or phytohormones. The cells were located either at the cut surface or within the explant after phytohormone pretreatment. In root explants, competent cells were present in dedifferentiating pericycle, and were produced only after phytohormone pretreatment. Irrespective of their origin, the competent cells were small, isodiametric with thin primary cell walls, small and multiple vacuoles, prominent nuclei and dense cytoplasm. In both cotyledon and root explants, histological enumeration and β-glucuronidase assays showed that the number of putatively competent cells was increased by preculture treatment, indicating that cell activation and cell division following wounding were insufficient for transformation without phytohormone treatment. Exposure of explants for 48 h to A. tumefaciens produced no characteristic stress response nor any gradual loss of viability nor cell death. However, in the competent cell, association between the polysaccharide of the host cell wall and that of the bacterial filament was frequently observed, indicating that transformation required polysaccharide-to-polysaccharide contact. Flow cytofluorometry and histological analysis showed that abundant transformation required not only cell activation (an early state exhibiting an increase in nuclear protein) but also cell proliferation (which in cotyledon tissue occurred at many ploidy levels). Noncompetent cells could be made competent with the appropriate phytohormone treatments before bacterial infection: this should aid analysis of critical steps in transformation procedures and should facilitate developing new strategies to transform recalcitrant plants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00192812
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  • 74
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    Agrobacterium-mediated transformation of tomatillo (Physalis ixocarpa) and tissue specific and developmental expression of the CaMV 35S promoter in transgenic tomatillo plants (1992)
    Springer
    Plant cell reports 11 (1992), S. 558-562 
    Details
    ISSN: 1432-203X
    Keywords: Agrobacterium ; Plant Transformation ; Gene Expression ; CaMV 35S Promoter ; Physalis ixocarpa
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon expiants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed formation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233092
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  • 75
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    Plant regeneration from long term suspension culture-derived protoplasts of hexaploid wheat (Triticum aestivum L.) (1992)
    Springer
    Plant cell reports 11 (1992), S. 262-265 
    Details
    ISSN: 1432-203X
    Keywords: Triticum aestivum ; embryogenic suspension cultures ; protoplasts ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Highly regenerable callus cultures have been obtained from immature embryos of hexaploid wheat cv. Oderzo. Friable fast growing calli were induced at high frequency. Suspensions were initiated from the most friable callus lines: they became established in about two months. Suspensions consisted of cell aggregates of 30 to 1000 um in diameter. Upon plating on MS hormone-free medium, suspensions regenerated green plantlets, and their regenerative capability was maintained for at least 10 months. Protoplasts were isolated from 7–8 day old suspension cultures with a yield of 4–6×106 protoplasts/g fresh weight cells. Protoplast culture was either in liquid medium or in a bead-type system with agarose beads. First divisions were detected at day 5. At day 14 visible colonies were detected and the plating efficiency was evaluated between 2 and 8% over the initial number of protoplasts plated. Protoplast-derived calli were cultured in the presence of 1 mg/l IAA and 0.5 mg/l zeatin and were used for reinitiating new suspension cultures. Upon plating onto MS hormone-free medium, with or without the addition of 0.1 mg/l GA3, calliclones were induced to differentiate. Regeneration of complete plantlets, with shoot and roots took about two months. Plantlets were grown in sterile conditions until 12–15 cm height, and were subsequently transplanted in soil.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00235078
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  • 76
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    Electronic Resource
    Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs, plant ecotypes and Agrobacterium strains (1992)
    Springer
    Plant cell reports 12 (1992), S. 7-11 
    Details
    ISSN: 1432-203X
    Keywords: Arabidopsis thaliana ; Transformation ; Agrobacterium ; T-DNA ; Shoot regeneration ; Transgenic plant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl expiants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the ß-glucuronidase (GUS) reporter gene showed that the gusA gene expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80–90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00232413
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  • 77
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    Electronic Resource
    Translation controls the expression level of a chimaeric reporter gene (1992)
    Springer
    Plant molecular biology 20 (1992), S. 921-938 
    Details
    ISSN: 1573-5028
    Keywords: β-D-glucuronidase ; mannopine synthase promoter ; Agrobacterium ; gene expression ; initiation of translation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transcriptional and translational fusions between the reading frame of the β-D-glucuronidase gene (gusA) and the 2′ as well as the 1′ promoter of mannopine synthase (mas), a TR locus of Agrobacterium tumefaciens, were made. The expression of these constructs was studied in the transgenic F1 offspring of independent tobacco transformants at the protein level by assaying for GUS activity and western blot analysis of the GUS protein and at the steady-state mRNA level. In leaves, stems and roots no correlation was found between steady-state levels of GUS mRNA and enzyme activity. In older tissues significantly higher GUS activities were found. This is explained by the stable character of the GUS protein together with an accumulation of protein upon ageing. Three to ten times higher GUS activities were found for in vitro grown plants than for greenhouse-grown plants of the same offspring, despite similar levels of GUS mRNA. Roots from in vitro grown plants display three to ten times higher GUS activities than stems and leaves. In transgenic plants grown in vitro, containing a translational fusion with two AUGs in phase, the initiation of translation in leaf material occurred at both AUGs. Initiation of translation at the first AUG, however, was ten times more frequent. In contrast, initiation in roots from in vitro grown plants occurred exclusively at the second AUG.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00027163
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  • 78
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    Electronic Resource
    Factors influencing Agrobacterium-mediated transient expression of gusA in rice (1992)
    Springer
    Plant molecular biology 20 (1992), S. 1037-1048 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; rice ; transformation ; Ti plasmid ; GUS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transient expression of GUS in rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens was characterized using binary vectors containing gusA genes that express minimal (pKIWI105 and pCNL1) or no (p35S-GUS-INT and pCNL56) GUS activity in bacteria. Four-day old seedlings obtained from seeds or immature embryos of rice were cut into shoot, root, and seed remnants and inoculated with various strains of A. tumefaciens. Transient GUS expression events were quantitated histochemically by determining the frequency of explants exhibiting blue spots indicative of GUS at four to six days after cocultivation with A. tumefaciens. A. tumefaciens strains that did not contain the gusA gene (At643) or a Ti-plasmid (At563 and At657) did not elicit any blue staining characteristic of GUS activity. Several parameters were important in obtaining efficient transient expression of GUS in rice mediated by A. tumefaciens. The growth regulator 2,4-D inhibited GUS expression if present during the seed germination period, but the presence of 6 mg/1 2,4-D during cocultivation of the explants with A. tumefaciens slightly enhanced GUS expression efficiency. All 21 rice cultivars tested expressed GUS after co-cultivation with A. tumefaciens. The GUS expression frequency was highest amongst the indica cultivars. The frequencies of GUS expression in japonica cultivars and in Oryza glaberrima cultivars (grown primarily in Africa) were generally one-half to one-third the level found for indica varieties. Leaf explants were more susceptible to A. tumefaciens-facilitated GUS expression than were roots or seed remnants. The vir genes of an agropine-type Ti-plasmid of A. tumefaciens were most effective in directing transient GUS expression in rice, whereas those of a nopaline-type and an octopine-type plasmid were less effective. We have also found that the frequency of transient expression of GUS was higher with pBIN19 as the precursor cloning vector than with pEND4K as the precursor cloning vector. Reasons for differences in effectiveness of these binary vectors are discussed. Using the conditions described here, A. tumefaciens-mediated frequencies of transient GUS expression in four-day old shoots of several rice cultivars were routinely in excess of 50%.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00028891
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  • 79
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    Electronic Resource
    A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome (1992)
    Springer
    Plant molecular biology 20 (1992), S. 1203-1207 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; binary vector ; CaMV 35S ; gene expression ; β-glucuronidase ; Nicotiana plumbaginifolia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A versatile gene expression cartridge and binary vector system was constructed for use in Agrobacterium-mediated plant transformation. The expression cartridge of the primary cloning vector, pART7, comprises of cauliflower mosaic virus Cabb B-JI isolate 35S promoter, a multiple cloning site and the transcriptional termination region of the octopine synthase gene. The entire cartridge can be removed from pART7 as a Not I fragment and introduced directly into the binary vector, pART27, recombinants being selected by blue/white screening for β-galactosidase. pART27 carries the RK2 minimal replicon for maintenance in Agrobacterium, the ColE1 origin of replication for high-copy maintenance in Escherichia coli and the Tn7 spectinomycin/streptomycin resistance gene as a bacterial selectable marker. The organisational structure of the T-DNA of pART27 has been constructed taking into account the right to left border, 5′ to 3′ model of T-DNA transfer. The T-DNA carries the chimaeric kanamycin resistance gene (nopaline synthase promoter-neomycin phosphotransferase-nopaline synthase terminator) distal to the right border relative to the lacZ′ region. Utilisation of these vectors in Agrobacterium-mediated transformation of tobacco demonstrated efficient T-DNA transfer to the plant genome.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00028910
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  • 80
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    Electronic Resource
    Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system (1992)
    Springer
    Plant molecular biology 18 (1992), S. 353-361 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00034962
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  • 81
    Electronic Resource
    Electronic Resource
    Effect of two consensus sequences preceding the translation initiator codon on gene expression in plant protoplasts (1992)
    Springer
    Plant molecular biology 18 (1992), S. 815-818 
    Details
    ISSN: 1573-5028
    Keywords: expression cassette ; gene expression ; protoplasts ; translation initiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression cassettes containing a duplicated cauliflower mosaic virus (CaMV) 35S promoter fused to a polylinker preceded by the CCACCATGG and AACAATGG sequences were constructed. These two sequences correspond to the consensus sequences around the translation start codons in vertebrates and plants respectively. Translational fusions were made with the β-glucuronidase-coding sequence and transient expression was recorded in tobacco mesophyll protoplasts. Approximately three times more GUS activity was found in protoplasts incubated with the constructs harbouring translational fusions as compared to a control harbouring a transcriptional fusion. No significant difference was observed between GUS activities obtained with the two consensus sequences.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00020027
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  • 82
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    Electronic Resource
    Functional analysis of the promoter region of a nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L. (1992)
    Springer
    Plant molecular biology 19 (1992), S. 837-846 
    Details
    ISSN: 1573-5028
    Keywords: glutamine synthetase ; hairy roots ; nodules ; Phaseolus vulgaris ; protoplasts ; transcriptional regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The 5′-flanking region of gln-γ, the nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L., has been analysed for cis-regulatory elements using a series of 5′ deletions and hybrid gln-γ: : CaMV 35S promoters. The promoters were fused to the uidA reporter gene and their activities tested in two heterologous expression systems. In the first system, the chimaeric genes were transferred to Lotus corniculatus L. using Agrobacterium rhizogenes and their expression was studied in nodulated hairy roots. In the second system, the constructs were electroporated into tobacco mesophyll protoplasts. The results of the 5′ deletion analysis showed that the sequence between −597 and −21 (relative to the ATG codon) was sufficient for nodule-specific expression of the chimaeric gene in nodulated hairy roots, and revealed the existence of at least two positive regulatory elements. Sequences located between −2000 and −597 were able to stimulate expression in nodules but not protoplasts, while the region from −597 to −354 enhanced expression in both nodules and protoplasts. Results obtained with the hybrid gln-γ: :35 S promoters showed that two overlapping restriction fragments (−516/−343 and −474/−293) were able to stimulate expression from a heterologous promoter in an orientation-dependent manner. Previous work has demonstrated the presence of conserved A/T-rich binding sites for nuclear proteins in the region between −516 and −446, and their possible role in regulating gln-γ expression is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00027079
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  • 83
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    Multiple ocs-like elements required for efficient transcription of the mannopine synthase gene of T-DNA in maize protoplasts (1992)
    Springer
    Plant molecular biology 20 (1992), S. 219-233 
    Details
    ISSN: 1573-5028
    Keywords: promoter ; electroporation ; protoplasts ; transient assay ; Agrobacterium ; Ti plasmid ; Zea mays
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Regulatory elements controlling transcriptional activity of the mannopine synthase 2′ promoter (mas 2′) were defined by analysis of deletion mutants in transient expression assays in maize protoplasts. Deletion of the region between −305 and −290 containing sequence similarity to the octopine synthase (ocs) promoter element reduced activity by 67% compared to wild type activity. Less than 1% of the activity remained in 5′ deletions downstream of −153. Inclusion of various heterologous enhancer-like sequences immediately upstream of position −325 increased activity by up to 7.5-fold. Insertion of the −325 to −275 sequence alone, or in combination with heterologous enhancer-like elements, restored activity of some of the 5′-deletion mutants. Restoration of activity was not obtained with mutants deleted past position −127. Our results suggest that a single class of nuclear proteins from maize interact with high affinity at elements designated mas b (−306 to −275; mas 1′ element), d (−127 to −108), and e (−82 to −39; mas 2′ element) as well as the 20 bp element from the ocs promoter. Although the binding site at mas d only appears to accommodate a single protein, this element has the potential to make a weak, but positive, contribution to the activity of the mas 2′ promoter. The binding of nuclear proteins could not be demonstrated at mas a and c, both of which showed limited homology to the ocs element. Mutational evidence suggested that mas a and c may also contribute to mas 2′ transcription.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00014490
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  • 84
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    Electronic Resource
    DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts (1992)
    Springer
    Plant molecular biology 18 (1992), S. 703-712 
    Details
    ISSN: 1573-5028
    Keywords: DNA methylation ; geminivirus ; protoplasts ; tomato golden mosaic virus ; transfection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m5C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m5C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00020012
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  • 85
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    Electronic Resource
    Expression of a chimaeric heat-shock-inducible Agrobacterium 6b oncogene in Nicotiana rustica (1992)
    Springer
    Plant molecular biology 18 (1992), S. 921-930 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; 6b gene ; oncogenes ; tumour induction ; wound-induced cell division
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The T-6b gene of Agrobacterium tumefaciens strain Tm4 induces tumours on Nicotiana rustica by an as yet unknown mechanism. These tumours cannot be regenerated into normal plants. To study the effect of the T-6b gene product on normal plant cells, the T-6b gene was placed under control of the Drosophila melanogaster hsp70 heat-shock promoter and introduced into N. rustica. Progeny of an hsp70-T-6b transformant developed into normal plants. The inducibility of the hsp70-T-6b construct was shown by northern analysis and by heat-shock-dependent growth alterations on the level of whole seedlings. Upon wounding at normal temperature conditions hsp70-T-6b plants formed small tumours on leaves and stems. Grafts between transformed plants and normal plants led to a wound callus which remained limited to transformed tissues, indicating that the T-6b gene product does not diffuse. Protoplasts of hsp70-T-6b plants divided in the same way as control protoplasts under standard culture conditions. However, when protoplast cultures were started in the absence of hormones, normal cells rapidly lost their sensitivity towards hormones, whereas hsp70-T-6b cells remained sensitive for a significantly longer period. Thus, the T-6b gene product alters hormone sensitivity during the initial phases of protoplast culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00019206
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  • 86
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    Electronic Resource
    Functional elements of the Arabidopsis Adh promoter include the G-box (1992)
    Springer
    Plant molecular biology 19 (1992), S. 859-862 
    Details
    ISSN: 1573-5028
    Keywords: luciferase ; cis-DNA element ; GUS ; protoplasts ; PEG
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The alcohol dehydrogenase (Adh) gene of Arabidopsis is expressed constitutively in immature seedlings and cells in suspension, and may be induced by hypoxic stress only in roots of mature plants. Deletions and G-box mutations of the Adh promoter were assayed in Arabidopsis protoplasts by PEG-mediated transient expression. Sequence domains necessary for full gene activity are confined to the 384 bp immediately 5′ to the transcription start site, and deletion to −177 results in 〉90% reduction in promoter activity. Site-specific mutations of G-box bases result in 〉60% reduction in activity and disrupt G-box factor binding in vitro.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00027081
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  • 87
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    Electronic Resource
    Potato granule-bound starch synthase promoter-controlled GUS expression: regulation of expression after transient and stable transformation (1992)
    Springer
    Plant molecular biology 20 (1992), S. 19-30 
    Details
    ISSN: 1573-5028
    Keywords: granule-bound starch synthase ; potato ; protoplasts ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the β-glucuronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient to result in tissue-dependent GUS expression: levels in stably transformed microtubers exceeded levels in corresponding leaves by orders of magnitude. GBSS-GUS constructs could be transiently expressed in leaf protoplasts from wild-type and amylose-free potato lines, etuberosumSolanum brevidens, Nicotiana tabacum andArabidopsis thaliana. Transient expression levels in potato leaf protoplasts were clearly lower than in corresponding suspension cell protoplasts. This lower expression in leaf protoplasts could not be elevated by increasing DNA concentrations during transfection. Light incubation of electroporated suspension cell protoplasts reduced transient GBSS-GUS expression, whereas incubation of transfected protoplasts in media with different sucrose concentrations did not affect transient expression levels. However, electroporated protoplasts, isolated from suspensions, which had been grown on media with increasing amounts of sucrose showed a sucrose concentration-dependent transient expression profile. This indicates that studying GBSS regulation by transient expression experiments needs pre-treatment of the protoplast source. Sequence data of the GBSS promoter were compared to those of two other potato alleles.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00029145
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  • 88
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    Induction of fast-growing and morphologically different strains through intergeneric protoplast fusions of Ulva and Enteromorpha (Ulvales, Chlorophyta) (1992)
    Springer
    Journal of applied phycology 4 (1992), S. 57-65 
    Details
    ISSN: 1573-5176
    Keywords: Ulva ; Enteromorpha ; protoplasts ; electrofusion ; intergeneric fusion ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Isolated protoplasts of Ulva pertusa and Enteromorpha prolifera were electrically fused. Treatment of protoplasts in 1% protease for 15–20 min prior to fusion enhanced fusion ability. Protoplasts from each fusion partner were mixed together in 1:1 ratio in low conductivity electrofusion solution at a density of 1 × 105 cells ml−1 before subjecting them to electrofusion. The protoplasts were aligned in AC field (1MHz, 25 V for 10–15 s) and subsequently fused by a high intensity single DC pulse of 250 V for 25 μs duration. Fusion buffer supplemented with 1 mM calcium and 1 mM magnesium yielded optimum fusion frequencies (about 18–24%). Entrapment of fusion treated cells inside agarose/agar plate facilitated marking and regeneration of fusion products. The regeneration patterns of fused protoplasts were similar to normal (unfused) protoplast development. Most of the regenerated plants from fusion products had a thallus similar to either U. pertusa type or E. prolifera type. Although some of the plants of the former were morphologically similar to U. pertusa, but most had a higher growth rate (1.9 to 1.5 times) than U. pertusa. Furthermore the thallus of some plants had a characteristic irregular and dentate margin, which was never observed in the parental type.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00003961
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  • 89
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    Electronic Resource
    Advances and prospects in potato virology with special reference to virus resistance (1992)
    Springer
    European journal of plant pathology 98 (1992), S. 1-12 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01974466
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  • 90
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    Electronic Resource
    Organization and functional analysis of three T-DNAs from the vitopine Ti plasmid pTiS4 (1992)
    Springer
    Molecular genetics and genomics 235 (1992), S. 292-303 
    Details
    ISSN: 1617-4623
    Keywords: Agrobacterium ; Ti plasmid ; T-DNA ; Vitopine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The vitopine Ti plasmid pTiS4 of Agrobacterium vitis has an unusual T-DNA organization. The pTiS4 oncogenes, localized by screening selected pTiS4 clones for growth-inducing activity, are localized on three T-DNAs, whereas in all other characterized Ti plasmids one or two T-DNAs are found. The nucleotide sequences and predicted amino acid sequences of the pTiS4 oncogenes set them apart from the corresponding genes from other Ti or Ri plasmids. The oncogenes induce the same type of reaction on various test plants as the well-known pTiAch5 oncogenes but the pTiS4 ipt gene induces considerably more shoots than its Ach5 homologue. We have also identified the gene coding for vitopine synthase as well as a vitopine synthase pseudogene. Both sequences show homology to the octopine synthase gene. In terms of both nucleotide sequence and overall organization, the pTiS4 T-DNAs appear to be only distantly related to previously characterized T-DNAs.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00279373
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  • 91
    Electronic Resource
    Electronic Resource
    The region essential for efficient autonomous replication of pSa in Escherichia coli (1992)
    Springer
    Molecular genetics and genomics 235 (1992), S. 55-63 
    Details
    ISSN: 1617-4623
    Keywords: RepA protein ; Agrobacterium ; recombination ; Recombinase ; Interons
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00286181
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  • 92
    Electronic Resource
    Electronic Resource
    Transfer of resistance to Verticillium dahliae Kleb. from Solanum torvum S.W. into potato (Solanum tuberosum L.) by protoplast electrofusion (1992)
    Springer
    Euphytica 64 (1992), S. 39-47 
    Details
    ISSN: 1573-5060
    Keywords: Solanum tuberosum ; S. torvum ; Verticillium dahliae ; protoplasts ; electrofusion ; somatic hybrids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Interspecific somatic hybrid plants were regenerated after electrofusion of mesophyll protoplasts with the objective of transferring resistance to Verticillium dahliae from Solanum torvum into potato. Early selection of the putative hybrids was based on differences in cultural behaviour of the parental and hybrid calli (particularly the ability of the latter to regenerate early) in combination with morphological markers. Four putative hybrids were recovered from hundreds of calli, probably resulting from complementation of the two parental genomes. The regenerates were tetraploids (2n=4×=48 chromosomes) and exhibited intermediate traits including leaf form, plant morphology and the presence of anthocyanin. The hybrid nature of the four selected plants was confirmed by examining isoenzyme patterns for isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), phosphoglucoisomerase (Pgi) and 6-phosphogluconate dehydrogenase (6-Pgd). While the hybrid plants rooted readily and grew vigorously under in vitro conditions, in the greenhouse their development and growth were retarded by difficulties in rooting. When grafted on potato or S. torvum rootstocks, the hybrid plants recovered normal development and growth. Again, they exhibited intermediate morphological traits. Tests for resistance realized in vitro with medium containing 50% Verticillium wilt filtrate showed that all the somatic hybrids were resistant to the fungus filtrate.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023536
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  • 93
    Electronic Resource
    Electronic Resource
    Stabilization of steroid 11-hydroxylation activity ofCunninghamella elegans protoplasts in organic osmotic stabilizers (1992)
    Springer
    World journal of microbiology and biotechnology 8 (1992), S. 500-504 
    Details
    ISSN: 1573-0972
    Keywords: Cunninghamella elegans ; hydroxylation ; protoplasts ; steroids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Protoplasts ofCunninghamella elegans, showing 11α-, and 11β-hydroxylating ability of Substance S, preserved high transformation activity when dispersed in glucose-enriched, organic osmotic stabilizers. A joint action of polyoxins and 2-deoxy-d-glucose was necessary to prevent regeneration of the cell wall in long-lasting experiments. Stabilized and active, dispersed protoplasts may be an alternative research model for studying the function of the cell wall and intracellular metabolic pool constituents in steroid hydroxylation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01201948
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  • 94
    Electronic Resource
    Electronic Resource
    Improved culture technique for strawberry (Fragaria × ananassa Duch.) protoplasts and the determination of DNA content in protoplast derived plants (1992)
    Springer
    Plant cell, tissue and organ culture 30 (1992), S. 127-133 
    Details
    ISSN: 1573-5044
    Keywords: flow cytometry ; plant regeneration ; protoplasts ; strawberry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A reproducible and efficient protoplast to plant-system has been developed for strawberry. By using young shoots growing on medium supplemented with cytokinin we were able to exclude the detrimental enzyme Pectolyase from our enzyme solution. The more gentle isolation procedure reported in this paper, together with the agarose-embedding system had a pronounced effect on protoplast viability and growth. Plant regeneration was enhanced by using thidiazuron instead of benzyladenine. Flow cytometric measurement of the DNA content in protoplast derived plants revealed that 27 out of 51 plants contained the amount of DNA corresponding to octoploid level. Fifteen plants exhibited chromosome doubling(16×), one was diplodecaploid(12×) and eight plants mixoploid.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00034306
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  • 95
    Electronic Resource
    Electronic Resource
    Transient gene expression in electroporated protoplasts of Eucalyptus citriodora Hook (1992)
    Springer
    Plant cell, tissue and organ culture 30 (1992), S. 69-75 
    Details
    ISSN: 1573-5044
    Keywords: chloramphenicol acetyltransferase ; electroporation ; Eucalyptus citriodora ; protoplasts ; transient gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts isolated from cotyledons of Eucalyptus citriodora were electroporated using a rectangular pulse, with plasmid carrying the cat gene. The levels of transient expression and protoplast viability were influenced by the voltage and pulse duration. At a field strength of 800 V cm-1 (1000 μs), a protoplast viability of 57%, and 47% conversion of 14C-chloramphenicol to its acetylated forms, were obtained. Expression levels were improved by an increase in plasmid concentration (up to 60 μg ml-1), and also by the addition of carrier DNA. Gene expression was further enhanced by the addition of 40% (w/v) PEG, in the presence of the carrier DNA, to the protoplasts after electroporation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00040003
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  • 96
    Electronic Resource
    Electronic Resource
    An ultrastructural investigation on the polyethylene glycol-induced adhesion of tobacco nuclei and uptake of micronuclei by soybean protoplasts (1992)
    Springer
    Plant cell, tissue and organ culture 29 (1992), S. 207-214 
    Details
    ISSN: 1573-5044
    Keywords: amiprophosmethyl ; isolated nuclei ; micronuclei ; organelle uptake ; polyethylene glycol ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Nuclei isolated from tobacco protoplasts were induced to be taken up by soybean protoplasts using a protocol involving polyethylene glycol (PEG), osmotic shock and pH shift. Transmission electron microscopy revealed that PEG treatment condensed the chromatin of the isolated nuclei. Close adhesion of isolated nuclei to the plasma membrane of protoplasts following PEG treatment, was observed by both scanning and transmission electron microscopic methods. Ultrastructural observations were also made on the formation of micronuclei in tobacco cells following the treatment with amiprophosmethyl (APM). Nuclei and micronuclei isolated from APM-treated cells were induced to be taken up by soybean protoplasts. A single case of uptake of an isolated micronucleus was observed by transmission electron microscopy. The observations on the effects of PEG on the isolated nuclei, micronuclei and protoplasts are discussed in relation to the possible mechanism of uptake of nuclei by protoplasts using PEG.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00034354
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  • 97
    Electronic Resource
    Electronic Resource
    High laccase producing mutants ofPleurotus florida (1992)
    Springer
    World journal of microbiology and biotechnology 8 (1992), S. 39-41 
    Details
    ISSN: 1573-0972
    Keywords: Pleurotus florida ; laccase ; para-constitutive ; protoplasts ; mutagenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Pleurotus florida produced high amounts of laccase (4.60 U/ml) in malt extract broth after 12 days' growth under stationary conditions. The production of laccase was semi-constitutive. Hyperlaccase mutants ofP. florida were obtained through mutagenesis of mycelial protoplasts usingN-methyl-N′-nitro-N-nitrosoguanidine (50 μg/ml) for 2 min. Three hyperlaccase mutants were selected showing growth and enzyme production responses similar to the parent.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01200681
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  • 98
    Electronic Resource
    Electronic Resource
    Protoplasts from the cyanobacterium,Spirulina platensis (1992)
    Springer
    World journal of microbiology and biotechnology 8 (1992), S. 385-386 
    Details
    ISSN: 1573-0972
    Keywords: Lysozyme ; protoplasts ; regeneration ; Spirulina platensis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Protoplasts were obtained from the filamentous blue-green algaSpirulina platensis by treating the filaments with 0.05% (w/v) lysozyme in 0.03m phosphate buffer. The protoplasts regenerated cell walls and formed colonies when plated on a regeneration medium. The highest percentage of regeneration, 40% was obtained after 21 days.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01198750
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  • 99
    Electronic Resource
    Electronic Resource
    Enhanced stable expression of aVibrio luciferase under the control of the Ω-translational enhancer in transgenic plants (1992)
    Springer
    World journal of microbiology and biotechnology 8 (1992), S. 638-644 
    Details
    ISSN: 1573-0972
    Keywords: Agrobacterium ; bioluminescence ; high-copyvir vector ; high-efficiency protein production ; high-efficiency transformation of plants ; T-vectors ; Vibrio fischeri ; Vibrio harveyi
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A fusion gene usingluxA andluxB genes ofVibrio species has been designed to express light autonomously in plants.LuxA:luxB was introduced into plants by a high-efficiency transformation system consisting of a high-copy virulence helper plasmid pUCD2614 and T-vector pUCD2715 containingluxA:luxB. The expression ofluxA:luxB fusion gene was optimized by adjusting the spacing between the genes and by placing the translational efficiency of its mRNA under the control of the Ω-3 translational enhancer. The resulting transgenic plants synthesized luciferase at levels greater than 1% of the total leaf protein. These plants produced light autonomously and light intensity was enhanced by the addition of aldehyde. That theluxA:luxB fusion has been optimized enables its use as a reporter for gene activity in plants during development and under various stress-inducing conditions. These results show that a specific protein from an introduced foreign gene can be produced with high efficiency in cultivated plants and such a system is therefore amenable for production of desired proteins through conventional farming methods.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01238805
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  • 100
    Electronic Resource
    Electronic Resource
    Molecular breeding for virus resistant potato plants (1992)
    Springer
    European journal of plant pathology 98 (1992), S. 29-36 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01974469
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