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  • 2020-2022
  • 1995-1999  (63)
  • 1985-1989  (30)
  • 1955-1959  (8)
  • 1920-1924
  • 1840-1849
  • particle bombardment
  • protoplasts
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  • 1
    ISSN: 1573-9368
    Schlagwort(e): low molecular weight glutenin promoter ; particle bombardment ; transgene expression ; Triticum aestivum
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Transgenic wheat plants from specific cultivars can now be routinely engineered in many laboratories. However, our understanding of the factors controlling transgene expression and stability in wheat compared to other cereals is rather limited. Only a few promoters have been tested in transgenic wheat, and relatively little is known of their relative activities and expression parameters. In the present study, the spatial and temporal properties of one heterologous constitutive promoter and one seed‐specific wheat promoter were investigated. We generated constructs with the reporter gene gusA (β‐glucuronidase) driven by: (a) the constitutive maize ubiquitin‐1 (ubi‐1) promoter, and (b) two different‐sized fragments of the seed‐specific low molecular weight glutenin (LMWG1D1) promoter from wheat. The activities of all three promoter constructs were comparable in endosperm tissue. A detailed analysis of spatial and temporal properties of the promoters is described. Heat shock treatment of transgenic plants carrying the ubi‐1: gusA construct resulted in a significant elevation in the levels of GUS activity. The inheritance of transgene expression levels and stability was evaluated over four generations, as a function of transgene integration patterns and copy number.
    Materialart: Digitale Medien
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  • 2
    Digitale Medien
    Digitale Medien
    Springer
    Molecular breeding 5 (1999), S. 11-20 
    ISSN: 1572-9788
    Schlagwort(e): plant breeding ; protoplasts ; somatic fusion ; ribosomal RNA genes
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract Ribosomal RNA genes were exploited as markers to identify somatic hybrids between Solanum tuberosum cv. Brodick and wild diploid Solanum species, S. megistacrolobum, S. sanctae-rosae and S. sparsipilum and DNA methylation as a possible regulatory factor in gene expression was investigated. Specific restriction enzyme/probe combinations revealed useful polymorphisms in the conserved coding and variable intergenic spacer regions of the ribosomal RNA genes. Some intermediate ribosomal RNA gene profiles indicate hybridity whereas others were characteristic of S. tuberosum cv. Brodick. This evidence is suggestive of somatic exchange/re-arrangement between the NOR region of S. sanctae-rosae and S. tuberosum cv. Brodick. Ribosomal RNA gene copy number analysis of the somatic hybrids did not reveal hexaploid values suggesting that these products are not symmetric hybrids derived from the parental diploid and tetraploid plants. The results indicate site-specific methylation of ribosomal RNA gene sequences for the parental plants; while some somatic hybrids display a reduction, others show an increase. The significance of the findings for somatic cell genetics and plant breeding studies is discussed.
    Materialart: Digitale Medien
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  • 3
    ISSN: 1572-9788
    Schlagwort(e): Basmati 370 and M7 varieties ; δ-endotoxins (Cry1Ac and Cry2A) ; particle bombardment ; snowdrop lectin (GNA) ; transgenic rice
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract We cotransformed indica rice (Oryza sativa L. cvs. Basmati 370 and M7) with three plasmids, carrying a total of four genes, by particle bombardment. The Bacillus thuringiensis (Bt) δ-endotoxin genes cry1Ac and cry2A were carried on separate vectors, while the gna (snowdrop lectin) and hpt (hygromycin phosphotransferase) genes were linked on the same, cointegrate vector. We evaluated the molecular and expression profiles of 29 independently derived transgenic lines over two generations. The gna and hpt genes cointegrated with a frequency of 100% as expected. Furthermore, 60% of the transgenic plants carried all four genes. Southern blot analysis showed that transgene copy number ranged from 1 to 15. We used western blots to determine protein expression levels in R0 and R1 plants. We observed wide variation in the expression levels of the nonselected transgenes among independently-derived lines, but expression levels within lines derived from the same clone were similar. Consistent with previous reports, we observed no correlation between transgene copy number and the level or stability of protein expression. We show that the introduction of multiple agronomically valuable genes into the rice genome by cotransformation is a practical strategy for engineering elite rice varieties.
    Materialart: Digitale Medien
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  • 4
    ISSN: 1573-5060
    Schlagwort(e): rice ; Oryza sativa L. ; photoperiod sensitive genic male sterility (PGMS) ; protoplasts ; flow cytometry ; tetraploid
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract Plants were regenerated from protoplasts isolated from embryonic suspension cultures of N5047S, a photoperiod sensitive genic male sterile (PGMS) Japonica rice line. Flow cytometric analyses of nuclear DNA content identified some tetraploid regenerates whose agronomic traits could be distinguished from diploid regenerates. Pollen and female fertility of diploid protoplast-derived clones grown under different light and temperature conditions was compared. A promising PGMS protoplast clone, ZAU11S, was developed from these clones. Its male sterility was confirmed as a photoperiod × temperature interaction type.
    Materialart: Digitale Medien
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  • 5
    ISSN: 1573-5028
    Schlagwort(e): enhancer ; flavonoids ; particle bombardment ; pericarp ; promoter ; Zea mays
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The maize P-rr gene conditions floral-specific flavonoid pigmentation, especially in the kernel pericarp and cob. We analyzed the P-rr promoter by transient expression assays, in which segments of the P-rr promoter were fused to the GUS reporter gene and introduced into maize cells by particle bombardment. A basal P-rr promoter fragment (−235 to +326) gave low, but significant, levels of GUS reporter gene expression. Interestingly, two widely spaced segments containing enhancer-like activity were found. When tested individually, both the proximal (−1252 to −236) and distal (−6110 to −4842) segments boosted expression of the basal P-rr promoter::GUS construct about five-fold. A 1.6 kb segment of the P-rr promoter (−1252 to +326) containing the proximal enhancer and the 5′-untranslated leader driving the GUS reporter gene showed preferential expression in BMS and embryogenic suspension cell cultures vs. endosperm-derived suspension cell cultures. These results demonstrate the application of transient assay techniques for the identification of regulatory elements responsible for floral-specific regulation of the complex P-rr gene promoter in maize.
    Materialart: Digitale Medien
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  • 6
    ISSN: 1573-5028
    Schlagwort(e): heartwood constituent ; phytoalexin ; pinosylvin ; Pinus sylvestris ; protoplasts ; stilbene synthase ; transient expression
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Young pine seedlings respond to environmental stress by induced synthesis of pinosylvin, a stilbene phytoalexin. Heartwood of pine trees is characterized by a high content of pinosylvin. The formation of pinosylvin from cinnamoyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase is typical of the genus Pinus. Its enzyme activity not detectable in unstressed seedlings is substantially increased upon application of stimuli like UV-light or infection with the phytopathogenic fungus Botrytis cinerea. A genomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a probe. Ten clones were isolated and grouped into five subclasses according to the size of their introns. After subcloning into plasmid T7T3, four different members of the five gene subclasses were characterized by sequencing. Emphasis was put on isolating various promoters and analyzing and comparing their responsiveness. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an overall identity of more than 95%. In gene PST-5, the putative translation start site ATG was replaced by CTG. While promoter regions near the TATAA box were almost identical PST-1, PST-2 and PST-3, further upstream sequences differed substantially. Differences in promoter strength were analysed both in transgenic tobacco plants and by transient expression in tobacco protoplasts. Constructs used contained the bacterial β-glucuronidase under the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-specific expression in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinosylvin formation in the heartwood.
    Materialart: Digitale Medien
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  • 7
    ISSN: 1573-5028
    Schlagwort(e): cellulase ; heterologous expression ; Hordeum vulgare L. ; particle bombardment ; thermotolerant endo-1.4-β-glucanase ; transgenic barley
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The malting quality of two barley cultivars, Kymppi and Golden Promise, was modified to better meet the requirements of the brewing process. The egl1 gene, coding for fungal thermotolerant endo-1,4-β-glucanase (EGI, cellulase), was transferred to the cultivars using particle bombardment, and transgenic plants were regenerated on bialaphos selection. Integration of the egl1 gene was confirmed by Southern blot hybridization. The transgenic seeds were screened for the expression of the heterologous EGI. Under the high-pI α-amylase promoter, the egl1 gene was expressed during germination. The heterologous enzyme was thermotolerant at 65 °C for 2 h, thus being suitable for mashing conditions. The amount of heterologous EGI produced by the seeds (ca. 0.025% of soluble seed protein), has been shown to be sufficient to reduce wort viscosity by decreasing the soluble β-glucan content. A decrease in the soluble β-glucan content in the wort improves the filtration rate of beer.
    Materialart: Digitale Medien
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  • 8
    ISSN: 1573-5044
    Schlagwort(e): adventive somatic embryo ; cell culture ; protoplasts ; regeneration
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Adventive somatic embryos were initiated from the cut edges of juvenile leaf explants of two cultivars of sugarcane (Saccharum spp. hybrid cv. CoL-54 and cv. CP-43/33). This response was achieved using MS medium containing 9 μmol (2 mg l-1) 2,4-D and 500 mg l-1 CH under either continuous or 16-h photoperiod. Regeneration from somatic embryos was achieved under either continuous or 16-h photoperiod on MS basal medium in 5–6 weeks. Using adventive somatic embryos of 20–25 days of age as an explant source, homogeneous cell suspension cultures were initiated in both AA and MS media supplemented with 2 mg l-1 2,4-D and 500 mg l-1 CH. Protoplasts were isolated from homogeneous cell suspension cultures, an average yield being 2.5×107 ml-1 for both the cultivars. The best division efficiency (1.5 and 0.80%) and microcalluses for cv. CoL-54 and cv. CP-43/33, respectively were achieved using modified KPR medium under dark conditions in 6–8 weeks. Microcalluses were proliferated and plant regeneration was achieved from protocalluses.
    Materialart: Digitale Medien
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  • 9
    ISSN: 1573-5060
    Schlagwort(e): gus expressing cells ; histological analysis ; japonica and indica rice ; particle bombardment ; primary callus ; regeneration capacity
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract The microprojectile bombardment procedure has allowed the stable transformation of indica and japonica rice varieties, although at different frequencies of transformation depending mainly on their regeneration capacity and on the specific parameters of the transformation protocol. A study of the process of regeneration to whole plants from primary calli derived from mature indica and japonica rice seeds, via embryogenesis, has shown that somatic embryos are produced by division and differentiation of the external cell layers of callus tissues. Adjusting the bombardment conditions to optimize gene delivery to those regenerable cells, we have evaluated the influence of parameters such as the target distance, particle penetration and the effect of osmotic treatment on the regeneration capacity of bombarded cells.
    Materialart: Digitale Medien
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  • 10
    ISSN: 1573-6776
    Schlagwort(e): β-glucuronidase ; Catharanthus roseus ; genetic transformation ; green fluorescent protein ; particle bombardment
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: Abstract In vitro propagation of Catharanthus roseus was achieved using nodal explants. Bud induction was best on medium containing 1.0 mg benzyl aminopurine l−1. Hardening of rooted shoots to soil was very successful with 98% survival. Genetically transformed C. roseus plantlets were obtained after bombardment of nodal explants, which were then micropropagated, with DNA coated particles with green fluorescent protein (GFP) or β-glucuronidase (GUS) reporter genes. Histological studies showed that the gene insertion method proved effective with many cells and different tissues displaying the reporter gene signals, showing that gene expressions were rather stable.
    Materialart: Digitale Medien
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  • 11
    ISSN: 1573-9368
    Schlagwort(e): Antirrhinum ; Arabidopsis ; green fluorescent protein (GFP) ; in vitro ; microspores ; particle bombardment ; tobacco
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The transient expression of three mutant forms of green fluorescent protein (GFP) genes, GFP4, GFP5ER, and GFP4S65C, under several constitutive and pollen‐specific promoters throughout pollen development in Nicotianatabacum, thaliana and Antirrhinummajus is described. Immature pollen of tobacco, Arabidopsis and snapdragon, isolated at different developmental stages, were bombarded with plasmids containing the GFP and cultured in vitro for several days until maturity. The expression of GFP was monitored every day during in vitro maturation, germination and pollination, as well as after in situ pollination. The expression pattern of each construct was compared in parallel experiments to that of ß‐glucuronidase (GUS) constructs expressed by the same promoters. The results show that the expression level of all three GFP mutant forms was dependent on the strength of the promoter used. The strongest promoter was the DC3 promoter, and no notable differences in the intensity and brightness of all three versions of GFP were observed. GFP‐expressing pollen from tobacco and snapdragon developed in vitro for several days until maturity and germinated in vitro as well as on the surface of stigmata, strongly suggesting that all three GFPs are not toxic for the development of functional pollen. Furthermore, stably transformed tobacco plants expressing GFP under the control of the strong pollen‐expressed DC3 and LAT52 promoters were not impaired in reproductive function, confirming that GFP can be used as a non‐destructive marker for plant reproductive biology and development.
    Materialart: Digitale Medien
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  • 12
    ISSN: 1573-9368
    Schlagwort(e): Oryza sativa ; particle bombardment ; transformation ; transgenic rice
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract We developed a practical and efficient gene transfer system for indica rice utilizing mature-seed derived explants and a simple bombardment device which uses compressed helium for accelerating DNA-coated metal particles. Unlike instruments which have been described in the literature previously, this new bombardment device, which is an improvement of the particle inflow concept, does not require vacuum. This attribute simplifies the transformation procedure significantly and it makes rice transformation technology accessible to laboratories which may not have the resources to invest in more expensive particle bombardment instruments. We determined experimentally that we could recover transgenic rice plants utilizing three different particle bombardment instruments at comparable frequencies.
    Materialart: Digitale Medien
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  • 13
    Digitale Medien
    Digitale Medien
    Springer
    Transgenic research 7 (1998), S. 463-471 
    ISSN: 1573-9368
    Schlagwort(e): particle bombardment ; transgene expression ; Triticum aestivum
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract A population of R0 transgenic wheat plants, generated by particle bombardment, was analyzed to define molecular, genetic and phenotypic properties resulting from transformation with a cointegrate vector, or cotransformation with two separate plasmids. By evaluating the progeny of 70 independently-derived transgenic plants, we also identified rare events such as chimerism and transgene elimination, which provide valuable information concerning the development of transgenic cereal plants following bombardment experiments. The frequency of chimerism in our transgenic wheat plants was very low. Furthermore, while transgene elimination did occur, this was also a very rare event. We determined the copy numbers of integrated transgenes and the levels of transgene expression. Comparisons to transgenic rice plants generated in the same manner demonstrated some similarities, but also important differences in transgene behavior. Whereas in rice there is no evidence for any direct relationship between transgene copy number and transgene expression or stability, multicopy populations in wheat demonstrated a bias towards higher levels of expression for the two genes and the maize ubiquitin promoter evaluated in the present study.
    Materialart: Digitale Medien
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  • 14
    ISSN: 1572-9788
    Schlagwort(e): transgenic rice ; particle bombardment ; cell electroporation ; RAPD ; AFLP ; AFRP ; RAMP
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract In the present work we utilised some of the most discriminative molecular tools, such as RAPD, AFLP, AFRP and RAMP, to analyse the genome of independently derived transgenic plants from three elite Italian cultivars (cv. Lido, Carnaroli and Thaibonnet) and found that two methods for direct gene transfer, namely particle bombardment and intact cell electroporation (the latter being a procedure set up in this work), result in transgenic rice (Oryza sativa L.) plants that exhibit negligible genomic changes. This is in contrast with recently published results showing relevant changes in the DNA of transgenic rice plants generated through protoplasts electroporation and of transgenic poplar plants engineered through Agrobacterium tumefaciens infection. Implications of these findings are discussed in the context of selecting appropriate gene transfer methodologies to produce transgenic plants expressing genes of interest while retaining their genomic integrity and, thus, their superior agronomic and/or industrial traits.
    Materialart: Digitale Medien
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  • 15
    ISSN: 1572-9788
    Schlagwort(e): Bacillus thuringiensis (Bt) ; cry2A ; particle bombardment ; rice leaf folder ; transgenic rice ; yellow stem borer
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract Transgenic rice indica varieties Basmati 370 and M 7 expressing the novel cry2A (Bt) insecticidal gene were generated by particle bombardment. Molecular and biochemical analyses in R0 and R1 populations confirmed stable integration and expression of this novel Bt transgene. We estimated that the gene product was expressed up to 5% of total leaf protein. Insect feeding bioassays demonstrated that the Cry2A protein was effective against the yellow stem borer and the rice leaf folder, two major rice pests in the Indian Subcontinent. This is the first report of the control of major rice pests using this specific Bt gene. The cry2A gene can now be used in combination with other insecticidal genes for pyramiding resistance against insect pests. This will delay, or perhaps in combination with integrated pest management practices, prevent evolution of insect populations resistant to single insecticidal genes.
    Materialart: Digitale Medien
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  • 16
    ISSN: 1572-9788
    Schlagwort(e): BGMV ; common bean ; particle bombardment ; plant transformation ; virus resistance
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract The genes Rep-TrAP-REn and BC1 from the Brazilian isolate bean golden mosaic geminivirus (BGMV-BR) were cloned in antisense orientation under the transcriptional control of the CaMV 35S promoter. This construct was used to transform common bean (Phaseolus vulgaris L.) using the biolistic method. Transgenic plants from the R3 and R4 generations were challenged by inoculation with a BGMV-BR viruliferous whitefly population. Of the four transgenic lines tested, two had both delayed and attenuated viral symptoms. Un-transformed plants or plants transformed with a construct containing only the gus-neo gene developed typical BGMV-BR symptoms 10–15 days after inoculation.
    Materialart: Digitale Medien
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  • 17
    ISSN: 1573-5079
    Schlagwort(e): C4 photosynthesis ; enzyme kinetics ; protein phosphorylation ; protoplasts
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Kinetic analyses were performed on the nonphosphorylated and in vitro phosphorylated forms of recombinant Sorghum C4 phospho enolpyruvate carboxylase (C4 PEPC). The native enzyme was purified by immunoaffinity chromatography and its integrity demonstrated by Western blot analyses using anti N- and C-terminus antibodies. At suboptimal pH (7.1 to 7.3) and PEP concentration (2.5 mM), phosphorylation, positive metabolite effectors e.g., glucose-6-phosphate, glycine and dihydroxyacetone-phosphate, or an increase in pH strongly activated the enzyme and lowered the inhibitory effect of L-malate. C4 PEPC phosphorylation strengthened the effect of the positive effectors thereby decreasing further the enzyme's sensitivity to this inhibitor. L-malate also decreased the phosphorylation rate of C4 PEPC, a process antagonized by positive metabolite effectors. This was shown both in vitro, in a reconstituted phosphorylation assay containing the catalytic subunit of a cAMP-dependent protein kinase or the Sorghum leaf PEPC-PK and in situ, during induction of C4 PEPC phosphorylation in mesophyll cell protoplasts.
    Materialart: Digitale Medien
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  • 18
    ISSN: 1573-5060
    Schlagwort(e): particle bombardment ; promoter ; tissue-specificity sucrose synthase ; transient expression ; Zea mays
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract The reporter gene encoding β-glucuronidase (GUS) driven by either of the two maize sucrose synthase gene (Sh1 and Sus 1) promoters was introduced and expressed in various maize tissues via particle bombardment. Transient gene expression was examined by histochemical assays. It was found that the two SS promoters directed differential GUS expression. In the developing kernel, the Sh1 promoter was active only in the upper and central parts of the endosperm. In contrast, strong GUS activity controlled by the Sus1 promoter was detected in various types of cells, including the aleurone cells, the subaleurone endosperm cells, the scutellar cells of the embryo and the pericarp cells. Both promoters showed similar expression patterns in vegetative tissues.
    Materialart: Digitale Medien
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  • 19
    Digitale Medien
    Digitale Medien
    Springer
    Plant growth regulation 25 (1998), S. 135-141 
    ISSN: 1573-5087
    Schlagwort(e): ABA synthesis ; Amaranthus ; fluridone ; osmotic stress ; protoplasts ; rose petal
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract All the protoplasts analysed in this study whatever the original plant material were able to accumulate ABA under osmotic stress. The time course of ABA accumulation strongly differed according to the plant material. In both rose petal or Amaranthus leaf protoplasts, the increase in ABA level was significant but transient. Protoplasts prepared from Amaranthus cell suspensions behaved differently, showing a late and durable accumulation of ABA. Similar patterns of changes in ABA accumulation were observed in the original plant material under osmotic stress. A pretreatment of plant material by fluridone induced a strong inhibition of ABA accumulation whatever the origin of protoplasts was. This result suggests that ABA could be synthesised via the carotenoid pathway in the absence of the cell wall.
    Materialart: Digitale Medien
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  • 20
    ISSN: 1573-9368
    Schlagwort(e): rice transformation ; GNA ; particle bombardment ; immunolocalisation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Transgenic rice (Oryza sativa L.) plants generated through particle bombardment expressed high levels of an insecticidal protein (the snowdrop lectin, GNA) directed against sap-sucking insects. Engineered plants expressed GNA either constitutively or in a tissue specific manner, depending on the nature of the promoter used to drive expression of the gene. We used specific antibodies raised against GNA to localize its expression in phloem tissue in plants engineered with the rice sucrose synthase promoter driving GNA expression. We report here molecular, biochemical and immunological analyses for fifteen independently-derived transformants out of more than 200 plants we generated.
    Materialart: Digitale Medien
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  • 21
    ISSN: 1573-5028
    Schlagwort(e): CaMV 35S promoter ; Catharanthus roseus ; FMV 34S promoter ; particle bombardment ; transformation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Activities of several CaMV 35S and FMV 34S promoter derivatives fused to the gusA reporter gene were compared in suspension-cultured Catharanthus roseus cells that were transiently and stably transformed using particle bombardment. Our data demonstrate that the 35S and a deletion derivative of the 34S promoter combined with particle bombardment form useful tools for genetic engineering of C. roseus cells. Our results disagree on several points with activities of 35S and 34S promoter derivatives reported for tobacco, indicating that absolute and relative promoter activities can differ between plant species.
    Materialart: Digitale Medien
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  • 22
    ISSN: 1573-5028
    Schlagwort(e): movement protein ; protoplasts ; resistance ; tomato spotted wilt virus (TSWV) ; tospovirus ; transgenic plants
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Transgenic Nicotiana tabacum plants expressing RNA sequences of the tomato spotted wilt virus NSM gene, which encodes the putative viral movement protein, were found to be highly resistant to infection with the virus. Expression of untranslatable as well as anti-sense RNA of the NSM gene resulted in resistance levels as high as those in plants expressing translatable RNA sequences. For all three types of transgenic plants resistance levels of up to 100% were reached in the S2 progeny. These results indicate that the resistance mediated by the NSM gene is accomplished by expression of transcripts rather than protein in transgenic plants, similar to previously observed N gene-mediated resistance. Protoplast inoculations revealed that resistant plants expressing NSM are, in contrast to N transgenic resistant plants, not resistant at the cellular level. This suggests the RNA-mediated resistance mechanism against TSWV targets viral mRNAs rather than the viral genome.
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  • 23
    Digitale Medien
    Digitale Medien
    Springer
    Transgenic research 6 (1997), S. 385-392 
    ISSN: 1573-9368
    Schlagwort(e): transgenic cotton ; bialaphos ; particle bombardment ; transformation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Resistance to bialaphos, a non-selective herbicide, was intro duced into cotton through genetic engineering. A gene encoding phosphinothric in acetyltransferase (bar) from Streptomyces hygroscopicus was inserted into elite varieties of cotton through particle bombardment. Based on the marker gene, β-glucuronidase (gus) expression, a total of 18 Pima (Gossypium barbadense), 45 DP50 (G. hirsutum L.), 20 Coker 312 (G. hirsutum) and 2 El Dorado (G. hirsutum) transgenic plants were recovered. Integration of the bar gene into cotton genomic DNA was confirmed by Southern blot analysis and gene expression was confirmed by northern blot and enzyme assays. Herbicide (Basta®) tolerance up to 15 000 ppm was demonstrated in greenhouse trials. The newly introduced herbicide tolerance trait is inherited in a Mendelian fashion in the progenies of germline transformants. This study demonstrates the potential for particle bombardment to introduce commerically important genes directly into elite varieties of cotton. This mode of gene transfer can expedite the introduction of transgenic cotton products into world markets
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  • 24
    ISSN: 1573-5117
    Schlagwort(e): transgenic fish ; gene transfer ; growth enhancement ; lopifection ; particle bombardment ; electroporation ; fish sperm ; fish embryos
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Recent advantages in transgenic fish research are reviewed, with special reference to the methods for gene transfer. These include microinjection, electroporation, particle bombardment, and lipofection. The success and problems associated with each of these methods, and the possible applications of transgenic fish research to aquaculture are discussed.
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  • 25
    Digitale Medien
    Digitale Medien
    Springer
    Plant and soil 192 (1997), S. 23-30 
    ISSN: 1573-5036
    Schlagwort(e): aluminium ; callose ; protoplasts ; resistance ; toxicity ; Zea mays
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract The screening of 37 Zea mays L. cultivars in nutrient solution using root elongation (24 h) as a parameter showed large genotypic differences in Al resistance among the genetic material evaluated. Callose concentrations in root tips were closely and positively related to Al-induced inhibition of root elongation. Therefore, Al-induced callose formation in root tips appears to be an excellent indicator of Al injury and can be used as a selection criteria for Al sensitivity. In contrast, aluminium concentrations in root tips were not related to Al-induced inhibition of root elongation, nor to Al-induced callose formation. Callose formation was also induced by short-term A1 treatment in root tip protoplasts, and the response of protoplasts clearly reflected the cultivar-specific response to Al of intact roots. This indicates that in maize, Al sensitivity is expressed on the protoplast level.
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  • 26
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 51 (1997), S. 43-47 
    ISSN: 1573-5044
    Schlagwort(e): Allium cepa ; onion ; organogenesis ; protoplasts ; suspension culture
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Tissue culture techniques involving cell suspension, protoplast fusion and culture in the genus Allium are different as Allium is recalcitrant due to the biological peculiarity of the genus. A procedure is described for the establishment of a regenerable suspension culture and for the isolation and culture of protoplasts of Allium cepa.
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  • 27
    ISSN: 1573-5060
    Schlagwort(e): Oryza sativa ; japonica rice cultivar ; mature embryo ; primary calli ; protoplasts ; green plant regeneration
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract A plant regeneration system from rice protoplasts using calli derived from mature embryos was established for the two Brazilian modern rice cultivars IAC-201 and IAC-165. After 30 to 40 days of in vitro culture it was possible to obtain on average 6 million protoplasts per gram of callus. Microscopic selection of embryogenic calli was a key step for protoplast isolation. The production of embryogenic calli increased when L-proline and casein hydrolysate were used in the callus induction medium. The Oc or IR52 nurse cell lines were essential for protoplast division. Different regeneration media were studied and 139 plants were regenerated which set seed. Some of the regenerated plants showed morphological variation such as the presence of awns in spite of the short time of the in vitro culture.
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  • 28
    Digitale Medien
    Digitale Medien
    Springer
    Plant growth regulation 21 (1997), S. 145-152 
    ISSN: 1573-5087
    Schlagwort(e): chloroplasts ; membrane dynamics ; Pisum ; photosymthesis ; protoplasts ; triacontanol
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract The effects of a long chain aliphatic alcohol 1-triacontanol (TRIA) on the photosynthesis and membrane properties of mesophyll protoplasts and chloroplasts isolated from pea leaves were studied. In vitro treatments of isolated protoplasts caused a large enhancement (166% ) of the CO2-fixation rate after 60 min of TRIA (10-6 M) application as compared to the control. An enhanced photosynthetic response was observed in in vitro treated leaf pieces. Application of octacosanol (OCTA) under the same experimental conditions did not result in any stimulating effects. In vivo treatments of pea seedlings also resulted in a significant increase of the net CO2 uptake to 109% and 119% in 10-8 M and 10-6 M TRIA-treated plants respectively. It was demonstrated that the incubation of both protoplasts and chloroplasts with TRIA resulted in a rise of the excimer/monomer (IE/IM ) ratio of pyrene (Py) fluorescence, thus indicating remarkable fluidization and/or disordering of the lipid matrix of their membranes. This effect depended on the incubation time and became evident at very low concentrations of TRIA (0.3 μM). The increase of membrane fluidity was accompanied by TRIA-induced alterations in the dielectric environment in the membrane regions where Py molecules are situated. The results are discussed in terms of specific concentration dependent TRIA-induced alterations of the dynamic properties of protoplast and chloroplast membranes and their possible involvement in the initiation of the integral physiological response to exogenous application of TRIA.
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  • 29
    Digitale Medien
    Digitale Medien
    Springer
    Plant molecular biology 35 (1997), S. 197-203 
    ISSN: 1573-5028
    Schlagwort(e): genetic engineering ; particle bombardment ; plant biotechnology ; transgenic rice ; Oryza sativa
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Bombardment-based methodology is responsible for the effective genetic manipulation of major cereals including rice. Many groups reported significant advances on various aspects of rice molecular biology and genetic engineering using procedures based on bombardment technology. Molecular and genetic characterization of large numbers of these plants (more than 500 independent transgenic plants) provided information on structure, expression and stability of integrated DNA through multiple generations. Such evaluations were carried out in the greenhouse and in the field. Stability of expression was found to be dependent on the nature of the promoter and the transgene, and in specific cases on gene copy number. Direct DNA transfer utilizing particle bombardment for the delivery of foreign DNA into rice tissue results in the recovery of large numbers of independently derived transgenic plants in a variety-independent fashion. Gene copy number, level and stability of expression of transgenes can be compared to other DNA delivery methods, direct or indirect, including Agrobacterium-mediated gene transfer. In this paper, the technology is summarized and discussed in terms of present and future applications, including field trials and potential commercialization of transgenic rice expressing a number of genes of agronomic interest such as pest and herbicide resistance.
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  • 30
    ISSN: 1573-5044
    Schlagwort(e): PEG-mediated transformation ; protoplasts ; transgene inheritance
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract A protocol for PEG-mediated transformation of protoplasts is described forA. thaliana ecotype Landsbergerecta and two marker lines derived from it, M4 and M10. The optimal transformation conditions were: 14 µg plasmid DNA and 28 µg carrier DNA per 6 x 105 protoplasts in 15% (w/v) PEG solution. Based on the hygromycin resistance conferred by the transgene, relative transformation frequencies of 2.5–3.2% and absolute transformation frequencies of 1–2 x 10−4, were obtained. Shoot regeneration frequencies of 40–60% were achieved, and fertile transgenic plants of the three tested lines were obtained. Southern blot hybridizations demonstrated multi-copy integration patterns in most cases. Hygromycin resistance segregation patterns of 3:1 and 15:1 were found, as well as unexpected segregation patterns, suggesting that modifications in gene expression took place and that these can progressively occur over successive generations.
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  • 31
    ISSN: 1573-5060
    Schlagwort(e): Sweet potato ; Ipomoea batatas ; plant regeneration ; somatic embryogenesis ; protoplasts ; genetic variation ; somaclonal variation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract The application of new techniques for improvement of sweet potato crops, particularly including the exploitation of somaclonal variation, gene transfer by genetic transformation and somatic hybridization, requires the control of plant regeneration from tissue cultures. Shoots can easily be regenerated from explants of stems, petioles, leaves and roots, while callus cultures do not produce any shoots. The potential of somatic embryogenesis and plant regeneration via embryogenesis was evaluated for 10 cultivars of sweet potato. Protocols for plant regeneration from cultured protoplasts have also been developed. Since mesophyll was resistant to enzyme digestion, fragments of stems and petioles, callus and cell suspensions were used as source of protoplasts of sweet potato. Series of transfers of protoplast-derived calluses, particularly those which had been obtained from in vitro plants, to media containing a high level of zeatin resulted in successful formation of shoots in only two sweet potato cultivars. In addition, the embryogenic potential was irreversibly lost through protoplast culture, since protoplasts isolated from embryogenic cell suspensions developed into non-embryogenic callus. Consequently, an alternative protocol is being successfully developed to improve plant regeneration from cultured protoplasts of sweet potato, involving first root formation from which shoots can then be regenerated. Preliminary evaluation in field conditions in Gabon revealed that plants regenerated from cultured protoplasts exhibited a great genetic variability in their growth and tuber formation in particular.
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  • 32
    Digitale Medien
    Digitale Medien
    Springer
    World journal of microbiology and biotechnology 13 (1997), S. 469-473 
    ISSN: 1573-0972
    Schlagwort(e): 2-Deoxy-d-glucose ; hydroxylation ; immobilization ; polyoxin ; protoplasts ; steroids
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Materialart: Digitale Medien
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  • 33
    Digitale Medien
    Digitale Medien
    Springer
    Biologia plantarum 39 (1997), S. 561-567 
    ISSN: 1573-8264
    Schlagwort(e): Beta vulgaris ; plating efficiency ; protoplasts ; putrescine ; spermidine ; spermine
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The influence of the exogenous polyamines: putrescine, spermidine and spermine, on the frequency of protoplast divisions for 2 genotypes of sugar beet (Beta vulgaris L.) was analyzed. Protoplasts were cultured by the agarose disk method on Saunders and Doley medium supplemented with either hormones or polyamines, or hormones combined with polyamines. The latter supplement led to a statistically significant increase in plating efficiency. The improvement in division index was caused mainly by spermine.
    Materialart: Digitale Medien
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  • 34
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell reports 15 (1996), S. 949-953 
    ISSN: 1432-203X
    Schlagwort(e): Ty1-copia group retrotransposons ; potato ; protoplasts ; expression
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The potato (Solanum tuberosum) genome contains a highly heterogeneous population of Ty1-copia group retrotransposons. Here we identify the first such transposable element known to be transcribed in this species. The elements are transcriptionally activated during protoplast isolation. The majority of the activated Ty1-copia sequences are similar to elements which are transcriptionally induced under the same conditions in tobacco (Nicotiana tabacum). We also show that a previously identified potato element M166, which has no known equivalent in tobacco is also transcribed under these conditions. It appears that the control of transcription of this particular Ty1-copia group retrotransposons has been broadly conserved between these two species.
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  • 35
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell reports 15 (1996), S. 737-741 
    ISSN: 1432-203X
    Schlagwort(e): Zoysia japonica ; embryogenic callus ; protoplasts ; plant regeneration
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Embryogenic callus of Japanese lawngrass (Zoysia japonica Steud.) was induced from sterile mature seeds on LS medium with 5 mg / l of 2,4-D. Embryogenic callus selected visually under microscope was proliferated in liquid N6 medium with amino acids (N6-AA medium). Protoplasts were isolated from suspension cells by the treatment of enzyme mixture containing pectolyase Y-23 and cultured in K8p medium with 2 mg / l of 2,4-D at the density of 106 / ml. Plants were regenerated by transferring the protoplasts derived callus to MS medium and incubating at 28 °C under light for two months. Plantlets were successfully transplanted in the soil.
    Materialart: Digitale Medien
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  • 36
    ISSN: 1432-203X
    Schlagwort(e): Transformation ; particle bombardment ; Agrobacterium ; Allium cepa.
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Particle bombardment and Agrobacterium-mediated DNA delivery into immature embryos and microbulbs were used to investigate the expression of the uidA gene in in vitro onion cultures. Both methods were successful in delivering DNA and subsequent uidA expression was observed. Optimal transient β-glucuronidase activity was observed in immature embryos that had been pre-cultured for three days and bombarded at a distance of 3 cm from the stopping plate, under 25 in Hg vacuum, using 900–1300 psi rupture discs. The CaMV35S-uidA gene construct gave five fold higher transient β-glucuronidase activity than the uidA gene construct regulated by any of four other promoters initially chosen for high experession in monocotyledonous tissues.
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  • 37
    Digitale Medien
    Digitale Medien
    Springer
    Plant molecular biology 30 (1996), S. 297-306 
    ISSN: 1573-5028
    Schlagwort(e): cotton fiber ; transgenics ; particle bombardment ; fiber properties ; cDNA and genes ; nucleotide sequence
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Two genes, each corresponding to fiber mRNA E6, were isolated from cotton cultivars Coker 312 (Gossypium hirsutum L.) and Sea Island (G. barbadense L.). E6 is one of the predominant fiber-specific mRNAs present during early fiber development. The distinguishing feature of the nucleotide-derived E6 protein is the presence of a motif where a dimer, Ser-Gly, is repeated several times. Two of the Sea Island genes contained a pentameric motif, Ser-Gly, while one of the Coker genes had one and the other had four motifs each. cDNA clones containing one or five Ser-Gly motifs were also identified. Thus, it appears that the strict conservation of this motif may not be critical to E6 protein function. Sequence characterizations of the genes and cDNAs showed that multiple members of the E6 family are transcribed in fiber and may result in proteins 238 to 246 amino acids long. The 3′ ends of the genes and cDNAs showed considerable heterology among them. Transgenic plants containing antisense genes were generated to decipher E6 function. Transgenic cotton with reduced E6 protein levels in the range of 60 to 98% were identified. However, no discernible phenotypic changes in fiber development or properties were apparent. This result leads to the conclusion that E6 is not critical to the normal development or structural integrity of cotton fibers.
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  • 38
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell reports 16 (1996), S. 88-91 
    ISSN: 1432-203X
    Schlagwort(e): Gentiana crassicaulis Duthie ex Burk ; protoplasts ; callus ; plant regeneration
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1–2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4–5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA3, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.
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  • 39
    ISSN: 1573-9368
    Schlagwort(e): cultured liverwort cells ; hygromycin phosphotransferase (HPT) ; Marchantia polymorpha ; particle bombardment ; stable transformation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×106 cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.
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  • 40
    ISSN: 1572-9788
    Schlagwort(e): bar gene ; glufosinate ; herbicide tolerance ; particle bombardment ; Oryza sativa L.
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract The commerical cultivars ‘Gulfmont’, ‘IR72’ and ‘Koshihikari’ were genetically engineered using electric discharge particle bombardment to express the bar gene which confers resistance to the broad-range herbicide glufosinate. Southern and northern blot analyses of transgenics material revealed stable integration and expression of introduced transgenes in the lines evaluated. In a few plants, silencing of the uidA marker gene was detected at the transcriptional level. Field studies were conducted in 1993 and 1994 at the Rice Research Station near Crowley, LA. This report summarizes results from the first two years of field trial for transgenic Gulfmont and Koshihikari. Transgenic cultivar IR72 was tested in 1995 and preliminary results are similar to those reported for transgenic Gulfmont. All 11 independently derived transgenic lines produced fertile, normal looking seed at maturity. Significant differences were observed in the absence of the herbicide between parental cultivars and transgenic Gulfmont-and Koshihikari-derived lines for days to 50% heading (20% of transgenic lines), plant height (13%), and grain yield (7%). Foliar application of glufosinate had little or no effect on agronomic performance of all transgenic Gulfmont and IR72 lines, while herbicide applications affected grain, yield and plant height of some transgenic Koshhikari. Non-transgenic plants of all three cultivars at the 4-leaf stage were killed within 7 days after 1.12 or 2.24 kg/ha glufosinate applications. Significant differences among certain transgenic lines were observed for agronomic traits after herbicide applications. These results demonstrate that the bar gene was effective in conferring field-level resistance to glufosinate in rice. Variation among transgenic lines required traditional breeding selection procedures to identify superior agronomic types with high levels of herbicide resistance and showed the necessity to generate several independent transgenic lines of each cultivar.
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  • 41
    Digitale Medien
    Digitale Medien
    Springer
    Plant growth regulation 18 (1996), S. 149-154 
    ISSN: 1573-5087
    Schlagwort(e): cell culture ; auxins ; cytokinins ; confocal laser scanning microscope ; protoplasts
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract The model system presented here is based on immobilised single cells, derived directly from tobacco mesophyll protoplasts. It allows the adequate steering of cell populations towards expansion, cell cycling or cell resting. Using this approach cells always have the same predictable response to auxins and cytokinins whatever their actual physiological status. This model system opens new ways to study cellular parameters governing these hormone responses, some of which have been explored so far; a) the cytokinin response can equally well be induced by endogenous as by exogenous cytokinins; b) at least two intracellular components, microtubuli and the ER, adapt their architecture to the hormone-induced status of the cell; c) addition of NAA to the cells does not induce a change in the cytoplasmic pH.
    Materialart: Digitale Medien
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  • 42
    Digitale Medien
    Digitale Medien
    Springer
    World journal of microbiology and biotechnology 12 (1996), S. 625-628 
    ISSN: 1573-0972
    Schlagwort(e): Mycorrhizae ; protoplasts ; Suillus
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: Abstract Experiments were performed with the mycorrhizal fungus Suillus granulatus to define the parameters for production and regeneration of protoplasts. Protoplasts were released at frequencies between 1 and 3×107/ml from mycelium 3 to 7 days old. The best osmotic stabilizer for protoplast release was MgSO4 (0.7 m). To optimize protoplast release and regeneration an enzyme (Novozym 234) concentration 1.7 mg/ml was chosen, with a digestion time of 1 to 2 h. Regenerated colonies formed mycorrhizae within 60 days after inoculation in Pinus caribaea var. hondurensis seedlings.
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  • 43
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 46 (1996), S. 35-41 
    ISSN: 1573-5044
    Schlagwort(e): Elaeis guineensis ; oil palm ; protoplasts
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Procedures are deseribed for the efficient isolation of protoplasts from a variety of oil palm (Elaeis guineensis Jacq.) tissues. Various factors including donor source, composition of enzyme mixture and culture medium affected the yield and viability of the protoplasts Polyembryogenic cultures of oil palm were the most suitable starting material in terms of yield, viability and metabolic competence. Pectolyase Y-23 in association with cellulase and hemicellulase was required for the efficient release of protoplasts from the oil palm tissues. Limited cell division to form microcallus was observed at very low frequency (〈0.01%) when glutathione and catalase were incorporated in the culture medium.
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  • 44
    ISSN: 1573-5060
    Schlagwort(e): protoplasts ; potato ; somatic hybrids ; sexual hybrids ; late blight ; Phytophthora infestans
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract Intraspecific tetraploid somatic and sexual hybrid plants have been resynthesised following protoplast fusion and by sexual crosses between two dihaploid potato (Solanum tuberosum) lines each possessing complementary agronomic traits. The dihaploid PDH 40 possesses good tuber shape and yield but has foliage susceptibility to late blight (Phytophthora infestans). On the other hand, the dihaploid PDH 727 possesses resistance to blight in the foliage but has a low yield of small and irregular shaped tubers. Since it was only possible to use a partial selection strategy based on culture media to facilitate recovery of somatic hybrid plants-further morphological and esterase isozyme based characterisations were performed to identify somatic hybrid plants from amongst the non-hybrid plant material. When the blight resistance of both the intraspecific somatic and sexual hybrid plants was assessed there was no significant difference in the mean resistance value and it was intermediate between those of their parents. However, the range of resistance was much wider among the sexual hybrids than among the plants derived from somatic fusion. An assessment of tuber yield between tetraploid sexual and somatic hybrids showed no significant difference and it was higher than that of either parent value. The implication of these results in the context of potato genetics and breeding is discussed.
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  • 45
    ISSN: 1573-5087
    Schlagwort(e): auxin receptor ; ion channels ; auxin-binding protein ; protoplasts ; electrophysiology
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract Studies of membrane electrical responses of isolated protoplasts to auxin have demonstrated the existence of elementary response chains to auxin at the plasma membrane, presently defined only by their uttermost ends. At one side, as demonstrated by several lines of evidence, the auxin perception unit involves proteins homologous to ZmER-abp1 (abp1), the most abundant auxin-binding protein from maize coleoptiles. At the other side, multiple ion transport proteins appear as targets of the auxin signal; the proton pump ATPase, an anion channel and potassium channels. We investigated early electrical responses to auxin at the plasma membrane of tobacco protoplasts. The work presented here will initially focus on abp1 and its functional role at the membrane. The C-terminus abp1 peptide (Pz151–163) was recently reported to modulate K+ currents at the plasma membrane of intact guard cells from broad bean [23] and induce plasma membrane hyperpolarisation of tobacco mesophyll protoplasts. These results further demonstrate that proteins involved in plasma membrane responses to auxin are related to maize abp1, and provide clues as to the region of the protein possibly involved in the interaction of abp1 with the plasma membrane. Secondly, this report concentrates on one of the targets of auxin, a voltage-dependent and ATP-regulated anion channel that we characterised on protoplasts from tobacco cell suspensions. This anion channel was specifically modulated by auxin, as already observed for the anion channel of guard cells [14]. Further work will be needed to assess if this auxin modulation involves a direct interaction between the hormone and the anion channel protein(s), or follows from the activation of a perception chain including abp1 homologues.
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  • 46
    ISSN: 1572-9788
    Schlagwort(e): cassava ; genetic modification ; luciferase ; particle bombardment
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract Cassava embroids derived from friable embryogenic callus of the genotype TMS60444 were bombarded with DNA of the constructs pJIT100 or pJIT64. Both constructs contain the non-invasive reporter gene luciferase from firefly driven by the CaMV 35S promoter. The influence of several particle gun machine parameters and pretreatment of plant material on transient luciferase activity were studied to determine the most essential conditions for stable transformation. Two weeks after bombardment pieces of friable calli with luciferase activity were selected. In total, 67 independent selected calli with luciferase activity (spots), derived from five different experiments, were further cultured either in liquid or on solid medium. Per plate or flask one spot was cultured. In subsequent selection rounds all spots of one individual plate or flask were cultured as one individual group. In this way different transformation events were separated and multiplied. Eight weeks after bombardment 34 cultures still contained luciferase activity. The mean number of luciferase spots per culture had increased from 1 to 4.6 spots in liquid and to 2.5 spots on solid medium. After two more months of subsequent culture and luciferase selection presence of the construct in these cultures was confirmed at the molecular level using the polymerase chain reaction assay and Southern analysis. Friable embryos derived from four transformation events were cultured for maturation. Between 3% and 21% of the mature embryos of the different transformation events were luciferase-positive. After multiplication of the luciferase-positive mature embryos by secondary somatic embryogenesis they were germinated. The plantlets analysed contained one to several copies of the inserted DNA. The method presented enables the transformation of this particular cassava genotype, thus allowing the genetic improvement of this important tropical crop by transgenesis.
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  • 47
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell reports 14 (1995), S. 738-742 
    ISSN: 1432-203X
    Schlagwort(e): Hyoscyamus muticus ; plant regeneration ; protoplasts ; transformed roots
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Summary Successful plant regeneration was achieved for the first time from hairy root-derived protoplasts of Hyoscyamus muticus. High yields (7 × 106 / g fresh weight) of protoplasts were isolated directly from the transformed roots of Hyoscyamus muticus using an enzyme mixture comprising 1 % macerozyme and 2 % cellulase in an osmoticum consisting of 0.2 M CaCl2 and 0.6 M mannitol. Protoplasts were first cultured in liquid NT/PRO I medium and further on semi-solid NT/PRO II agar medium. The procedure permits highly efficient formation of colonies. The plating efficiency varied from 1–9 %. The small individual colonies regenerated easily into shoots and roots at frequencies of 18 % and 70 %, respectively. The time required for the development of small plantlets from protoplasts was 8–11 weeks. The regenerated plants contained rolB from Ri-T-DNA and exhibited an altered phenotype compared to the control plants.
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  • 48
    ISSN: 1432-203X
    Schlagwort(e): Barley ; Hordeum vulgare ; immature embryo ; particle bombardment ; particle gun ; transformation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Fertile transgenic barley (Hordeum vulgare L.) plants were obtained by high velocity particle bombardment. The plasmid pBCl was used to deliver the selectable hph gene and reporter Gus gene into immature embryo. After the selection culture 18 hygromycin resistant plants were obtained. Samples for Southern hybridization and enzymatic Gus assay were obtained from 11 plants. Southern hybridization confirmed the presence of the hph gene in the 11 hygromycin resistant plants(T0). Enzymatic assay indicated that all the t0 plants that showed hph positive in Southern analysis possessed detectable amount of Gus activity. To date all the 11 t0 plants reached maturity and mature seeds were obtained Transmission of the hph gene to progeny(T1) of two independent t0 plants was confirmed by Southern hybridization.
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  • 49
    Digitale Medien
    Digitale Medien
    Springer
    Plant molecular biology 28 (1995), S. 123-136 
    ISSN: 1573-5028
    Schlagwort(e): Agrobacterium rhizogenes ; auxin biosynthesis genes ; plant cell division ; protoplasts
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Plant oncogenes aux1 and aux2 carried by the TR-DNA of Agrobacterium rhizogenes strain A4 encode two enzymes involved in the auxin biosynthesis pathway in transformed plant cells. The short divergent promoter region between the two aux-coding sequences contains the main regulatory elements. This region was fused to the uidA reporter gene and introduced into Nicotiana tabacum in order to investigate the regulation and the tissue specificity of these genes. Neither wound nor hormone induction could be detected on transgenic leaf discs. However, phytohormone concentration and auxin/cytokinin balance controlled the expression of the chimaeric genes in transgenic protoplasts. The expression was localised in apical meristems, root tip meristems, lateral root primordia, in cells derived from transgenic protoplasts and in transgenic calli. Histological analysis showed that the expression was located in cells reactivated by in vitro culture. Experiments using cell-cycle inhibitors such as hydroxyurea or aphidicolin on transgenic protoplast cultures highly decreased the β-glucuronidase activity of the chimaeric genes. These results as well as the histological approach suggest a correlation between expression of the aux1 and aux2 genes and cell division.
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  • 50
    ISSN: 1573-5028
    Schlagwort(e): Canavalia gladiata ; canavalin ; concanavalin A ; particle bombardment ; transgenic tobacco ; 5′-upstream sequence
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract A number of A/T-rich sequences and a CATGCAT/A sequence are contained in the 5′-upstream regions of the genes encoding concanavalin A (Con A) and canavalin, two major seed storage proteins of Canavalia gladiata D.C. To study the role of these sequences in the seed-specific gene expression, we constructed 5′-deletion mutants and examined the transient expression of β-glucuronidase reporter gene by particle bombardment and the stable expression by Agrobacterium-mediated transformation of tobacco plants. Positive regulatory elements were located in the −894/−602 and −602/−74 regions of the Con A gene, and in the −428/−376, −281/−155 and −155/−50 regions of the canavalin gene. In addition, the results suggested that the A/T-rich sequences in the 5′-upstream region of the Con A gene play a role in transcriptional activation, but that those of the canavalin gene have little effect on the gene expression. The CATGCAT/A sequence was not sufficient by itself for high levels of expression of both the Con A and canavalin genes. The canavalin polypeptide amounted to about 1% of the total extractable protein in the transgenic tobacco seeds, but the Con A polypeptide was not detected in the extractable protein.
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  • 51
    ISSN: 1573-5028
    Schlagwort(e): Lilium longiflorum ; Freesia refracta ; Tulipa gesneriana ; GUS mRNA ; particle bombardment ; pollen
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Gold particles coated with β-glucuronidase (GUS) mRNA with a 5′ cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.
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  • 52
    Digitale Medien
    Digitale Medien
    Springer
    Plant molecular biology 28 (1995), S. 949-955 
    ISSN: 1573-5028
    Schlagwort(e): Liliaceae ; tulip ; lily ; leek ; monocot intron ; particle bombardment ; promoter activity ; transient expression
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract In this paper we first review literature on the performance of various promoters in monocotyledonous species. In general, promoters isolated from monocots show a higher activity in monocot species. Moreover, the presence of an intron between the promoter and reporter gene increases transcription levels. We used the same approach to study gene expression in Liliaceae. The activities of the CaMV 35S, maize Adh1-based pEmu, rice Act1 and maize Ubi promoters, coupled to the β-glucuronidase (gus) reporter gene, were evaluated for transient gene expression upon particle bombardment of tissues of tobacco, rice, tulip, lily and leek. Although monocot promoters performed very well in rice tissues, the results of this study show that this cannot be generalized for other monocot species. The transcription inducing effects of monocot promoters were less pronounced or even absent in tissues of Liliaceae, while the presence of an intron between promoter and gus gene reduced promoter activity.
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  • 53
    ISSN: 1573-5028
    Schlagwort(e): Agrobacterium tumefaciens ; extensin ; hydroxyproline-rich cell wall protein ; Nicotiana sylvestris ; protoplasts ; wounding
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.
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  • 54
    ISSN: 1432-203X
    Schlagwort(e): garlic ; Allium sativum L. ; shoot primordia ; protoplasts ; plant regeneration
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x104 protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants.
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  • 55
    Digitale Medien
    Digitale Medien
    Springer
    Transgenic research 4 (1995), S. 44-51 
    ISSN: 1573-9368
    Schlagwort(e): Oryza sativa ; transgenic rice plants ; particle bombardment ; selection parameters ; hygromycin resistance
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Transgenic rice embryogenic callus and plants were recovered from experiments involving electric discharge particle acceleration (AccellTM technology). Critical parameters influencing successful delivery and stable integration of exogenous DNA into organized rice tissue had been identified previously. We report here on the effects of one selective agent (hygromycin B) on the phenotype and genotype of recovered callus and plants. The nature, timing and culture practices appeared to be more critical for the successful recovery of transgenic callus and plants than the level of selection used. By utilizing the procedures described in this report, transformation frequencies well in excess of 10% were obtained routinely in all varieties of rice tested. The combination of AccellTM technology with a selectable and prolific regeneration culture system resulted in the development of a variety-independent and highly efficient method for the routine introduction of any gene into any rice variety.
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  • 56
    ISSN: 1573-9368
    Schlagwort(e): transgenic haploid plants ; pollen culture ; particle bombardment ; β-glucuronidase ; neomycin phosphotransferase II
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Immature pollen fromNicotiana rustica was bombarded with gold particles coated with plasmid DNA encoding neomycin phosphotransferase II (NPTII) and β-glucuronidase (GUS) genes which, respectively, are under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator in the plasmid. Kanamycin-resistant pollen embryoids were selected from the bombarded pollen cells and two independent lines of transgenic plants were regenerated. Enzyme assays showed that one has both NPTII and GUS activities and the other only weak NPTII activity. Southern blot analyses indicated that the former has a DNA fragment corresponding to the intact expression cassettes for both genes in its genome; whereas the latter lacks intact expression cassettes for both genes and has only the intactnptII coding sequence in its genome. The transgenic plants of both lines have 24 chromosomes, confirming haploidy, and they are infertile. These results indicate that transgenic haploid plants can be produced directly by the bombardment-mediated transformation of immature pollen.
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  • 57
    Digitale Medien
    Digitale Medien
    Springer
    Journal of applied phycology 7 (1995), S. 413-420 
    ISSN: 1573-5176
    Schlagwort(e): protoplasts ; flow cytometry ; seaweed ; biotechnology
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The ability to rapidly distinguish viable sub-populations of cells within populations of macroalgal protoplast isolations was demonstrated using flow cytometry. Viable protoplasts from Ulva sp. and Porphyra perforata J. Ag. were distinguished from non-viable protoplasts based on differential fluorescein accumulation. The identities of cortical and epidermal protoplasts from Macrocystis pyrifera (L.) C. Ag. were inferred based on light-scattering and chlorophyll a autofluorescence. Three cell types could be distinguished among protoplasts released from thalli of P. perforata based on chlorophyll a and phycoerythrin autofluorescence. Mixed protoplast populations of Ulva sp. and P. perforata were also discernable based on relative chlorophyll a and phycoerythrin autofluorescence. The ability to screen heterogenous protoplast populations rapidly, combined with the cell sorting capabilities of many flow cytometers, should prove valuable for seaweed biotechnology.
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  • 58
    ISSN: 1573-5044
    Schlagwort(e): Hordeum vulgare ; plasmid uptake ; polyethylene glycol (PEG) ; protoplasts ; transient gene expression ; stable transformation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 μg ml−1 or kanamycin sulphate at 250 μg ml−1. Absolute transformation frequencies of 28.9×10−5 and 21.3×10−5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10−5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.
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  • 59
    ISSN: 1573-5036
    Schlagwort(e): Al tolerance ; 1 ; 3-β-glucan ; K leakage ; plasma membrane ; protoplasts
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Abstract Experiments were carried out to identify the primary site for aluminium (Al) toxicity in roots. Al accumulated in large amounts in the younger and outer cells in roots of pea and was retarded when the ionic strength of the Al solution was high. Cell destruction was extensive in the regions with high Al accumulation. The accumulation of Al in, and potassium (K) leakage from, the root tip were in the order pea〉maize〉rice, the same order as their sensitivity to Al. The protoplasts from the root tip portion of pea incubated with Al showed a wrinkled and uneven surface. The protoplasts progressively shrank and eventually collapsed. Viability decreased in this process. In the control protoplasts of maize, β-glucan formation was uniform on the spherical surfaces, whereas it was spotty in the Al-treated protoplasts; the cell wall material of the latter contained partly 1, 3-β-glucan which is known to be synthesised by 1, 3-β-glucan synthase embedded in the plasma membrane. These results suggest that the specific site for Al toxicity is the plasma membrane of younger and outer cells in roots and that Al tolerance depends largely on the integrity of the plasma membrane.
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  • 60
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 41 (1995), S. 41-48 
    ISSN: 1573-5044
    Schlagwort(e): callus ; Medicago littoralis ; plant regeneration ; protoplasts
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Plant regeneration from explant and protoplast derived callus has been achieved in Medicago littoralis cv. Harbinger 1886, an annual legume resistant to the fungus Pseudopeziza medicaginis. Callus was induced from different tissue explants and the fastest growth rate was observed for hypocotyls in B5 medium with 2 mg l−1 2,4-dichlorophenoxyacetic acid and 0.5 mg l−1 N6-benzyladenine. Protoplasts were isolated from cotyledons and leaves of sterile plants and from callus; the first two kinds of protoplasts showed a plating efficiency of 5.6% and 5%, respectively, when embedded in agarose. Plant regeneration occurred on media containing % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9qq-f0-yqaqVeLsFr0-vr% 0-vr0db8meaabaqaciGacaGaaeqabaWaaeaaeaaakeaacaqGobWaaW% baaSqabeaacaqG2aaaaOGaaeOVfiaabs5adaahaaWcbeqaaiaaikda% aaGccaqG+waaaa!3F97!\[{\text{N}}^{\text{6}} {\text{\Delta }}^2 {\text{}}\]isopentenyl-adenine combined with indole-3-acetic acid or 1,2-benzisoxazole-3-acetic acid, and on media with N6-benzyladenine plus α-naphtaleneacetic acid; a cytokinin/auxin ratio higher than 1 induced embryos while a ratio around 1 stimulated shoot formation. Embryo development and rooting of shoots were performed in RL medium without growth regulators.
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  • 61
    Digitale Medien
    Digitale Medien
    Springer
    Journal of industrial microbiology and biotechnology 15 (1995), S. 397-400 
    ISSN: 1476-5535
    Schlagwort(e): mycolytic enzymes ; Trichoderma harzianum ; protoplasts ; Rhizopus nigricans ; induction
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: Abstract The effect of the growth form of the inducing microorganism on specificTrichoderma harzianum mycolytic enzyme production was studied. The pelleted form ofRhizopus nigricans gave a better product concerning protoplast formation ability. The maximum yield of protoplasts from the target fungusCochliobolus lunatus was 1×108 ml−1. Analysis of individual specific enzyme activities inTrichoderma mycolytic enzyme preparations confirms the importance of high chitinase and low protease activity for high protoplast yields. Supplementation of the production medium with chitin increased the chitinase activity in theTrichoderma exoenzyme mixture.
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  • 62
    ISSN: 1573-5044
    Schlagwort(e): potato ; protoplasts ; regeneration ; Solanum
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The response to different in vitro methods for use in potato breeding has been evaluated in 11 genotypes of 5 Solanum species, S. etuberosum, S. lycopersicoides, S. maglia, S. rickii, and S. tuberosum. Callus induction and growth, and shoot regeneration were strongly influenced by the genotype, explant source, and medium utilized. Furthermore, considerable differences among the 11 genotypes were found both in plating efficiency and shoot regeneration from protoplast culture. Some interesting correlations were found between different tissue culture responses, suggesting linkage and/or pleiotropic effect of genes. The potential application to potato breeding of the in vitro techniques analyzed is discussed.
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  • 63
    Digitale Medien
    Digitale Medien
    Springer
    World journal of microbiology and biotechnology 11 (1995), S. 409-415 
    ISSN: 1573-0972
    Schlagwort(e): Breeding ; embryo culture ; haploids ; micropropagation ; protoplasts ; synthetic seed ; transformation ; wide hybridization
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: Abstract Plant tissue culture comprises a set of in vitro techniques, methods and strategies that are part of the group of technologies called plant biotechnology. Tissue culture has been exploited to create genetic variability from which crop plants can be improved, to improve the state of health of the planted material and to increase the number of desirable germplasms available to the plant breeder. Tissue-culture protocols are available for most crop species, although continued optimization is still required for many crops, especially cereals and woody plants. Tissueculture techniques, in combination with molecular techniques, have been successfully used to incorporate specific traits through gene transfer. In vitro techniques for the culture of protoplasts, anthers, microspores, ovules and embryos have been used to create new genetic variation in the breeding lines, often via haploid production. Cell culture has also produced somaclonal and gametoclonal variants with crop-improvement potential. The culture of single cells and meristems can be effectively used to eradicate pathogens from planting material and thereby dramatically improve the yield of established cultivars. Large-scale micropropagation laboratories are providing millions of plants for the commercial ornamental market and the agricultural, clonally-propagated crop market. With selected laboratory material typically taking one or two decades to reach the commercial market through plant breeding, this technology can be expected to have an ever increasing impact on crop improvement as we approach the new millenium.
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  • 64
    ISSN: 1573-0832
    Schlagwort(e): dermatophyte ; Microsporum gypseum ; Novozyme ; protoplasts ; regeneration ; viability
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Medizin
    Notizen: Abstract Factors affecting high yields, regeneration frequencies, and viability of protoplasts from clonal cultures of Microsporum gypseum were investigated. Maximum yields of protoplasts were obtained after 6 hrs digestion of 2–4 days old mycelium with Novozyme 234 using CaCl2 (0.4 M) as an osmotic stabilizer and glycine + HCl (pH 4.5) as the buffer system. Mercaptoethanol + dithiothreitol (0.01 M) proved to be the best pretreatment of mycelium prior to digestion with enzyme. A regeneration frequency of 94.4% was obtained using the top agar method with complete medium (pH 6.5) containing 0.5% agar and 0.4 M CaCl2 as an osmoticum. Colonies from regenerated protoplasts on medium containing CaCl2 were pigmented and completely powdery with high sporulation. Protoplast viability was studied in osmotic stabilizer supplemented with glucose or glutamine. After 24 hrs, glucose (2%) and glutamine (2%) enhanced protoplast viability by 22% and 23%, respectively. Protein synthesis, as measured by 3H-lysine uptake, matched the viability profile determined by fluorescence microscopy.
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  • 65
    Digitale Medien
    Digitale Medien
    Springer
    Methods in cell science 12 (1989), S. 127-133 
    ISSN: 1573-0603
    Schlagwort(e): polyethylene glycol ; transformation ; protoplasts ; Nicotiana
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Summary Polyethylene glycol can be used to induce DNA uptake into plant protoplasts. Procedures for isolation, culture and transformation ofN. tabacum protoplasts are described and can be adapted for other dicot and monocot species. Criteria for proof of transformation are discussed.
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  • 66
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell reports 8 (1989), S. 313-316 
    ISSN: 1432-203X
    Schlagwort(e): Zea mays L. ; supersweet corn ; protoplasts ; haploids ; plant regeneration
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Summary Plants were regenerated from maize (Zea mays L.) protoplasts isolated from embryogenic cell suspensions. The donor maize suspension cultures were established from friable callus initiated from microspores of a commercial supersweet hybrid (sh2sh2). The frequency of cell colony formation was higher when protoplasts were cultured on feeder layers of maize cells as compared with a liquid thin layer method. It was demonstrated that haploid and dihaploid soil-grown plants can be regenerated from maize protoplasts isolated from haploid cell cultures.
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  • 67
    ISSN: 1432-203X
    Schlagwort(e): sunflower ; protoplasts ; direct gene transfer
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Helianthus annuus protoplasts were transformed with the plasmid pCaMVNEO (Frommet al. 1986) conferring kanamycin resistance to plant. Transformed calli were selected with a frequency of 4 calli for 106 treated protoplasts. DNA was extracted from kanamycin resistant calli. Analysis of this DNA shows the presence of the NPTII gene.
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  • 68
    ISSN: 1573-5028
    Schlagwort(e): aleurone ; barley ; protoplasts ; transient expression ; wheat
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Methods have been developed for the isolation of aleurone protoplasts from developing caryopses of Hordeum vulgare and Triticum aestivum in order to study transient expression of introduced genes. Chimaeric gene constructs were introduced into aleurone protoplasts by polyethylene glycol (PEG). Transient expression directed by the 35S promoter from cauliflower mosaic virus (CaMV) of the reporter gene encoding chloramphenicol acetyl transferase (CAT) was detected in aleurone protoplasts from developing barley and wheat grains. Using a similar construct, CAT activity increased when the alcohol dehydrogenase intron 1 fragment from maize was ligated between the 35S promoter and the CAT coding region. The demonstration of transient expression in protoplasts from developing aleurone layers indicates that they may be useful for investigating tissue and developmental control of genes coding for cereal seed proteins.
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  • 69
    Digitale Medien
    Digitale Medien
    Springer
    Plant molecular biology 13 (1989), S. 503-511 
    ISSN: 1573-5028
    Schlagwort(e): electroporation ; patatin genes, potato ; protoplasts ; transient gene expression
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts −250 V/cm; Désirée mesophyll protoplasts −225 V/cm; Désirée suspension culture protoplasts −225 V/cm; and Désirée tuber protoplasts −150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36–48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the β-glucuronidase (gus) gene, showed expression (at DNA concentrations between 0–10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20–30 pmol/ml) the patatin promoter directed 4–5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.
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  • 70
    ISSN: 1573-5028
    Schlagwort(e): field inversion gel electrophoresis ; DNA isolation ; protoplasts ; nuclear DNA ; chloroplast DNA ; tomato
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The Schwartz and Cantor technique for releasing and fractionating megabase-sized DNA from agarose-embedded cells is beginning to bridge the gap in resoluation between classical genetics and current molecular DNA techniques, particularly in mammalian systems. As yet no conditions have been described for preparing plant DNA that is of sufficient length to allow similar long-range restriction mapping experiments in plant systems. In this report, we describe the application of the Schwartz and Cantor technique for preparing high molecular weight DNA from embedded tomato leaf protoplasts, as well as conditions for generating and fractionating large restriction fragments, by field inversion gel electrophoresis (FIGE). The bulk of DNA released from lysed protoplasts was at least 2 Mb in size and amenable to restriction digestion as shown by hybridizing Southern blots with, among others, a probe for the Adh-2 gene of tomato. Restriction fragments as large as 700 kb were detected. Chloroplast DNA is isolated intact, amenable to restriction analysis and, in its native form, not mobile in FIGE.
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  • 71
    Digitale Medien
    Digitale Medien
    Springer
    Plant molecular biology 13 (1989), S. 151-161 
    ISSN: 1573-5028
    Schlagwort(e): β-glucuronidase (gusA) gene ; maize ; protoplasts ; stable co-transformation ; transformation ; Zea mays L.
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract An efficient co-transformation protocol using polyethylene glycol was developed for Zea mays L. (cv. A188 × BMS) protoplasts isolated from suspension culture cells. Co-transformation was accomplished by using plasmid constructions containing β-glucuronidase (gusA) or neomycin phosphotransferase (neo) gene coding sequences; both were under control of the CaMV 35S promoter. Protoplast culture and transformation conditions were optimized to assure efficient recovery of transformed cells. The overall efficiency of transformation was 1 × 10−4 (calculated per viable protoplast plated). Among kanamycin-resistant lines, 50% showed a high level of GUS activity (above one unit). Southern blot hybridization confirmed the presence of numerous gusA and neo coding sequences in the maize genome. In two analyzed lines, integrated sequences appeared to be organized in tandem head-to-tail repeats. Results also indicated that the integrated sequences were partially methylated.
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  • 72
    ISSN: 1573-5060
    Schlagwort(e): Lycopersicon ; tomato ; haploids ; chromosomal instability ; chloroplast number ; callus culture ; protoplasts
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Summary The effect of the tissue culture system, the genotype and the ploidy level of the plant material used as explant source on the stability of the ploidy level of plants regenerated fromcell and tissue cultures of tomato was investigated. In addition the use of the chloroplast number in guard cells as a measure for ploidy level was evaluated. Haploids of tomato were very instable, which instability was observed already in somatic root tip and leaf cells. The number of regenerated plants that retained the original ploidy level differed significantly between the tested haploids. The plants that were regenerated from leaf explants of diploids were predominantly diploid in contrast to the plants regenerated from established callus cultures and protoplast where the majority was tetraploid.
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  • 73
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 19 (1989), S. 103-111 
    ISSN: 1573-5044
    Schlagwort(e): amino acids ; ammonium ; growth factors ; light ; mannitol ; nitrate ; organogenesis ; polyamines ; potato ; protoplasts ; Solanum tuberosum ; sucrose ; tissue culture
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The influence of several media components and environmental factors on shoot formation in protoplast-derived calli of Solanum tuberosum (a Rosamunda cross) were studied. Low sucrose concentration (3–15 mM) was beneficial for optimal shoot induction. Several concentrations of NO3 - and NH4 + were suitable for shoot induction as long as the concentration of NO3 - was about twice the concentration of NH4 + or higher. No stimulatory effect of glutamine, proline, putrescine, spermidine, spermine or adenine sulphate at 0.5 and 2 mM were found. White light promoted shoot induction compared with red and blue light or darkness. The intensity of light was shown to be a critical factor for good shoot induction. Lower light intensity (30 μE m-2 s-1) resulted in doubling of the number of calli producing shoots compared with higher (110 μE m-2 s-1) light intensity. A temperature of 20°C promoted shoot regeneration compared to 25°C. Based on these results improved conditions for regeneration of S. tuberosum are suggested, and shown to enhance shoot formation in five other genotypes tested.
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  • 74
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 19 (1989), S. 113-127 
    ISSN: 1573-5044
    Schlagwort(e): somatic embryogenesis ; plant regeneration ; protoplasts ; Trifolium pratense ; red clover ; protoclonal variation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Protoplasts are useful for subcellular studies, in vitro selection, somatic hybridization and transformation. Whole plant regeneration from protoplasts is a prerequisite to producing altered crop plants using these methods. Whole plant regeneration was achieved from leaf- and suspension culture-derived protoplasts of T. pratense. Regeneration was most dependent upon identifying genotypes with genetic capacity to regenerate. Additional factors that were used to select genotypes, but which proved to be less important, were a high rate of cell growth in culture and a high plating efficiency of protoplasts. One genotype was identified which had a regeneration response equivalent to that of T. rubens and which regenerated from both leaf- and suspension culture-derived protoplasts.
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  • 75
    Digitale Medien
    Digitale Medien
    Springer
    Methods in cell science 12 (1989), S. 139-144 
    ISSN: 1573-0603
    Schlagwort(e): microinjection ; genetic transformation ; protoplasts ; microspores ; Brassica napus
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Summary This paper describes a general method suitable for the microinjection ofBrassica napus protoplasts, unicellular microspores, and multicellular microspores. By incorporating components taken from other methods, manual operations frequently involved in the microinjection of plant cells have been simplified and microinjection rates increased. The embedding of cells in agarose provides a simple alternative to the variety of sophisticated immobilization strategies devised for different plant cell types thereby reducing the manipulations often involved in the culture of microinjected cells. Use of an automatic microinjector eliminated the operation of fine control systems on manual injectors; however, precision in sample delivery was reduced. Analyses indicate that transformed tissues can be recovered from microinjected protoplasts and microspores at high frequencies.
    Materialart: Digitale Medien
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  • 76
    Digitale Medien
    Digitale Medien
    Springer
    Methods in cell science 12 (1989), S. 157-161 
    ISSN: 1573-0603
    Schlagwort(e): protoplasts ; fusion ; somatic ; hybridization ; cybridization ; asymmetric hybridization
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Summary This report describes several techniques for plant gene transfer by protoplast fusion. These detailed procedures are adaptable to a wide variety of plant species for the transfer of either the entire or partial genomes of nuclear, mitochondrial, and chloroplast origins between species. Detailed procedures should allow researchers to modify and adapt these techniques to suit their own requirements. Effort has been made to describe the protoplast fusion techniques used in the authors' laboratory as comprehensively as possible while citing the many alternatives and modifications that other workers have successfully employed.
    Materialart: Digitale Medien
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  • 77
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 19 (1989), S. 213-224 
    ISSN: 1573-5044
    Schlagwort(e): Brassica napus ; B. oleracea ; rapid-cycling brassica populations ; protoplasts ; regeneration ; maltose
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Protoplasts were isolated from aseptic shoot cultures of commercial cultivars ofBrassica napus, B. oleracea andB. campestris, and from the six ‘rapid-cycling brassica species’. Of the rapid-cycling species, onlyB. napus responded well to the culture conditions used; 2% of protoplasts formed calli and up to 5% of calli regenerated shoots. Regeneration was also achieved from commercial cultivars ofB. napus andB. oleracea. For these two species the plating density, time of dilution with fresh medium and the composition of the shoot-inducing medium were all found to have an important influence on the efficiency of plant regeneration. Both responded better to maltose than to sucrose-based media. Under the optimum conditionsB. napus showed a plating efficiency of 7.8% and shooting efficiency of 17%; forB. oleracea the figures were 2% and 56%, respectively.
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  • 78
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 17 (1989), S. 91-100 
    ISSN: 1573-5044
    Schlagwort(e): amino acids ; Brassica oleracea ; light ; nitrate ; organogenesis ; polyamines ; protoplasts ; regeneration ; sucrose
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The effect of media components and environmental factors on growth and organogenesis of protoplast-derived calli of curly kale and cabbage were tested. Optimal growth (fresh weight increase of calli, shoots and roots) was found at 60 mM sucrose. Lower sucrose concentrations (3–30 mM) were favourable for shoot formation. Nitrate concentrations from 23 to 100 mM in combination with 8 or 21 mM ammonium were optimal for shoot formation. However, growth was reduced by high (100 mM) nitrate concentration. The effects of various organic nitrogen compounds at 0.5 and 2 mM were tested. Glutamine did not influence shoot formation and barely growth. Proline at 0.5 mM stimulated growth of cabbage calli but decreased growth of curly kale calli, and at 2 mM, proline also inhibited shoot production. Adenine sulphate decreased growth of cabbage calli at 0.5 mM, and at 2 mM shoot production was also reduced. Spermidine and spermine inhibited both growth and differentiation. Putrescine resulted in about 50% higher fresh weights, and also increased the number of calli producing shoots by about 35%. More calli produced shoots in white light than in blue or red light or in darkness. The length of the photoperiod or intensity of light was not critical for shoot production.
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  • 79
    Digitale Medien
    Digitale Medien
    Springer
    Cellular and molecular life sciences 44 (1988), S. 348-351 
    ISSN: 1420-9071
    Schlagwort(e): L. lactis ; protoplasts ; protoplast regeneration
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Medizin
    Notizen: Summary More than 99% ofL. lactis cells have been converted to protoplasts upon digestion of cell walls with mutanolysin (N-(acetyl)muramidase). Functional protoplasts were obtained even with the lowest level of the enzyme that was used (0.1 U·ml−1 of the cell suspension) and after incubation at 37°C for 2 min. The regeneration of the polymerized cell wall appears to be induced by a cell homogenate of the same organism.
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  • 80
    ISSN: 1573-5028
    Schlagwort(e): Gene transfer ; Glycine max ; Oryza sativa ; Triticum monococcum ; transient expression ; particle bombardment ; beta-glucuronidase ; chloramphenicol acetyltransferase
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract The development of an efficient transformation system is a prerequisite for the molecular analysis of gene expression in plants. In crop plants, this development has been hindered by difficulties encountered both in whole plant regeneration from protoplasts and in the general insusceptibility of monocots to Agrobacterium-mediated transformation. We have circumvented these difficulties by transferring foreign genes directly into the intact cells (with cell walls) of three important crop plants including rice, wheat and soybean by a particle bombardment device. Oryza sativa and Triticum monococcum cells were bombarded with accelerated tungsten particles coated with plasmids containing a β-glucuronidase gene as the reporter. Blue transformed cells were detected in an in situ enzyme assay. The number of blue cells was next used as a convenient criterion to study several factors affecting gene transfer efficiency. After optimal conditions were defined, gene transfer into intact cells of O. sativa, T. monococcum and Glycine max was successfully carried out with chloramphenicol acetyltransferase (CAT) gene as the reporter.
    Materialart: Digitale Medien
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  • 81
    ISSN: 1573-5044
    Schlagwort(e): Brassica napus ; protoplasts ; Ficoll ; agarose
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract A method is described for regenerating callus from mesophyll protoplasts of a winter variety of Brassica napus. The method combines the use of Ficoll in an initial liquid medium, enhancing early protoplast division and cell colony formation, with a transfer to an agarose system after 10 days culture to give rapid microcalli formation. Further transfers resulted in callus regeneration and the initiation of organogenesis.
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  • 82
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 14 (1988), S. 15-24 
    ISSN: 1573-5044
    Schlagwort(e): Brassica ; genetics of regeneration ; protoplasts ; totipotency
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Factors affecting the division of cells derived from leaf and cotyledon protoplasts from Brassica oleracea L. var. italica (‘Green Comet’ hybrid broccoli) were examined to optimize conditions for plant regeneration and to determine whether there was a genetic basis for improved regeneration from protoplasts derived from plants previously regenerated from tissue cultures [15]. When leaf protoplasts from different plants grown from hybrid seed were isolated and cultured simultaneously, division efficiencies of 1–95% were obtained. Cells from some plants showed high division efficiencies in consecutive experiments while cells from other plants had consistently low division rates. More plants from hybrid seed gave high division efficiencies when cotyledon protoplasts were used. However, cotyledon or leaf protoplasts from selfed progeny of regenerated plants produced more vigorous calli and more shoots than protoplasts from hybrid seed. These results suggest that there may be a genetic component to the increased totipotency of Brassica oleracea protoplasts.
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  • 83
    Digitale Medien
    Digitale Medien
    Springer
    Potato research 30 (1987), S. 371-380 
    ISSN: 1871-4528
    Schlagwort(e): dihaploids ; protoclones ; protoplasts ; Solanum tuberosum ; somaclonal variation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Beschreibung / Inhaltsverzeichnis: Zusammenfassung Die Verwendung dihaploider Kartoffelklone (2n=2x=24) in Züchtungsprogrammen, vor allem die Rückkehr zum tetraploiden Grad, wird sehr oft durch deren fehlende Fertilität verhindert. Dieses Problem dürfte am elegantesten durch somatische Fusion der Dihaploiden überwunden werden. Als Ergebnis einer solchen Prozedur würden sich, ausser der Paarung beider Ploidiestufen, die Addition qualitativer und quantitativer Merkmale ergeben. Grösstes Hindernis für die Anwendung der Protoplastenfusion ist das Fehlen eines brauchbaren Selektionssystems für die Separierung von homo- und heterokaryotischen Fusionsprodukten. In der hier beschriebenen Methode wurde der sehr kräftige Wuchs einiger Kalli für die Vorselektion einiger vermuteter Hybriden verwendet. Nach Anwendung der Polyethylenglykol-Fusions-methode (PEG, Tabelle 1) konnten von fünf selektierten dihaploiden Klonen (P1–P5) grosse Zahlen von Kalli und Pflanzen regeneriert werden, obwohl die PEG-Behandlung einen negativen Einfluss auf die Regenerationsrate hatte (Tabelle 2). Insgesamt wurden nach PEG-Behandlung 115 Kalli als vermutliche Hybriden, ihrer extremen Wuchsleistung entsprechend, selektiert. Tabelle 3 vergleicht die Ploidiestufen dieser selektierten Klone mit der von unbehandelten Elternklonen. Wegen der somaklonalen Variation wurden auch von nicht fusionierten Protoplasten viele tetraploide Klone gefunden. Ihre Zahl war allerdings signifikant kleiner, und unter den nicht PEG-behandelten Protoplasten waren immer einige diploide vorhanden. Die Tabellen 4 und 5 zeigen die Merkmale von 9 und 10 selektierten Klonen (Hy 1–10) in vitro und in vivo für Sprosslänge, Zahl der Nodien, Blattfläche, Blattform, Zahl der Wurzeln und allgemeiner Wachstumsleistung. In allen Fällen waren die gemessenen Parameter bei den selektierten Klonen signifikant grösser als bei den Kontrollen. Folglich kann das stärkere Wachstum der selektierten Klone nicht nur mit somaklonaler Variation erklärt werden. Es ist ein starkes Indiz für die Hybridnatur. Das Isoenzym-Muster der Esterasen unterstreicht diese Schlussfolgerung. Den Ergebnissen zufolge ist es möglich, somatische Hybriden anhand ihrer hybriden Vitalität vorzuselektieren. Dies sollte die Möglichkeit zur Entdeckung somatischer Hybriden in ausreichender Häufigkeit für praktische Züchtungsprogramme erhöhen.
    Kurzfassung: Résumé L'utilisation de clônes dihaploïdes (2n=2x=24) dans les programmes d'hybridation, et particulièrement le retour au niveau tétraploïde, est entravée par leur manque de fertilité. Ce problème pourrait être maitrisé élégamment par la fusion somatique de dihaploïdes. D'un tel procédé résulterait de plus l'héritage de caractères qualitatifs et quantitatifs apportés par le doublement de ploïdie. Le principal obstacle pour utiliser la fusion de protoplastes est l'absence d'un système de sélection approprié pour la séparation des homo et hétérokaryotes produits par la fusion. Par la méthode décrite dans cet article la grande vigueur de croissance de quelques cals a été mise à profit pour la présélection des présumés hybrides. Après l'adaptation du procédé de fusion au polyéthylène-glycol (PEG, tableau 1) sur cinq clônes dihaploïdes sélectionnés (P1–P5) un grand nombre de cals et de plantes pourrait être régénéré, bien que le traitement PEG ait une influence négative sur le taux de régénération (tableau 2). Au total 115 cals étaient sélectionnés après le traitement comme de présumés hybrides, en raison de leur extrème vigueur. Le tableau 3 compare les niveaux de ploïdie de ces clônes sélectionnés avec ceux des parents non traités. La variation somatique de protoplastes non fusionnés est également trouvée dans de nombreaux clônes tétraploïdes, leur nombre est cependant significativement plus petit, et parmi les protoclônes régénérés de protoplastes non traités quelques diploïdes sont toujours présents. Les tableaux 4 et 5 montrent les caractéristiques de 9 et 10 clônes sélectionnés (Hy 1–10) in vitro et in vivo, respectivement pour la longueur de pouses, le nombre de noeuds, la surface et la forme des feuilles, le nombre de racines et la vigueur générale. Dans tous les cas, les paramètres mesurés ont des valeurs significativement plus élevées dans les clônes sélectionnés que dans les témoins. En conséquence, leur vigoureuse croissance ne peut être expliquée seulement par la variation somatique mais elle constitue une indication sur la vigueur hybride. L'analyse des estérases souligne cette conclusion (figure 1). Ces résultats montrent qu'il est possible de présélectionner des hybrides somatiques par leur vigueur, ce qui augmente les possibilités de les détecter en quantité suffisante dans les programmes d'hybridation.
    Notizen: Summary Tetraploid potato plants were regenerated after polyethylene-glycol-induced protoplast fusion between dihaploids. Hybrid vigour of the regenerated calli was used for preselection of fusion products. Nearly all the selected vigorous clones possessed chromosome counts at the tetraploid level. Fusion products were compared to the parental material to auto-fused plants of and to three protoclones expressing different degrees of somaclonal variation. The selected clones, where grown in vitro in growth rooms and in pots in the glasshouse, showed increased vigour compared to their parents, to auto-fused and to 4x protoclones. Plants of clones from very vigorous calli, when assessed by height, the number of nodes per plant, leaf morphology and tuber production, showed hybrid vigour. The hypothesis that superior clones result from heterokaryons after protoplast fusion or that they arise from other in vitro events such as somaclonal variation is discussed.
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  • 84
    ISSN: 1573-5044
    Schlagwort(e): Humulus lupulus ; micro-calli ; protoplasts
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract A cell-wall digestion medium has been devised to isolate protoplasts from suspension cultures of Humulus lupulus. Conditions have been developed for colony formation from protoplasts and the plating efficiency determined in three types of agar and by two culture methods. Viable calli were produced only when protoplasts embedded in Seaplaque agarose were incubated in a defined liquid medium. HPLC analysis showed that none of the isolated colonies accumulated α-acids.
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  • 85
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 8 (1987), S. 225-233 
    ISSN: 1573-5044
    Schlagwort(e): Brassica napus ; thin cell layer ; protoplasts ; plant regeneration ; rapeseed ; organogenesis ; callus
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Cell layer strips composed of the epidermis and 7–9 layers of subepidermal cells were isolated from the 3–4 terminal internodes of Brassica napus cv Westar plants at the early flowering stage. The strips were precultured for one day in modified liquid MS [11] medium and subsequently incubated for 17–18 h in a 0.4 M mannitol solution containing 1% Macerozyme and 1% Cellulase ‘Onozuka’ R-10. Protoplast yield was 2–2.8×106 per 1.0g of tissue. Protoplasts were cultured at 1×105/ml in three different media: S1 [13], B [12] and L[8]. The first cell divisions occurred after 2–8 days of culture at frequencies of 20–54%. The highest growth rate of colonies was obtained in L medium containing 0.4 M sucrose and 2% Ficoll. After 4 weeks, green calli, 1–2 mm in diameter were transferred onto B5 [2] medium with 3 mgl-1 zeatin, 1% sucrose, 0.1 M mannitol and 0.5% agarose for shoot regeneration. Up to 20% of the calli regenerated shoots which subsequently were rooted and established in soil in the greenhouse.
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  • 86
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 10 (1987), S. 187-196 
    ISSN: 1573-5044
    Schlagwort(e): protoplasts ; Azolla ; Sporophytes ; ferns ; Cellulysin ; Pectolyase
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract We have developed a method for producing protoplasts from the heterosporous water fern Azolla using a combination of Cellulysin (4.0%) and Pectolyase (0.025%) in 0.6 M mannitol containing 6 mM CaCl2 2H2O. These protoplasts regenerate new cell walls within 48 hours when cultured on modified Gamborg B-5 medium.
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  • 87
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 11 (1987), S. 179-188 
    ISSN: 1573-5044
    Schlagwort(e): Solanum melongena ; protoplasts ; lamina ; petioles ; stems
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Plants of Solanum melongena were propagated under in vitro conditions (27°C, 12h/day illumination at 62 μEm-2s-1, 60% humidity) by subculture of terminal and lateral cuttings on MS medium +20 gl-1 sucrose + Morel and Wetmore vitamins at 1/8 strength and 7 gl-1 agar. Lamina, petioles and stems of 3-week-old cuttings were used as sources of protoplasts. The best mean yield of protoplasts was obtained from the lamina with 9,030×103 protoplasts per gram of tissue. Petioles and stems yielded respectively 3,144×103 and 1,220.4×103 protoplasts per gram of tissue. first division of petiole and stem protoplasts occurred within 48 h, while lamina protoplasts underwent division after 3–4 days of culture in KM8p medium +2,4-D(0.2 gl-1) + zeatin (0.5 mgl-1) + NAA (1 mgl-1) and 0.35M glucose as osmoticum. The highest percentage of dividing cells was obtained from petiole material, estimated at 33.4% after 7 days, compared to 23.8% and 19.4% respectively for stem and lamina protoplasts. When BAP replaced zeatin in KM8p, the division percentage of lamina protoplasts was reduced to 10–15%. When transferred to regeneration medium, all calli derived from KM8p + zeatin formed deep-green spots identified as embryo-like structures, while only few calli from KM8p + BAP underwent shoot organogenesis without formation of green spots. Some of embryo-like structure developed into plantlets with a frequency of 1–2 plantlets per callus especially on MS medium + zeatin (4 mgl-1) + IAA (0.2 mgl-1). Maintaining protoplast-derived calli on MS + BAP (0.5 mgl-1) + NAA (0.5 mgl-1) for more than 3 weeks resulted in a decrease and loss of cell totipotency.
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  • 88
    Digitale Medien
    Digitale Medien
    Springer
    Plant molecular biology 8 (1987), S. 363-373 
    ISSN: 1573-5028
    Schlagwort(e): transformation ; MgCl2-PEG-electroporation ; competence ; protoplasts
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Direct gene transfer into plant protoplasts has been recently developed, and conditions for high frequency transformation of SR1 tobacco protoplasts established. In this paper we analyse numerous transformation parameters in a comparative study on SR1 Nicotiana tabacum and N. plumbaginifolia, and report on a simple chemical technique for very efficient protoplast transformation. It is based on the synergistic interaction of MgCl2 and PEG. The technique yielded up to 1400 transformants per 3×105 treated N. tabacum protoplasts (up to 4.8% of the survivors, late selected clones). Using N. plumbaginifolia, the frequencies were 10-fold lower, indicating that the ‘competence’ for transformation has a species-specific component.
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  • 89
    ISSN: 1573-5079
    Schlagwort(e): carbon dioxide fixation ; freezing stress ; photophosphorylation ; photosynthetic electron transport ; protoplasts ; Valerianella locusta
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Mesophyll protoplasts were isolated from unhardened and cold-acclimated leaves of Valerianella locusta L. and subjected to freeze-thaw treatment. To evaluate the extent and course of freezing injury, photosynthetic reactions of whole protoplasts and of free thylakoid membranes, liberated from protoplasts by osmotic lysis, were measured. In addition, the integrity of the protoplasts was determined by microscopy. The results reveal an increased frost tolerance of protoplasts isolated from acclimated leaves with respect to all parameters measured. CO2-dependent O2 evolution (representing net photosynthetic CO2 fixation of protoplasts) was the most freezing-sensitive reaction; its inhibition due to freeze-thaw treatment of protoplasts was neither correlated with disintegration of the plasma membrane, nor was it initiated by inactivation of the thylakoid membranes. The frost-induced decline of protoplast integrity was not closely correlated to thylakoid damage either. Freezing injury of the thylakoid membranes was manifested by inhibition of photosynthetic electron transport and photophosphorylation. Both photosystems were affected by freezing and thawing with strongest inhibition occurring in the water-oxidation system or at the oxidizing site of photosystem II. Photophosphorylation responded more sensitively to freezing stress than electron transport, although uncoupling (increased permeability of the thylakoid membranes to protons) was not a conspicuous effect. The data are discussed in relation to freezing injury in leaves and seem to indicate that frost damage in vivo is initiated at multiple sites.
    Materialart: Digitale Medien
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  • 90
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 6 (1986), S. 47-59 
    ISSN: 1573-5044
    Schlagwort(e): cotton ; ovule epidermis ; protoplasts ; cell wall regeneration ; β-1,3-glucans ; β-1,4-glucans
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Culture protocols were developed and characterization of the regenerated cell walls was performed for protoplasts of cotton (Gossypium hirsutum L., L., var. Acala SJ-2) ovule epidermal cells. This work was undertaken in order to extend studies concerning nutritional effects and regulation of nucleotide sugar incorporation into β-1,3- and β-1,4-glucan components of cotton fiber cell walls. Protein and carbohydrate polymers and recovered from the culture medium. Analysis of a cellular fraction indicated that the majority of 14C incorporated from [14C] glucose was present in the hot-water-soluble fraction of the cells. The majority of label incorporated into cell wall material could be solubilized with acetic-nitric reagent, indicative of noncellulosic material, and characterized as β-1,3-linked glucans. Only 5 to 15% of the regenerated cell wall could be characterized as β-1,4-linked glucose indicative of cellulose.
    Materialart: Digitale Medien
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  • 91
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 7 (1986), S. 11-19 
    ISSN: 1573-5044
    Schlagwort(e): Solanum etuberosum ; protoplasts ; plating efficiency ; shoot regeneration
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract A procedure for the culture of Solanum etuberosum mesophyll protoplasts with subsequent shoot regeneration is described. Several factors affected protoplast yield, colony formation, and shoot regeneration from in vitro plants. A protoplast isolation medium with 0.6 M sucrose produced twice the yield as one with 0.3 M sucrose. uowever, a higher concentration of osmoticum was inhibitory to colony development unless it was diluted into a lower osmoticum medium in a bilayer system. A 16 hour light/8 hour dark photoperiod for stock plants allowed twice the protoplast yield compared to plants grown under continuous light but no effect was found on subsequent colony formation or shoot regeneration. The concentrations of four major salts in the protoplast plating medium were critical for a high frequency of colony formation from protoplasts. Levels of 0.25 × or 1 × were considerably better than 4 ×. Fast colony formation, but at a lower efficiency, was obtained with a monolayer plating method. A bilayer plating system allowed a higher efficiency but colonies developed more slowly. For the best treatments, the frequency of colony formation from protoplasts ranged from 2.4 to 3.6 × 10-3 with 37% to 66% of the colonies producing shoots ten weeks after protoplast isolation.
    Materialart: Digitale Medien
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  • 92
    Digitale Medien
    Digitale Medien
    Springer
    Plant cell, tissue and organ culture 4 (1985), S. 171-182 
    ISSN: 1573-5044
    Schlagwort(e): potato ; protoplasts ; regeneration ; aneuploidy ; karyotypic changes
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Abstract Over two hundred plants were regenerated from shoot-culture derived proto-plasts of potato (Solanum tuberosum L. cv. Majestic). Some had grossly aberrant phenotypes but the majority were similar to, or indistinguishable from normal control Majestic. Cytological examination showed that on average, 57% of the regenerants had the normal chromosome number (2n=4x=48). The remainder were aneuploids and fell into two classes in approximately equal numbers. The first class was limited at about the euploid level (ie, 2n=44−49). The second class contained plants with higher chromosome numbers ranging from 2n=73 to the octaploid level (2n=8x=96). The overall results represent an improvement over our earlier studies on chromosome variation in protoplast-derived potato plants. In addition, three cases of structural chromosome variation were observed.
    Materialart: Digitale Medien
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  • 93
    ISSN: 1573-5028
    Schlagwort(e): cauliflower mosaic virus ; DNA synthesis ; kinetics ; protoplasts ; replication intermediates
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie
    Notizen: Summary Turnip protoplasts infected with cauliflower mosaic virus (CaMV) have been used to examine the kinetics of CaMV DNA synthesis, and the different classes of CaMV DNA found in vivo partially characterised. Differential extraction techniques for DNA from infected protoplasts has identified several distinct classes of viral DNA. The same approach applied to virus preparations revealed that while the majority of virion DNA was stably encapsidated, some small DNAs and a heterogeneous population 3.8-ca. 5.0 Kb were not. The structural relationship of sa-DNA (3) with the particle is such that only its 5′ RNA moeity is susceptible to nuclease attack. Two-dimensional gel electrophoresis of total CaMV DNA from infected protoplasts revealed all the DNA species found in virion DNA, those species representing the ‘free’ DNA class and a further class of molecules, rich in DNA of (−) polarity (24), to which the role of reverse transcription intermediates has been ascribed. ‘Free’ DNA contains 8 Kb supercoiled DNA (Form I DNA), an 8 Kb open circle (Form II), an 8 Kb linear (Form III) and a truncated molecule with an extension of the (−) strand previously observed from infected plants (10). Kinetic experiments show that the accumulation of total CaMV-DNA parallels the accumulation of progeny virions to reach a maximum around 72 h post-inoculation and that there is not a separation of CaMV-DNA synthesis into clearly defined early and late stages.
    Materialart: Digitale Medien
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  • 94
    ISSN: 1573-5060
    Schlagwort(e): protoplasts ; protoclonal variation ; somatic hybridization ; top-fruit trees ; woody ornamentals
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Summary Until recently, temperate fruit trees and woody ornamentals have been regarded as recalcitrant to biotechnological breeding approaches based on protoplasts. This however should no longer be the case, as procedures are now available, not only for the regeneration of complete plants from protoplasts of various tissues of such species, but also for the exploitation of protoplast technology for their genetic manipulation. This paper will examine the recent advances and state of the art in this domain, with particular attention to the use of protoplast technology as a novel tool in the breeding of rosaceous top-fruit tree species and woody ornamentals. Problems and their solutions within the context of regenerating plants from isolated protoplasts of stone (Prunus spp.), pome (Pyrus spp., Malus spp.) and small (Rubus spp.) fruits, and of several shrubby ornamental genotypes (Lonicera spp., Weigela spp., Forsythia spp., Cotoneaster spp.) will be addressed. Interspecific (Prunus spinosa + Prunus cerasifera) and intergeneric (Forsythia spp. + Syringa spp.) somatic hybridization within this group of species, as well as the use of protoplasts for host/pathogen interaction studies (Pyrus/Erwinia amylovora) will also be discussed.
    Materialart: Digitale Medien
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  • 95
    ISSN: 1573-5060
    Schlagwort(e): Hordeum vulgare ; isolated microspores ; particle bombardment ; transformation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Summary A highly regenerable, isolated microspore system for barley, Hordeum vulgare L. cv. Igri, has been developed which is amenable to transformation studies using particle bombardment. The system allows DNA to be delivered to microspores at the single cell stage and both transient and stable transformation events have been demonstrated. The potential advantages of using isolated microspores as the target tissue in routine transformation systems are discussed.
    Materialart: Digitale Medien
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  • 96
    ISSN: 1573-5060
    Schlagwort(e): gene transfer ; Hordeum vulgare ; neomycin phosphotransferase II ; particle bombardment ; transgenic barley
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Summary Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for β-glucuronidase (GUS). Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T0). This chimeric plant passed the transferred nptII gene to its T1 progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T0 spikes and 15 T1 offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T2 and T3 plants were produced by isolating embryos from green grains of transgenic T1 and T2 plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T2 offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed. Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticin R or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile.
    Materialart: Digitale Medien
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  • 97
    ISSN: 1573-5060
    Schlagwort(e): barley ; electroporation ; PEG-mediated DNA uptake ; promoter analysis ; protoplasts ; transient expression
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Summary In some cereal species that are still recalcitrant to stable transformation and regeneration, transient expression in isolated protoplasts is a useful tool for the study of gene expression and regulation. We have successfully applied these techniques to barley protoplasts derived from developing endosperm, aleurone, leaves and roots in order to characterize functionally cis-acting motives in two gene promoters, corresponding to trypsin inhibitor BTI-CMe and to sucrose synthase Ss1. Gene specificity is maintained in transient expression assays with protoplasts isolated from these different barley tissues and the pattern of expression parallels the mRNA levels observed for the corresponding genes in the same tissues.
    Materialart: Digitale Medien
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  • 98
    ISSN: 1573-5060
    Schlagwort(e): Oryza sativa L. cv. Taipei 309 ; rice ; protoplasts ; direct DNA uptake ; kanamycin-resistant transgenic plants ; field trial ; glasshouse trial ; neomycin phosphotransferase II (npt II) gene ; gene expression and inheritance
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Summary The phenotypes of seed progeny (R2 generation) of Oryza sativa L. cv. Taipei 309, which carried the neomycin phosphotransferase II (npt II) gene, were compared with those of non-transformed, protoplast-derived plants of the same generation and non-transformed, seed-derived plants under field and glasshouse conditions. Under both conditions the transgenic plants were generally smaller, took longer to flower and had reduced fertility. Significant differences were observed between individuals within the group of transgenic plants. The npt II gene was present in most of the transgenic plants, but NPT II activity was only detected in a minority of individuals.
    Materialart: Digitale Medien
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  • 99
    ISSN: 1573-5060
    Schlagwort(e): bleomycin ; direct gene transfer ; expression ; irradiation ; petunia ; protoplasts
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Summary The production of transgenic plants by means of direct gene transfer to protoplasts is now a widely-used technique. The biological mechanisms underlying the transformation are still poorly understood, but many investigations have attempted to shed light on some components of this process. Varying the experimental conditions has in some cases led to better transformation rates, but further improvements of the protocols are possible. Such improvements will require a better understanding of how the alien DNA enters the cells, becomes integrated into the chromosomes and is treated as a part of the plant genome. Irradiation with sublethal doses of X-rays or UV-light has been shown to increase the transformation frequency, while certain drugs have been shown to act in a similar manner. The effects of these and other factors are discussed.
    Materialart: Digitale Medien
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  • 100
    Digitale Medien
    Digitale Medien
    Springer
    Euphytica 85 (1955), S. 35-44 
    ISSN: 1573-5060
    Schlagwort(e): cereals ; protoplast transformation ; tissue electroporation ; particle bombardment
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Land- und Forstwirtschaft, Gartenbau, Fischereiwirtschaft, Hauswirtschaft
    Notizen: Summary Many aspects of basic and applied problems in plant biology can be investigated by transformation techniques. In dicotyledonous species, the ability to generate transgenic plants provides the tools for an understanding of plant gene function and regulation as well as for the directed transfer of genes of agronomic interest. For many dicotyledonous plants Agrobacterium tumefaciens can be routinely used to introduce foreign DNA into their genome. However, cereals seem to be recalcitrant to Agrobacterium-mediated transformation. In cereals, many efforts have been made in recent years to establish reliable transformation techniques. Several transformation techniques have been developed but to date only three methods have been found to be suitable for obtaining transgenic cereals: transformation of totipotent protoplasts, particle bombardment of regenerable tissues and, more recently, tissue electroporation. The current state of transformation methods used for cereals will be reviewed.
    Materialart: Digitale Medien
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