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  • 1990-1994  (63)
  • 1985-1989  (29)
  • 1955-1959  (4)
  • 1890-1899
  • 1880-1889
  • protoplasts
  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell reports 13 (1994), S. 394-396 
    ISSN: 1432-203X
    Keywords: Rice ; α-amylase ; protoplasts ; aleurone ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Protoplasts isolated from the aleurone have been used extensively in molecular studies focusing on hormone-mediated regulation of gene expression in barley seed. To extend the use of aleurone protoplasts to other species, we have determined the conditions necessary for the isolation of protoplasts from rice aleurone layers of germinated seed. Many of the common cell wall degrading enzymes used in making protoplasts were tested for their ability to release protoplasts from rice aleurone layers. Cellulysin was found to be the most effective. Transformation of these aleurone protoplasts was accomplished using polyethylene glycol and DNA constructs containing the firefly luciferase reporter gene under the control of two different promoters were tested. Luciferase expression was 24-fold greater when the reporter gene was under the control of the CaMV 35S promoter than when the promoter from the alcohol dehydrogenase 1 gene was used. With the isolation and transformation of aleurone protoplasts from rice, it is now possible to investigate molecular events occurring in this tissue during germination.
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  • 2
    ISSN: 1432-203X
    Keywords: Vigna sublobata ; protoplasts ; microcalli ; shoot bud formation ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Viable protoplasts of Vigna sublobata L. were isolated enzymatically from hypocotyls of axenic seedlings. Protoplast yields were dependent upon seedling age, with maximum yields (2.25 ± 0.35 × 106 g fwt−1) from seedlings aged 6 d. Protoplasts regenerated cell walls and underwent sustained divisions when cultured in either agarose-solidified or liquid K8P medium. The plating density affected the division frequency and plating efficiency; the division frequency (68 ±0 6.0%) was maximum at 4.0 × 104 ml−1 while plating efficiency was maximum (1.3 ± 0.1%) at 5.0 × 104 ml−1. Dividing protoplasts developed into microcalli, which produced glossy green compact nodular calli on transfer to 8.0 gl−1 w/v agar-solidified medium containing MS salts, B5 organic components, 30 g l−1 sucrose, NAA (0.2–0.5 mg l−1), zeatin riboside (0.5–2.0 mg l−1) and GA3 (0.5–1.0 mg l−1). These calli, after sub-culture on the same medium, produced shoot buds which underwent elongation following transfer of tissues to 6.0 g l−1 agar-solidified B5 medium containing 30g l−1 sucrose, IBA (0.01 mg l−1) and BAP (1.0 mg l−1). Elongated shoots developed roots after transfer to 8.0g l−1 agar-solidified, hormone-free MS medium with 30 g l−1 sucrose.
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  • 3
    ISSN: 1432-203X
    Keywords: Petunia hybrida ; protoplasts ; oxygen delivery ; perfluorochemicals ; Pluronic F-68 ; surfactant ; cell division
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Cell suspension-derived protoplasts of albino Petunia hybrida were grown for 10 d at the interface between aqueous culture medium (KM8P) and an oxygenated (10 mbar for 15 min) perfluorocarbon liquid, perfluorodecalin. Protoplasts synthesised new cell walls and divided normally at the perfluorodecalin/culture medium interface, with a mean viability after 10 d of 〉 92.0%. The mean plating efficiency of protoplasts was elevated by 37% (P〈0.05) following culture at the perfluorodecalin/medium interface, but was unaltered by perfluorodecalin or oxygen separately. The mean plating efficiency of protoplasts cultured at the interface was further increased to a maximium of 52% above control, in the presence of oxygenated perfluorodecalin and KM8P medium supplemented with the non-ionic, co-polymer surfactant, Pluronic F-68 at 0.01% (w/v). These findings demonstrate the effectiveness of oxygenated perfluorodecalin for promoting protoplast growth, by facilitating oxygen delivery. The finding that Pluronic F-68 further increased the plating efficiency of protoplasts cultured at the perfluorocarbon/aqueous interface suggests that these agents improve growth through separate, but cumulative, mechanisms.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 38 (1994), S. 181-188 
    ISSN: 1573-5044
    Keywords: Allium cepa ; biosynthesis ; compartmentation ; γ-glutamyl peptides ; onion ; protoplasts ; S-alkenyl-L-cysteine sulphoxide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Pulse labelling experiments with 35SO4 2- fed for 24h to intact plants (shooted onion sets)of Allium cepa (onion) showed that 〉70% of the label appeared in the S-alkenyl-L-cysteine sulphoxides within 18h, reached a maximum at 48h and thereafter decreased. The amount of label detected in the γ-glutamyl peptide fractions was below 20% of the total label at any time. It is concluded that in intact plants (at the growth stage used) the γ-glutamyl peptides are not the immediate precursors of the S-alkenyl-L-cysteine sulphoxides. The major S-alkenyl-L-cysteine sulphoxide in onion was found to be compartmentalized mainly within the endoplasmatic reticulum.
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  • 5
    ISSN: 1573-5044
    Keywords: electrofusion ; L. pennellii ; protoplasts ; S. tuberosum ; salt tolerance ; somatic hybrids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Mesophyll protoplasts of Lycopersicon pennelli Corr., a wild relative of tomato, were electrofused with those from a dihaploid potato clone, cv Nicola, with the objectives of transferring saline tolerance from L. pennellii to cultivated potato. 150 calli were selected from the fusion experiments, finally giving 2 hybrid shoots. Their hybrid nature was verified by examining isoenzyme patterns for esterases (EST), peroxidase (PRX), phosphogluconate dehydrogenase (6-PGD), and glutamate oxaloacetate transaminase (GOT). The hybrid plants had an intermediate morphology, and grew vigorously in vitro. When transplanted to soil, they were less vigorous, due to difficulties in rooting, but were still capable of flowering, and forming short stolons and mishaped tubers, probably resulting from the effects of gene dosage due to the novel association of two genomes from a tuberizing (potato) and a non tuberizing species (L. pennellii). The characteristics of such mishaped tubers provided strong evidence of a hybrid nature for the selected plants. The hybrid plants were highly sterile, producing only 3–7% viable pollen. Tests for salt tolerance showed that the growth of the somatic hybrid plants was reduced by 50% as for L. pennellii, whilst potato did not grow at all under saline conditions.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 99-105 
    ISSN: 1573-5044
    Keywords: cell division ; peach ; protoplasts ; Prunus persica ; tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Yields of 106–108 peach mesophyll cells and protoplasts · gfw-1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4–10 mm in length, were a superior source for protoplast isolation than medium or big expanded leaves, 22–30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml-1. Purification of protoplasts on a sucrose gradient yielded about 107 protoplasts · gfw-1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 255-258 
    ISSN: 1573-5044
    Keywords: tomato ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three Indian cultivars ofL. esculentum were assessed for shoot regeneration from protoplast-derived calli. Consistent yields of viable protoplasts (〉9.0×106 g f.wt.-1) were obtained from leaflets of 14 days old cultured shoots. Protoplast viability (88–94%) and planting efficiency (55–70%) were recorded for the three cultivars. Up to 71% of the protoplast-derived tissues regenerated shoots.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 197-204 
    ISSN: 1573-5044
    Keywords: interferon ; Lipofectin ; protoplasts ; rice ; transgenic plant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The plasmid pIG3031 containing human α-interferon cDNA and the neomycin phosphotransferase II coding sequence was successfully transferred into rice protoplasts (Indica type rice) by Lipofectinmediated transformation. Assays for NPT II enzyme activity indicated that the transformation frequency was 10%. Transgenic plants were regenerated from transformed calli. Southern blots showed that the human α-interferon cDNA sequence was present in rice DNA. RNA slot blots indicated apparent transcription of human α-interferon cDNA under the control of the plant-active promoter PI'. Extracts of transgenic cell cultures and plants contained apparent interferon activity as measured by resistance of a human amniotic cell line to viral infection in the presence of plant extracts. These studies demonstrate that human α-interferon cDNA may be correctly expressed in rice cells.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 237-242 
    ISSN: 1573-5044
    Keywords: aseptic culture ; female gametophyte ; heterospory ; male gametophyte ; protoplasts ; Salvinia natans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Sporophytes were aseptically obtained by co-culture of female and male gametophytes derived from two types of spores (megaspores and microspores) of the heterosporous fernSalvinia natans All. Protoplasts isolated enzymatically from juvenile leaflets of sporophytes were cultured in a 1/10 Murashige and Skoog's medium containing 2.2 μM naphthalene acetic acid, 2.2 μM 6-benzyl-aminopurine, 0.35 M mannitol, and 0.05 M sucrose. Cell division took place within 6 days of culture, and cell-clusters composed of 9–10 cells were observed after 30 days of culture.
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  • 10
    ISSN: 1573-5044
    Keywords: Catharanthus roseus ; crown gall tumor ; heterokaryons ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts enzymatically isolated from cell line of Catharanthus roseus G. Don crown gall, were cultured at high density (105 P ml-1) in modified B5 liquid medium (Gamborg et al. 1976). In the absence of growth regulators C. roseus protoplasts were able to regenerate a cell-wall, divide and, subsequently, yield very numerous clones in the absence of growth regulators. After two weeks, the cultures were greatly diluted in order to obtain clones of single-cell origin. Most of the clones individually transferred onto solid medium can proliferate indefinitely, without growth regulators. Among analyzed clones, 90% were nopaline positive. Their ajmalicine and serpentine content was compared with that of the parental crown gall line, and was found to be low. The CR10 protoplasts were very easy to grow, they were an interesting model for the development of pure tumorous lines. Moreover, we found that the tumorous protoplasts were useful for cell fusion experiments or for the delicate culture of tree protoplasts.
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  • 11
    Electronic Resource
    Electronic Resource
    Springer
    Transgenic research 3 (1994), S. 13-19 
    ISSN: 1573-9368
    Keywords: gene transfer ; electroporation ; stable transformation ; protoplasts ; transgenic trees ; Populus tremula x P. alba
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts from leaflets ofin vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying thenptII gene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene,als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 105 to 104 protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 μM paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS ofArabidopsis and 25 μM phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated. Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.
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  • 12
    ISSN: 1432-203X
    Keywords: Solanum tuberosum ; anthocyanins ; gamma-irradiation ; protoplasts ; protoclones
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Callus cell lines of potato (Solanum tuberosum L. cv. Zarevo) were obtained from seedlings germinated from gamma-irradiated seeds (200 Gy). Some of these cell lines produce red-violet pigments which were identified as acylated anthocyanins. The major anthocyanin was determined to be peonidin 3-O-[6-O-(4-O-E-p-coumaroyl-rhamnosyl)-glucoside]-5-O-glucoside (“peonanin”). Single cell-derived protoclones from non-pigmented protoplasts sometimes also gave rise to pigmented cell clusters thus indicating that the changes in the expression of the anthocyanin pathway can also occur after the stage of initial callus induction.
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  • 13
    ISSN: 1432-203X
    Keywords: Arabidopsis thaliana ; ecotypes and mutant lines ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of four ecotypes (Col C24, Per-1, Bur-0, Landsberg erecta) and two marker lines (M4 and M10) of Ardbidopsis thaliana is described. The different lines showed plating efficiencies between 1.0 and 3.9% using Nitsch medium or this medium supplemented with coconut water. For the differentiation of callus into normal shoots a single shoot regeneration medium was applicable to all ecotypes, but depending on the line other regeneration media showed to be more suitable. The results indicated that the protoplast culture procedure is applicable, with minor modifications, to all tested genotypes but the most suitable shoot regeneration medium should be established for each A. thaliana line.
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  • 14
    ISSN: 1432-203X
    Keywords: forage grasses ; Festuca pratensis ; suspension cultures ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Suspension cultures from mature embryo-derived compact callus were initiated in seven meadow fescue (Festuca pratensis Huds.) cultivars. Four to six months after initiation, embryogenic suspension cultures with a moderate growth rate were established from three of them (cvs. Barmondo, Belimo and Leopard). These suspension cultures showed the capacity, maintained over six months, to regenerate green plants which could be grown to maturity under greenhouse conditions. Morphogenic suspension cultures from single genotypes of three F. pratensis cultivars (cvs. Barmondo, Belimo and Leopard) yielded large numbers of protoplasts, which upon culture in agarose beads using nurse cells formed microcalli with an overall plating efficiency in the range of 10-3 to 10-4. Mature plants were reproducibly regenerated and established in soil, from such protoplasts during a period of six months. The regeneration of fertile plants from protoplasts derived from suspension cultures of meadow fescue and its implications on gene transfer technology for this species are discussed.
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  • 15
    ISSN: 1432-203X
    Keywords: Festuca pratensis ; suspension cultures ; protoplasts ; plant regeneration ; somaclonal variation ; genetic fidelity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials. The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.
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  • 16
    ISSN: 1432-203X
    Keywords: Auxin ; benzisoxazole-3-acetic acid ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Benzisoxazole-3-acetic acid, a new synthetic growth regulator, was administered to protoplast cultures from Nicotiana tabacum and subsequently to the developed microcalluses, to test its activity on plant regeneration from protoplasts in different culture conditions. Such activity, compared to that of naphthalene-acetic acid, proved to be rather low in the stage of cellular division and microcallus formation but particulary high in the stage of shoot induction from microcallus, thus confirming that the activity of this compound is mainly morphogenetic.
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  • 17
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell reports 12 (1993), S. 260-263 
    ISSN: 1432-203X
    Keywords: sunflower ; protoplasts ; somatic embryogenesis ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A sunflower genotype (Helianthus annuus L. cv. Florom-328) able to regenerate plants from in vitro cultures was identified by screening hybrids and inbred lines. Protoplasts of this genotype were isolated from dark grown hypocotyls and were cultured in droplets of agarose-solidified V-KM medium covered by liquid V-KM supplemented with naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). One week later colonies were subjected to 2,4-dichlorophenoxyaceticacid for a one week period. Further culture in V-KM with reduced concentrations of NAA and BAP resulted in the appearence of somatic embryos. Maturation of embryos was achieved by culture on MS medium supplemented with NAA, BAP, gibberellic acid A3 and the ethylene inhibitor AgNO3. Embryos were then transferred onto hormone free MS medium for germination. The frequency of shoot formation in the best case was 9.6 percent of viable colonies (1.3 percent of protoplasts plated). Some of the shoots with roots could be transplanted into soil, others were grafted on hypocotyls of in vivo germinated seedlings. Eighty percent of grafted shoots and over 95 percent of rooted shoots survived. The plants flowered and produced 5 to 10 seeds each. Factors affecting the frequency of embryo formation and plant regeneration are discussed.
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  • 18
    ISSN: 1573-5028
    Keywords: Key words ; antisense RNA ; β-glucuronidase ; protoplasts ; transient gene expression ; PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Antisense RNA-mediated inhibition of gene expression is a valuable tool to induce mutant phenotypes. We are interested in the application of antisense gene fragments with the aim to improve the efficiency of inhibition and to be able to selectively suppress gene family members in plants. Protoplasts may provide a rapid system to screen the efficiency of antisense gene segments. As a first step, we set up a transient expression system for leaf protoplasts of Petunia hybrida and used as a model system the inhibition of β-glucuronidase (uidA) expression by uidA antisense gene segments. Both GUS enzyme activities and uidA RNA levels were measured. Co-introducing equal amounts of a full-length uidA antisense gene and a uidA sense gene reduced GUS activity by 60–70%. Various uidA antisense fragments also inhibited expression although with different efficiencies and we show that strong antisense fragments can be retrieved from weak antisense gene fragments. A promoter-less antisense gene did not reduce uidA expression indicating that the inhibition is mediated by antisense transcripts. Using quantitative PCR on first-strand cDNA we show that expression of functional antisense genes lead to reduced levels of uidA mRNA. This suggests that the mechanism of antisense RNA inhibition in protoplasts is similar to that in transgenic plants and that the protoplast system in combination with PCR can be used to preselect antisense fragments of any gene.
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  • 19
    ISSN: 1573-5028
    Keywords: cis-acting elements ; intron conservation ; intron secondary structure ; pre-mRNA splicing ; CpG suppression ; protoplasts ; transcript levels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Our previous phylogenetic analysis based on cDNA sequences of chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPDH; genes GapA and GapC, respectively) of the red alga Chondrus crispus suggested that rhodophytes and green plants are sister groups with respect to plastids and mitochondria and diverged at about the same time or somewhat later than animals and fungi. Here we characterize the genomic sequences of genes GapC and GapA of C. crispus with respect to promotor structures, intron/exon organization, genomic complexity, G+C content, CpG suppression and their transcript levels in gametophytes and protoplasts, respectively. To our knowledge this is the first report on nuclear protein genes of red algae. The GapC gene is G+C-rich, contains no introns and displays a number of classic sequence motifs within its promotor region, such as TATA, CAAT, GC boxes and several elements resembling the plant-specific G-box palindrome. The GapA gene has a moderate G+C content, a single CAAT box motif in its promotor region and a single intron of 115 bp near its 5′ end. This intron occupies a conserved position corresponding to that of intron 1 in the transit peptide region of chloroplast GAPDH genes (GapA and GapB) of higher plants. It has consensus sequences similar to those of yeast introns and folds into a conspicuous secondary structure of - 61.3 kJ. CpG profiles of genes GapC and GapA and their flanking sequences show no significant CpG depletion suggesting that these genomic sequences are not methylated. Genomic Southern blots hybridized with generic and gene specific probes indicate that both genes are encoded by single loci composed of multiple polymorphic alleles. Northern hybridizations demonstrate that both genes are expressed in gametophytes but not in protoplasts where appreciable amounts of transcripts can only be detected for GapC.
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  • 20
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell reports 12 (1993), S. 339-342 
    ISSN: 1432-203X
    Keywords: Alginate embedding ; Beta vulgaris ; feeders ; protoplasts ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The effects of NaCl, feeder cells and the embedding of protoplasts in calcium alginate have been investigated in an attempt to improve culture conditions of recalcitrant sugar beet (Beta vulgaris L.) mesophyll protoplasts. While the use of NaCl in all instances proved detrimental to protoplast development, the other two treatments had clear beneficial effects. Minimum plating densities, necessary to sustain cell division, could be reduced to 〈5% (〈4000 protoplasts / ml) of the control levels and plating efficiencies could be significantly enhanced by approx. 10 fold. Plants could still be regenerated from soft calli derived from mesophyll protoplasts cultured under the modified conditions at a frequency of 20–30 %. In particular, the use of alginate is considered of potentially great importance for the further application of beet protoplasts for other aims e.g. asymmetric hybridization.
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  • 21
    Electronic Resource
    Electronic Resource
    Springer
    Plant and soil 155-156 (1993), S. 143-146 
    ISSN: 1573-5036
    Keywords: barley ; cell wall ; Hordeum vulgare ; pollen selection ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The nature of expression of the tolerance of barley to high levels of B at the cellular level was investigated with a view to identifying ways by which this level of expression might be exploited in a breeding programme. Using protoplasts derived from leaf tissue, it was found that genetic differences between B tolerant and intolerant barleys were not expressed in the absence of cell walls. Barley genotypes differing in their tolerance to B were subjected to high levels of B in the growth medium from pollen formation onwards. The genetic distribution of segregating populations in the next generation was not changed for tolerance to high B. Results also suggested that genetic tolerance to B is expressed by pollen in vitro.
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  • 22
    ISSN: 1573-5044
    Keywords: cell suspension ; Lycopersicon chilense ; plant regeneration ; protoplasts ; wild tomato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protocol has been established for rapid, high frequency plant regeneration from protoplasts of the wild tomato species Lycopersicon chilense Dun. Cell suspension cultures were obtained from calli initiated from seedling stem explants. Protoplasts were isolated from cell suspensions by an overnight one-step enzyme digestion, purified by washing in salts solution and cultured in liquid medium. Dilution of liquid medium every 3 days, with medium containing low levels of growth regulators and sucrose, was critical for sustained colony formation. Up to 70% of protoplast-derived calli regenerated shoots when cultured on agar-solidified medium with Murashige & Skoog (1962) salts and vitamins, 2.0 mg l-1 zeatin and 0.1 mg l-1 indole acetic acid for 21 days, followed by transfer to the same medium lacking indole acetic acid.
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  • 23
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 34 (1993), S. 19-25 
    ISSN: 1573-5044
    Keywords: Ca-alginate ; citrus ; plating efficiency ; protoplasts ; somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts were enzymatically isolated from log phase embryogenic sweet orange [Citrus sinensis (L.) Osbeck cv. Hamlin] suspension cultures, embedded in 3-mm diameter Ca-alginate beads, and cultured in a growth cabinet (15–20 μ mol m-2 s-1, 4-h photoperiod, 27°C). Plating efficiency exceeded 90% for Ca-alginate embedded protoplasts vs. 30% for protoplasts cultured in a liquid medium. Embryoids formed from protoplasts were recovered after 20 days by dissolving the Ca-alginate matrix with a calcium sequestrant. Embryoids readily formed shoots that were rooted on MS+0.01 μM NAA+5% sucrose. Potential applications are discussed.
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  • 24
    Electronic Resource
    Electronic Resource
    Springer
    Hydrobiologia 260-261 (1993), S. 429-436 
    ISSN: 1573-5117
    Keywords: Gracilaria ; protoplasts ; callus-like ; regeneration ; plant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A large number of viable protoplasts were produced by enzymatic digestion of Gracilaria asiatica vegetative tissue. The protoplasts underwent initial division after 5–7 d in culture and developed into callus-like cell-masses. Many filaments grew from the periphery of these cell-masses and disappeared after about one month in culture. Simultaneously, the central part of the callus-like cell-masses thickened and its color deepened. The first buds appeared from the center of the cell-masses and developed into whole plants after three months in aerated culture. Many new buds formed around the first plant and more than 20 plants grew per callus-like cell-masses in less than four months. Filaments taken from callus-like cell-masses developed into young plants after about 20 d of culture.
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  • 25
    Electronic Resource
    Electronic Resource
    Springer
    Hydrobiologia 260-261 (1993), S. 167-172 
    ISSN: 1573-5117
    Keywords: Phaeophyceae ; protoplasts ; apical cells ; immunofluorescence ; microtubules ; cell cycle ; polarity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The growth of the filamentous brown alga Sphacelaria depends on a large, strongly polarized, apical cell. The protoplast derived from this cell can be distinguished in a heterogeneous suspension by cytological markers, so it is possible to study development of the cytoskeleton during protoplast isolation and the first steps of regeneration. In the initial cell, microtubules show an asymmetric distribution along the axis; they are mainly located at the distal part around the physodes. After protoplast isolation, this polarity initially seems to be maintained; subsequently, the microtubules radiate from the two centrioles and spread out to the plasmalemma. This experimental model is suitable for investigating the development of the polarity of the initial cell, and the sequence of the first morphogenetic events leading to protoplast regeneration.
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  • 26
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    Hydrobiologia 260-261 (1993), S. 421-427 
    ISSN: 1573-5117
    Keywords: protoplasts ; Gelidium robustum ; agarophyte ; Rhodophyta
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Viable protoplasts were isolated from apices of the agarophyte Gelidium robustum (Gardn.) Hollenb. & Abb. using a combination of commercial cell-wall degrading enzymes and extracellular wall-degrading enzymes isolated from a marine bacterium. The protoplasts were approximately 8–15 µm in diameter, liberated mainly from the surface cell layers and from cells at the distal ends of medullary filaments. The bacterial enzyme alone was not sufficient to liberate significant numbers of protoplasts. Maximum yield was 9 × 105 protoplasts/g tissue (wet wt.). Optimum osmolality occurred between 1750–1950 mOs kg−1; yield and viability were severely diminished at osmolalities less than 1350 mOs kg−1. Viability, as determined by flurorescein diacetate staining and Evans Blue exclusion 1 hr after removal from the enzyme solution, was approximately 80–95%. Roughly 80% of the cells did not show Calcofluor fluorescence, while 40% stained positively for the presence of sulfated polysaccharides. Cell wall regeneration was observed with inconsistent reproducibility, and no cell division was observed when the protoplasts were placed in culture medium.
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  • 27
    ISSN: 1572-9699
    Keywords: Candida albicans ; chitin synthetase ; digitonin ; proenzyme activation ; protoplasts ; solubilization ; zymogen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) fromC. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen,in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processesin vivo andin vitro or, alternatively, that both enzyme activities (activein vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml−1 of digitonin and 64 µg ml−1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 µg ml−1 of protease, whereas activity strongly decreases in the presence of 64 µg ml−1 of trypsin. Digitonin does not produce zymogen activationper se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.
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  • 28
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    Plant cell, tissue and organ culture 33 (1993), S. 315-320 
    ISSN: 1573-5044
    Keywords: protoplasts ; plant regeneration ; woody ornamentals ; Weigela
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Strategies were developed for the successful isolation of large numbers of highly viable protoplasts from the leaves, stems and roots of axenic plants of the hybrid ornamental shrubWeigela ×florida cv Bristol Ruby. Protoplasts, of all sources, were cultured on different media, leading to the establishment of sustained divisions, and coupled with the production of multi-celled (〉50 cells) colonies. However, those colonies derived from mesophyll protoplasts only were capable of a further proliferation to the callus stage. Upon transfer to a regeneration medium consisting of MS salts and organics plus a range of concentrations of NAA and BAP, such calli underwent caulogenesis, with optimum responses for a medium with 1.0 mg l−1 NAA and 1.0 mg l−1 BAP. The protoplast-derived shoots thus obtained were multiplied on MS medium with 0.1 mg l−1 IBA, 0.5 mg l−1 BAP and 0.1 mg l−1 GA3. Individual shoots were subsequently rooted on a half-strength MS medium plus 3.0 mg l−1 IBA, and complete protoplast-derived plants were finally transferred to the glasshouse for acclimatization.
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  • 29
    ISSN: 1573-5044
    Keywords: Arachis species ; nurse culture ; plant regeneration ; protoplasts ; tissue culture ; wild peanut
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An efficient protocol has been developed for protoplast culture and plant regeneration from wild peanut (A. paraguariensis) using a nurse culture method. Protoplasts were isolated from suspension cultures initiated from leaf-derived callus, imbedded in agarose blocks and co-cultured with nurse cells of the same species. Up to 10% of the protoplasts divided and formed compact callus colonies. The protoplast plating efficiency was correlated with both the length of the nurse cell co-cultivation period and the protoplast plating density. The optimal nurse culture duration was 14 d. The optimal plating density was 2×104 protoplasts/ml plating medium. Multiple shoots (up to 10 shoots per colony) were readily regenerated from protoplast-derived callus after transfer of callus to semi-solid modified MS medium containing 0.5 mg l-1 NAA and 1 mg l-1 BA. Plantlets with normal leaflets were obtained by rooting shoots on porous rootcubes saturated with modified MS medium containing 1 mg l-1 NAA.
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  • 30
    ISSN: 1432-203X
    Keywords: Triticum aestivum ; embryogenic suspension cultures ; protoplasts ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Highly regenerable callus cultures have been obtained from immature embryos of hexaploid wheat cv. Oderzo. Friable fast growing calli were induced at high frequency. Suspensions were initiated from the most friable callus lines: they became established in about two months. Suspensions consisted of cell aggregates of 30 to 1000 um in diameter. Upon plating on MS hormone-free medium, suspensions regenerated green plantlets, and their regenerative capability was maintained for at least 10 months. Protoplasts were isolated from 7–8 day old suspension cultures with a yield of 4–6×106 protoplasts/g fresh weight cells. Protoplast culture was either in liquid medium or in a bead-type system with agarose beads. First divisions were detected at day 5. At day 14 visible colonies were detected and the plating efficiency was evaluated between 2 and 8% over the initial number of protoplasts plated. Protoplast-derived calli were cultured in the presence of 1 mg/l IAA and 0.5 mg/l zeatin and were used for reinitiating new suspension cultures. Upon plating onto MS hormone-free medium, with or without the addition of 0.1 mg/l GA3, calliclones were induced to differentiate. Regeneration of complete plantlets, with shoot and roots took about two months. Plantlets were grown in sterile conditions until 12–15 cm height, and were subsequently transplanted in soil.
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  • 31
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    Plant molecular biology 18 (1992), S. 815-818 
    ISSN: 1573-5028
    Keywords: expression cassette ; gene expression ; protoplasts ; translation initiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression cassettes containing a duplicated cauliflower mosaic virus (CaMV) 35S promoter fused to a polylinker preceded by the CCACCATGG and AACAATGG sequences were constructed. These two sequences correspond to the consensus sequences around the translation start codons in vertebrates and plants respectively. Translational fusions were made with the β-glucuronidase-coding sequence and transient expression was recorded in tobacco mesophyll protoplasts. Approximately three times more GUS activity was found in protoplasts incubated with the constructs harbouring translational fusions as compared to a control harbouring a transcriptional fusion. No significant difference was observed between GUS activities obtained with the two consensus sequences.
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  • 32
    ISSN: 1573-5028
    Keywords: glutamine synthetase ; hairy roots ; nodules ; Phaseolus vulgaris ; protoplasts ; transcriptional regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The 5′-flanking region of gln-γ, the nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L., has been analysed for cis-regulatory elements using a series of 5′ deletions and hybrid gln-γ: : CaMV 35S promoters. The promoters were fused to the uidA reporter gene and their activities tested in two heterologous expression systems. In the first system, the chimaeric genes were transferred to Lotus corniculatus L. using Agrobacterium rhizogenes and their expression was studied in nodulated hairy roots. In the second system, the constructs were electroporated into tobacco mesophyll protoplasts. The results of the 5′ deletion analysis showed that the sequence between −597 and −21 (relative to the ATG codon) was sufficient for nodule-specific expression of the chimaeric gene in nodulated hairy roots, and revealed the existence of at least two positive regulatory elements. Sequences located between −2000 and −597 were able to stimulate expression in nodules but not protoplasts, while the region from −597 to −354 enhanced expression in both nodules and protoplasts. Results obtained with the hybrid gln-γ: :35 S promoters showed that two overlapping restriction fragments (−516/−343 and −474/−293) were able to stimulate expression from a heterologous promoter in an orientation-dependent manner. Previous work has demonstrated the presence of conserved A/T-rich binding sites for nuclear proteins in the region between −516 and −446, and their possible role in regulating gln-γ expression is discussed.
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  • 33
    ISSN: 1573-5028
    Keywords: promoter ; electroporation ; protoplasts ; transient assay ; Agrobacterium ; Ti plasmid ; Zea mays
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Regulatory elements controlling transcriptional activity of the mannopine synthase 2′ promoter (mas 2′) were defined by analysis of deletion mutants in transient expression assays in maize protoplasts. Deletion of the region between −305 and −290 containing sequence similarity to the octopine synthase (ocs) promoter element reduced activity by 67% compared to wild type activity. Less than 1% of the activity remained in 5′ deletions downstream of −153. Inclusion of various heterologous enhancer-like sequences immediately upstream of position −325 increased activity by up to 7.5-fold. Insertion of the −325 to −275 sequence alone, or in combination with heterologous enhancer-like elements, restored activity of some of the 5′-deletion mutants. Restoration of activity was not obtained with mutants deleted past position −127. Our results suggest that a single class of nuclear proteins from maize interact with high affinity at elements designated mas b (−306 to −275; mas 1′ element), d (−127 to −108), and e (−82 to −39; mas 2′ element) as well as the 20 bp element from the ocs promoter. Although the binding site at mas d only appears to accommodate a single protein, this element has the potential to make a weak, but positive, contribution to the activity of the mas 2′ promoter. The binding of nuclear proteins could not be demonstrated at mas a and c, both of which showed limited homology to the ocs element. Mutational evidence suggested that mas a and c may also contribute to mas 2′ transcription.
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  • 34
    ISSN: 1573-5028
    Keywords: DNA methylation ; geminivirus ; protoplasts ; tomato golden mosaic virus ; transfection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m5C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m5C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.
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  • 35
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    Plant molecular biology 19 (1992), S. 859-862 
    ISSN: 1573-5028
    Keywords: luciferase ; cis-DNA element ; GUS ; protoplasts ; PEG
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The alcohol dehydrogenase (Adh) gene of Arabidopsis is expressed constitutively in immature seedlings and cells in suspension, and may be induced by hypoxic stress only in roots of mature plants. Deletions and G-box mutations of the Adh promoter were assayed in Arabidopsis protoplasts by PEG-mediated transient expression. Sequence domains necessary for full gene activity are confined to the 384 bp immediately 5′ to the transcription start site, and deletion to −177 results in 〉90% reduction in promoter activity. Site-specific mutations of G-box bases result in 〉60% reduction in activity and disrupt G-box factor binding in vitro.
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  • 36
    ISSN: 1573-5028
    Keywords: granule-bound starch synthase ; potato ; protoplasts ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the β-glucuronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient to result in tissue-dependent GUS expression: levels in stably transformed microtubers exceeded levels in corresponding leaves by orders of magnitude. GBSS-GUS constructs could be transiently expressed in leaf protoplasts from wild-type and amylose-free potato lines, etuberosumSolanum brevidens, Nicotiana tabacum andArabidopsis thaliana. Transient expression levels in potato leaf protoplasts were clearly lower than in corresponding suspension cell protoplasts. This lower expression in leaf protoplasts could not be elevated by increasing DNA concentrations during transfection. Light incubation of electroporated suspension cell protoplasts reduced transient GBSS-GUS expression, whereas incubation of transfected protoplasts in media with different sucrose concentrations did not affect transient expression levels. However, electroporated protoplasts, isolated from suspensions, which had been grown on media with increasing amounts of sucrose showed a sucrose concentration-dependent transient expression profile. This indicates that studying GBSS regulation by transient expression experiments needs pre-treatment of the protoplast source. Sequence data of the GBSS promoter were compared to those of two other potato alleles.
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  • 37
    ISSN: 1573-5176
    Keywords: Ulva ; Enteromorpha ; protoplasts ; electrofusion ; intergeneric fusion ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Isolated protoplasts of Ulva pertusa and Enteromorpha prolifera were electrically fused. Treatment of protoplasts in 1% protease for 15–20 min prior to fusion enhanced fusion ability. Protoplasts from each fusion partner were mixed together in 1:1 ratio in low conductivity electrofusion solution at a density of 1 × 105 cells ml−1 before subjecting them to electrofusion. The protoplasts were aligned in AC field (1MHz, 25 V for 10–15 s) and subsequently fused by a high intensity single DC pulse of 250 V for 25 μs duration. Fusion buffer supplemented with 1 mM calcium and 1 mM magnesium yielded optimum fusion frequencies (about 18–24%). Entrapment of fusion treated cells inside agarose/agar plate facilitated marking and regeneration of fusion products. The regeneration patterns of fused protoplasts were similar to normal (unfused) protoplast development. Most of the regenerated plants from fusion products had a thallus similar to either U. pertusa type or E. prolifera type. Although some of the plants of the former were morphologically similar to U. pertusa, but most had a higher growth rate (1.9 to 1.5 times) than U. pertusa. Furthermore the thallus of some plants had a characteristic irregular and dentate margin, which was never observed in the parental type.
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  • 38
    ISSN: 1573-5060
    Keywords: Solanum tuberosum ; S. torvum ; Verticillium dahliae ; protoplasts ; electrofusion ; somatic hybrids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Interspecific somatic hybrid plants were regenerated after electrofusion of mesophyll protoplasts with the objective of transferring resistance to Verticillium dahliae from Solanum torvum into potato. Early selection of the putative hybrids was based on differences in cultural behaviour of the parental and hybrid calli (particularly the ability of the latter to regenerate early) in combination with morphological markers. Four putative hybrids were recovered from hundreds of calli, probably resulting from complementation of the two parental genomes. The regenerates were tetraploids (2n=4×=48 chromosomes) and exhibited intermediate traits including leaf form, plant morphology and the presence of anthocyanin. The hybrid nature of the four selected plants was confirmed by examining isoenzyme patterns for isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), phosphoglucoisomerase (Pgi) and 6-phosphogluconate dehydrogenase (6-Pgd). While the hybrid plants rooted readily and grew vigorously under in vitro conditions, in the greenhouse their development and growth were retarded by difficulties in rooting. When grafted on potato or S. torvum rootstocks, the hybrid plants recovered normal development and growth. Again, they exhibited intermediate morphological traits. Tests for resistance realized in vitro with medium containing 50% Verticillium wilt filtrate showed that all the somatic hybrids were resistant to the fungus filtrate.
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  • 39
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    World journal of microbiology and biotechnology 8 (1992), S. 500-504 
    ISSN: 1573-0972
    Keywords: Cunninghamella elegans ; hydroxylation ; protoplasts ; steroids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Protoplasts ofCunninghamella elegans, showing 11α-, and 11β-hydroxylating ability of Substance S, preserved high transformation activity when dispersed in glucose-enriched, organic osmotic stabilizers. A joint action of polyoxins and 2-deoxy-d-glucose was necessary to prevent regeneration of the cell wall in long-lasting experiments. Stabilized and active, dispersed protoplasts may be an alternative research model for studying the function of the cell wall and intracellular metabolic pool constituents in steroid hydroxylation.
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  • 40
    ISSN: 1573-5044
    Keywords: flow cytometry ; plant regeneration ; protoplasts ; strawberry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A reproducible and efficient protoplast to plant-system has been developed for strawberry. By using young shoots growing on medium supplemented with cytokinin we were able to exclude the detrimental enzyme Pectolyase from our enzyme solution. The more gentle isolation procedure reported in this paper, together with the agarose-embedding system had a pronounced effect on protoplast viability and growth. Plant regeneration was enhanced by using thidiazuron instead of benzyladenine. Flow cytometric measurement of the DNA content in protoplast derived plants revealed that 27 out of 51 plants contained the amount of DNA corresponding to octoploid level. Fifteen plants exhibited chromosome doubling(16×), one was diplodecaploid(12×) and eight plants mixoploid.
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  • 41
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    Plant cell, tissue and organ culture 30 (1992), S. 69-75 
    ISSN: 1573-5044
    Keywords: chloramphenicol acetyltransferase ; electroporation ; Eucalyptus citriodora ; protoplasts ; transient gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts isolated from cotyledons of Eucalyptus citriodora were electroporated using a rectangular pulse, with plasmid carrying the cat gene. The levels of transient expression and protoplast viability were influenced by the voltage and pulse duration. At a field strength of 800 V cm-1 (1000 μs), a protoplast viability of 57%, and 47% conversion of 14C-chloramphenicol to its acetylated forms, were obtained. Expression levels were improved by an increase in plasmid concentration (up to 60 μg ml-1), and also by the addition of carrier DNA. Gene expression was further enhanced by the addition of 40% (w/v) PEG, in the presence of the carrier DNA, to the protoplasts after electroporation.
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  • 42
    ISSN: 1573-5044
    Keywords: amiprophosmethyl ; isolated nuclei ; micronuclei ; organelle uptake ; polyethylene glycol ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Nuclei isolated from tobacco protoplasts were induced to be taken up by soybean protoplasts using a protocol involving polyethylene glycol (PEG), osmotic shock and pH shift. Transmission electron microscopy revealed that PEG treatment condensed the chromatin of the isolated nuclei. Close adhesion of isolated nuclei to the plasma membrane of protoplasts following PEG treatment, was observed by both scanning and transmission electron microscopic methods. Ultrastructural observations were also made on the formation of micronuclei in tobacco cells following the treatment with amiprophosmethyl (APM). Nuclei and micronuclei isolated from APM-treated cells were induced to be taken up by soybean protoplasts. A single case of uptake of an isolated micronucleus was observed by transmission electron microscopy. The observations on the effects of PEG on the isolated nuclei, micronuclei and protoplasts are discussed in relation to the possible mechanism of uptake of nuclei by protoplasts using PEG.
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  • 43
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    World journal of microbiology and biotechnology 8 (1992), S. 39-41 
    ISSN: 1573-0972
    Keywords: Pleurotus florida ; laccase ; para-constitutive ; protoplasts ; mutagenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Pleurotus florida produced high amounts of laccase (4.60 U/ml) in malt extract broth after 12 days' growth under stationary conditions. The production of laccase was semi-constitutive. Hyperlaccase mutants ofP. florida were obtained through mutagenesis of mycelial protoplasts usingN-methyl-N′-nitro-N-nitrosoguanidine (50 μg/ml) for 2 min. Three hyperlaccase mutants were selected showing growth and enzyme production responses similar to the parent.
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  • 44
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    World journal of microbiology and biotechnology 8 (1992), S. 385-386 
    ISSN: 1573-0972
    Keywords: Lysozyme ; protoplasts ; regeneration ; Spirulina platensis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Protoplasts were obtained from the filamentous blue-green algaSpirulina platensis by treating the filaments with 0.05% (w/v) lysozyme in 0.03m phosphate buffer. The protoplasts regenerated cell walls and formed colonies when plated on a regeneration medium. The highest percentage of regeneration, 40% was obtained after 21 days.
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  • 45
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992), S. 39-45 
    ISSN: 0749-503X
    Keywords: Flow cytometry ; autolytic mutants ; protoplasts ; yeast ; viability assay ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Simple methods, based on the technique of flow cytometry, have been developed for the phenotypic characterization of yeast autolytic mutants and for the analysis of the formation and regeneration of the yeast protoplasts. The expression of lytic mutations determined uptake of the fluorescent dye propidium iodide, which could be carefully monitored by flow cytometry. Mixed populations of lysed and viable cells were precisely quantified and sorted, and the technique was also applied to demonstrate protection from lysis of mutant cells with cell wall defects, in the presence of osmotic stabilizers. Protoplast formation and regeneration was monitored by analysing relative cell size; this was facilitated by the preparation of homogeneous protoplast preparations. The technique of flow cytometry proved superior to other conventional methods for these types of study.
    Additional Material: 6 Ill.
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  • 46
    ISSN: 1573-5028
    Keywords: methylation ; Oryza sativa ; protoplasts ; transformation ; β-D-glucuronidase ; methotrexate ; hygromycin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cell suspension-derived rice (Oryza sativa L.) protoplasts were transformed by direct gene uptake. PEG-mediated transformation was more efficient than electroporation. Plasmid DNA containing a hygromycin phosphotransferase (HPT) gene (which confers hygromycin resistance) driven by the CaMV 35S promoter and a β-D-glucuronidase (GUS) gene under control of the 1′, 2′ double promoter of the mannopine synthase (mas) locus of Agrobacterium tumefaciens was introduced into rice protoplasts. Southern analysis of DNA from transformed cell lines showed that the HPT and GUS genes were present intact. Both genes were expressed in transgenic cell suspensions. GUS activity was detected by histochemical staining of the cells and by enzyme assays. During a 12-day culture period the proportion of stained cells rose to a maximum and then decreased again. Considerably higher numbers of blue-stained cells were obtained when the transgenic cell lines were grown in the presence of 5-azacytidine. Transcripts of the GUS gene could not be detected, in contrast with the HPT gene. Plantlets were regenerated from one transgenic cell line. GUS activity was found in both leaf and root tissues of these plants, particularly, but not exclusively, in vascular bundles. A mouse dihydrofolate reductase coding sequence (DHFR), conferring methotrexate resistance, fused to the CaMV 35S promotor and the wild-type nopaline synthase (NOS) gene of A. tumefaciens were also introduced into rice protoplasts. Stable integration of both genes was confirmed by Southern analysis. Expression of the DHFR gene was demonstrated by high levels of resistance to methotrexate of the transgenic cell suspensions and by the presence of DHFR transcripts. Expression of the NOS gene at enzyme or RNA level was not detected. Southern analysis suggests that this gene was probably either methylated or scrambled in these lines.
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  • 47
    ISSN: 1573-5028
    Keywords: Actinidia deliciosa ; chloramphenicol acetyl transferase ; gas chromatography-mass spectrometry ; ion trap detector ; polyethylene glycol ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 °C, 5 min) prior to transfection.
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  • 48
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    Journal of applied phycology 3 (1991), S. 265-275 
    ISSN: 1573-5176
    Keywords: Enteromorpha ; protoplasts ; regeneration ; cell density ; axenic culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract High yields of protoplasts have been obtained from vegetative thalli of three species ofEnteromorpha by enzymatic degradation of the cell wall. Several commercial and crude enzymes prepared from the digestive system and hepatopancrease of abalone and top-shell were tested at different concentrations and combinations to evaluate the yield. Commercial enzymes in combination with either abalone or top-shell crude enzymes, consistently produced a high yield of protoplasts from all three species. High regeneration rate (85–95%) occurred in the protoplasts cultured at a density greater than 1.72 × 103 cells cm−2 at 20 and 25°C. Light intensities tested in the present study did not affect protoplast wall formation and regeneration. Protoplasts, after regenerating the cell wall, followed different types of developmental patterns under identical culture conditions. In one type some cells underwent repeated cell divisions and formed a round and oval shaped hollow thallus with a single layer of cells. In the second type many cells underwent one or two cell divisions (occasionally no division) and soon matured and discharged many motile spores, which on germination grew into normal plantlets. In the third type some cells divided irregularly to form a mass of callus-like cells (exceptE. prolifera). Culture medium supplemented with either mannitol, sorbitol, dextrose, saccharose or NaCl at higher concentrations (〉 0.4 M) inhibited cell division and further differentiation in all species.
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  • 49
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    Plant cell, tissue and organ culture 24 (1991), S. 43-47 
    ISSN: 1573-5044
    Keywords: Brassica napus ; protoplasts ; purification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effects of three different general purification protocols have been assessed quantitatively using mesophyll protoplasts of Brassica napus. Within the initial sample two distinct sub-populations were determined. The methods used influenced the ratio of the vacuolated to chloroplastic type protoplast sub-populations. Overall recovery rates of the initial sample varied according to the method used from 38% to 27%, but the relative recovery of the sub-populations varied considerably with a purified ratio of between 1.0:0.78 to 1.0:7.0. Size distribution profiles of the initial and purified populations are also presented.
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  • 50
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    Plant cell, tissue and organ culture 25 (1991), S. 27-33 
    ISSN: 1573-5044
    Keywords: cytology ; leaf explants ; Lotus corniculatus ; protoplasts ; regeneration ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Regenerants from three types of tissue, leaf explants (132 plants), leaf protoplasts (68 plants) and cotyledonary protoplasts (119 plants) of L. corniculatus cv Leo differed both morphologically and cytologically from control plants grown from seed. Four categories of chromosome number were found. The frequency and type of variation found in the chromosome numbers of regenerants reflected the method of plant regeneration. Regenerants with both normal and abnormal numbers of chromosomes produced progeny which were cytologically normal and showed only minor morphological changes when compared with control plants.
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  • 51
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    Plant cell, tissue and organ culture 27 (1991), S. 243-248 
    ISSN: 1573-5044
    Keywords: callus culture ; cell suspensions ; protoplasts ; Salix viminalis ; Salix schwerinii
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts were isolated from cell suspensions of Salix viminalis (basket willow) clone 78-0-90 and S. schwerinii clone 77-0-77, using cellulysin and macerase in modified Woody Plant medium. For clone 78-0-90, 6.3 · 106 ± 1.9 · 106 protoplasts were obtained per gram fresh weight. Cell divisions started two days after protoplast isolation and gave rise to callus which has been maintained in culture for up to four years. Protoplast yield from the clone 77-0-77 was lower (less than 106 protoplasts per gram cells), cell division was infrequent and no callus was obtained. Protoplasts were also isolated from the leaves of willow shoot cultures using cellulysin and pectolyase, but these did not show cell divisions.
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  • 52
    ISSN: 1432-203X
    Keywords: Somatic embryogenesis ; protoplasts ; Brassica nigra ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A method to obtain plants from embryogenic callus of Brassica nigra and protoplasts of hypocotyl expiants is described. Callus was initiated on Murashige and Skoog medium containing kinetin (kn) and 2,4-dichlorophenoxy acetic acid (2,4-D). Lowering of auxin induced embryo formation. Supplementation with gibberellic acid (GA3) enhanced embryogenic response tenfold. Passage through liquid medium devoid of growth regulators was essential for the growth of embryos. Secondary embryos were produced on transfer to solid basal medium. Embryogenic callus retained its morphogenic ability even after 12 subcultures. Both primary and secondary embryos produced fertile plants. Hypocotyl-derived protoplasts were also regenerated to plants following the same protocol. The survival of plants on transfer to soil was about 80%. The seeds from plants derived from callus and protoplasts were viable.
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  • 53
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    Plant molecular biology 15 (1990), S. 485-496 
    ISSN: 1573-5028
    Keywords: protoplasts ; P.R. proteins ; tobacco ; wounding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have used 2-dimensional (2D) non-equilibrium pH gradient gel electrophoresis (NEPHGE) of in vitro synthesized proteins and northern hybridization with labelled cDNAs coding for three pathogenesis related (P.R.) proteins, to analyze the shift in mRNA content induced by the isolation and culture of tobacco mesophyll protoplasts. The in vitro protein pattern of mRNAs from freshly isolated protoplasts is characterized by the absence of most leaf spots and the appearance of 19 new spots. After 6 hours of culture, the mRNAs coding for the P.R. proteins become detectable and after 12 hours the protoplasts contain an mRNA population almost typical of callus cells. The different steps involved in the isolation and culture of protoplasts were analysed. Cutting off the leaf and sterilization do not change the mRNA set. In contrast, the mechanical injury applied to the leaf in order to facilitate the penetration of the enzymatic mixture induces a modification of the mRNA content identical to that resulting from protoplast isolation. Wounding is the essential event inducing dedifferentiation. Varying the culture medium and conditions leads to only limited modifications of the mRNA pattern. These results are discussed on the basis of present knowledge of the reaction of the plant to wounding and we suggest that wound healing callus and in vitro callus correspond to the same differentiation state.
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  • 54
    ISSN: 1573-5028
    Keywords: maize gene expression ; protoplasts ; transient assays ; transcriptional regulation ; zein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three DNA regions required for high levels of transcription were identified by transient gene expression analysis of the 5′ flanking region of a 19 kDa α-zein gene. For these analyses, the zein promoter region was fused to the β-glucuronidase (GUS) gene and assayed by transient expression in carrot protoplasts. A 107-bp sequence (-114/-8) containing the TATA box resulted in low levels of GUS activity. Addition of the proximal 75 bp (-189/-114) doubled the level of GUS expression, and a further increase in expression was obtained when additional upstream sequences (-483/-226) were placed 5′ of the zein promoters. Zein upstream sequences enhanced transcription independently of the-189/-114 region. Although the-189/-114 region was not essential for transcription, it was important to obtain maximum GUS activity. A 121 bp upstream sequence (-347/-226) that contains the conserved TGTAAAG sequence gave high levels of GUS activity when placed in either orientation 5′ of the zein promoter sequences. However, nucleotides-347 to-309, containing the TGTAAAG sequence, could be deleted from this fragment without a significant change in GUS activity. Zein upstream sequences did not promote transcription of the GUS gene in somatic maize protoplasts. The upstream activating sequence from the cauliflower mosaic virus (CaMV) 35S promoter placed 5′ of deletion mutants of the zein promoter also failed to produce GUS activity above background.
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  • 55
    ISSN: 1573-5028
    Keywords: inheritance ; NPTII gene ; protoplasts ; S phase ; 3-aminobenzamide ; UV irradiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Freshly isolated haploid mesophyll protoplasts of Nicotiana plumbaginifolia were transformed for kanamycin resistance. In 38% of the 224 transformants analysed, transmission of the NPTII gene occurred as a homozygous trait, while 62% of the transformants were heterozygous for the trait. In the first case, the foreign DNA integration predominantly (95%) resulted in monogenic inheritance. The second group was characterized by a significant (46%) proportion of multiple insertions. However, there was no clear-cut difference in the integration pattern between the two groups. Furthermore, transformation rates were increased by 4- to 10-fold when transformed diploid protoplasts were treated with UV light or with 3-aminobenzamide. The number of insertion sites was also increased by these treatments. These results shed further light on the fate of the foreign DNA in transformed plants and on means to control or manipulate the integration event(s).
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  • 56
    ISSN: 1573-5028
    Keywords: expression vector ; heat shock ; protoplasts ; transient assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A heat-inducible expression cassette has been constructed to study the conditional expression of sense or antisense orientations of any sequence of interest in transgenic plants or plant tissues. The construct includes the promoter and all but 5 bases of the mRNA leader from the soybeanGmhsp17.5-E gene, the polylinker from pUC18 (modified to remove the ATG), and a fragment that contains the polyadenylation signal and site from the nopaline synthase gene. Analysis of transient expression of a construct containing the β-glucuronidase (GUS) coding sequence cloned in the cassette and introduced intoNicotiana plumbaginifolia protoplasts by electroporation shows that the promoter has high expression at heat shock temperatures. This construct is expressed at a roughly 80-fold higher level per unit time than a cauliflower mosaic virus 35S gene promoter-GUS construction. The heat shock promoter is regulated positively by supercoiling in this transient assay system. The level of expression of HS-GUS constructions with the polyadenylation sites from either the nopaline synthase gene or theGmhsp17.5-E gene was similar. Constructs with a perfect fusion at the 5′ end had higher levels of expression than those with the corresponding nonperfect transcriptional fusion.
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  • 57
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    Euphytica 47 (1990), S. 39-44 
    ISSN: 1573-5060
    Keywords: Vitis vinifera ; grape vine ; leaves ; protoplasts ; cell wall regeneration ; division
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Experimental conditions were established that resulted in high yields and good viability of the protoplasts obtained from leaves of Vitis vinifera, cv. Chardonnay regenerated in vitro by somatic embryogenesis. The effect of factors of the culture medium and various environmental conditions upon the frequency of cell division has been examined, and a method of culture is described by which protoplasts were induced to begin division. Most protoplasts obtained in this way regenerated cell walls within the first few days and cell division occurred after 10 days of culture in a liquid medium. Some cells have divided two or even three times. Nevertheless, the cells did not continue dividing beyond this stage.
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  • 58
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    Hydrobiologia 204-205 (1990), S. 409-413 
    ISSN: 1573-5117
    Keywords: amoeba ; grazing ; enzymatic induction ; protoplasts ; seaweeds ; Trichosphaerium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A crude enzyme preparation from the marine amoeba Trichosphaerium was used to produce protoplasts from Sargassum muticum, Macrocystis pyrifera Porphyra perforata, and other red and brown marcroalgae. Cortical and medullary protoplasts of Sargassum, which were impossible to obtain using mixtures of previously available enzymes have now been prepared. Intact inner cortical and medullary protoplasts of Macrocystis, which were not observed in past isolations were obtained. Improved protoplast yields of as much as 500 fold resulted from feeding the amoebae on specific seaweeds. Cuticles of live Sargassum and Macrocystis were digested easily by the amoebae. However cuticles of autoclaved Macrocystis and those of Porphyra (fresh or autoclaved) were eaten last. In spite of the absence of identifiable extracellular enzymatic activity in the medium the amoebae were able to ‘cut’ and consume live fronds and blocks of gelled agars carrageenans, and alginates.
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  • 59
    ISSN: 1573-5044
    Keywords: Brassica oleracea ; protoplasts ; regeneration ; cytoplasmic traits ; mitochondrial DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Nearly 1000 plants have been regenerated from leaf protoplasts of two cauliflower (Brassica oleracea ssp.botrytis) alloplasmic inbred lines. One line (7642A) carried the Ogura (R1) cms cytoplasm derived from radish; the other line (7642B) carried a normalBrassica cytoplasm and was the fertile maintainer for the cms line. The majority of regenerated plants displayed normal vegetative morphology; they formed normal cauliflower heads and retained the floral characteristics of seed-grown plants from which they were derived. We found no change in either male sterility or in the low temperature-induced chlorosis associated with the 7642A line. Mitochondrial DNA analysis by hybridization with five cloned mtDNA probes revealed no apparent alteration in 75 regenerated plants of both lines. These results indicate that cytoplasmic traits inBrassica oleracea are stable after one cycle of in vitro culture and regeneration.
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  • 60
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    Plant cell, tissue and organ culture 23 (1990), S. 181-186 
    ISSN: 1573-5044
    Keywords: heritability ; protoplasts ; RFLPs ; Solanum tuberosum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The genetics of tissue culture response in potato has been examined by analysing a sample of dihaploids (2n=2x=24) extracted from tetraploid parents (4n=4x=48). The genotypes were screened for rate of nodal multiplication, in vitro tuberisation, regeneration from leaf discs and protoplast plating efficiency. Significant differences were detected between dihaploids for the traits measured and this indicates that tissue culture response in the tetraploid parents must be in the heterozygous condition. Estimates of the broad sense heritabilities were calculated together with the number of genes or effective factors involved in the control of the traits. These estimates indicate that tissue culture response in potato is under relatively simple genetic control and “blocks of genes” may be located on specific chromosomes. The inheritance of RFLP markers in the segregating dihaploid population was also monitored and the potential of using molecular markers linked to gene(s) controlling tissue culture response is discussed.
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  • 61
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    Plant cell, tissue and organ culture 20 (1990), S. 75-79 
    ISSN: 1573-5044
    Keywords: Lens culinaris ; protoplasts ; osmoticum ; agarose ; callus formation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts of Lens culinaris Medik. cv. Eston were isolated from epicotyl tissues of seedlings grown on Murashige & Skoog basal medium. For isolating the protoplasts, epicotyl tissues were digested for 12–14 h at 25°C in an isolation mixture (pH 5.7) containing 1% Cellulase RS, 0.5% Driselase, 0.25% Pectolyase Y23, 0.2M calcium chloride, 10 mM mannitol and 10 mM MES. Protoplasts were purified by flotation over 20% sucrose and washed with 0.2 M calcium chloride solution supplemented with 10 mM mannitol. Purified protoplasts were cultured at a density of 105 ml-1 in agarose (Seaplaque, 0.6%) blocks which were suspended in an identical but liquid KM8P culture medium lacking amino acids, ammonium nitrate, and coconut water but containing 0.35 M glucose and a growth regulator complement of either 2.2 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 2.7 μM naphthaleneacetic acid (NAA), 2.3 μM N-(2-furanylmethyl)-1H-purine-6-amine (kinetin), 2.2 μM benzylamino purine (BAP), 2.3 μM 2-methyl-4-(1H-purine-6-ylamino)-2-buten-1-ol (zeatin), and 1.4 μM gibberellic acid (GA3), or 5.4 μM NAA and 2.2 μM each of 2,4-D and BAP. The osmotic potential of the liquid culture medium was gradually reduced over a period of 3 weeks by replacing the spent medium with a fresh medium containing 0.25, 0.1 and 0 M glucose at weekly intervals. About 6% of the dividing protoplasts developed into cell colonies after 3 weeks of culture at 25°C in diffuse light (10 μE m-2s-1). In 35–42 days the microcolonies were about 1 mm in diameter and developed into calli on transfer to agar-solidified B5 medium supplemented with growth regulators used in the protoplast culture medium and 5 mM glutamine. Attempts to regenerate plants from protoplast-derived calli have so far been unsuccessful.
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  • 62
    ISSN: 1573-5044
    Keywords: calcium ; Lycopersicon esculentum ; myo-inositol ; photoperiod ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Plants from four cultivars of Lycopersicon esculentum were grown under different conditions, in controlled environment chambers. Low light intensity, long photoperiod (16 h), 25° C/17°C temperature alternance (day/night) were found to be the most convenient conditions for obtaining viable protoplasts. The use of myo-inositol as an osmoticum in the digestion medium and the adjustment of the pH to 6.5, instead of the usual 5.8, for this medium increased the yield of viable protoplasts and enhanced their stability. Under these conditions neither pretreatment (dark and cold treatments), nor preplasmolysis of leaf tissues, were required before protoplast isolation. The concentrations of ammonium nitrate, calcium chloride, myo-inositol, and sucrose were found to be critical for the success of protoplast culture. A medium containing 5 mM ammonium nitrate, 40 mM calcium chloride, 10 mg l-1 adenine sulfate, 0.5% myo-inositol and 6% sucrose gave sustained protoplast divisions. Under these conditions, plating efficiency ranged from 5% for the cultivar Lukulus to 15% for the cultivar Golden Sunrise.
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  • 63
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    Plant cell, tissue and organ culture 23 (1990), S. 107-114 
    ISSN: 1573-5044
    Keywords: electrofusion ; grape ; polyethylene glycol ; protoplasts ; vacuoles
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cultured grape cells accumulate anthocyanins in vacuoles rather than secreting them into the nutrient medium. Therefore, grape cells that contain tonoplast segments in their plasmalemma should be capable of excreting anthocyanins rather than sequestering them in their vacuoles. In initial attempts to construct such ‘novel’ cells, small vacuoles were fused with the plasmalemma of cultured plant cells. Protoplasts were isolated from grape calluses that produce and accumulate anthocyanins. Small vacuoles were formed by gently rupturing vacuoles isolated from grape protoplasts. Although small vacuoles and protoplasts became aligned in an AC field, the tonoplast and plasmalemma did not readily fuse when subjected to 3 DC pulses of 1200 V cm−1 for 50 μs each. Changes in the intensity, number and/or duration of the DC pulses had no effect on the fusion process. When 1.0% polyethylene glycol was added to the electrofusion buffer, however, small vacuoles and protoplasts fused within a few minutes after the DC pulses were applied. These ‘novel’ grape cells remained viable for several hours.
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  • 64
    ISSN: 1573-0832
    Keywords: dermatophyte ; Microsporum gypseum ; Novozyme ; protoplasts ; regeneration ; viability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: 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
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    Methods in cell science 12 (1989), S. 127-133 
    ISSN: 1573-0603
    Keywords: polyethylene glycol ; transformation ; protoplasts ; Nicotiana
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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
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    Plant cell reports 8 (1989), S. 313-316 
    ISSN: 1432-203X
    Keywords: Zea mays L. ; supersweet corn ; protoplasts ; haploids ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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
    Keywords: sunflower ; protoplasts ; direct gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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
    Keywords: aleurone ; barley ; protoplasts ; transient expression ; wheat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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
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    Plant molecular biology 13 (1989), S. 503-511 
    ISSN: 1573-5028
    Keywords: electroporation ; patatin genes, potato ; protoplasts ; transient gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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
    Keywords: field inversion gel electrophoresis ; DNA isolation ; protoplasts ; nuclear DNA ; chloroplast DNA ; tomato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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
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    Plant molecular biology 13 (1989), S. 151-161 
    ISSN: 1573-5028
    Keywords: β-glucuronidase (gusA) gene ; maize ; protoplasts ; stable co-transformation ; transformation ; Zea mays L.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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
    Keywords: Lycopersicon ; tomato ; haploids ; chromosomal instability ; chloroplast number ; callus culture ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: 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
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    Plant cell, tissue and organ culture 19 (1989), S. 103-111 
    ISSN: 1573-5044
    Keywords: amino acids ; ammonium ; growth factors ; light ; mannitol ; nitrate ; organogenesis ; polyamines ; potato ; protoplasts ; Solanum tuberosum ; sucrose ; tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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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    Plant cell, tissue and organ culture 19 (1989), S. 113-127 
    ISSN: 1573-5044
    Keywords: somatic embryogenesis ; plant regeneration ; protoplasts ; Trifolium pratense ; red clover ; protoclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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
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    Methods in cell science 12 (1989), S. 139-144 
    ISSN: 1573-0603
    Keywords: microinjection ; genetic transformation ; protoplasts ; microspores ; Brassica napus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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.
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  • 76
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    Methods in cell science 12 (1989), S. 157-161 
    ISSN: 1573-0603
    Keywords: protoplasts ; fusion ; somatic ; hybridization ; cybridization ; asymmetric hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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.
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  • 77
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    Plant cell, tissue and organ culture 19 (1989), S. 213-224 
    ISSN: 1573-5044
    Keywords: Brassica napus ; B. oleracea ; rapid-cycling brassica populations ; protoplasts ; regeneration ; maltose
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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
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    Plant cell, tissue and organ culture 17 (1989), S. 91-100 
    ISSN: 1573-5044
    Keywords: amino acids ; Brassica oleracea ; light ; nitrate ; organogenesis ; polyamines ; protoplasts ; regeneration ; sucrose
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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
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    Cellular and molecular life sciences 44 (1988), S. 348-351 
    ISSN: 1420-9071
    Keywords: L. lactis ; protoplasts ; protoplast regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: 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: 1573-5044
    Keywords: Brassica napus ; protoplasts ; Ficoll ; agarose
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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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  • 81
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    Plant cell, tissue and organ culture 14 (1988), S. 15-24 
    ISSN: 1573-5044
    Keywords: Brassica ; genetics of regeneration ; protoplasts ; totipotency
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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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  • 82
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    Potato research 30 (1987), S. 371-380 
    ISSN: 1871-4528
    Keywords: dihaploids ; protoclones ; protoplasts ; Solanum tuberosum ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Description / Table of Contents: 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.
    Abstract: 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.
    Notes: 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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  • 83
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    Keywords: Humulus lupulus ; micro-calli ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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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    Plant cell, tissue and organ culture 8 (1987), S. 225-233 
    ISSN: 1573-5044
    Keywords: Brassica napus ; thin cell layer ; protoplasts ; plant regeneration ; rapeseed ; organogenesis ; callus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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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    Plant cell, tissue and organ culture 10 (1987), S. 187-196 
    ISSN: 1573-5044
    Keywords: protoplasts ; Azolla ; Sporophytes ; ferns ; Cellulysin ; Pectolyase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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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    Plant cell, tissue and organ culture 11 (1987), S. 179-188 
    ISSN: 1573-5044
    Keywords: Solanum melongena ; protoplasts ; lamina ; petioles ; stems
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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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  • 87
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    Plant molecular biology 8 (1987), S. 363-373 
    ISSN: 1573-5028
    Keywords: transformation ; MgCl2-PEG-electroporation ; competence ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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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  • 88
    ISSN: 1573-5079
    Keywords: carbon dioxide fixation ; freezing stress ; photophosphorylation ; photosynthetic electron transport ; protoplasts ; Valerianella locusta
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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.
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  • 89
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    Plant cell, tissue and organ culture 6 (1986), S. 47-59 
    ISSN: 1573-5044
    Keywords: cotton ; ovule epidermis ; protoplasts ; cell wall regeneration ; β-1,3-glucans ; β-1,4-glucans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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.
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    Plant cell, tissue and organ culture 7 (1986), S. 11-19 
    ISSN: 1573-5044
    Keywords: Solanum etuberosum ; protoplasts ; plating efficiency ; shoot regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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.
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  • 91
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    Plant cell, tissue and organ culture 4 (1985), S. 171-182 
    ISSN: 1573-5044
    Keywords: potato ; protoplasts ; regeneration ; aneuploidy ; karyotypic changes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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.
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  • 92
    ISSN: 1573-5028
    Keywords: cauliflower mosaic virus ; DNA synthesis ; kinetics ; protoplasts ; replication intermediates
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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.
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  • 93
    ISSN: 1573-5060
    Keywords: protoplasts ; protoclonal variation ; somatic hybridization ; top-fruit trees ; woody ornamentals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Until recently, temperate fruit trees and woody ornamentals have been regarded as recalcitrant to biotechnological breeding approaches based on protoplasts. This however should no longer be the case, as procedures are now available, not only for the regeneration of complete plants from protoplasts of various tissues of such species, but also for the exploitation of protoplast technology for their genetic manipulation. This paper will examine the recent advances and state of the art in this domain, with particular attention to the use of protoplast technology as a novel tool in the breeding of rosaceous top-fruit tree species and woody ornamentals. Problems and their solutions within the context of regenerating plants from isolated protoplasts of stone (Prunus spp.), pome (Pyrus spp., Malus spp.) and small (Rubus spp.) fruits, and of several shrubby ornamental genotypes (Lonicera spp., Weigela spp., Forsythia spp., Cotoneaster spp.) will be addressed. Interspecific (Prunus spinosa + Prunus cerasifera) and intergeneric (Forsythia spp. + Syringa spp.) somatic hybridization within this group of species, as well as the use of protoplasts for host/pathogen interaction studies (Pyrus/Erwinia amylovora) will also be discussed.
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  • 94
    ISSN: 1573-5060
    Keywords: barley ; electroporation ; PEG-mediated DNA uptake ; promoter analysis ; protoplasts ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary In some cereal species that are still recalcitrant to stable transformation and regeneration, transient expression in isolated protoplasts is a useful tool for the study of gene expression and regulation. We have successfully applied these techniques to barley protoplasts derived from developing endosperm, aleurone, leaves and roots in order to characterize functionally cis-acting motives in two gene promoters, corresponding to trypsin inhibitor BTI-CMe and to sucrose synthase Ss1. Gene specificity is maintained in transient expression assays with protoplasts isolated from these different barley tissues and the pattern of expression parallels the mRNA levels observed for the corresponding genes in the same tissues.
    Type of Medium: Electronic Resource
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  • 95
    ISSN: 1573-5060
    Keywords: Oryza sativa L. cv. Taipei 309 ; rice ; protoplasts ; direct DNA uptake ; kanamycin-resistant transgenic plants ; field trial ; glasshouse trial ; neomycin phosphotransferase II (npt II) gene ; gene expression and inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The phenotypes of seed progeny (R2 generation) of Oryza sativa L. cv. Taipei 309, which carried the neomycin phosphotransferase II (npt II) gene, were compared with those of non-transformed, protoplast-derived plants of the same generation and non-transformed, seed-derived plants under field and glasshouse conditions. Under both conditions the transgenic plants were generally smaller, took longer to flower and had reduced fertility. Significant differences were observed between individuals within the group of transgenic plants. The npt II gene was present in most of the transgenic plants, but NPT II activity was only detected in a minority of individuals.
    Type of Medium: Electronic Resource
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  • 96
    ISSN: 1573-5060
    Keywords: bleomycin ; direct gene transfer ; expression ; irradiation ; petunia ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The production of transgenic plants by means of direct gene transfer to protoplasts is now a widely-used technique. The biological mechanisms underlying the transformation are still poorly understood, but many investigations have attempted to shed light on some components of this process. Varying the experimental conditions has in some cases led to better transformation rates, but further improvements of the protocols are possible. Such improvements will require a better understanding of how the alien DNA enters the cells, becomes integrated into the chromosomes and is treated as a part of the plant genome. Irradiation with sublethal doses of X-rays or UV-light has been shown to increase the transformation frequency, while certain drugs have been shown to act in a similar manner. The effects of these and other factors are discussed.
    Type of Medium: Electronic Resource
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