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  • 1
    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.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    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.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    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.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    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.
    Type of Medium: Electronic Resource
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  • 7
    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.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    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.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    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.
    Type of Medium: Electronic Resource
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  • 11
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 12
    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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  • 13
    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.
    Type of Medium: Electronic Resource
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  • 14
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 15
    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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  • 16
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 17
    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.
    Type of Medium: Electronic Resource
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  • 18
    Electronic Resource
    Electronic Resource
    Springer
    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.
    Type of Medium: Electronic Resource
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  • 19
    Electronic Resource
    Electronic Resource
    Springer
    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.
    Type of Medium: Electronic Resource
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  • 20
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 21
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 22
    ISSN: 1573-5044
    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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  • 23
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 24
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 25
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 26
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 27
    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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  • 28
    Electronic Resource
    Electronic Resource
    Springer
    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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  • 29
    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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  • 30
    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.
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  • 31
    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.
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  • 32
    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.
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  • 33
    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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