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  • 2010-2014
  • 1990-1994  (63)
  • 1980-1984  (8)
  • 1955-1959  (4)
  • protoplasts
  • 1
    Electronic Resource
    Electronic Resource
    Synergistic enhancement of protoplast growth by oxygenated perfluorocarbon and Pluronic F-68 (1994)
    Springer
    Plant cell reports 13 (1994), S. 251-255 
    Details
    ISSN: 1432-203X
    Keywords: Petunia hybrida ; protoplasts ; oxygen delivery ; perfluorochemicals ; Pluronic F-68 ; surfactant ; cell division
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Cell suspension-derived protoplasts of albino Petunia hybrida were grown for 10 d at the interface between aqueous culture medium (KM8P) and an oxygenated (10 mbar for 15 min) perfluorocarbon liquid, perfluorodecalin. Protoplasts synthesised new cell walls and divided normally at the perfluorodecalin/culture medium interface, with a mean viability after 10 d of 〉 92.0%. The mean plating efficiency of protoplasts was elevated by 37% (P〈0.05) following culture at the perfluorodecalin/medium interface, but was unaltered by perfluorodecalin or oxygen separately. The mean plating efficiency of protoplasts cultured at the interface was further increased to a maximium of 52% above control, in the presence of oxygenated perfluorodecalin and KM8P medium supplemented with the non-ionic, co-polymer surfactant, Pluronic F-68 at 0.01% (w/v). These findings demonstrate the effectiveness of oxygenated perfluorodecalin for promoting protoplast growth, by facilitating oxygen delivery. The finding that Pluronic F-68 further increased the plating efficiency of protoplasts cultured at the perfluorocarbon/aqueous interface suggests that these agents improve growth through separate, but cumulative, mechanisms.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233314
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  • 2
    Electronic Resource
    Electronic Resource
    Isolation and transformation of rice aleurone protoplasts (1994)
    Springer
    Plant cell reports 13 (1994), S. 394-396 
    Details
    ISSN: 1432-203X
    Keywords: Rice ; α-amylase ; protoplasts ; aleurone ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Protoplasts isolated from the aleurone have been used extensively in molecular studies focusing on hormone-mediated regulation of gene expression in barley seed. To extend the use of aleurone protoplasts to other species, we have determined the conditions necessary for the isolation of protoplasts from rice aleurone layers of germinated seed. Many of the common cell wall degrading enzymes used in making protoplasts were tested for their ability to release protoplasts from rice aleurone layers. Cellulysin was found to be the most effective. Transformation of these aleurone protoplasts was accomplished using polyethylene glycol and DNA constructs containing the firefly luciferase reporter gene under the control of two different promoters were tested. Luciferase expression was 24-fold greater when the reporter gene was under the control of the CaMV 35S promoter than when the promoter from the alcohol dehydrogenase 1 gene was used. With the isolation and transformation of aleurone protoplasts from rice, it is now possible to investigate molecular events occurring in this tissue during germination.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00234145
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  • 3
    Electronic Resource
    Electronic Resource
    A reproducible procedure for plant regeneration from seedling hypocotyl protoplasts of Vigna sublobata L. (1994)
    Springer
    Plant cell reports 14 (1994), S. 175-179 
    Details
    ISSN: 1432-203X
    Keywords: Vigna sublobata ; protoplasts ; microcalli ; shoot bud formation ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Viable protoplasts of Vigna sublobata L. were isolated enzymatically from hypocotyls of axenic seedlings. Protoplast yields were dependent upon seedling age, with maximum yields (2.25 ± 0.35 × 106 g fwt−1) from seedlings aged 6 d. Protoplasts regenerated cell walls and underwent sustained divisions when cultured in either agarose-solidified or liquid K8P medium. The plating density affected the division frequency and plating efficiency; the division frequency (68 ±0 6.0%) was maximum at 4.0 × 104 ml−1 while plating efficiency was maximum (1.3 ± 0.1%) at 5.0 × 104 ml−1. Dividing protoplasts developed into microcalli, which produced glossy green compact nodular calli on transfer to 8.0 gl−1 w/v agar-solidified medium containing MS salts, B5 organic components, 30 g l−1 sucrose, NAA (0.2–0.5 mg l−1), zeatin riboside (0.5–2.0 mg l−1) and GA3 (0.5–1.0 mg l−1). These calli, after sub-culture on the same medium, produced shoot buds which underwent elongation following transfer of tissues to 6.0 g l−1 agar-solidified B5 medium containing 30g l−1 sucrose, IBA (0.01 mg l−1) and BAP (1.0 mg l−1). Elongated shoots developed roots after transfer to 8.0g l−1 agar-solidified, hormone-free MS medium with 30 g l−1 sucrose.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233785
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  • 4
    Electronic Resource
    Electronic Resource
    The biosynthetic pathway of the S-alk(en)yl-L-cysteine sulphoxides (flavour precursors) in species of Allium (1994)
    Springer
    Plant cell, tissue and organ culture 38 (1994), S. 181-188 
    Details
    ISSN: 1573-5044
    Keywords: Allium cepa ; biosynthesis ; compartmentation ; γ-glutamyl peptides ; onion ; protoplasts ; S-alkenyl-L-cysteine sulphoxide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Pulse labelling experiments with 35SO4 2- fed for 24h to intact plants (shooted onion sets)of Allium cepa (onion) showed that 〉70% of the label appeared in the S-alkenyl-L-cysteine sulphoxides within 18h, reached a maximum at 48h and thereafter decreased. The amount of label detected in the γ-glutamyl peptide fractions was below 20% of the total label at any time. It is concluded that in intact plants (at the growth stage used) the γ-glutamyl peptides are not the immediate precursors of the S-alkenyl-L-cysteine sulphoxides. The major S-alkenyl-L-cysteine sulphoxide in onion was found to be compartmentalized mainly within the endoplasmatic reticulum.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00033876
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  • 5
    Electronic Resource
    Electronic Resource
    Single-cell cloning of a crown gall from protoplasts regenerated in hormone-free medium. Establishment of pure transformed cell-lines of Catharanthus roseus (1994)
    Springer
    Plant cell, tissue and organ culture 37 (1994), S. 25-30 
    Details
    ISSN: 1573-5044
    Keywords: Catharanthus roseus ; crown gall tumor ; heterokaryons ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts enzymatically isolated from cell line of Catharanthus roseus G. Don crown gall, were cultured at high density (105 P ml-1) in modified B5 liquid medium (Gamborg et al. 1976). In the absence of growth regulators C. roseus protoplasts were able to regenerate a cell-wall, divide and, subsequently, yield very numerous clones in the absence of growth regulators. After two weeks, the cultures were greatly diluted in order to obtain clones of single-cell origin. Most of the clones individually transferred onto solid medium can proliferate indefinitely, without growth regulators. Among analyzed clones, 90% were nopaline positive. Their ajmalicine and serpentine content was compared with that of the parental crown gall line, and was found to be low. The CR10 protoplasts were very easy to grow, they were an interesting model for the development of pure tumorous lines. Moreover, we found that the tumorous protoplasts were useful for cell fusion experiments or for the delicate culture of tree protoplasts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00048113
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  • 6
    Electronic Resource
    Electronic Resource
    Isolation of cells and protoplasts from leaves of in vitro propagated peach (Prunus persica) plants (1994)
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 99-105 
    Details
    ISSN: 1573-5044
    Keywords: cell division ; peach ; protoplasts ; Prunus persica ; tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Yields of 106–108 peach mesophyll cells and protoplasts · gfw-1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4–10 mm in length, were a superior source for protoplast isolation than medium or big expanded leaves, 22–30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml-1. Purification of protoplasts on a sucrose gradient yielded about 107 protoplasts · gfw-1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00048320
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  • 7
    Electronic Resource
    Electronic Resource
    Highly efficlent plant regeneration from mesophyll protoplasts of Indian field cultivars of tomato (Lycopersicon esculentum) (1994)
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 255-258 
    Details
    ISSN: 1573-5044
    Keywords: tomato ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three Indian cultivars ofL. esculentum were assessed for shoot regeneration from protoplast-derived calli. Consistent yields of viable protoplasts (〉9.0×106 g f.wt.-1) were obtained from leaflets of 14 days old cultured shoots. Protoplast viability (88–94%) and planting efficiency (55–70%) were recorded for the three cultivars. Up to 71% of the protoplast-derived tissues regenerated shoots.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00037728
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  • 8
    Electronic Resource
    Electronic Resource
    Production and characterization of intergeneric somatic hybrids through protoplast electrofusion between potato (Solanum tuberosum) and Lycopersicon pennellii (1994)
    Springer
    Plant cell, tissue and organ culture 37 (1994), S. 137-144 
    Details
    ISSN: 1573-5044
    Keywords: electrofusion ; L. pennellii ; protoplasts ; S. tuberosum ; salt tolerance ; somatic hybrids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Mesophyll protoplasts of Lycopersicon pennelli Corr., a wild relative of tomato, were electrofused with those from a dihaploid potato clone, cv Nicola, with the objectives of transferring saline tolerance from L. pennellii to cultivated potato. 150 calli were selected from the fusion experiments, finally giving 2 hybrid shoots. Their hybrid nature was verified by examining isoenzyme patterns for esterases (EST), peroxidase (PRX), phosphogluconate dehydrogenase (6-PGD), and glutamate oxaloacetate transaminase (GOT). The hybrid plants had an intermediate morphology, and grew vigorously in vitro. When transplanted to soil, they were less vigorous, due to difficulties in rooting, but were still capable of flowering, and forming short stolons and mishaped tubers, probably resulting from the effects of gene dosage due to the novel association of two genomes from a tuberizing (potato) and a non tuberizing species (L. pennellii). The characteristics of such mishaped tubers provided strong evidence of a hybrid nature for the selected plants. The hybrid plants were highly sterile, producing only 3–7% viable pollen. Tests for salt tolerance showed that the growth of the somatic hybrid plants was reduced by 50% as for L. pennellii, whilst potato did not grow at all under saline conditions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00043607
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  • 9
    Electronic Resource
    Electronic Resource
    Expression of human α-interferon cDNA in transgenic rice plants (1994)
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 197-204 
    Details
    ISSN: 1573-5044
    Keywords: interferon ; Lipofectin ; protoplasts ; rice ; transgenic plant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The plasmid pIG3031 containing human α-interferon cDNA and the neomycin phosphotransferase II coding sequence was successfully transferred into rice protoplasts (Indica type rice) by Lipofectinmediated transformation. Assays for NPT II enzyme activity indicated that the transformation frequency was 10%. Transgenic plants were regenerated from transformed calli. Southern blots showed that the human α-interferon cDNA sequence was present in rice DNA. RNA slot blots indicated apparent transcription of human α-interferon cDNA under the control of the plant-active promoter PI'. Extracts of transgenic cell cultures and plants contained apparent interferon activity as measured by resistance of a human amniotic cell line to viral infection in the presence of plant extracts. These studies demonstrate that human α-interferon cDNA may be correctly expressed in rice cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00037720
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  • 10
    Electronic Resource
    Electronic Resource
    Isolation and culture of protoplasts from young sporophytes ofSalvinia natans aseptically obtained by co-culture of female and male gametophytes (1994)
    Springer
    Plant cell, tissue and organ culture 36 (1994), S. 237-242 
    Details
    ISSN: 1573-5044
    Keywords: aseptic culture ; female gametophyte ; heterospory ; male gametophyte ; protoplasts ; Salvinia natans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Sporophytes were aseptically obtained by co-culture of female and male gametophytes derived from two types of spores (megaspores and microspores) of the heterosporous fernSalvinia natans All. Protoplasts isolated enzymatically from juvenile leaflets of sporophytes were cultured in a 1/10 Murashige and Skoog's medium containing 2.2 μM naphthalene acetic acid, 2.2 μM 6-benzyl-aminopurine, 0.35 M mannitol, and 0.05 M sucrose. Cell division took place within 6 days of culture, and cell-clusters composed of 9–10 cells were observed after 30 days of culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00037725
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  • 11
    Electronic Resource
    Electronic Resource
    Recovery of transgenic trees after electroporation of poplar protoplasts (1994)
    Springer
    Transgenic research 3 (1994), S. 13-19 
    Details
    ISSN: 1573-9368
    Keywords: gene transfer ; electroporation ; stable transformation ; protoplasts ; transgenic trees ; Populus tremula x P. alba
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts from leaflets ofin vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying thenptII gene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene,als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 105 to 104 protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 μM paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS ofArabidopsis and 25 μM phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated. Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01976022
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  • 12
    Electronic Resource
    Electronic Resource
    Selection of anthocyanin-accumulating potato (Solanum tuberosum L.) cell lines from calli derived from seedlings produced by gamma-irradiated seeds (1993)
    Springer
    Plant cell reports 12 (1993), S. 555-558 
    Details
    ISSN: 1432-203X
    Keywords: Solanum tuberosum ; anthocyanins ; gamma-irradiation ; protoplasts ; protoclones
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Callus cell lines of potato (Solanum tuberosum L. cv. Zarevo) were obtained from seedlings germinated from gamma-irradiated seeds (200 Gy). Some of these cell lines produce red-violet pigments which were identified as acylated anthocyanins. The major anthocyanin was determined to be peonidin 3-O-[6-O-(4-O-E-p-coumaroyl-rhamnosyl)-glucoside]-5-O-glucoside (“peonanin”). Single cell-derived protoclones from non-pigmented protoplasts sometimes also gave rise to pigmented cell clusters thus indicating that the changes in the expression of the anthocyanin pathway can also occur after the stage of initial callus induction.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233059
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  • 13
    Electronic Resource
    Electronic Resource
    Plant regeneration from mesophyll-protoplasts of four different ecotypes and two marker lines from Arabidopsis thaliana using a unique protocol (1993)
    Springer
    Plant cell reports 12 (1993), S. 569-572 
    Details
    ISSN: 1432-203X
    Keywords: Arabidopsis thaliana ; ecotypes and mutant lines ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of four ecotypes (Col C24, Per-1, Bur-0, Landsberg erecta) and two marker lines (M4 and M10) of Ardbidopsis thaliana is described. The different lines showed plating efficiencies between 1.0 and 3.9% using Nitsch medium or this medium supplemented with coconut water. For the differentiation of callus into normal shoots a single shoot regeneration medium was applicable to all ecotypes, but depending on the line other regeneration media showed to be more suitable. The results indicated that the protoplast culture procedure is applicable, with minor modifications, to all tested genotypes but the most suitable shoot regeneration medium should be established for each A. thaliana line.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233062
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  • 14
    Electronic Resource
    Electronic Resource
    Fertile plant regeneration from protoplasts of meadow fescue (Festuca pratensis Huds.) (1993)
    Springer
    Plant cell reports 12 (1993), S. 95-100 
    Details
    ISSN: 1432-203X
    Keywords: forage grasses ; Festuca pratensis ; suspension cultures ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Suspension cultures from mature embryo-derived compact callus were initiated in seven meadow fescue (Festuca pratensis Huds.) cultivars. Four to six months after initiation, embryogenic suspension cultures with a moderate growth rate were established from three of them (cvs. Barmondo, Belimo and Leopard). These suspension cultures showed the capacity, maintained over six months, to regenerate green plants which could be grown to maturity under greenhouse conditions. Morphogenic suspension cultures from single genotypes of three F. pratensis cultivars (cvs. Barmondo, Belimo and Leopard) yielded large numbers of protoplasts, which upon culture in agarose beads using nurse cells formed microcalli with an overall plating efficiency in the range of 10-3 to 10-4. Mature plants were reproducibly regenerated and established in soil, from such protoplasts during a period of six months. The regeneration of fertile plants from protoplasts derived from suspension cultures of meadow fescue and its implications on gene transfer technology for this species are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00241942
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  • 15
    Electronic Resource
    Electronic Resource
    Analysis of genetic stability of plants regenerated from suspension cultures and protoplasts of meadow fescue (Festuca pratensis Huds.) (1993)
    Springer
    Plant cell reports 12 (1993), S. 101-106 
    Details
    ISSN: 1432-203X
    Keywords: Festuca pratensis ; suspension cultures ; protoplasts ; plant regeneration ; somaclonal variation ; genetic fidelity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials. The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00241943
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  • 16
    Electronic Resource
    Electronic Resource
    Effect of 1,2-benzisoxazole-3-acetic acid on plant regeneration from protoplasts of Nicotiana tabacum (1993)
    Springer
    Plant cell reports 12 (1993), S. 121-124 
    Details
    ISSN: 1432-203X
    Keywords: Auxin ; benzisoxazole-3-acetic acid ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Benzisoxazole-3-acetic acid, a new synthetic growth regulator, was administered to protoplast cultures from Nicotiana tabacum and subsequently to the developed microcalluses, to test its activity on plant regeneration from protoplasts in different culture conditions. Such activity, compared to that of naphthalene-acetic acid, proved to be rather low in the stage of cellular division and microcallus formation but particulary high in the stage of shoot induction from microcallus, thus confirming that the activity of this compound is mainly morphogenetic.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00239090
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  • 17
    Electronic Resource
    Electronic Resource
    Somatic embryogenesis and plant regeneration from hypocotyl protoplasts of sunflower (Helianthus annum L.) (1993)
    Springer
    Plant cell reports 12 (1993), S. 260-263 
    Details
    ISSN: 1432-203X
    Keywords: sunflower ; protoplasts ; somatic embryogenesis ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A sunflower genotype (Helianthus annuus L. cv. Florom-328) able to regenerate plants from in vitro cultures was identified by screening hybrids and inbred lines. Protoplasts of this genotype were isolated from dark grown hypocotyls and were cultured in droplets of agarose-solidified V-KM medium covered by liquid V-KM supplemented with naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). One week later colonies were subjected to 2,4-dichlorophenoxyaceticacid for a one week period. Further culture in V-KM with reduced concentrations of NAA and BAP resulted in the appearence of somatic embryos. Maturation of embryos was achieved by culture on MS medium supplemented with NAA, BAP, gibberellic acid A3 and the ethylene inhibitor AgNO3. Embryos were then transferred onto hormone free MS medium for germination. The frequency of shoot formation in the best case was 9.6 percent of viable colonies (1.3 percent of protoplasts plated). Some of the shoots with roots could be transplanted into soil, others were grafted on hypocotyls of in vivo germinated seedlings. Eighty percent of grafted shoots and over 95 percent of rooted shoots survived. The plants flowered and produced 5 to 10 seeds each. Factors affecting the frequency of embryo formation and plant regeneration are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00237131
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  • 18
    Electronic Resource
    Electronic Resource
    Improvement of protoplast culture protocols for Beta vulgaris L. (sugar beet) (1993)
    Springer
    Plant cell reports 12 (1993), S. 339-342 
    Details
    ISSN: 1432-203X
    Keywords: Alginate embedding ; Beta vulgaris ; feeders ; protoplasts ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The effects of NaCl, feeder cells and the embedding of protoplasts in calcium alginate have been investigated in an attempt to improve culture conditions of recalcitrant sugar beet (Beta vulgaris L.) mesophyll protoplasts. While the use of NaCl in all instances proved detrimental to protoplast development, the other two treatments had clear beneficial effects. Minimum plating densities, necessary to sustain cell division, could be reduced to 〈5% (〈4000 protoplasts / ml) of the control levels and plating efficiencies could be significantly enhanced by approx. 10 fold. Plants could still be regenerated from soft calli derived from mesophyll protoplasts cultured under the modified conditions at a frequency of 20–30 %. In particular, the use of alginate is considered of potentially great importance for the further application of beet protoplasts for other aims e.g. asymmetric hybridization.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00237431
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  • 19
    Electronic Resource
    Electronic Resource
    An efficient protoplast-to-plant system for the hybrid ornamental shrub,Weigela ×florida cv. Bristol Ruby (Caprifoliaceae) (1993)
    Springer
    Plant cell, tissue and organ culture 33 (1993), S. 315-320 
    Details
    ISSN: 1573-5044
    Keywords: protoplasts ; plant regeneration ; woody ornamentals ; Weigela
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Strategies were developed for the successful isolation of large numbers of highly viable protoplasts from the leaves, stems and roots of axenic plants of the hybrid ornamental shrubWeigela ×florida cv Bristol Ruby. Protoplasts, of all sources, were cultured on different media, leading to the establishment of sustained divisions, and coupled with the production of multi-celled (〉50 cells) colonies. However, those colonies derived from mesophyll protoplasts only were capable of a further proliferation to the callus stage. Upon transfer to a regeneration medium consisting of MS salts and organics plus a range of concentrations of NAA and BAP, such calli underwent caulogenesis, with optimum responses for a medium with 1.0 mg l−1 NAA and 1.0 mg l−1 BAP. The protoplast-derived shoots thus obtained were multiplied on MS medium with 0.1 mg l−1 IBA, 0.5 mg l−1 BAP and 0.1 mg l−1 GA3. Individual shoots were subsequently rooted on a half-strength MS medium plus 3.0 mg l−1 IBA, and complete protoplast-derived plants were finally transferred to the glasshouse for acclimatization.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02319017
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  • 20
    Electronic Resource
    Electronic Resource
    Efficient plant regeneration from protoplasts of Arachis paraguariensis Chod. et Hassl. using a nurse culture method (1993)
    Springer
    Plant cell, tissue and organ culture 34 (1993), S. 83-90 
    Details
    ISSN: 1573-5044
    Keywords: Arachis species ; nurse culture ; plant regeneration ; protoplasts ; tissue culture ; wild peanut
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An efficient protocol has been developed for protoplast culture and plant regeneration from wild peanut (A. paraguariensis) using a nurse culture method. Protoplasts were isolated from suspension cultures initiated from leaf-derived callus, imbedded in agarose blocks and co-cultured with nurse cells of the same species. Up to 10% of the protoplasts divided and formed compact callus colonies. The protoplast plating efficiency was correlated with both the length of the nurse cell co-cultivation period and the protoplast plating density. The optimal nurse culture duration was 14 d. The optimal plating density was 2×104 protoplasts/ml plating medium. Multiple shoots (up to 10 shoots per colony) were readily regenerated from protoplast-derived callus after transfer of callus to semi-solid modified MS medium containing 0.5 mg l-1 NAA and 1 mg l-1 BA. Plantlets with normal leaflets were obtained by rooting shoots on porous rootcubes saturated with modified MS medium containing 1 mg l-1 NAA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00048467
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  • 21
    Electronic Resource
    Electronic Resource
    Effect of digitonin on membrane-bound and chitosomal chitin synthetase activity in protoplasts from yeast cells ofCandida albicans (1993)
    Springer
    Antonie van Leeuwenhoek 64 (1993), S. 67-74 
    Details
    ISSN: 1572-9699
    Keywords: Candida albicans ; chitin synthetase ; digitonin ; proenzyme activation ; protoplasts ; solubilization ; zymogen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) fromC. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen,in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processesin vivo andin vitro or, alternatively, that both enzyme activities (activein vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml−1 of digitonin and 64 µg ml−1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 µg ml−1 of protease, whereas activity strongly decreases in the presence of 64 µg ml−1 of trypsin. Digitonin does not produce zymogen activationper se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00870923
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  • 22
    Electronic Resource
    Electronic Resource
    Regeneration of whole plants from Gracilaria asiatica Chang et Xia protoplasts (Gracilariaceae, Rhodophyta) (1993)
    Springer
    Hydrobiologia 260-261 (1993), S. 429-436 
    Details
    ISSN: 1573-5117
    Keywords: Gracilaria ; protoplasts ; callus-like ; regeneration ; plant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A large number of viable protoplasts were produced by enzymatic digestion of Gracilaria asiatica vegetative tissue. The protoplasts underwent initial division after 5–7 d in culture and developed into callus-like cell-masses. Many filaments grew from the periphery of these cell-masses and disappeared after about one month in culture. Simultaneously, the central part of the callus-like cell-masses thickened and its color deepened. The first buds appeared from the center of the cell-masses and developed into whole plants after three months in aerated culture. Many new buds formed around the first plant and more than 20 plants grew per callus-like cell-masses in less than four months. Filaments taken from callus-like cell-masses developed into young plants after about 20 d of culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00049052
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  • 23
    Electronic Resource
    Electronic Resource
    Conditional inhibition of β-glucuronidase expression by antisense gene fragments in petunia protoplasts (1993)
    Springer
    Plant molecular biology 23 (1993), S. 45-55 
    Details
    ISSN: 1573-5028
    Keywords: Key words ; antisense RNA ; β-glucuronidase ; protoplasts ; transient gene expression ; PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Antisense RNA-mediated inhibition of gene expression is a valuable tool to induce mutant phenotypes. We are interested in the application of antisense gene fragments with the aim to improve the efficiency of inhibition and to be able to selectively suppress gene family members in plants. Protoplasts may provide a rapid system to screen the efficiency of antisense gene segments. As a first step, we set up a transient expression system for leaf protoplasts of Petunia hybrida and used as a model system the inhibition of β-glucuronidase (uidA) expression by uidA antisense gene segments. Both GUS enzyme activities and uidA RNA levels were measured. Co-introducing equal amounts of a full-length uidA antisense gene and a uidA sense gene reduced GUS activity by 60–70%. Various uidA antisense fragments also inhibited expression although with different efficiencies and we show that strong antisense fragments can be retrieved from weak antisense gene fragments. A promoter-less antisense gene did not reduce uidA expression indicating that the inhibition is mediated by antisense transcripts. Using quantitative PCR on first-strand cDNA we show that expression of functional antisense genes lead to reduced levels of uidA mRNA. This suggests that the mechanism of antisense RNA inhibition in protoplasts is similar to that in transgenic plants and that the protoplast system in combination with PCR can be used to preselect antisense fragments of any gene.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00021418
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  • 24
    Electronic Resource
    Electronic Resource
    Investigations of boron uptake at the cellular level (1993)
    Springer
    Plant and soil 155-156 (1993), S. 143-146 
    Details
    ISSN: 1573-5036
    Keywords: barley ; cell wall ; Hordeum vulgare ; pollen selection ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The nature of expression of the tolerance of barley to high levels of B at the cellular level was investigated with a view to identifying ways by which this level of expression might be exploited in a breeding programme. Using protoplasts derived from leaf tissue, it was found that genetic differences between B tolerant and intolerant barleys were not expressed in the absence of cell walls. Barley genotypes differing in their tolerance to B were subjected to high levels of B in the growth medium from pollen formation onwards. The genetic distribution of segregating populations in the next generation was not changed for tolerance to high B. Results also suggested that genetic tolerance to B is expressed by pollen in vitro.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00025004
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  • 25
    Electronic Resource
    Electronic Resource
    Culturing embryogenic protoplasts of ‘Hamlin’ sweet orange in calcium alginate beads (1993)
    Springer
    Plant cell, tissue and organ culture 34 (1993), S. 19-25 
    Details
    ISSN: 1573-5044
    Keywords: Ca-alginate ; citrus ; plating efficiency ; protoplasts ; somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts were enzymatically isolated from log phase embryogenic sweet orange [Citrus sinensis (L.) Osbeck cv. Hamlin] suspension cultures, embedded in 3-mm diameter Ca-alginate beads, and cultured in a growth cabinet (15–20 μ mol m-2 s-1, 4-h photoperiod, 27°C). Plating efficiency exceeded 90% for Ca-alginate embedded protoplasts vs. 30% for protoplasts cultured in a liquid medium. Embryoids formed from protoplasts were recovered after 20 days by dissolving the Ca-alginate matrix with a calcium sequestrant. Embryoids readily formed shoots that were rooted on MS+0.01 μM NAA+5% sucrose. Potential applications are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00048459
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  • 26
    Electronic Resource
    Electronic Resource
    Plant regeneration from protoplasts isolated from cell suspension cultures of the wild tomato, Lycopersicon chilense Dun. (1993)
    Springer
    Plant cell, tissue and organ culture 32 (1993), S. 311-317 
    Details
    ISSN: 1573-5044
    Keywords: cell suspension ; Lycopersicon chilense ; plant regeneration ; protoplasts ; wild tomato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protocol has been established for rapid, high frequency plant regeneration from protoplasts of the wild tomato species Lycopersicon chilense Dun. Cell suspension cultures were obtained from calli initiated from seedling stem explants. Protoplasts were isolated from cell suspensions by an overnight one-step enzyme digestion, purified by washing in salts solution and cultured in liquid medium. Dilution of liquid medium every 3 days, with medium containing low levels of growth regulators and sucrose, was critical for sustained colony formation. Up to 70% of protoplast-derived calli regenerated shoots when cultured on agar-solidified medium with Murashige & Skoog (1962) salts and vitamins, 2.0 mg l-1 zeatin and 0.1 mg l-1 indole acetic acid for 21 days, followed by transfer to the same medium lacking indole acetic acid.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00042294
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  • 27
    Electronic Resource
    Electronic Resource
    The GAPDH gene system of the red alga Chondrus crispus: promotor structures, intron/exon organization, genomic complexity and differential expression of genes (1993)
    Springer
    Plant molecular biology 23 (1993), S. 981-994 
    Details
    ISSN: 1573-5028
    Keywords: cis-acting elements ; intron conservation ; intron secondary structure ; pre-mRNA splicing ; CpG suppression ; protoplasts ; transcript levels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Our previous phylogenetic analysis based on cDNA sequences of chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPDH; genes GapA and GapC, respectively) of the red alga Chondrus crispus suggested that rhodophytes and green plants are sister groups with respect to plastids and mitochondria and diverged at about the same time or somewhat later than animals and fungi. Here we characterize the genomic sequences of genes GapC and GapA of C. crispus with respect to promotor structures, intron/exon organization, genomic complexity, G+C content, CpG suppression and their transcript levels in gametophytes and protoplasts, respectively. To our knowledge this is the first report on nuclear protein genes of red algae. The GapC gene is G+C-rich, contains no introns and displays a number of classic sequence motifs within its promotor region, such as TATA, CAAT, GC boxes and several elements resembling the plant-specific G-box palindrome. The GapA gene has a moderate G+C content, a single CAAT box motif in its promotor region and a single intron of 115 bp near its 5′ end. This intron occupies a conserved position corresponding to that of intron 1 in the transit peptide region of chloroplast GAPDH genes (GapA and GapB) of higher plants. It has consensus sequences similar to those of yeast introns and folds into a conspicuous secondary structure of - 61.3 kJ. CpG profiles of genes GapC and GapA and their flanking sequences show no significant CpG depletion suggesting that these genomic sequences are not methylated. Genomic Southern blots hybridized with generic and gene specific probes indicate that both genes are encoded by single loci composed of multiple polymorphic alleles. Northern hybridizations demonstrate that both genes are expressed in gametophytes but not in protoplasts where appreciable amounts of transcripts can only be detected for GapC.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00021813
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  • 28
    Electronic Resource
    Electronic Resource
    Microtubule organization in the apical cell of Sphacelaria (Phaeophyceae) and its related protoplast (1993)
    Springer
    Hydrobiologia 260-261 (1993), S. 167-172 
    Details
    ISSN: 1573-5117
    Keywords: Phaeophyceae ; protoplasts ; apical cells ; immunofluorescence ; microtubules ; cell cycle ; polarity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The growth of the filamentous brown alga Sphacelaria depends on a large, strongly polarized, apical cell. The protoplast derived from this cell can be distinguished in a heterogeneous suspension by cytological markers, so it is possible to study development of the cytoskeleton during protoplast isolation and the first steps of regeneration. In the initial cell, microtubules show an asymmetric distribution along the axis; they are mainly located at the distal part around the physodes. After protoplast isolation, this polarity initially seems to be maintained; subsequently, the microtubules radiate from the two centrioles and spread out to the plasmalemma. This experimental model is suitable for investigating the development of the polarity of the initial cell, and the sequence of the first morphogenetic events leading to protoplast regeneration.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00049016
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  • 29
    Electronic Resource
    Electronic Resource
    Protoplasts of Gelidium robustum (Rhodophyta) (1993)
    Springer
    Hydrobiologia 260-261 (1993), S. 421-427 
    Details
    ISSN: 1573-5117
    Keywords: protoplasts ; Gelidium robustum ; agarophyte ; Rhodophyta
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Viable protoplasts were isolated from apices of the agarophyte Gelidium robustum (Gardn.) Hollenb. & Abb. using a combination of commercial cell-wall degrading enzymes and extracellular wall-degrading enzymes isolated from a marine bacterium. The protoplasts were approximately 8–15 µm in diameter, liberated mainly from the surface cell layers and from cells at the distal ends of medullary filaments. The bacterial enzyme alone was not sufficient to liberate significant numbers of protoplasts. Maximum yield was 9 × 105 protoplasts/g tissue (wet wt.). Optimum osmolality occurred between 1750–1950 mOs kg−1; yield and viability were severely diminished at osmolalities less than 1350 mOs kg−1. Viability, as determined by flurorescein diacetate staining and Evans Blue exclusion 1 hr after removal from the enzyme solution, was approximately 80–95%. Roughly 80% of the cells did not show Calcofluor fluorescence, while 40% stained positively for the presence of sulfated polysaccharides. Cell wall regeneration was observed with inconsistent reproducibility, and no cell division was observed when the protoplasts were placed in culture medium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00049051
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  • 30
    Electronic Resource
    Electronic Resource
    Plant regeneration from long term suspension culture-derived protoplasts of hexaploid wheat (Triticum aestivum L.) (1992)
    Springer
    Plant cell reports 11 (1992), S. 262-265 
    Details
    ISSN: 1432-203X
    Keywords: Triticum aestivum ; embryogenic suspension cultures ; protoplasts ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Highly regenerable callus cultures have been obtained from immature embryos of hexaploid wheat cv. Oderzo. Friable fast growing calli were induced at high frequency. Suspensions were initiated from the most friable callus lines: they became established in about two months. Suspensions consisted of cell aggregates of 30 to 1000 um in diameter. Upon plating on MS hormone-free medium, suspensions regenerated green plantlets, and their regenerative capability was maintained for at least 10 months. Protoplasts were isolated from 7–8 day old suspension cultures with a yield of 4–6×106 protoplasts/g fresh weight cells. Protoplast culture was either in liquid medium or in a bead-type system with agarose beads. First divisions were detected at day 5. At day 14 visible colonies were detected and the plating efficiency was evaluated between 2 and 8% over the initial number of protoplasts plated. Protoplast-derived calli were cultured in the presence of 1 mg/l IAA and 0.5 mg/l zeatin and were used for reinitiating new suspension cultures. Upon plating onto MS hormone-free medium, with or without the addition of 0.1 mg/l GA3, calliclones were induced to differentiate. Regeneration of complete plantlets, with shoot and roots took about two months. Plantlets were grown in sterile conditions until 12–15 cm height, and were subsequently transplanted in soil.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00235078
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  • 31
    Electronic Resource
    Electronic Resource
    An ultrastructural investigation on the polyethylene glycol-induced adhesion of tobacco nuclei and uptake of micronuclei by soybean protoplasts (1992)
    Springer
    Plant cell, tissue and organ culture 29 (1992), S. 207-214 
    Details
    ISSN: 1573-5044
    Keywords: amiprophosmethyl ; isolated nuclei ; micronuclei ; organelle uptake ; polyethylene glycol ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Nuclei isolated from tobacco protoplasts were induced to be taken up by soybean protoplasts using a protocol involving polyethylene glycol (PEG), osmotic shock and pH shift. Transmission electron microscopy revealed that PEG treatment condensed the chromatin of the isolated nuclei. Close adhesion of isolated nuclei to the plasma membrane of protoplasts following PEG treatment, was observed by both scanning and transmission electron microscopic methods. Ultrastructural observations were also made on the formation of micronuclei in tobacco cells following the treatment with amiprophosmethyl (APM). Nuclei and micronuclei isolated from APM-treated cells were induced to be taken up by soybean protoplasts. A single case of uptake of an isolated micronucleus was observed by transmission electron microscopy. The observations on the effects of PEG on the isolated nuclei, micronuclei and protoplasts are discussed in relation to the possible mechanism of uptake of nuclei by protoplasts using PEG.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00034354
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  • 32
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    Electronic Resource
    Transient gene expression in electroporated protoplasts of Eucalyptus citriodora Hook (1992)
    Springer
    Plant cell, tissue and organ culture 30 (1992), S. 69-75 
    Details
    ISSN: 1573-5044
    Keywords: chloramphenicol acetyltransferase ; electroporation ; Eucalyptus citriodora ; protoplasts ; transient gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts isolated from cotyledons of Eucalyptus citriodora were electroporated using a rectangular pulse, with plasmid carrying the cat gene. The levels of transient expression and protoplast viability were influenced by the voltage and pulse duration. At a field strength of 800 V cm-1 (1000 μs), a protoplast viability of 57%, and 47% conversion of 14C-chloramphenicol to its acetylated forms, were obtained. Expression levels were improved by an increase in plasmid concentration (up to 60 μg ml-1), and also by the addition of carrier DNA. Gene expression was further enhanced by the addition of 40% (w/v) PEG, in the presence of the carrier DNA, to the protoplasts after electroporation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00040003
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  • 33
    Electronic Resource
    Electronic Resource
    High laccase producing mutants ofPleurotus florida (1992)
    Springer
    World journal of microbiology and biotechnology 8 (1992), S. 39-41 
    Details
    ISSN: 1573-0972
    Keywords: Pleurotus florida ; laccase ; para-constitutive ; protoplasts ; mutagenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Pleurotus florida produced high amounts of laccase (4.60 U/ml) in malt extract broth after 12 days' growth under stationary conditions. The production of laccase was semi-constitutive. Hyperlaccase mutants ofP. florida were obtained through mutagenesis of mycelial protoplasts usingN-methyl-N′-nitro-N-nitrosoguanidine (50 μg/ml) for 2 min. Three hyperlaccase mutants were selected showing growth and enzyme production responses similar to the parent.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01200681
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  • 34
    Electronic Resource
    Electronic Resource
    Protoplasts from the cyanobacterium,Spirulina platensis (1992)
    Springer
    World journal of microbiology and biotechnology 8 (1992), S. 385-386 
    Details
    ISSN: 1573-0972
    Keywords: Lysozyme ; protoplasts ; regeneration ; Spirulina platensis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Protoplasts were obtained from the filamentous blue-green algaSpirulina platensis by treating the filaments with 0.05% (w/v) lysozyme in 0.03m phosphate buffer. The protoplasts regenerated cell walls and formed colonies when plated on a regeneration medium. The highest percentage of regeneration, 40% was obtained after 21 days.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01198750
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  • 35
    Electronic Resource
    Electronic Resource
    Stabilization of steroid 11-hydroxylation activity ofCunninghamella elegans protoplasts in organic osmotic stabilizers (1992)
    Springer
    World journal of microbiology and biotechnology 8 (1992), S. 500-504 
    Details
    ISSN: 1573-0972
    Keywords: Cunninghamella elegans ; hydroxylation ; protoplasts ; steroids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Protoplasts ofCunninghamella elegans, showing 11α-, and 11β-hydroxylating ability of Substance S, preserved high transformation activity when dispersed in glucose-enriched, organic osmotic stabilizers. A joint action of polyoxins and 2-deoxy-d-glucose was necessary to prevent regeneration of the cell wall in long-lasting experiments. Stabilized and active, dispersed protoplasts may be an alternative research model for studying the function of the cell wall and intracellular metabolic pool constituents in steroid hydroxylation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01201948
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  • 36
    Electronic Resource
    Electronic Resource
    Functional analysis of the promoter region of a nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L. (1992)
    Springer
    Plant molecular biology 19 (1992), S. 837-846 
    Details
    ISSN: 1573-5028
    Keywords: glutamine synthetase ; hairy roots ; nodules ; Phaseolus vulgaris ; protoplasts ; transcriptional regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The 5′-flanking region of gln-γ, the nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L., has been analysed for cis-regulatory elements using a series of 5′ deletions and hybrid gln-γ: : CaMV 35S promoters. The promoters were fused to the uidA reporter gene and their activities tested in two heterologous expression systems. In the first system, the chimaeric genes were transferred to Lotus corniculatus L. using Agrobacterium rhizogenes and their expression was studied in nodulated hairy roots. In the second system, the constructs were electroporated into tobacco mesophyll protoplasts. The results of the 5′ deletion analysis showed that the sequence between −597 and −21 (relative to the ATG codon) was sufficient for nodule-specific expression of the chimaeric gene in nodulated hairy roots, and revealed the existence of at least two positive regulatory elements. Sequences located between −2000 and −597 were able to stimulate expression in nodules but not protoplasts, while the region from −597 to −354 enhanced expression in both nodules and protoplasts. Results obtained with the hybrid gln-γ: :35 S promoters showed that two overlapping restriction fragments (−516/−343 and −474/−293) were able to stimulate expression from a heterologous promoter in an orientation-dependent manner. Previous work has demonstrated the presence of conserved A/T-rich binding sites for nuclear proteins in the region between −516 and −446, and their possible role in regulating gln-γ expression is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00027079
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  • 37
    Electronic Resource
    Electronic Resource
    Functional elements of the Arabidopsis Adh promoter include the G-box (1992)
    Springer
    Plant molecular biology 19 (1992), S. 859-862 
    Details
    ISSN: 1573-5028
    Keywords: luciferase ; cis-DNA element ; GUS ; protoplasts ; PEG
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The alcohol dehydrogenase (Adh) gene of Arabidopsis is expressed constitutively in immature seedlings and cells in suspension, and may be induced by hypoxic stress only in roots of mature plants. Deletions and G-box mutations of the Adh promoter were assayed in Arabidopsis protoplasts by PEG-mediated transient expression. Sequence domains necessary for full gene activity are confined to the 384 bp immediately 5′ to the transcription start site, and deletion to −177 results in 〉90% reduction in promoter activity. Site-specific mutations of G-box bases result in 〉60% reduction in activity and disrupt G-box factor binding in vitro.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00027081
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  • 38
    Electronic Resource
    Electronic Resource
    Potato granule-bound starch synthase promoter-controlled GUS expression: regulation of expression after transient and stable transformation (1992)
    Springer
    Plant molecular biology 20 (1992), S. 19-30 
    Details
    ISSN: 1573-5028
    Keywords: granule-bound starch synthase ; potato ; protoplasts ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the β-glucuronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient to result in tissue-dependent GUS expression: levels in stably transformed microtubers exceeded levels in corresponding leaves by orders of magnitude. GBSS-GUS constructs could be transiently expressed in leaf protoplasts from wild-type and amylose-free potato lines, etuberosumSolanum brevidens, Nicotiana tabacum andArabidopsis thaliana. Transient expression levels in potato leaf protoplasts were clearly lower than in corresponding suspension cell protoplasts. This lower expression in leaf protoplasts could not be elevated by increasing DNA concentrations during transfection. Light incubation of electroporated suspension cell protoplasts reduced transient GBSS-GUS expression, whereas incubation of transfected protoplasts in media with different sucrose concentrations did not affect transient expression levels. However, electroporated protoplasts, isolated from suspensions, which had been grown on media with increasing amounts of sucrose showed a sucrose concentration-dependent transient expression profile. This indicates that studying GBSS regulation by transient expression experiments needs pre-treatment of the protoplast source. Sequence data of the GBSS promoter were compared to those of two other potato alleles.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00029145
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  • 39
    Electronic Resource
    Electronic Resource
    Effect of two consensus sequences preceding the translation initiator codon on gene expression in plant protoplasts (1992)
    Springer
    Plant molecular biology 18 (1992), S. 815-818 
    Details
    ISSN: 1573-5028
    Keywords: expression cassette ; gene expression ; protoplasts ; translation initiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression cassettes containing a duplicated cauliflower mosaic virus (CaMV) 35S promoter fused to a polylinker preceded by the CCACCATGG and AACAATGG sequences were constructed. These two sequences correspond to the consensus sequences around the translation start codons in vertebrates and plants respectively. Translational fusions were made with the β-glucuronidase-coding sequence and transient expression was recorded in tobacco mesophyll protoplasts. Approximately three times more GUS activity was found in protoplasts incubated with the constructs harbouring translational fusions as compared to a control harbouring a transcriptional fusion. No significant difference was observed between GUS activities obtained with the two consensus sequences.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00020027
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  • 40
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    Electronic Resource
    DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts (1992)
    Springer
    Plant molecular biology 18 (1992), S. 703-712 
    Details
    ISSN: 1573-5028
    Keywords: DNA methylation ; geminivirus ; protoplasts ; tomato golden mosaic virus ; transfection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m5C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m5C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00020012
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  • 41
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    Transfer of resistance to Verticillium dahliae Kleb. from Solanum torvum S.W. into potato (Solanum tuberosum L.) by protoplast electrofusion (1992)
    Springer
    Euphytica 64 (1992), S. 39-47 
    Details
    ISSN: 1573-5060
    Keywords: Solanum tuberosum ; S. torvum ; Verticillium dahliae ; protoplasts ; electrofusion ; somatic hybrids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Interspecific somatic hybrid plants were regenerated after electrofusion of mesophyll protoplasts with the objective of transferring resistance to Verticillium dahliae from Solanum torvum into potato. Early selection of the putative hybrids was based on differences in cultural behaviour of the parental and hybrid calli (particularly the ability of the latter to regenerate early) in combination with morphological markers. Four putative hybrids were recovered from hundreds of calli, probably resulting from complementation of the two parental genomes. The regenerates were tetraploids (2n=4×=48 chromosomes) and exhibited intermediate traits including leaf form, plant morphology and the presence of anthocyanin. The hybrid nature of the four selected plants was confirmed by examining isoenzyme patterns for isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), phosphoglucoisomerase (Pgi) and 6-phosphogluconate dehydrogenase (6-Pgd). While the hybrid plants rooted readily and grew vigorously under in vitro conditions, in the greenhouse their development and growth were retarded by difficulties in rooting. When grafted on potato or S. torvum rootstocks, the hybrid plants recovered normal development and growth. Again, they exhibited intermediate morphological traits. Tests for resistance realized in vitro with medium containing 50% Verticillium wilt filtrate showed that all the somatic hybrids were resistant to the fungus filtrate.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023536
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  • 42
    Electronic Resource
    Electronic Resource
    Improved culture technique for strawberry (Fragaria × ananassa Duch.) protoplasts and the determination of DNA content in protoplast derived plants (1992)
    Springer
    Plant cell, tissue and organ culture 30 (1992), S. 127-133 
    Details
    ISSN: 1573-5044
    Keywords: flow cytometry ; plant regeneration ; protoplasts ; strawberry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A reproducible and efficient protoplast to plant-system has been developed for strawberry. By using young shoots growing on medium supplemented with cytokinin we were able to exclude the detrimental enzyme Pectolyase from our enzyme solution. The more gentle isolation procedure reported in this paper, together with the agarose-embedding system had a pronounced effect on protoplast viability and growth. Plant regeneration was enhanced by using thidiazuron instead of benzyladenine. Flow cytometric measurement of the DNA content in protoplast derived plants revealed that 27 out of 51 plants contained the amount of DNA corresponding to octoploid level. Fifteen plants exhibited chromosome doubling(16×), one was diplodecaploid(12×) and eight plants mixoploid.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00034306
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  • 43
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    Electronic Resource
    Multiple ocs-like elements required for efficient transcription of the mannopine synthase gene of T-DNA in maize protoplasts (1992)
    Springer
    Plant molecular biology 20 (1992), S. 219-233 
    Details
    ISSN: 1573-5028
    Keywords: promoter ; electroporation ; protoplasts ; transient assay ; Agrobacterium ; Ti plasmid ; Zea mays
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Regulatory elements controlling transcriptional activity of the mannopine synthase 2′ promoter (mas 2′) were defined by analysis of deletion mutants in transient expression assays in maize protoplasts. Deletion of the region between −305 and −290 containing sequence similarity to the octopine synthase (ocs) promoter element reduced activity by 67% compared to wild type activity. Less than 1% of the activity remained in 5′ deletions downstream of −153. Inclusion of various heterologous enhancer-like sequences immediately upstream of position −325 increased activity by up to 7.5-fold. Insertion of the −325 to −275 sequence alone, or in combination with heterologous enhancer-like elements, restored activity of some of the 5′-deletion mutants. Restoration of activity was not obtained with mutants deleted past position −127. Our results suggest that a single class of nuclear proteins from maize interact with high affinity at elements designated mas b (−306 to −275; mas 1′ element), d (−127 to −108), and e (−82 to −39; mas 2′ element) as well as the 20 bp element from the ocs promoter. Although the binding site at mas d only appears to accommodate a single protein, this element has the potential to make a weak, but positive, contribution to the activity of the mas 2′ promoter. The binding of nuclear proteins could not be demonstrated at mas a and c, both of which showed limited homology to the ocs element. Mutational evidence suggested that mas a and c may also contribute to mas 2′ transcription.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00014490
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  • 44
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    Electronic Resource
    Induction of fast-growing and morphologically different strains through intergeneric protoplast fusions of Ulva and Enteromorpha (Ulvales, Chlorophyta) (1992)
    Springer
    Journal of applied phycology 4 (1992), S. 57-65 
    Details
    ISSN: 1573-5176
    Keywords: Ulva ; Enteromorpha ; protoplasts ; electrofusion ; intergeneric fusion ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Isolated protoplasts of Ulva pertusa and Enteromorpha prolifera were electrically fused. Treatment of protoplasts in 1% protease for 15–20 min prior to fusion enhanced fusion ability. Protoplasts from each fusion partner were mixed together in 1:1 ratio in low conductivity electrofusion solution at a density of 1 × 105 cells ml−1 before subjecting them to electrofusion. The protoplasts were aligned in AC field (1MHz, 25 V for 10–15 s) and subsequently fused by a high intensity single DC pulse of 250 V for 25 μs duration. Fusion buffer supplemented with 1 mM calcium and 1 mM magnesium yielded optimum fusion frequencies (about 18–24%). Entrapment of fusion treated cells inside agarose/agar plate facilitated marking and regeneration of fusion products. The regeneration patterns of fused protoplasts were similar to normal (unfused) protoplast development. Most of the regenerated plants from fusion products had a thallus similar to either U. pertusa type or E. prolifera type. Although some of the plants of the former were morphologically similar to U. pertusa, but most had a higher growth rate (1.9 to 1.5 times) than U. pertusa. Furthermore the thallus of some plants had a characteristic irregular and dentate margin, which was never observed in the parental type.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00003961
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  • 45
    Electronic Resource
    Electronic Resource
    Flow cytometric analysis of Saccharomyces cerevisiae autolytic mutants and protoplasts (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992), S. 39-45 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320080104
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  • 46
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    Electronic Resource
    Callus production from willow (Salix viminalis L.) protoplasts (1991)
    Springer
    Plant cell, tissue and organ culture 27 (1991), S. 243-248 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00157587
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  • 47
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    A comparative assessment of purification techniques for mesophyll protoplasts of Brassica napus L. (1991)
    Springer
    Plant cell, tissue and organ culture 24 (1991), S. 43-47 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00044264
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  • 48
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    Lotus corniculatus L.: Morphological and cytological analysis of regenerants from three sources of tissue and selected progeny (1991)
    Springer
    Plant cell, tissue and organ culture 25 (1991), S. 27-33 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00033909
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  • 49
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    Electronic Resource
    Transgenic rice cell lines and plants: expression of transferred chimeric genes (1991)
    Springer
    Plant molecular biology 16 (1991), S. 807-820 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00015073
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  • 50
    Electronic Resource
    Electronic Resource
    Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method (1991)
    Springer
    Plant molecular biology 17 (1991), S. 235-242 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00039498
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  • 51
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    Regeneration of plantlets fromEnteromorpha (Ulvales, Chlorophyta) protoplasts in axenic culture (1991)
    Springer
    Journal of applied phycology 3 (1991), S. 265-275 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00003585
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  • 52
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    Plant regeneration from callus and protoplasts of Brassica nigra (IC 257) through somatic embryogenesis (1990)
    Springer
    Plant cell reports 9 (1990), S. 427-430 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00232265
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  • 53
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    Isolation and culture of Lens culinaris Medik. cv. Eston epicotyl protoplasts to calli (1990)
    Springer
    Plant cell, tissue and organ culture 20 (1990), S. 75-79 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00034762
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  • 54
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    Improved protoplast culture and stability of cytoplasmic traits in plants regenerated from leaf protoplasts of cauliflower (Brassica oteracea ssp.botrytis) (1990)
    Springer
    Plant cell, tissue and organ culture 21 (1990), S. 227-236 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00047615
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  • 55
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    Electronic Resource
    Genetical analysis of in vitro cell and tissue culture response in potato (1990)
    Springer
    Plant cell, tissue and organ culture 23 (1990), S. 181-186 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00034429
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  • 56
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    Importance of myo-inositol, calcium, and ammonium for the viability and division of tomato (Lycopersicon esculentum) protoplasts (1990)
    Springer
    Plant cell, tissue and organ culture 23 (1990), S. 27-37 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00116086
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  • 57
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    PEG-enhanced, electric field-induced fusion of tonoplast and plasmalemma of grape protoplasts (1990)
    Springer
    Plant cell, tissue and organ culture 23 (1990), S. 107-114 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00035830
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  • 58
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    Digestion of seaweeds by the marine amoeba Trichosphaerium (1990)
    Springer
    Hydrobiologia 204-205 (1990), S. 409-413 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00040264
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  • 59
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    Isolation and culture of grape vine cv. Chardonnay leaf protoplasts (1990)
    Springer
    Euphytica 47 (1990), S. 39-44 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00040361
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  • 60
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    Development of a heat shock inducible expression cassette for plants: Characterization of parameters for its use in transient expression assays (1990)
    Springer
    Plant molecular biology 14 (1990), S. 949-967 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00019392
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  • 61
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    Analysis of promoter activity from an α-zein gene 5′ flanking sequence in transient expression assays (1990)
    Springer
    Plant molecular biology 15 (1990), S. 755-764 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00016125
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  • 62
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    Hybrid genes in the analysis of transformation conditions. 3. Temporal/spatial fate of NPTII gene integration, its inheritance and factors affecting these processes in Nicotiana plumbaginifolia (1990)
    Springer
    Plant molecular biology 14 (1990), S. 687-696 
    Details
    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).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00016501
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  • 63
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    Electronic Resource
    mRNAs newly synthesized by tobacco mesophyll protoplasts are wound-inducible (1990)
    Springer
    Plant molecular biology 15 (1990), S. 485-496 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00019165
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  • 64
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    Electronic Resource
    Studies on the preparation and on the properties of sea snail enzymes (1984)
    Springer
    Hydrobiologia 116-117 (1984), S. 319-320 
    Details
    ISSN: 1573-5117
    Keywords: seaweed ; enzyme ; protoplasts ; decomposition ; preparation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00027694
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  • 65
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    Electronic Resource
    Improved protoplast culture and agarose media (1983)
    Springer
    Plant cell, tissue and organ culture 2 (1983), S. 217-226 
    Details
    ISSN: 1573-5044
    Keywords: protoplasts ; agarose ; Nicotiana tabacum ; Nicotiana plumbaginifolia ; Daucus carota ; Hyoscyamus muticus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Media solidified with agarose resulted in higher plating efficiency of protoplasts than commonly used agar media. Improved culture efficiency was observed with mesophyll protoplasts of Nicotiana tabacum and N. plumbaginifolia and with protoplasts isolated from cell lines of Daucus carota, Hyoscyamus muticus and two lines of N. tabacum. The improvement with agarose was consistent over a wide cell density range and also for different media. The positive effect was not due to the lower temperature at which the protoplasts could be plated. Culture experiments with mixtures of different agar types generally gave intermediate division frequencies. There was no obvious effect on plating efficiency in experiments where diffusion was permitted between agar and other agar types.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00033560
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  • 66
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    Flow cytometric analysis of tobacco and cowpea protoplasts infected in vivo and in vitro with alfalfa mosaic virus (1983)
    Springer
    Plant molecular biology 2 (1983), S. 19-25 
    Details
    ISSN: 1573-5028
    Keywords: alfalfa mosaic virus ; protoplasts ; viral antigen detection flow cytometry ; fluorescence-activated cell sorter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Immunofluorescence flow cytometry was used to study the distribution of viral antigen in protoplast populations. Protoplasts were isolated from healthy and alfalfa mosaic virus (AMV) infected tobacco leaves (designated in vivo infected). Furthermore isolated tobacco and cowpea protoplasts were infected in vitro with AMV. The FITC-conjugated antibodies could penetrate formaldehyde fixed protoplasts. The flow cytometric measurements were rapid and reproducible. Comparable immunofluorescence patterns were found for all infected samples (per sample 104 protoplasts were measured). Infectious virus could only be detected in in vivo infected tobacco protoplasts and in in vitro infected cowpea protoplasts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00187572
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  • 67
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    An alfalfa mosaic virus RNA 2 mutant, which does not induce a hypersensitive reaction in cowpea plants, is multiplied to a high concentration in cowpea protoplasts (1983)
    Springer
    Plant molecular biology 2 (1983), S. 85-88 
    Details
    ISSN: 1573-5028
    Keywords: alfalfa mosaic virus ; mutant ; protoplasts ; supplementation ; repression ; host response ; hypersensitive reaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A mutant of alfalfa mosaic virus (AMV), which in contrast to wild type (wt) can invade cowpea plants systemically, is replicated more efficiently in cowpea protoplasts than the wt. Mutant preparations isolated from infected cowpea protoplasts contained a higher amount of middle component (M, containing RNA 2) than wt preparations. Both in cowpea plants and in cowpea protoplasts a wt phenotype is obtained upon addition of wt M to this mutant, suggesting a correlation between the type of plant reaction evoked by the virus infection and the regulation of viral RNA synthesis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01595169
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  • 68
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    Electronic Resource
    Rotation of cells in an alternating electric field theory and experimental proof (1982)
    Springer
    The journal of membrane biology 67 (1982), S. 13-26 
    Details
    ISSN: 1432-1424
    Keywords: cell rotation ; polarization ; protoplasts ; dielectric breakdown
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Protoplasts ofAvena sativa rotate in an alternating electric field provided that at least two cells are located close to each other. An optimum frequency range (20 to 30 kHz) exists where rotation of all cells exposed to the field is observed. Below and above this frequency range, rotation of some cells is only occasionally observed. The angular velocity of rotation depends on the square of the electric field strength. At field strengths above the value leading to electrical breakdown of the cell membrane, rotation is no longer observed due to deterioration of the cells. The absolute value of the angular velocity of rotation at a given field strength depends on the arrangement of the cells in the electric field. A maximum value is obtained if the angle between the field direction and the line connecting the two cells is 45o. With increasing distance between the two cells the rotation speed decreases. Furthermore, if two cells of different radii are positioned close to each other the cell with the smaller radius will rotate with a higher speed than the larger one. Rotation of cells in an alternating electric field is described theoretically by interaction between induced dipoles is adjacent cells. The optimum frequency range for rotation is related to the relaxation of the polarization process in the cell. The quadratic dependence of the angular velocity of rotation on the field strength results from the fact that the torque is the product of the external field and the induced dipole moment which is itself proportional to the external field. The theoretical and experimental results may be relevant for cyclosis (rotational streaming of cytoplasm) in living cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01868644
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  • 69
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    Sugarcane tissue and protoplast culture (1981)
    Springer
    Plant cell, tissue and organ culture 1 (1981), S. 149-164 
    Details
    ISSN: 1573-5044
    Keywords: Saccharum ; cell culture ; protoplasts ; embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Experiments are described which improve the protocols for initiating in vitro cultures of sugarcane and allowing efficient regeneration of plants even after 30 months of callus proliferation. Procedures adopted included use of leaf base explants, CS medium with 3 mg/l 2, 4-D and 0.25 mg/l kinetin for callus initiation and growth, MS medium with 0.5 mg/l IAA and 1 mg/l BAP for shoots, MS medium with 5 mg/l NAA and 7% (wt/vol) sucrose for rooting of shoots. Casein hydrolysate (N-Z amine) significantly shortened the lag period in the growth of sugarcane suspension cultures, but did not increase the rate of growth following the lag phase. Protoplasts isolated from two types of cultures could be grown to re-establish cell cultures but no plants have yet been regenerated derived from isolated protoplasts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02318913
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  • 70
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    Winged-bean protoplasts: Isolation and culture to callus (1981)
    Springer
    Plant cell, tissue and organ culture 1 (1981), S. 201-209 
    Details
    ISSN: 1573-5044
    Keywords: protoplasts ; suspension cells ; callus formation ; winged bean ; Psophocarpus tetragonolobus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A system was developed for protoplast isolation and culture from suspension cultured cells of winged bean,Psophocarpus tetragonolobus. Cells from a three-day-old suspension were incubated in an enzyme mixture containing 6% cullulysin, 1% Macerase, 1% desalted Rhozyme, 0.4M sorbitol, and 0.1M CaCl2 at pH 5.5. Average yields of protoplasts were 6.5 × 106 per gram fresh weight of cells. Protoplasts were cultured in modified B5 medium containing 68.4 g/l glucose, 250 mg/l xylose, 0.1 mg/l 2,4-D, 0.5 mg/l BAP, 250 mg/l N-Z amine type AS, and 20 ml/l coconut water. After 24 h of culture, the protoplasts had synthesized a new wall, and in three days had begun division. The optimum plating density was 1–2 × 103 protoplasts/ml. The division frequency ranged between 40%–60% for most experiments with a high of 72% in one experiment. After three weeks, cell colonies could be transferred to solid MS medium containing N-Z amine and coconut water where callus developed. This protoplast system is technically comparable to soybean for experiments concerned with genetic manipulation involving legumes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02318916
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  • 71
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    factors increasing the fusion efficiency ofBacillus subtilis (1980)
    Springer
    Bulletin of experimental biology and medicine 89 (1980), S. 666-668 
    Details
    ISSN: 1573-8221
    Keywords: protoplasts ; fusion of protoplasts ; regeneration ; “primary prototrophs.”
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00835815
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  • 72
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    The phenotypic characterisation of R2 generation transgenic rice plants under field and glasshouse conditions (1955)
    Springer
    Euphytica 85 (1955), S. 395-401 
    Details
    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
    URL: http://dx.doi.org/10.1007/BF00023972
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  • 73
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    Studies of the mechanism of transgene integration into plant protoplasts: improvement of the transformation rate (1955)
    Springer
    Euphytica 85 (1955), S. 53-61 
    Details
    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
    URL: http://dx.doi.org/10.1007/BF00023930
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  • 74
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    Gene specificity is maintained in transient expression assays with protoplasts derived from different tissues of barley (1955)
    Springer
    Euphytica 85 (1955), S. 203-207 
    Details
    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
    URL: http://dx.doi.org/10.1007/BF00023949
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  • 75
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    Protoplast technology for the breeding of top-fruit trees (Prunus, Pyrus, Malus, Rubus) and woody ornamentals (1955)
    Springer
    Euphytica 85 (1955), S. 287-294 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023958
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