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Showing papers on "Protoplast published in 2001"


Journal ArticleDOI
Jen Sheen1
TL;DR: The development of defined protoplast transient expression systems for high-throughput screening and systematic characterization of gene functions has greatly contributed to elucidating plant signal transduction pathways, in combination with genetic, genomic, and transgenic approaches.
Abstract: Plant protoplasts show physiological perceptions and responses to hormones, metabolites, environmental cues, and pathogen-derived elicitors, similar to cell-autonomous responses in intact tissues and plants. The development of defined protoplast transient expression systems for high-throughput screening and systematic characterization of gene functions has greatly contributed to elucidating plant signal transduction pathways, in combination with genetic, genomic, and transgenic approaches.

628 citations


Journal ArticleDOI
TL;DR: The results indicate that the auxin signal for protoplast swelling is perceived by extracellular ABP1.
Abstract: Protoplasts of corn coleoptiles and Arabidopsis hypocotyls respond to the plant hormone auxin with a rapid change in volume. We checked the effect of antibodies directed against epitopes of auxin-binding protein 1 from Arabidopsis thaliana (AtERabp1) and Zea mays (ZmERabp1), respectively. Antibodies raised against the C-terminus of AtERabp1 inhibited the response to auxin, while antibodies raised against a part of box a, the putative auxin-binding domain, induced a swelling response similar to that caused by auxin treatment. Synthetic C-terminal oligopeptides of ZmERabp1 also caused a swelling response. These effects occurred regardless of whether the experiments were carried out with homologous (anti-AtERabp1 antibodies on Arabidopsis protoplasts or anti-ZmERabp1 antibodies in maize protoplasts) or heterologous immunological tools. The results indicate that the auxin signal for protoplast swelling is perceived by extracellular ABP1.

112 citations


Journal ArticleDOI
TL;DR: The stress-induced amplification of the tobacco Tnt1 element is analyzed to provide direct evidence that factors of microbial origin are able to induce retrotransposon amplification in plants, and strengthen the hypothesis that stress modulation of transposable elements might play a role in generating host genetic plasticity in response to environmental stresses.
Abstract: We have analyzed the stress-induced amplification of the tobacco Tnt1 element, one of the rare active plant retrotransposons. Tnt1 mobility was monitored using the retrotransposon-anchored SSAP strategy that allows the screening of multiple insertion sites of high copy number elements. We have screened for Tnt1 insertion polymorphisms in plants regenerated from mesophyll leaf cells, either via explant culture or via protoplast isolation. The second procedure includes an overnight exposure to fungal extracts known to induce high levels of Tnt1 transcription. Newly transposed Tnt1 copies were detected in nearly 25% of the plants regenerated via protoplast isolation, and in less than 3% of the plants derived from explant culture. These results show that Tnt1 transcription is followed by transposition, and that fungal extracts efficiently activate Tnt1 mobility. Transcription appears to be the key step to controlling Tnt1 amplification, as newly transposed Tnt1 copies show high sequence similarities to the subpopulations of transcribed Tnt1 elements. Our results provide direct evidence that factors of microbial origin are able to induce retrotransposon amplification in plants, and strengthen the hypothesis that stress modulation of transposable elements might play a role in generating host genetic plasticity in response to environmental stresses.

111 citations


Journal ArticleDOI
TL;DR: Papadakis et al. as discussed by the authors showed that the expression of totipotency in protoplasts is related to the activity of cellular antioxidant machinery during protoplast culture.
Abstract: We previously showed that during protoplast isolation, an oxidative burst occurred and the generation of active oxygen species was differentially mediated in tobacco (Nicotiana tabacum) and grapevine (Vitis vinifera), accompanied by significant quantitative differences (A.K. Papadakis, K.A. Roubelakis-Angelakis [1999] Plant Physiol 127: 197–205). We have now further tested if the expression of totipotency in protoplasts is related to the activity of cellular antioxidant machinery during protoplast culture. Totipotent (T) tobacco protoplasts had 2-fold lower contents of intracellular O2.− and H2O2 and 7-fold lower levels of O2.− and H2O2 in the culture medium, compared with non-totipotent (NT) tobacco protoplasts. Addition of alkaline dimethylsulfoxide, known to generate O2.−, resulted in isolation of tobacco protoplasts with reduced viability and cell division potential during subsequent culture. Active oxygen species levels decreased in tobacco and grapevine protoplasts during culturing, although higher contents of O2.− and H2O2 were still found in NT- compared with T-tobacco protoplasts, after 8 d in culture. In T-tobacco protoplasts, the reduced forms of ascorbate and glutathione predominated, whereas in NT-tobacco and grapevine protoplasts, the oxidized forms predominated. In addition, T-tobacco protoplasts exhibited severalfold lower lipid peroxidation than NT-tobacco and grapevine protoplasts. Furthermore, several antioxidant enzyme activities were increased in T-tobacco protoplasts. Superoxide dismutase activity increased in tobacco, but not in grapevine protoplasts during culturing due to the increased expression of cytoplasmic Cu/Zn-superoxide dismutase. The increase was only sustained in T-tobacco protoplasts for d 8. Together, these results suggest that suppressed expression of totipotency in protoplasts is correlated with reduced activity of the cellular antioxidant machinery.

89 citations


Journal ArticleDOI
TL;DR: Protoplast culture and plant regeneration of the dessert banana cultivar Grande Naine were achieved through somatic embryogenesis and the transfer of microcalli and protoplast-derived cell suspensions onto regeneration medium containing plant growth regulators slightly increased the number of embryos relative to those maintained on a feeder layer with growth regulators.
Abstract: Protoplast culture and plant regeneration of the dessert banana cultivar Grande Naine (Musa spp., Cavendish sub-group AAA) were achieved through somatic embryogenesis. Protoplasts were isolated from cell suspensions at a yield of 3×107 protoplasts/ml packed cell volume (0.5 g). For the induction of cell divisions, two banana cell suspensions, SF265 (AA) and IRFA903 (AA), were used as feeder layers. SF265 (AA) was found to be more efficient for inducing cell divisions than IRFA903 (AA). The first embryogenic cell suspensions were established from protoplast-derived microcalli. The transfer of microcalli and protoplast-derived cell suspensions onto regeneration medium containing plant growth regulators slightly increased the number of embryos relative to those maintained on a feeder layer with growth regulators. Plant regeneration was achieved in the same regeneration medium.

66 citations


Journal ArticleDOI
TL;DR: A remarkable similarity was observed in the inhibitory effect of CO(2) on the respiration rate was almost identical for protoplasts and intact pears, suggesting that protoplast suspensions are useful for the study of other aspects of the resppiration metabolism.
Abstract: The influence of the O2 and CO2 concentration and the temperature on the O2 uptake rate of cool-stored intact pears and pear cell protoplasts in suspension was compared. Protocols to isolate pear cell protoplasts from pear tissue and two methods to measure protoplast respiration have been developed. Modified Michaelis–Menten kinetics were applied to describe the effect of the O2 and the CO2 concentration on the O2 uptake rate and temperature dependence was analysed with an Arrhenius equation. Both systems were described with a non-competitive type of CO2 inhibition. Due to the inclusion of gas diffusion properties, the Michaelis–Menten constant for intact pears (2.5 mM) was significantly larger than the one for protoplasts in suspension (3 mM), which was in turn larger than the Michaelis–Menten constant obtained in mitochondrial respiration measurements described in the literature. It was calculated that only 3.6% of the total diffusion effect absorbed in the Michaelis–Menten constant for intact pears, could be attributed to intracellular gas diffusion. The number of cells per volume of tissue was counted microscopically to establish a relationship between the pear cell protoplast and intact pear O2 uptake rate. A remarkable similarity was observed: values of 61.8 nmol kg � 1 s � 1 for protoplasts and 87.1 nmol kg � 1 s � 1 for intact pears were obtained. Also, the inhibitory effect of CO2 on the respiration rate was almost identical for protoplasts and intact pears, suggesting that protoplast suspensions are useful for the study of other aspects of the respiration metabolism.

59 citations


Book
19 Nov 2001
TL;DR: This book discusses the origins, nature, and Significance of Variation in Tissue Culture, and the application of Molecular Markers in Plant Breeding.
Abstract: Contents * Foreword * Preface * Chapter 1. Introduction * Types of In Vitro Culture * Applications of Plant Tissue Culture * Chapter 2. Morphogenesis/Organogenesis * Introduction * Plant Growth * Cellular Differentiation * Morphogenesis * Chapter 3. Micropropagation * Definition * Stages in Micropropagation * Commercial Micropropagation * Applications of Micropropagation * Chapter 4. Haploid Plant Production In Vitro * Anatomy of Anther * Anther Culture * Androgenesis * Chapter 5. In Vitro Pollination and Fertilization * Development of Female Gametophyte * Pollination * Fertilization * Embryo Culture * Chapter 6. Somatic Hybridization Using Protoplast Technology * Introduction * Uses of Protoplast Technology * Obtaining Protoplasts * The Culture of Protoplasts * The Cytoplasmic Genomes * Common Potential of Protoplast Fusion * Chapter 7. Cell Culture and Selection of Desirable Traits * Selection of Naturally Occurring Variants in Culture * General Selection Strategies * Chapter 8. In Vitro Mutagenesis * Types of Mutagens * Determining the Type and Suitable Concentration of Mutagens * The Choice of Plant Tissues for In Vitro Mutagensis * Chapter 9. The Origin, Nature, and Significance of Variation in Tissue Culture * Introduction * The Basis of Somaclonal Variations * Causes of Somaclonal Variations * Use of Somaclonal Variation in Breeding * Prevention of Somaclonal Variation * Chapter 10. Cryopreservation and Plant Breeding * Introduction * Theory and Technology * Cryopreservation Protocols for Cold-Hardy and Non-Cold-Hardy Species * Storage and Thawing * Equipment for Cryopreservation * Practical Issues and Strategies Toward Improved Cryoprotection * Chapter 11. In Vitro Micrografting * Definition of Micrografting * Analysis of Compatibility and Incompatibility Phenomena * Chapter 12. In Vitro Flowering: Its Relevance to Plant Breeding * Factors Influencing In Vitro Flowering * Plant Growth Regulators * Mineral Nutrients and Other Medium Components * Explant, Light, and Other Variables * Application of In Vitro Flowering to Plant Breeding * Chapter 13. In Vitro Tuberization * Introduction * Factors Controlling Microtuber Production * Practical Aspects of In Vitro Tuberization * Chapter 14. Molecular Plant Breeding * Types of Molecular Markers * Major Objectives of Molecular Breeding * Applications of Molecular Markers in Plant Breeding * Case Study: Application of Molecular Markers in Barley (Hordeum vulgare) Breeding * References * Index

58 citations


Journal ArticleDOI
TL;DR: In this paper, the nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP) patterns of diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents.
Abstract: Somatic hybridization offers the possibility of manipulating chloroplast and mitochondrial genomes and evaluating their role on cultivar qualities in citrus. Numerous associations between Willow-leaf mandarin (Citrus deliciosa Ten.), as embryogenic parent, and sweet orange cv. Valencia (Citrus sinensis (L.) Osb.), as mesophyll parent, and between Willow-leaf mandarin (embryogenic parent) and grapefruit cv. Duncan (Citrus paradisi Macf.) (mesophyll parent) were obtained by the fusion of protoplasts induced by polyethylene glycol. Regenerated plants were characterized by flow cytometry and nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP). All plants were diploid. Diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents, while only grapefruit nuclear types were found in the mandarin + grapefruit progeny. The diploid plants with the nuclear profile of the mesophyll parent originated systematically from cells formed through spontaneous association of the nuclear genome of the mesophyll parent and the mitochondrial genome of the embryogenic parent. These plants are assumed to be alloplasmic hybrids or cybrids. They were viable and have been propagated for field testing.

54 citations


Journal ArticleDOI
TL;DR: Direct transformation, polyethyleneglycol (PEG) induced protoplast transformation and conjugal transfer was established for A. japonicum MG417-CF17, the ethylenediaminedisuccinic acid (EDDS) producer and it was demonstrated that conjugalTransfer of the plasmid pSET152 from Escherichia coli ET12567 (pUB307) to Amycolatopsis spores is possible.

42 citations


Journal ArticleDOI
TL;DR: Virulence tests of the transformants showed that there was no significant loss in the pathogenicity toward Helicoverpa armigera third instar larvae, and this method of transformation should prove useful with entomopathogenic fungal species in which a genetic transformation system has not yet been established.
Abstract: Beauveria bassiana transformants were obtained by conventional protoplasting and transformed by eletroporation and polyethylene glycol (PEG) treatment. These displayed mitotic stability in Beauveria bassiana . Strains transformed with pSV50 harbouring the g -tubulin gene of Neurospora crassa grew well on benomyl concentrations of 10 w g ml -1 unlike the recipient strain. The transformants were mitotically stable on either selective or non-selective medium. The efficiency of transformation by linear and circular pSV50 cosmid was 8 and 10 transformants per mug DNA per ml viable protoplast by electroporation, respectively, and 4 and 6 by the protoplast PEG method, respectively. Southern blot and hybridization of undigested fungal DNA of wild type and four transformants, probed with g -tubulin sequence of pSV50, showed hybridization at high M r region of genomic DNA in four transformants, whereas in wild type genomic DNA, no homology of the sequence was observed. Digested genomic DNA, of four tran...

40 citations


Journal ArticleDOI
TL;DR: Plant regeneration from protoplast culture of Crocus cancellatus was investigated using regenerable embryogenic calli obtained from shoot meristem culture on LS on medium supplemented with 4 mg l+1 kinetin and 1 mg l−1 2,4-dichlorophenoxyacetic acid.
Abstract: Plant regeneration from protoplast culture of Crocus cancellatus was investigated using regenerable embryogenic calli obtained from shoot meristem culture on LS (Linsmaier and Skoog, 1965) medium containing 4 mg l−1 kinetin and 1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D). Protoplasts were isolated directly from embryogenic calli. The best protoplast growth was found on those embedded in Ca-alginate beads and cultured with nurse cells in MS (Murashige and Skoog, 1962) medium supplemented with 2 mg l−1 kinetin, 1 mg l−1 2,4-D and 100 mg l−1 ascorbic acid at 25 °C in darkness. After 4–5 weeks of culture, microcalli appeared on the surface of the Ca-alginate beads, but the protoplasts without immobilization in Ca-alginate beads showed very low cell division. Growth of the microcalli in the medium with nurse cells was much better than in the medium without nurse cells. Transferring beads onto half strength MS medium supplemented with 0.2 mg l−1 kinetin and 0.1 mg l−1 2,4-D, increased the growth of embryogenic calli. Somatic embryo development was observed either on half strength MS medium growth regulator free or with 1 mg l−1 abscisic acid. Matured embryos germinated on half strength MS medium containing 25 mg l−1 of gibberelic acid. Plantlet formation was obtained on half strength MS medium containing 1 mg l−1 6-benzyladenine and 1 mg l−1 α-naphthaleneacetic acid at 20 °C in a 16/8 h light/dark cycle.

Journal ArticleDOI
01 Feb 2001
TL;DR: Efficient protoplast culture procedures have been worked out from cell suspension as a source, however, regeneration of plants posed a developmental block in both parental and hybrid calli.
Abstract: Damask rose, Rosa damascena, an important species among the scented roses, yields a highly fragrant commercially important essential oil. It is commonly used in perfumery industry, beverages, soft drinks, medicines etc. Besides rose oil, the other products are rose water, rose concrete, rose absolute. "gulkand" (a sugary preparation) etc. R. bourboniana, a related species, is also used also used for rose oil extraction. For achieving faster rates of multiplication, tissue culture methods are best employed and may be of great commercial value in establishing plantations. Micropropagation protocols using nodal segments were established in R. damascena and R. bourboniana. Rooted plants were transferred to field. In addition protoplast culture studies were also carried out in the two species of scented rose. Friable callus was initiated from stem and leaf segments inoculated on Murashige and Skoog (1962) medium supplemented with varying concentrations of 2,4-D (1-10 muM), NAA (1-10 muM), BAP (1-10 muM). Efficient protoplast culture procedures have been worked out from cell suspension as a source, however, regeneration of plants posed a developmental block in both parental and hybrid calli. Alternative explants for their regeneration potential are under assessment and promising results are obtained for genetic manipulation of these important essential oil bearing rose species.

Journal ArticleDOI
TL;DR: The construction of inulin-assimilating and sorbitol-producing fusants was achieved by intergeneric protoplast fusion between Kluyveromyces sp.
Abstract: The construction of inulin-assimilating and sorbitol-producing fusants was achieved by intergeneric protoplast fusion between Kluyveromyces sp. Y-85 and Saccharomyces cerevisiae E-15. The cells of parental strains were pretreated with 0.1% EDTA (w/v) and 2-mercaptoethanol (0.1%, v/v) and then exposed to 2.0% (w/v) Zymolase at 30 °C for 30–40 min. The optimized fusion condition demonstrated that with the presence of 30% (w/v) polyethylene glycol 6000 (PEG-6000) and 10 mM CaCl2 for 30 min, the fusion frequency reached 2.64 fusants/106 parental cells. The fusants were screened by different characters between two parental strains and further identified by DNA contents, inulinase activity and sorbitol productivity. One of the genetically stable fusants, Strain F27, reached a maximal sorbitol production of 4.87 g/100 ml under optimal fermentation condition.

Journal ArticleDOI
TL;DR: A protoplast fusion system for A. alternata was used to analyze the genetics of HST production and its relation to the specific pathogenicity of these pathogens, finding that all fusants pathogenic to apple maintained this gene.
Abstract: The genetic controls of host-specific toxin (HST) biosynthesis and the pathogenicity of A. alternata pathogens have been limited by the asexual nature of the life cycle of these fungi. We used a protoplast fusion system for A. alternata to analyze the genetics of HST production and its relation to the specific pathogenicity of these pathogens. Drug-resistant transformants were isolated by genetic transformation, using vectors conferring resistance to hygromycin B and geneticin, for the A. alternata apple pathotype (AM-toxin producer) and A. alternata tomato pathotype (AAL-toxin producer), respectively. Protoplasts of the respective transformants were fused by electrofusion. The majority of resultant stable fusants produced both AM- and AAL-toxins and were pathogenic to susceptible cultivars of both apple and tomato. Pulsed-field gel electrophoresis analysis demonstrated that these fusants (or hybrids) carried small 1.7and 1.1-Mb chromosomes, characteristic of the parental strains of the apple and tomato pathotypes, respectively. Detection of the AMT gene, involved in AM-toxin biosynthesis, by polymerase chain reaction revealed that all fusants pathogenic to apple maintained this gene. Microfluorimetry analysis using propidium iodide staining suggested that the fusants might be diploid. (Received Nobember 14, 2000 ; Accepted December 11, 2000)

Journal ArticleDOI
TL;DR: The whole chromosomal DNA must be certainly incorporated into competent cells from the following reasons; purB is opposite to trpC on the chromosome, and 2.7×103 protoplasts per ml is about 100 times lower than 3.2×105 competent cells per ml, and the cotransfer ratio is constant at all the concentrations.
Abstract: Competent cells of Bacillus subtilis AC870 (purB, leuB, trpC, ald-1) were transformed to Ade+, Trp+, or Ade+ Trp+ with DNA in protoplast lysates of B. subtilis AC819 (hisH, tet-1, rpsL, smo-1). The contransfer ratio of purB to trpC was constant at 7-9% (Ade+ Trp+/Trp+) or 3% (Ade+ Trp+/Ade+) at protoplast concentrations of 2.7×103∼2.7×106 per ml. The whole chromosomal DNA must be certainly incorporated into competent cells from the following reasons; (1) purB is opposite to trpC on the chromosome, (2) 2.7×103 protoplasts per ml is about 100 times lower than 3.2×105 competent cells per ml, and (3) the cotransfer ratio is constant at all the concentrations. Similar results were obtained with the cotransfer ratio of purA to trpC. The transformation requires several Com proteins including ComK.

Journal ArticleDOI
TL;DR: It is concluded that transference and recombination of nuclear DNA can be achieved effectively by symmetric and asymmetric fusion, hybrids with small fragment translocation which are valuable in plant breeding can be obtained directly by asymmetrical fusion.
Abstract: Symmetric and asymmetric protoplast fusion between long term cell suspension-derived protoplasts ofTriticum aestivum (cv. Jinan 177) and protoplasts ofHaynaldia villosa prepared from one-year-old embryogeneric calli was performed by PEG method. In asymmetric fusion, donor calli were treated with gamma ray at a dose of 40, 60, 80 Gy (1.3 Gy/min) respectively and then used to isolate protoplasts. Results of morphological, cytological, biochemical (isozyme) and 5S rDNA spacer sequence analysis revealed that we obtained somatic hybrid lines at high frequency from both symmetric and asymmetric fusion. Hybrid plants were recovered from symmetric and low dose γ-fusion combinations. GISH (genomicin situ hybridization) analysis proved exactly the existence of both parental chromosomes and the common occurrence of several kinds of translocation between them in the hybrid clones regenerated from symmetric and asymmetric fusion. And the elimination of donor DNA in hybrid clones regenerated from asymmetric fusion combinations was found to increase with the increasing gamma doses. It is concluded that transference and recombination of nuclear DNA can be achieved effectively by symmetric and asymmetric fusion, hybrids with small fragment translocation which are valuable in plant breeding can be obtained directly by asymmetric fusion.

Journal ArticleDOI
TL;DR: The Rhizoctonia strain was highly sensitive to hygromycin B, so transformation of protoplasts was achieved with a polyethylene glycol-based procedure, using plasmid pES200 containing the bacterial hygromcin B phosphotransferase gene driven by a trpC promoter from Aspergillus nidulans.

Journal ArticleDOI
TL;DR: Protoplast viability was determined by fluorescein diacetate staining and transient gene expression was studied after electroporation of protoplasts with plasmid DNA containing a β-glucuronidase (GUS) reporter gene.
Abstract: Establishment of protoplast techniques for conifers is of crucial importance to forest biotechnology. Protoplasts can be used to regenerate whole trees and to study transcriptional regulation in woody plants by promoter analysis. Here we describe a technique for isolating protoplasts fromPinus pinaster shoots. Protoplast viability was determined by fluorescein diacetate staining. Transient gene expression was studied after electroporation of protoplasts with plasmid DNA containing a β-glucuronidase (GUS) reporter gene. The 35S promoter and a pine (cytosolic glutamine synthetase) promoter both were able to driveGUS expression, indicating applicability of conifer protoplasts as a model to study gene promoters using transient expression.

Dissertation
30 Nov 2001
TL;DR: Efficient regeneration systems and methods of DNA transfer were established for rapeseed and sugarbeet and straightened the way for successful plastid transformation in either species.
Abstract: In the current study tissue cultures of rapeseed (cv. “Drakkar”, cv. “Westar”) and sugarbeet (cv. ”Viktoria”, cv. “VRB”, cv. ”31-188”, cv. ”7T1308” and 47 other breeding lines, Appendix 1) have been investigated for the establishment of conditions that make possible plastid transformation in both species. Tobacco leaf protoplasts (cv. ”petite Havana”, cv. ”Wisconsin 38”) were used to develop a novel technique – the TAL (thin-alginate-layers) technique. The TAL technique in combination with new culture media resulted in very rapid protoplast development and fast shoot regeneration (in less than two weeks). This method was also successfully applied to improve protoplast culture of rapeseed and of the extremely recalcitrant species sugarbeet. Factors, which included protoplast source, mineral and organic composition of isolation and culture media, influence of growth regulators etc. were investigated and conditions for protoplast culture and regeneration were established for both species. According to reports in the literature, only protoplasts from guard cells could be regenerated into plants. Thus, an alternative and reproducible method of shoot regeneration from protoplasts isolated from hypocotyl derived callus was successfully developed. While no shoot regeneration was observed from guard cell protoplasts in our experiments, plant regeneration (efficiencies up to 30%) from callus protoplasts could be achieved for the first time in this study. The influence of different parameters on the efficiency of callus formation from etiolated hypocotyl explants was investigated. Protoplasts from callus and hypocotyl derived callus were used for the experiments on nuclear transformation in sugarbeet. Both, the PEG method and the biolistic method were successfully applied to obtain nuclear transformants as confirmed by molecular methods (PCR analysis and Southern blot hybridisation). The biolistic method was applied for plastid transformation experiments in sugarbeet. Species specific vectors containing the aadA cassette were constructed for plastid transformation in rapeseed and sugarbeet. However, difficulties to select plastid transformants were observed due to a high natural resistance to spectinomycin and streptomycin in rapeseed. In sugarbeet spectinomycin at a concentration of 100 mg/l was found efficient for selection and spectinomycin and streptomycin resistant colonies were obtained after callus bombardment. The presence of the aadA gene in antibiotic-resistant lines was proven by PCR analysis, but an integration of DNA into the plastome could not be verified so far. Efficient regeneration systems and methods of DNA transfer were established for rapeseed and sugarbeet and straightened the way for successful plastid transformation in either species.

Journal ArticleDOI
TL;DR: Protoplast yield was enhanced with higher enzyme concentrations, longer digestion times and treatment of cells with β‐mercaptoethanol, and could be useful in the genetic manipulation of yeast of industrial importance.
Abstract: Release of viable protoplasts of Saccharomyces cerevisiae and Candida tropicalis was achieved using fresh crude enzyme extracts of the giant African snail Achatina achatina. Optimum results of 2.8 x 10(6) protoplast ml(-1) were obtained when 1 g (wet wt) of cell slurry from the yeast strains was first treated with 1% beta-mercaptoethanol for 10 min and incubated with the undiluted crude enzyme for 120 min using 1.0 mol l(-1) sorbitol as osmotic stabilizer. Protoplast yield was enhanced with higher enzyme concentrations, longer digestion times and treatment of cells with beta-mercaptoethanol. Percentage regeneration of protoplast to viable cells in isotonic medium containing 0.01 mol l(-1) CaCl2 was in the range of 52-77%. These findings could be useful in the genetic manipulation of yeast of industrial importance.

Journal ArticleDOI
TL;DR: Electrofused protoplasts of Primula malacoides cv ‘Lovely Tokyo’ were electrofused with cell suspension-derived protoplast of P. obconica to produce somatic hybrids, suggesting that the elimination of genetic materials or polyploidization had occurred in some of the hybrid plants obtained.

Journal ArticleDOI
TL;DR: A continuous micropropagation was established from protoplasts of the green alga Enteromorpha intestinalis to optimise the product quality and a promising storage process has been developed which involves including protoplast in beads of alginic acid gel.
Abstract: A continuous micropropagation was established from protoplasts of thegreen alga Enteromorpha intestinalis. The effects of two differentcrude enzymes and the osmolarity at different concentrations of the enzymesolution on algal protoplast yields were tested. The optimal enzymecomposition for cell wall digestion and protoplast viability was 2%cellulase R 10 Onozuka and 2% Aplysie with 0.5 m mannitol. Largenumbers of Enteromorpha protoplasts were released (10.0 × 106protoplasts from 1 g fresh thalli) and settled on a rangeof substrata. Regeneration of the protoplasts followed the normal patternfor this species. Conditions for pure cultures and efficient systems offloating supports with nets were determined to optimise the product qualityof plantlets of Enteromorpha. A promising storage process has beendeveloped which involves including protoplasts in beads of alginic acid gel.Plants regenerated from protoplasts may also be used as seedstock tofacilitate propagation for macroalgal culture.

01 Jan 2001
TL;DR: Regenerated plants were obtained from cultured protoplasts in a series of media based on the MS medium and showed some degrees of aneuploid but basically were similar to the original potato plants.
Abstract: The use of in vitro potato shoot cultures for protoplast isolation is desirable for consistent protoplast qualities. Ethylene build-up in the culture vessels causes problems for leaf growth (e.g. decreasing leaf surface, dry weight and chlorophyll) in shoot culture. Fifty to one hundred µM Silver thiosulfate (STS) produced a larger leaf area as a source of plant material for protoplast isolation. STS also decreased the internode length of potato shoots but increased dry weight and chlorophyll. Regenerated plants were obtained from cultured protoplasts in a series of media based on the MS medium. Protoplasts of Delaware showed a better response in cell division and colony formation in agarose-solidified culture medium. Regenerated plants showed some degrees of aneuploid but basically were similar to the original potato plants.

Journal ArticleDOI
TL;DR: The union of potato monoploid genotypes through protoplast fusion may result in vigorous somatic hybrids due to a reduction of the “genetic load” normally present in this highly heterozygous tetraploid crop.
Abstract: The union of potato monoploid genotypes (2n=1x =12) through protoplast fusion may result in vigorous somatic hybrids due to a reduction of the “genetic load” normally present in this highly heterozygous tetraploid (2n=4x=48) crop. More than 100 androgenic monoploids derived from diploid (2n=2x=24)Solanum phureja Juz. & Buk. andS. chacoense Bitt. xS. phureja clones were evaluated in field trials during 1996 and 1997 to identify the most promising genotypes for protoplast fusion experiments. Compared to the total population, the 1996 selected genotypes had higher means for tuber number (30.1 vs 11.2 tubers/plant), average tuber weight (3.0 vs 1.8 g/tuber) and total yield (66.1 vs 20.4 g/plant). Similarly, the 1997 selected genotypes had higher means for tuber number (42.8 vs 25.4 tubers/plant), average tuber weight (3.6 vs 2.5 g/tuber) and total yield (114.0 vs 63.4 g/plant) compared to the total population. The 31 selected monoploid genotypes from 1996-97 varied in their response to protoplast isolation and culture from no growth (9), cell enlargement (5), limited cellular divisions (8), callus formation (5) to plant regeneration from callus (4). Chemical fusion and electrofusion produced three groups of intermonoploid somatic hybrids. Polymorphic simple sequence repeat (SSR) loci enabled distinction of somatic hybrids from parental somaclones. Rapid DNA extraction with SSR analysis enabled screening of calluses to identify somatic hybrid tissue prior to plant regeneration. The somatic hybrids were highly polyploid, mostly hexaploid (2n=6x=72), possibly due to fusion of endopolyploid protoplasts and/or chromosomal doubling during plant regeneration.

Journal ArticleDOI
TL;DR: A simple protocol for regenerating plants from leaf protoplasts of vegetable Brassicas, viz., cabbage, cauliflower and broccoli, which occurred within 6-6 weeks of culture initiation and was easily rooted on MS medium without growth regulators.
Abstract: We report here a simple protocol for regenerating plants from leaf protoplasts of vegetable Brassicas, viz., cabbage, cauliflower and broccoli. Protoplasts from in vitro grown leaf material were cultured in Kao’s medium with a supplementation of 2,4-D, NAA, BAP and glucose, initially in dark for 3d and subsequently in light. Dilution of protoplast cultures was effected on the 7th, 10th and 13th day of culture initiation with Kao’s medium supplemented with sucrose, and reduced 2,4-13 content; NAA was omitted. Micro-colonies were plated on a K3 medium having 2,4-D, BAP and sucrose gelled with agarose. Transfer of calli to another K3 medium with zeatin regenerated shoots from cauliflower protoplast derived calli, whereas a medium with kinetin and zeatin supported shoot regeneration in cabbage and broccoli. Shoot regeneration occurred within 6-6 weeks of culture initiation. Shoots were easily rooted on MS medium without growth regulators.

Journal ArticleDOI
TL;DR: A cucurbit protoplast isolation protocol is applicable to both cucumber and squash leaf tissue with significant increases in yields of viable protoplasts suitable for electroporation, compared to other published methods.

Journal ArticleDOI
TL;DR: An efficient and rapid procedure has been developed to establish embryogenic cell suspension cultures of two Japonica Chinese commercial rice cultivars, Zhonghua 8 and Eryi 105, and reproducible plant regeneration from 14–26% of the protoplast-derived tissues was achieved without the requirement for nurse cells.
Abstract: An efficient and rapid procedure has been developed to establish embryogenic cell suspension cultures of two Japonica Chinese commercial rice cultivars, Zhonghua 8 and Eryi 105. Embryogenic cell suspensions of both varieties were established from 0.5–1.0 g fresh weight of embryogenic callus in AA medium within 2.5 months of the initiation of callus from sterilised seeds. The previously reported subculture of callus on semi-solid medium for 4–8 weeks prior to transfer into liquid medium was unnecessary and caused delay in the establishment of embryogenic cell suspensions. Protoplasts were isolated reproducibly from cell suspensions up to 18 months after their initiation, with protoplast plating efficiencies attaining 0.15–0.37%. Reproducible plant regeneration from 14–26% of the protoplast-derived tissues was achieved without the requirement for nurse cells.

Journal ArticleDOI
TL;DR: The results show that GFP gene expression can occur in various sweet potato tissues, and that it may be a useful sereenable marker to improve transformation efficiency and obtain transgenic sweet potato plants.
Abstract: The expression of the green-fluorescent protein (GFP) gene from Aequorea victoria (jellyfish) was analyzed by transient and stable expression in sweet potato Ipomoea batatas L. (Lam.) ev. Beauregard tissues by electroporation and particle bombardment. Leaf and petiole segments from in vitro-raised young plantlets were used for protoplast isolation and electroporation. Embyrogenic callus was also produced from leaf segments for particle bombardment experiments. A buffer solution containing 1×106 protoplasts ml−1 was mixed with plasmid DNA containing the GFP gene, and electroporated at 375 V cm−1. Approximately 25–30% of electroporated mesophyll cell protoplasts subsequently cultured in KM8P medium regenerated cell walls after 48 h. Of these, 3% emitted bright green fluorescence when exposed to UV-blue light at 395 nm. Transformed cells continued to grow after embedding in KM8P medium solidifed with 1.2% SeaPlaque agarose. Stable expression of GFP was observed after 4 wk of culture in approximately 1.0% of the initial GFP positive cells (27.5 GFP positive micro callases out of 3024 cells which transiently expressed GFP 48 h after electroporation). In a separate experiment, 600–700 bright green spots were observed per plate 48 h after bombarding leaf segments or embryogenic cellus. In bombarded cultures, several stable GEP-expressing sectors were observed in leafderived embryogenic callus grown without selection for 4 wk. These results show that GFP gene expression can occur in various sweet potato tissues, and that it may be a useful sereenable marker to improve transformation efficiency and obtain transgenic sweet potato plants.

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TL;DR: The development of a protoplast fusion system and its use for genetic analysis of HST production and specific pathogenicity of the tomato pathotype of A. alternata that produces AAL-toxin as a HST is reported.
Abstract: Several pathotypes of Alternaria alternata are known to produce host-specific toxins (HSTs) as agents of pathogenicity or virulence. However, investigations into the genetic controls of HST biosynthesis and pathogenicity of Alternaria pathogens have been limited by the lack of a sexual stage in the life cycle of these pathogens. We report here the development of a protoplast fusion system and its use for genetic analysis of HST production and specific pathogenicity of the tomato pathotype of A. alternata that produces AAL-toxin as a HST. Drug-resistant transformants have been isolated by genetic transformation of nonpathogenic A. alternata (strain O-94) and A. alternata tomato pathotype (strain As-27) with vectors conferring resistance to hygromycin B and geneticin, respectively. Protoplasts of the respective transformants were fused by polyethylene glycol treatment or electrofusion. Fusion products were selected by culturing in the presence of both hygromycin B and geneticin, then confirmed by amplification using a polymerase chain reaction with specific primers to the transforming drug-resistance genes. Stable fusants were purified by successive subcultures on selective medium and single-spore isolation. The resultant stable fusants, probably inter-strain hybrids, had the same pathogenicity and toxin production as the wild-type strain As-27. These results suggest that protoplast fusion has potential applications for genetic analysis of A. alternata pathogens.

Journal ArticleDOI
TL;DR: In this method, the initial pH of culture medium was elucidated as a critical factor and optimized for both transformation efficiency and avermectin productivity.
Abstract: The optimal pH conditions for efficient transformation of protoplasts and intact cells were established in avermectin high-producing mutants, ATCC31780 and L-9. Among all factors tested, protoplast buffer pH was elucidated as the most important factor influencing transformation efficiency. The optimal pH of the protoplast buffer for the regeneration of ATCC31780 was 6.5, and using this condition, 4.5 × 106 transformants per μg of pIJ702 were produced. At pH 6.3, the maximal number of L-9 transformants was 1.6 × 105 per μg of the same plasmid. However, the protoplasting process decreased avermectin productivity to half or one-sixth of ATCC31780 or L-9, respectively. To avoid the productivity loss, electroporation of intact cells without lysozyme treatment was developed for these mutant strains even though this method was approximately 100-fold less efficient. In this method, the initial pH of culture medium was elucidated as a critical factor and optimized for both transformation efficiency and avermectin productivity.