Showing papers on "Protoplast published in 1991"
TL;DR: It is shown that a wide variety of diverse cultivars can be transformed and it is possible to produce transgenic monocoty–ledonous plants by transforming scutellar tissue of immature embryos by using electric discharge particle acceleration.
Abstract: We have recovered transgenic rice plants from a number of commercially important cultivars, including until now recalcitrant Indica varieties, using electric discharge particle acceleration Immature embryos from greenhouse–grown plants were bombarded with gold particles carrying DNA, and transgenic plants were recovered following a simple culture protocol Mendelian segregation of foreign genes was observed in R1 progeny and stable integration was demonstrated by Southern blot analysis of genomic DNA isolated from progeny plants Alternative transformation protocols that are dependent on the development of protoplast and suspension culture systems are no longer necessary as we have shown that a wide variety of diverse cultivars can be transformed Transgenic plants expressing agronomically useful traits such as herbicide resistance have been obtained and are currently undergoing further evaluation This report also demonstrates that it is possible to produce transgenic monocoty–ledonous plants by transforming scutellar tissue of immature embryos
640 citations
TL;DR: For most species the efficiency of plantlet production from somatic embryos is poor and remains a problem for the commercial utilization of this technology, so improvements in knowledge about conifer somatic embryogenesis are needed.
Abstract: There is currently considerable interest in developing techniques for producing somatic embryos from conifers. Somatic embryos could be used for micropropagation of trees with desirable characteristics such as improved dimension increment, better quality wood and disease resistance. The availability of somatic embryos would also provide excellent experimental material for basic studies of conifer embryo development. Finally, embryogenic callus and suspension cultures should facilitate the development of a protoplast regeneration system to be used in future genetic manipulation studies. The use of embryogenic material has previously proved successful for developing protoplast systems with other recalcitrant species (e.g. Vasil and Vasil 1986).
299 citations
TL;DR: Isolated egg cells of maize showed protoplasmic streaming during 22 days of culture, but they did not divide, but after fusion of the sperm with the egg cells, these fused cells did develop, with a mean division frequency of 83%, and grew to multicellular structures.
Abstract: Electrofusion-mediated in vitro fertilization of maize using single sperm and egg cells was performed. Sperm cells were released from pollen grains after rupture of the latter by osmotic shock in the fusion medium (0.55 M mannitol). Egg cells were isolated by enzyme treatment (pectinase, pectolyase, hemicellulase, and cellulase) followed by mechanical isolation. The conditions generally used for the electrical fusion of protoplasts of somatic cells were also applied to the protoplasts of gametic cells of maize. Electrofusion was performed with single pairs of gametes under microscopic observation. The mean fusion frequency was 79%. Isolated egg cells of maize showed protoplasmic streaming during 22 days of culture, but they did not divide. However, after fusion of the sperm with the egg cells, these fused cells did develop, with a mean division frequency of 83%, and grew to multicellular structures. Egg cells and fusion products were cultivated with a maize feeder-cell system.
212 citations
TL;DR: Tnt1 transcription features provide a first example of tissue culture‐induced mutagenesis in plants and a molecular basis for some of the somaclonal variation events.
Abstract: The Tnt1 transposable element of tobacco belongs to the retrotransposon family and shares the structural features of viral retroelements including two long terminal repeats (LTRs) which are known to contain promoter regions. We show that two Tnt1 RNAs of 5.2 and 6.5 kb can be found. The 5.2 kb RNA matches with the size of the Tnt1 elements so far isolated (5.3 kb), whilst the evidence suggests that the 6.5 kb RNA could be a chimaeric RNA initiated in a gene in which Tnt1 has inserted. The Tnt1 5.2 kb RNA starts in the LTR, and the LTR can promote the expression of a translational LTR-beta-glucuronidase (GUS) fusion at a high level in transient expression assays. The Tnt1 5.2 kb RNA and the LTR-GUS fusion of transgenic tobacco plants are specifically expressed in leaf-derived protoplasts whereas they are not expressed in leaf tissue. The 5.2 kb RNA is also transcribed at low levels in roots. This RNA is induced after 2 h of maceration in the protoplast isolation medium, and its level declines rapidly after protoplast isolation. The induction requires only the presence of cell wall hydrolases, and is independent of wounding and plasmolysis. The induction of Tnt1 expression is not mediated by typical oligosaccharide elicitors released from the cell wall known to mediate defense gene responses. Tnt1 transcription features provide a first example of tissue culture-induced mutagenesis in plants and a molecular basis for some of the somaclonal variation events.
185 citations
TL;DR: In vitro techniques are presently being applied to Eucalyptus to achieve genetic transformations, and suspension and protoplast cultures have been achieved and plants have been regenerated from protoplasts.
Abstract: Micropropagation has the potential to provide very high multiplication rates of selected tree genotypes, with resulting short-term silvicultural gains. Aseptic cultures have been established from seeds, seedlings, shoots, flowers and lignotubers. Callus cultures have been established from a wide range of tissue sources for at least 30 species of Eucalyptus. Plant regeneration from callus was successful for 12 of these species. Micropropagation through axillary proliferation, or adventitious shoot proliferation on nodal explants, or both, has been successful. An agar-based medium of Murashige and Skoog with a low auxin/cytokinin ratio is most commonly used for shoot multiplication. Vitrification and shoot senescence remain problems. Gibberellic acid was added in some media to stimulate shoot elongation. Various media are used for in vitro root initiation. Suspension and protoplast cultures have been achieved and plants have been regenerated from protoplasts. In vitro techniques are presently being applied to Eucalyptus to achieve genetic transformations.
111 citations
TL;DR: Proline accumulation in the halophyte Distichlis spicata L occurred rapidly following initiation of NaCl-induced stress and stabilized at an elevated level within 48 h of exposure to NaCl, suggesting that mRNA translation but not mRNA transcription may be necessary prior to production of proline in stressed cells.
92 citations
TL;DR: Hopes that the transfer of desirable traits between sexually incompatible species will prove to be a practical approach for the genetic improvement of crops are stimulated by successful plant protoplast culture and fusion techniques.
90 citations
Book•
01 Nov 1991
TL;DR: This book discusses cellular techniques for extraction, transformation, and analysis of Structural and Functional Analysis of Genomes and Genes in Plants, using Arabidopsis thaliana as a model.
Abstract: Foreword.- 1 Cellular Techniques - General Introduction.- 1.1 Isolation and Culture of Protoplasts.- 1.2 Mutagenic Treatments on Isolated Cells.- 1.3 Somatic Hybridization.- 1.3.1 Protoplast Fusion - The PEG System.- 1.3.2 Electrofusion of Protoplasts.- 1.4 Dilution Series as a Tool to Improve Medium Composition.- 1.5 Plating Efficiency Evaluation in a Peroxidase Assay.- 1.6 Production of Haploid Plants.- 1.6.1 Pollen Culture for Haploid Production in Tobacco.- 1.6.2 Haploid Induction Via Anther Culture as a Tool to Study Developmental Processes.- 1.7 Isolation of Viable Microspores and Immature Pollen Grains from Cereal Inflorescences.- 1.8 Isolation of Viable Sperm Cells from Corn (Zea mays) Pollen Grains.- 1.9 Embryo Rescue in Nicotiana plumbaginifolia.- 1.10 Use of Iodide Ions for Chemical Reduction of the Oxidative Agent H2O2 and Hypochlorites after Application as Decontaminating Agents for Plant Tissues.- 2 Transformation Techniques - General Introduction.- 2.1 Agrobacterium Transformation of Various Arabidopsis Expiants.- 2.2 Direct Gene Transfer.- 2.2.1 Direct Gene Transfer into Protoplasts - The Chemical Approach.- 2.2.2 Direct Gene Transfer - Electroporation for Transient Expression in Protoplasts.- 3 Extraction Techniques - General Introduction.- 3.1 Isolation of DNA and RNA from Arabidopsis thaliana.- 3.1.1 Large-Scale Extraction of Arabidopsis Genomic DNA.- 3.1.2 Mini-Scale DNA Extraction Procedure.- 3.1.3 Extraction of Nuclei and Nuclear DNA.- 3.1.4 RNA Extraction Procedure.- 3.1.5 Isolation of Poly (A+)-RNA.- 3.2 Total DNA Extraction - Alternative Protocols.- 3.3 Characterization of Mitochondrial-DNA from Minute Quantities of Plant Material.- 3.4 Generation of Large Amounts of cDNA by Polymerase Chain Reaction from Small Amounts of Total RNA.- 3.5 Isolation of Nuclei from Plant Tissues.- 3.6 Extraction of Amino Acids from Plant Samples and Their Analysis Using Ion-Exchange Chromatography.- 3.7 Electroelution of Proteins from Plant Tissues.- 3.8 Extraction, Purification and Analysis of Endogenous Indoleacetic Acid and Abscisic Acid.- 4 Aspects of Structural and Functional Analysis of Genomes and Genes - General Introduction.- 4.1 Southern Blot Analysis of Transgenic Nicotiana sp.- 4.2 Northern Blot Analysis of ADH (Alcohol Dehydrogenase) Mutants in Arabidopsis.- 4.3 Western Blot Detection of Proteins Synthesized Transiently in Transfected Plant Protoplasts.- 4.4 Cloning Nuclear Single Copy Sequences for RFLP Analysis.- 4.5 Run-on Transcription in Isolated Plant Nuclei.- 4.6 Preparation of Nuclear Extracts, Gel-Retardation Assay and DNAase I Footprinting.- 4.6.1 Preparation of Nuclear Extracts from Plant Nuclei.- 4.6.2 Gel Retardation Assay Using Large DNA Probes.- 4.6.3 DNAse I Analysis of Retarded Complexes.- 4.6.4 Gel Retardation Using Oligonucleotide Probes.- 4.7 Pulsed-Field Gel Electrophoresis of Plant DNA.- 4.8 Assessing Methylation of Inserted DNA by Restriction with Isoschizomeric Enzymes and Inducing Demethylation with 5-Azacytidine.- 5 Cytological Techniques - General Introduction.- 5.1 Karyotyping with Protoplast Procedures.- 5.2 In situ Hybridization.- 5.2.1 Gene targeting in Plant Metaphase Chromosomes by In situ Hybridization with Tritiated Probe DNA.- 5.2.2 Protocols for In situ Hybridization - The Biotinylation Technique.- 5.3 How Cytometry of Nuclei for Ploidy and Cell Cycle Analysis.- 5.4 Control of Cell Cycle Progression.- 5.5 Induction and Isolation of Micronuclei and Microprotoplasts.- 6 Appendices.- 6.1 Culture Media and Basic Stock Solution.- 6.4 Extraction and Purification of IAA and ABA.- 7 Subject Index.
70 citations
TL;DR: The results suggest that transgenic rice plants carring the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.
Abstract: In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1–2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carring the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.
69 citations
TL;DR: It was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion and could be proved by Southern hybridization to high molecular weight DNA.
Abstract: A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basis layer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l-1 kanamycin or 5 mg l-1 paromomycin. Single resistant cells can be recovered from about 10,000 sensitive cells in one alginate layer. Injection of the neo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.
67 citations
TL;DR: The analysis of the elimination of potato DNA from potato-tomato somatic cell hybrids and the absence of true cybrids is described.
Abstract: This paper describes the analysis of the elimination of potato DNA from potato-tomato somatic cell hybrids. The hybrids were obtained by fusion of protoplasts of a cytoplasmic albino tomato genotype with leaf mesophyll protoplasts of a potato genotype carrying the β-glucuronidase (GUS) gene of Escherichia coli. The potato protoplasts were either isolated from unirradiated plants or from plants irradiated with 50 or 500 Gy of γ-rays. Green calli were selected as putative fusion products. The hybridity of these calli was confirmed by isoenzyme analysis. All of the green calli tested contained a potato-specific chloroplast DNA restriction fragment, and most of the calli analysed were positive for β-glucuronidase activity. In 72 of the hybrid calli we determined the percentage of potato nuclear DNA using species-specific probes. All of the tested green calli contained a considerable amount of potato genomic DNA, irrespective of the dose of irradiation of the potato parent. The limited degree of potato DNA elimination and the absence of true cybrids are discussed.
TL;DR: Protoplast-derived colonies grew slowly into calli and regenerated plants were transferred to the greenhouse and seed was harvested within 7 months of the initial protoplast isolation.
Abstract: Sunflower hypocotyl protoplasts (Helianthus annuus L.) from 5 PIONEER genotypes (PT024, SMF3, EMIL, HA300*PT024, VK5F) and 1 public line (RHa 274) formed colonies at frequencies of up to 60% when plated in 0.25ml agarose beads in a modified L4 medium (Lenee and Chupeau 1986) containing 3mg/l NAA, 1mg/l BA and 0.1mg/l 2,4-D, and 1000mg/l casamino acids. Protoplast-derived colonies grew slowly into calli. Organogenesis was obtained from callus of PT024 on a MS medium containing NAA and BA at 1mg/l and GA at 0.1mg/l. Freshly excised shoots were induced to root by an IAA treatment. Regenerated plants were transferred to the greenhouse and seed was harvested within 7 months of the initial protoplast isolation.
TL;DR: Rose breeding is now open to the full range of in vitro genetic manipulation techniques involving protoplast technology, with high yields of protoplasts isolated from embryogenic suspension cultures of Rosa persica x xanthina and Rosa wichuraiana.
Abstract: High yields of protoplasts were isolated from embryogenic suspension cultures of Rosa persica x xanthina and Rosa wichuraiana using an enzyme mixture comprising 20 g l-1 cellulase ‘Onozuka’ R10, 1 g l-1 Pectolyase Y-23 and 10 g l-1 hemicellulase. Agarose-immobilized protoplasts gave the most consistent growth at a plating density of 5×104 protoplasts ml-1 on the basic medium of Kao & Michayluk (KM8p) containing 2 mg l-1 naphthaleneacetic acid and 1 mg l-1 benzylaminopurine. At 25°C in the dark, 0.004% of R. persica x xanthina protoplasts developed into colonies. Using similar culture conditions, but with a plating density of 9×104 protoplasts ml-1, 0.017% of R. wichuraiana protoplasts developed into colonies. On transfer of R. persica x xanthina colonies to Schenk & Hildebrandt's medium containing 3 mg l-1 2,4-dichlorophenoxyacetic acid, globular and later stage embryos were formed. Approximately 30% of these embryos developed into plantlets on transfer to basal Schenk & Hildebrandt's medium. Further development of the plantlets took place on cellulose plugs (Sorbarods) soaked in Murashige & Skoog's medium containing 0.05 mg l-1 naphthaleneacetic acid, 0.05 mg l-1 indole-3-butyric acid and 0.1 mg l-1 benzylaminopurine. Rose breeding is now open to the full range of in vitro genetic manipulation techniques involving protoplast technology.
Book•
01 Jan 1991
TL;DR: Tobacco protoplast isolation, culture and regeneration, I.M. Negrutiu culture, regeneration and transformation of barley protoplasts, P.S. Johri and P. Srivastava.
Abstract: Tobacco protoplast isolation, culture and regeneration, I. Negrutiu culture, regeneration and transformation of barley protoplasts, P. Lazzeri et al in vitro fertilization of maize, E. Kranz monoclonal antibodies against marker protein for somatic embryogenesis, S. Stirn and H-J. Jacobsen endosperm culture, B.M. Johri and P.S. Srivastava.
TL;DR: Somatic hybrid plants produced after protoplast electrofusion with the objective of transferring insect resistance, which is associated with the presence of glandular hairs, into potato lines exhibited several morphological traits intermediate to both parents including plant habit, leaf form, and particularly short four-lobed glandular trichomes inherited from the wild parent.
TL;DR: Cryopreserved cells proliferated after plating on solid medium and new cell suspensions could be initiated within 15 days and viable protoplasts could be obtained 15–21 days after thawing.
Abstract: A method is described for cryopreservation of cell suspension lines of rice (Oryza sativa L.) for use in protoplast research and as a way of retaining desirable characteristics of cell lines. The procedure involves pre-culture with mannitol, addition of a cryoprotectant solution of sucrose, dimethyl sulfoxide, glycerol and L-proline, two step freezing and storage in liquid nitrogen. Cells have been preserved for up to 14 months (the longest period tried in these experiments). Cryopreserved cells proliferated after plating on solid medium and new cell suspensions could be initiated within 15 days. Viable protoplasts, capable of divisions and callus formation, could be obtained 15-21 days after thawing. Variation between cell lines in terms of recovery rate after cryopreservation occurred. Differences between cell lines in plating efficiencies on solidified medium, however, contributed to this variation. Protoplasts from cryopreserved regenerable cell lines gave rise to embryogenic callus from which plants could be regenerated. These plants developed to maturity. A transformed cell line was also cryopreserved and it had retained the hygromycin resistance and regenerative capacity of the original cell line.
TL;DR: In this paper, B. juncea cv. RLM-198 was fused with mesophyll protoplasts of B. spinescens using polyethylene glycol to produce interspecific hybrids.
Abstract: Hypocotyl derived protoplasts of B. juncea cv. RLM-198 were fused with mesophyll protoplasts of B. spinescens using polyethylene glycol to produce interspecific hybrids. Fusion products could be microscopically identified by characteristics of the protoplasts of both parents in the hybrid cells; they are colourless and vacuolated like the hypocotyl protoplasts and possess chloroplasts of the mesophyll protoplasts. The heterokaryotic fusion frequency was around 5%. However, the frequency of calli regenerating hybrid shoots was more than 10% of the regenerating calli. Putative somatic hybrids had morphological features characteristic of both the parents. Twelve plants analysed cytologically, possessed 52 chromosomes (26II) at meiosis representing the complete genomes of B. juncea (18II) and B. spinescens (8II). For esterase isozymes, the hybrids had bands of Doth the parents. Hybrid nature of some of the plants was confirmed by their close resemblance to B. juncea, chromosome number and isozyme bands of B. spinescens as in Rsp-19. Somatic hybrids had rudimentary, non-dehiscent anthers and completely sterile pollen. However, on back crossing with B. juncea, 10 out of 12 plants produced seeds and about 100 plants were realized.
TL;DR: Additive isozyme patterns and chromosome numbers as well as the expression of parental morphological characters have proved the hybrid origin of the selected regenerants, which have exhibited an intermediate degree of frost tolerance compared to the parents.
Abstract: Somatic hybrids between Solanum tuberosum L. cv. Gracia (2n=4x=48) and Solanum brevidens Phil. (2n=2x=24) were produced via fusion of mesophyll protoplasts. Selection of the protoplast derived putative hybrid calli was based on their vigorous growth. Additive isozyme patterns and chromosome numbers as well as the expression of parental morphological characters have proved the hybrid origin of the selected regenerants. Extensive chromosome loss during the regeneration process resulted in aneuploid hybrids with high frequency. Genomic instability could not be detected in these plants during the period of vegetative propagation. Regenerants from hybrid tissues exhibited wide morphological variation especially in tuber formation. The detailed morphological analysis based on the use of multivariate method (principal component analysis, PCA) enabled to identify morphological groups among the hybrid clones. The positioning of hybrid clones in the PCA space could not be correlated with chromosome numbers. The genomic ratio represented by the tetraploid and diploid parents influenced the morphology of somatic hybrid population according to the applied analytical system. Two selected hybrid clones have exhibited an intermediate degree of frost tolerance compared to the parents, based on the recovery of plants from lower buds after freezing of potted plants.
TL;DR: A transformation system for Ophiostoma ulmi (Buism.) Nannf.
Abstract: A transformation system for Ophiostoma ulmi (Buism.) Nannf. was developed and analyzed. Protoplasts were generated from actively budding yeastlike cells by digestion with NovoZym 234 in MgSO4 after pretreatment with 2-mercaptoethanol. Protoplast regeneration was most efficient when 0.6 M sucrose was used as the osmoticum. Several plasmids containing fusions between fungal promoters and a bacterial gene for hygromycin phosphotransferase successfully transformed O. ulmi to hygromycin resistance. One of these vectors, pPS57, which contains a promoter for isopenicillin N synthetase from Penicillium chrysogenum, consistently conferred the greatest resistance to hygromycin. Linearization of the vector and inclusion of 2-mercaptoethanol in the transformation reaction resulted in enhanced transformation efficiency. Approximately 4 × 103 transformants/μg DNA per 107 protoplasts were obtained using the optimized procedure. Southern hybridization after alternating field and standard electrophoresis suggested random insertion of tandem repeats (some greater than 250 kb) into the fungal chromosomes. Antibiotic resistance was stable through mitosis. However, expression of the transforming DNA after meiosis was highly variable.
TL;DR: Somatic hybrid plants were produced by protoplast fusion of navel orange and ‘Murcott’ tangor and showed hybridity of the plants was confirmed by the restriction endonuclease analysis of nuclear ribosomal DNA.
Abstract: Somatic hybrid plants were produced by protoplast fusion of navel orange and ‘Murcott’ tangor. Hybridity of the plants was confirmed by the restriction endonuclease analysis of nuclear ribosomal DNA. All of the plants (16 clones) were normal, uniform, and had the amphidiploid chromosome number of 36 (2n=2x=18 for each parent). The cpDNA analysis showed that each of the 16 somatic hybrids contained either one parental chloroplast genome or the other. In all cases, the mitochondrial genomes of the regenerated somatic hybrids were of the navel orange type.
Book•
01 Jan 1991
TL;DR: Plant cell culture genetics of cultured plant cells protoplast technology regeneration of plants improvements of plants via plant cell culture natural products and metabolites from plants and plant cell cultures bio-transformations by plants cell cultures strategies of plant cell immobilization plant cell reactor technology economic and strategic considerations.
Abstract: Plant cell culture genetics of cultured plant cells protoplast technology regeneration of plants improvements of plants via plant cell culture natural products and metabolites from plants and plant cell cultures bio-transformations by plants cell cultures strategies of plant cell immobilization plant cell reactor technology economic and strategic considerations.
TL;DR: A friable and fast-growing type of callus was isolated from a long term shoot-competent cell culture of wheat which consisted of small, densely cytoplasmic cells which divided more rapidly but with a lower plant regeneration frequency than the original culture.
Abstract: A friable and fast-growing type of callus was isolated from a long term shoot-competent cell culture of wheat. The suspension cultures established from this callus consisted of small, densely cytoplasmic cells which divided more rapidly but with a lower plant regeneration frequency than the original culture. A high yield of protoplasts was released from suspension cells (2 to 3×107 protoplasts per ml packed cell volume) when treated with enzyme mixtures. The isolated protoplasts divided at a relatively high frequency (20% to 50%) in both liquid and agarose-solidified KM8p medium. Up to 0.21% of the dividing protoplasts continued to divide and form micro-calli. Sixty-eight plants were regenerated from micro-calli, and among the 30 plants which were transplanted to the greenhouse, 3 have survived.
TL;DR: Protoplast fusion was used to combine the cytoplasmic traits of atrazine resistance and male sterility in Brassica oleracea var.
Abstract: Protoplast fusion was used to combine the cytoplasmic traits of atrazine resistance and male sterility in Brassica oleracea var. italica (broccoli). Leaf protoplasts from broccoli with the petaloid B. nigra type of cytoplasmic male sterility were fused with hypocotyl protoplasts from an atrazine-resistant biotype of B. campestris var. oleifera cv Candle (oilseed rape). A total of 19 colonies regenerated shoots, all of which were broccolilike in phenotype, i.e., lacked trichomes. Four shoots, all from one colony, were atrazine resistant, surviving and growing in the presence of 25 μM atrazine. A leaf piece assay also confirmed that they were atrazine resistant. Molecular analysis showed that they contain chloroplasts from the atrazine-resistant B. campestris parent and mitochondria from the B. nigra parent. No recombination or rearrangement of the mitochondrial genomes in the fusion products was detected. These four plants and their progeny all showed the petaloid B. nigra type of male sterility.
TL;DR: Protoplasts of Panax ginseng were isolated from embryos obtained from the 4-year old embryogenic cell line KCTC PCL 49031 which was derived from a zygotic embryo and myo-inositol (6%) was found to be the most suitable osmoticum.
Abstract: Protoplasts of Panax ginseng were isolated from embryos obtained from the 4-year old embryogenic cell line KCTC PCL 49031 which was derived from a zygotic embryo. High protoplast yields of 22-25 × 10(6) protoplast / g tissue were obtained following 5-6 h digestion with 2% Cellulysin, 1% Pectinase and 1% Macerasae in half strength Murashige and Skoog's medium containing 12% mannitol. A plating density of 1×10(5) protoplasts /ml was found optimal for protoplast culture. An initial division frequency of 10% was obtained in an agarosegelled defined medium. Myo-inositol (6%) was found to be the most suitable osmoticum. Somatic embryos were formed from protoplast derived embryogenic callus, which regenerated into plantlets.
TL;DR: Protoplasts were isolated from an embryogenic cell suspension culture derived from microspores of Brassica napus cv.
Abstract: Protoplasts were isolated from an embryogenic cell suspension culture derived from microspores of Brassica napus cv. Jet Neuf. Protoplast yield varied with the cell suspension growth medium. Optimization of protoplast plating density, manipulation of culture medium, carbon source and medium matrix, and inclusion of Ficoll resulted in protoplast plating efficiencies close to 30%. Placement of the protoplasts close to the gas interface contributed greatly to the elevated plating efficiency. Low density cultures could be induced to regenerate calli at optimum plating efficiencies if grown in the presence of nurse culture. This is of great advantage for manipulation of individual protoplasts or for microinjection. Plants were regenerated directly from the cell suspension or from the protoplast cultures.
TL;DR: Somatic hybrids between cultivated lettuce and a wild species L. virosa were produced by protoplast electrofusion and had more vigorous growth than either parent, and had normal flower morphology, but all were sterile.
Abstract: Somatic hybrids between cultivated lettuce (Lactuca sativa) and a wild species L. virosa were produced by protoplast electrofusion. Hybrid selection was based on inactivation of L. sativa with 20mM iodoacetamide for 15 min, and the inability of L. virosa protoplasts to divide in the culture conditions used. Protoplasts were cultured in agarose beads in a revised MS media. In all 71 calli were formed and 21 of them differentiated shoots on LS medium containing 0.1mg/l NAA and 0.2mg/l BA. Most regenerated plants exhibited intermediate morphology. These plants were confirmed as hybrids by isoenzyme analysis. The majority of somatic hybrids had 2n=4x=36 chromosomes, and had more vigorous growth than either parent. Hybrids had normal flower morphology, but all were sterile.
TL;DR: Transgenic R0 and R! plants were shorter, required longer to flower, and had reduced pollen viability compared to non-transformed R, protoplast-derived plants, but NPTII activity was detected in only 8 of these plants.
Abstract: Thirty eight green and 2 albino plants were regenerated from 400 kanamycin-resistant colonies derived from protoplasts isolated from cell suspensions of Oryza sativa variety Taipei 309 and electroporated with pCaMVNEO carrying the neomycin phosphotransferase II {nptll) gene. Twenty of the green transgenic R0 plants were transferred to the glasshouse, where 3 flowered after 7 months. Of 15 plants analysed by DNA hybridization, all carried the nptll gene, but only 2 of 11 plants assayed for NPTII activity expressed the nptll gene. One transgenic R0 plant produced 59 seeds following self-pollination. The seeds, when germinated on medium containing kanamycin sulphate, gave 16 green transgenic R, plants. Five transgenic Rt plants flowered and set seed, 7 flowered but failed to produce seeds, while 4 did not produce panicles. Transgenic R0 and R! plants were shorter, required longer to flower, and had reduced pollen viability compared to non-transformed R, protoplast-derived plants. The nptll gene was present in all 16 transgenic R, plants, but NPTII activity was detected in only 8 of these plants.
TL;DR: It is shown that tetraploid somatic hybrid potato plants can be obtained by the fusion and selection method presented and the majority of the hexaploid hybrids contained 2 genomes of the bleached parent and one genome of the mesophyll parent.
TL;DR: Coffee plants were regenerated from protoplasts isolated from embryogenic cell suspension cultures derived from somatic embryos of Coffea arabica L. cv.
Abstract: Coffee plants were regenerated from protoplasts isolated from embryogenic cell suspension cultures derived from somatic embryos of Coffea arabica L. cv. caturra. Yields of viable protoplasts ranged from 1×105 to 6×105 protoplast/g fresh weight. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the total. Plating efficiencies of protoplast ranged from 1 to 10%. Embryogenic protocolonies obtained after several subcultures in a medium supplemented with 0.5 mg/l each of benzylaminopurine, 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid, were transferred to a medium lacking plant growth regulators. Well differentiated embryos were formed in selected protocolonies that contained many embryos-like structures. Approximately 70% of the somatic embryos developed into green rooted plantlets which were succesfully transferred to vessels containing sterilized scoria. Plants grown for two months in scoria were finally transferred to greenhouse.