Showing papers on "Protoplast published in 1985"

Gene transfer to cereal cells mediated by protoplast transformation

Abstract: Direct gene transfer to cereal cells was achieved by transformation of protoplasts with naked DNA. Protoplasts isolated from cultured cells of Triticum monococcum were incubated in the presence of polyethylene glycol (PEG) with circular and linear plasmid DNA. The pBR322-derived plasmid, pBL1103-4, contained a selectable chimeric gene comprising the protein coding region of the Tn5 aminogly-coside phosphotransferase type II gene (NPT II), the nopaline synthase promoter (pNOS) and the polyadenylation signal of the octopine synthase gene. Transformed cells were selected in medium containing kanamycin and identified by detection of aminoglycoside phosphotransferase II activity.

Plant cell culture : a practical approach

Abstract: 1: Isolation and maintenance of callus and cell suspension cultures. 2: Isolation, culture, and regeneration of protoplasts. 3: Applications of protoplast technology. 3.A: Fusion and selection of somatic hybrids. 3.B: Transient gene expression and stable transformation. 3.C: Studies with viruses. 4: Selection of plant cells for desirable characteristics. 4.A: Inhibitor resistance. 4.B: Cold tolerance. 4.C: In vitro selection for salt tolerance. 4.D: In vitro selection for disease/toxin resistance. 5: Plant regeneration via embryonic suspension cultures. 6: Applied aspects of plant regeneration. 6.A: Micropropagation. 6.B: Virus-free plants. 6.C: Artificial seeds. 7: Cryopreservation. 8: Secondary products from cultured cells and organs I: molecular and cellular approaches. 9: Secondary products from cultured cells and organs II: large scale culture

Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata.

Abstract: Somatic hybrid plants of Rutaceae were obtained by protoplast fusion between Citrus sinensis Osb. ('Trovita' orange) and Poncirus trifoliata. Protoplasts isolated from embryogenic cells of C. sinensis and from leaves of P. trifoliata, and the culture of fusion products in the presence of high concentrations of sucrose were essential requirements for the selection of hybrids. Green globular embryoids derived from protoplasts resulted in the regeneration of trifoliate plants. Other morphological characters of these plants were intermediate between both parents. The chromosome number in one of the hybrid plants was 36, which was the sum of C. sinensis (2n=18) and P. trifoliata (2n=18). EcoRI restriction analysis of rDNA confirmed the presence of parental nuclear DNAs in the hybrid.

A procedure for plant regeneration from immature cotyledon tissue of soybean

Abstract: For some time, soybean (Glycine max (L.) Merr.) has been conspicuous among major crops in lacking an in vitro regeneration system. This deficiency has precluded the application of many nonconventional techniques of crop improvement (See Abelson, 1983; Lea and Stewart, 1984; Hildebrand et al., 1985 for reviews). Although soybean is recalcitrant in vitro, some wild Glycine species are more responsive. Of these, Glycine canescens F. J. Herm appears most amenable; shoot production has been obtained from hypocotyl (Kameya and Widholm, 1981; Widholm and Rick, 1983), cotyledon (Widholm and Rick, 1983; Grant, 1984), leaf and flower tissues (Lazzeri, Hildebrand and Collins, unpublished) and also from protoplasts (Newell and Luu, 1985; Myers, Lazzeri and Collins, unpublished). Shoot regeneration has also been reported in two other wild species, G. tomentella Mayata (Kameya and Widholm, 1981) and G.clandestina Wendl. (Hymowitz et al., 1986).

Totipotency of tomato protoplasts

Abstract: An efficient and reliable protocol for tomato protoplast isolation, culture, and plant regeneration has been developed. Fourteen diverse cultivars were tested. Fertile plants were regenerated from all 14 cultivars without any modification in the protocol. Plating efficiency (percentage of the protoplasts that formed minicalli) of up to 50% was achieved. Those mini-calli rapidly regenerated shoots at high frequencies. Regenerated shoots can be easily rooted on a basal medium with the appropriate auxin, and have been set to soil within two months after the isolation of the protoplasts.

Efficient plasmid transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts.

Abstract: A procedure for efficient transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts with plasmid DNA was developed. Transformation frequencies with S. fradiae protoplasts were strongly influenced by the temperatures for cell growth, protoplast formation, and protoplast regeneration. Transformation frequencies for both species were also influenced by the culture age before protoplast formation, the source and concentration of polyethylene glycol, the transformation-inducing agent, the concentration of protoplasts used in the transformation procedure, and the number of protoplasts added to regeneration plates. Transformation frequencies were substantially higher for both species when calf thymus DNA and protamine sulfate were added to the transformation mix. With S. fradiae, transformation frequencies were much lower with plasmid DNA prepared from other species than with the same plasmids prepared from S. fradiae, suggesting that S. fradiae expresses restriction and modification. With the modified transformation procedures using DNA prepared from homologous hosts, S. ambofaciens and S. fradiae are now transformed routinely at frequencies of 10(6) to 10(7) transformants per micrograms of plasmid DNA.

Protoplast water content of bacterial spores determined by buoyant density sedimentation.

Abstract: Protoplast wet densities (1.315 to 1.400 g/ml), determined by buoyant density sedimentation in Metrizamide gradients, were correlated inversely with the protoplast water contents (26.4 to 55.0 g of water/100 g of wet protoplast) of nine diverse types of pure lysozyme-sensitive dormant bacterial spores. The correlation equation provided a precise method for obtaining the protoplast water contents of other spore types with small impure samples and indicated that the average protoplast dry density was 1.460 g/ml.

Conditions for high frequency embryogenesis from orange (Citrus sinensis Osb. ) protoplasts

Abstract: Using ‘Trovita’ orange (Citrus sinensis Osb.) protoplasts isolated from 6-year-old nucellar callus, the effects of protoplast density and mannitol concentration on cell divisions and embryoid formation were examined.

Culture of plant somatic hybrids following electrical fusion.

Abstract: Electrically-induced protoplast fusion has been used to produce somatic hybrids between Nicotiana plumbaginifolia and Nicotiana tabacum. Following fusion of suspension culture protoplasts (N. plumbaginifolia) with mesophyll protoplasts (N. tabacum) heterokaryons were identified visually and their development was followed in culture. Because electrical fusion is a microtechnique, procedures were developed for culturing the heterokaryons in small numbers and at low density. The fusion and culture procedures described are rapid, uncomplicated and repeatable. Good cell viabilities indicate that the fusion procedure is not cytotoxic. Fused material was cultured 1–2 days at high density in modified K3 medium (Nagy and Maliga 1976). The heterokaryons were isolated manually and grown, at low density in conditioned media. Calli have been regenerated. Esterase isozyme patterns confirm the hybrid character of calli and clonally-derived plantlets recovered from these fusions.

Plant regeneration through somatic embryogenesis in protoplast cultures of sandalwood (Santalum album L.)

Abstract: Protoplasts were isolated from embryogenic cell suspension cultures derived from proliferating shoot segments of a 20-year-old sandalwood tree (Santalum album Linn.). Under appropriate conditions, isolated protoplasts divided in liquid culture medium and produced embryogenic cell aggregates and globular embryos. Plating of cell aggregates on a fresh medium facilitated the differentiation of somatic emryos which further developed into plantlets.

Electric field mediated stable transformation of carrot protoplasts with naked DNA

Abstract: We have developed an electroporation procedure for the transformation of carrot protoplasts with Ti-plasmid DNA from Agrobacterium tumefaciens. The uptake of pTiC58 into carrot protoplasts was mediated by high voltage electrical pulses at field strengths from 0.5 to 3.8 kV/cm. Protoplast regeneration, somatic embryogenesis and plantlet regeneration were unaffected by the electroporation conditions selected for DNA uptake. Uptake of plasmid pTiC58 resulted in hormone independent regeneration of carrot protoplasts. Transformed somatic embryos were detected in carrot cultures 45 days after electroporation. The transformed somatic embryos developed into teratomas which synthesized nopaline. Hybridization was obtained between a labeled T-DNA fragment from pTiC58 and DNA fragments from 4 month old teratomas regenerated from electro-transformed protoplasts. Based on the number of somatic embryos regenerated after electro-transformation, a frequency of 1.6×102 transformants/104 somatic embryos/μg pTiC58 DNA was obtained.

High frequency callus formation from maize protoplasts.

Abstract: A solid feeder layer technique was developed to improve callus formation of Black Mexican Sweet maize (Zea mays L.) suspension culture protoplasts. Protoplasts were plated in 0.2 ml liquid media onto a cellulose nitrate filter on top of agarose-solidified media in which Black Mexican Sweet suspension feeder cells were embedded. Callus colony formation frequencies exceeding 10% of the plated protoplasts were obtained for densities of 103–105 protoplasts/ 0.2 ml, which was 100- to 1,000-fold higher than colony formation frequencies obtained for conventional protoplast plating methods such as liquid culture or embedding in agarose media. Compared with conventional methods, the feeder layer method gave higher colony formation frequencies for three independently maintained Black Mexican Sweet suspension lines. Differences among the three lines indicated that colony formation frequencies might also be influenced by the suspension culture maintenance regime and length of time on different 2,4-dichlorophenoxyacetic acid concentrations. The callus colony formation frequency reported is an essential prerequesite for recovering rare mutants or genetically transformed maize protoplasts.

Electrical Control of Shoot Regeneration in Plant Tissue Cultures

Abstract: Minute electric currents of the order of 1–2 microamps stimulate shoot differentiation up to 5-fold in tobacco callus cultures. The location of the first-formed shoots depends on the direction of the current. Similar stimulations of both root and shoot formation have been observed in wheat. It is suggested that the effect is due to the artificial currents controlling the natural electric currents which flow through plant cells. The role of these currents in regulating eukaryotic differentiation is discussed. Possible applications include the rapid regeneration of plantlets from somaclonal variants and genetically engineered protoplast cultures.

Efficient induction and selection of chloroplast-encoded antibiotic-resistant mutants in Nicotiana

Abstract: A high rate of plastome-encoded mutations was induced in Nicotiana by exposing seeds to N-nitroso-N-methylurea. Seeds then were subjected to nutritional and in vitro selection procedures for systematic isolation of plastome-dependent antibiotic-resistant plants. Multiple flowering lines resistant to streptomycin, spectinomycin, lincomycin, or chloramphenicol were obtained. A detailed analysis of the streptomycin-resistant lines is presented. Sexual hybridization, cybrid formation following protoplast fusion, and in organello protein synthesis were used to rigorously assign the mutations to the chloroplast genome. The efficient rates of mutagenesis combined with the in vitro mass-screening procedures described here should facilitate investigation of fundamental aspects of chloroplast genetics in higher plants.

Intra-specific fusions in Solanum tuberosum.

Abstract: Plants were regenerated from callus arising from protoplast fusion of two S. tuberosum diploids. Tetraploid progeny from the fusion of the two diploid partners had increased vigor. Isozyme analysis confirmed the presence of proteins from both partners in the fusion progeny. Pigmentation of tubers and anthers was heightened substantially in the fusion products. This fusion, the first intra-specific fusion within S. tuberosum, indicates that somatic fusion may be useful for transferring traits within this group.

Fungal Protoplasts: Applications in Biochemistry and Genetics

Abstract: Preface Contributors THE PROTOPLAST SYSTEM The Fungal Cell Wall Vladimir Farkas Mycolytic Enzymes John F. Peberdy Factors Influencing Protoplast Isolation Barry Davis Protoplasts from Fungal Spores Cees J. Bos Protoplasts from Entomophthorales Richard A. Nolan PROTOPLASTS AS TOOLS IN PHYSIOLOGICAL AND BIOCHEMICAL STUDIES Cell Wall Regeneration and Protoplast Reversion Oldrich Necas and Augustin Svoboda The Protoplast Membrane and Antifungal Drugs David Kerridge Metabolic Properties of Protoplasts Susan Isaac Protoplasts and Secondary Metabolism Joan W. Bennett PROTOPLASTS AS GENETIC TOOLS Protoplast Fusion and Genetic Analysis in Cephalosporium John A. Birkett and Paul F. Hamlyn Protoplast Fusion and Incompatibility in Aspergillus James H. Croft Interspecies Hybridization after Protoplast Fusion in Aspergillus Ferenc Kevei and John F. Peberdy Protoplast Fusion and Interspecies Hybrization in Penicillium Jozef Anne and John F. Peberdy Protoplast Fusion in Yeasts Lajos Ferenczy Transfer of Cytoplasmic Genetic Elements by Protoplast Fusion Lajos Ferenczy Some Aspects of Yeast Transformation Albert Hinnen Perspectives for Protoplasts in Genetics of Industrially Important Fungi Christopher Ball Index

Somatic hybridization between Lycopersicon esculentum and Lycopersicon pennellii.

Abstract: Selection and screening methods were devised which resulted in the identification of a number of somatic hybrid callus clones following fusion of Lycopersicon esculentum protoplasts and L. pennellii suspension culture protoplasts. Visual selection for callus morphology combined with a high fusion frequency and irradiation of one parental protoplast type ((137)Cs source, 1.5 Krads) resulted in selection of a callus clone population containing a high proportion of somatic hybrids. Analysis of a dimeric isozyme for the presence of a heterodimeric form was found to be satisfactory for distinguishing parental-type calli, somatic hybrid calli, and mixed calli derived from both types of unfused parental cells. No somatic hybrid calli produced shoots, although the sexual hybrid between L. esculentum and L. pennellii regenerated well under the culture conditions employed. This result suggests that the non-regenerable growth habit of the L. pennellii suspension culture was dominant in the somatic hybrid. The culture conditions described here are suitable for obtaining regenerated plants from L. esculentum mesophyll protoplasts. L. esculentum protoplast calli from fusion cultures gave rise to shoots with L. esculentum phenotype at higher frequency than calli from control unfused L. esculentum mesophyll protoplast cultures. The use of probes for species-specific organelle DNA fragments allowed identification of organelle DNA restriction fragments in digests of total DNA from small samples of individual callus clones. The callus clones analyzed either carried predominantly one parental plastid DNA type or mixtures of both types. Use of a mitochondrial DNA (mtDNA) probe which distinguishes two parental mtDNA fragments revealed that the L. pennellii-specific fragment was present in all clones examined, but the L. esculentum fragment was absent or in low proportion.

Amino acid auxotrophs from protoplast cultures of Nicotiana plumbaginifolia , Viviani

Abstract: Several amino acid requiring auxotrophs have been isolated from unsupplemented protoplast cultures of haploid Nicotiana plumbaginifolia following incubation with BUdR (1-5x10-5, 2 days) and recovery on complete medium. The auxotrophic lines required the following amino acid(s) for growth: his, ile, leu, ile+val, met or try. Met− is a new type isolated in higher plants. The same absolute amino acid requirement was observed in plants regenerated from auxotrophic cultures. Precursor feeding tests, enzyme assays, and/or metabolic complementation through protoplast fusion were used to identify the genetic lesion leading to auxotrophy. Mutant seeds were obtained from supplemented Met− plants. Seeds were also collected from selfed plants regenerated from various complementing fusion products, and a His− revertant. Genetic analysis indicated that under natural conditions of seed formation amino acid auxotrophy-in contrast to NR deficiency-failed to segregate in progeny tests.

The physiological properties of plant protoplasts

Abstract: Introduction: The Use of Plant Protoplasts in Physiological Research (With 1 Figure).- Applications of Protoplast Technology.- Properties of Some Enzymes Used for Protoplast Isolation (With 3 Figures).- Isolation of Maize Protoplasts from the Root Cap and Apex (With 2 Figures).- Plant Protoplast Viability (With 2 Figures).- Isolation of Plasma Membrane from Ryegrass (Lolium multiflorum) Endosperm Protoplasts (With 2 Figures).- The Use of Protoplasts in the Study of Coated Vesicles (With 3 Figures).- Membrane Transport in Protoplasts (With 2 Figures).- The Binding of Anion Transport Inhibitors on the Plasmalemma Isolated from Corn Root Protoplasts (With 6 Figures).- Intracellular Transport of Metabolites in Protoplasts: Transport Between Cytosol and Vacuole (With 1 Figure).- Compartmentation of Metabolite Pools in Protoplasts: Chloroplasts, Mitochondria, Cytosol/Vacuole (With 1 Figure).- Protoplast Evacuolation (With 2 Figures).- Protoplasts in Studies of Vacuolar Storage Compounds (With 9 Figures).- Distribution of Saccharides Between Cytoplasm and Vacuole in Protoplasts (With 3 Figures).- Anthocyanin Containing Vacuoles Isolated from Protoplasts of Daucus carota Cell Cultures (With 1 Figure).- Vacuolar pH Variability in a Protoplast Population (With 4 Figures).- Mitotic Cycle of Mesophyll Protoplasts (With 2 Figures).- The Use of Guard Cell Protoplasts to Study Stomatal Physiology (With 6 Figures).- Regulation of Volume Changes in Guard Cell Protoplasts (With 3 Figures).- Wall Regeneration in Protoplasts of Higher Plants (With 3 Figures).- Glucan Synthases and Cell-Wall Regeneration in Fungal Protoplasts (With 3 Figures).- Fatty Acids in Protoplasts (With 3 Figures).- Proline in Protoplasts: The Chemical Potential of Proline and Stress Sensitivity of Cells (With 2 Figures).- The Biosynthesis and Catabolism of Indole-3-Acetic Acid in Protoplasts (With 3 Figures).- Auxin Receptors in Tobacco Leaf Protoplasts (With 3 Figures).- Some Physiological Properties of Protoplasts from Gravireacting Maize Roots (With 2 Figures).- Protoplasts and Gravireactivity (With 6 Figures).- Proton Extrusion in Protoplasts: Fusicoccin and Cytokinin Effects.- Protoplast Growth and Photoregulation (With 1 Figure).- Photorespiratory Metabolism in Protoplasts (With 3 Figures).

Protoplast dehydration correlated with heat resistance of bacterial spores.

Abstract: Water content of the protoplast in situ within the fully hydrated dormant bacterial spore was quantified by use of a spore in which the complex of coat and outer (pericortex) membrane was genetically defective or chemically removed, as evidenced by susceptibility of the cortex to lysozyme and by permeability of the periprotoplast integument to glucose. Water content was determined by equilibrium permeability measurement with 3H-labeled water (confirmed by gravimetric measurement) for the entire spore, with 14C-labeled glucose for the integument outside the inner (pericytoplasm) membrane, and by the difference for the protoplast. The method was applied to lysozyme-sensitive spores of Bacillus stearothermophilus, B. subtilis, B. cereus, B. thuringiensis, and B. megaterium (four types). Comparable lysozyme-resistant spores, in which the outer membrane functioned as the primary permeability barrier to glucose, were employed as controls. Heat resistances were expressed as D100 values. Protoplast water content of the lysozyme-sensitive spore types correlated with heat resistance exponentially in two distinct clusters, with the four B. megaterium types in one alignment, and with the four other species types in another. Protoplast water contents of the B. megaterium spore types were sufficiently low (26 to 29%, based on wet protoplast weight) to account almost entirely for their lesser heat resistance. Corresponding values of the other species types were similar or higher (30 to 55%), indicating that these spores depended on factors additional to protoplast dehydration for their much greater heat resistance.

Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana.

Abstract: The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·105 protoplasts (0.5 ml of cells at 106/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.

Vacuolar localization of 1-sinapolglucose: L-malate sinapoyltransferase in protoplasts from cotyledons of Raphanus sativus.

Abstract: The distribution of l-malate, sinapic acid esters and 1-sinapoylglucose: l-malate sinapoyltransferase (SMT) which catalyzes the synthesis of sinapoyl-l-malate were examined in preparations of protoplasts obtained from cotyledons of red radish (Raphanus sativus L. var. sativus). Vacuoles isolated from the protoplasts contained all of the SMT activity, all of the accumulated sinapic acid esters and about 50% of free l-malate present initially in the protoplasts. An esterase activity, acting on 1-sinapoyglucose, was found to be exclusively localized in the cytoplasm and a large proportion was found to be recoverable in a 100 000-g pellet obtained from protoplast lysates. The vacuoles were obtained after lysis of the protoplasts by osmotic shock and purification on a Ficoll gradient. The cytoplasmic contamination of vacuole preparations was found to be about 10%, as judged by enzymatic markers and microscopic inspection. No SMT activity was found in a 100 000-g pellet obtained from vacuole lysates. The results indicate that biosynthesis of sinapoyl-l-malate takes place within the central vacuoles of redradish cotyledons.

Protoplast transformation of group N streptococci with cryptic plasmids

Abstract: By using an extension to group N streptococci of a contransformation procedure we have introduced 4 different-sized cryptic plasmids for Streptococcus lactis into the plasmid-free S. lactis IL1403. A mixture of 4 cryptic plasmids with an indicator plasmid (pHV1301) conferring erythromycin resistance was used for IL1403 protoplast transformation. Under such conditions, 41.5% of the erythromycin-resistant transformants were contransformed with one of the cryptic plasmids in addition to pHV1301. Indicator plasmid pHV1301 was later spontaneously segregated from doubly transformed cells. This protocol should be very useful for constructing lactic streptococcal strains bearing any phenotypically cryptic plasmid.

In vitro culture of Cucumis sativus L. V. Stabilizing effect of glycine on leaf protoplasts

Abstract: A method for leaf mesophyll protoplast isolation and plant regeneration of cucumber (Cucumis sativus L.) is described. Using an isolation solution complemented with 0.1 M glycine, 8.2·106 viable protoplasts were isolated from 1 g of fresh leaves. The effect of the growth substances indole-3-acetic acid, naphthalene acetic acid, 2,4,-dichlorophenoxy acetic acid, 6-benzylaminopurine, 2-isopentenyladenine and kinetin at concentrations from 0.5 to 5 mg·1−1 was studied using the multi-hanging drop technique. The optimal growth substance combination, namely 5 mg·1−1 naphthalene acetic acid and 3 mg·1−1 2-isopentenyladenine, together with agarose medium in a so-called bead culture resulted in a plating efficiency of 21%. Some of the colonies obtained regenerated to plantlets which developed to plants.

Polyamine levels as related to growth, differentiation and senescence in protoplast-derived cultures of Vigna aconitifolia and Avena sativa

Abstract: We have previously reported that aseptically cultured mesophyll protoplasts of Vigna divide rapidly and regenerate into complete plants, while mesophyll protoplasts of Avena divide only sporadically and senesce rapidly after isolation. We measured polyamine titers in such cultures of Vigna and Avena, to study possible correlations between polyamines and cellular behavior. We also deliberately altered polyamine titer by the use of selective inhibitors of polyamine biosynthesis, noting the effects on internal polyamine titer, cell division activity and regenerative events. In Vigna cultures, levels of free and bound putrescine and spermidine increased dramatically as cell division and differentiation progressed. The increase in bound polyamines was largest in embryoid-forming callus tissue while free polyamine titer was highest in root-forming callus. In Avena cultures, the levels of total polyamines decreased as the protoplast senesced. The presence of the inhibitors alpha-difluoromethyl-arginine (specific inhibitor of arginine decarboxylase), alpha-difluoromethylornithine (specific inhibitor of ornithine decarboxylase) and dicyclohexylamine (inhibitor of spermidine synthase) reduced cell division and organogenesis in Vigna cultures. Addition of low concentration of polyamines to such cultures containing inhibitors or removal of inhibitors from the culture medium restored the progress of growth and differentiation with concomitant increase in polyamine levels.