Showing papers on "Protoplast published in 1984"

Plant Tissue Culture: Theory and Practice

Abstract: 1. Introductory History. 2. Laboratory Requirements and General Techniques. 3. Tissue Culture Media. 4. Cell Culture. 5. Cellular Totipotency. 6. Somatic Embryogenesis. 7. Haploid Production. 8. Triploid Production. 9. Cytogenetic Studies. 10. In Vitro Pollination. 11. Zygotic Embryo Culture. 12. Protoplast Isolation and Culture. 13. Somatic Hybridization. 14. Production of Pathogen-Free Plants. 15. Clonal Propagation. 16. Germplasm Storage. References. Indexes.

Expression of foreign genes in regenerated plants and in their progeny.

Abstract: Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non-oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co-integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co-cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non-oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (Km and Cm). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non-oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells.

High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae

Abstract: Protoplasts isolated from 4-day-old hypocotyls of various species of Brassica (Brassica napus, B. campestris and B. oleracea) produced callus with high efficiency in media containing casein hydrolysate and high concentrations of the auxin 2,4-D (4.5 μM). Cell division began after 24 h and 60% of the cells had divided after 48 h. In contrast, protoplasts isolated from stem and mesophyll of plants grown in vitro or in the greenhouse began to divide after a delay of 3–5 days. In these cases 40–50% of the cells had divided after 5 days as compared to 70% for hypocotyl protoplasts. To obtain a high frequency of regeneration, rapidly growing calli were transferred to media having a high cytokinin:auxin ratio as early as possible, usually 3 weeks after protoplast isolation. The average regeneration frequency for calli obtained from mesophyll protoplasts was 50%, while as many as 70% of the calli derived from hypocotyl protoplasts of B. napus regenerated plantlets on a medium containing zeatin (9.1 μM) and IAA (0.6 μM). On the same medium regeneration of Brassica oleracea was obtained. A low percentage of calli (1%) from Brassica campestris formed shoots when cultured on a combination of zeatin (4.6 μM), BA (4.4 μM) and IAA (0.6 μM).

Protoplast transformation of glutamate-producing bacteria with plasmid DNA.

Abstract: A method for polyethylene glycol-induced protoplast transformation of glutamate-producing bacteria with plasmid DNA was established. Protoplasts were prepared from cells grown in the presence of penicillin by treatment with lysozyme in a hypertonic medium. The concentration of penicillin during growth affected the efficiency of formation, regeneration, and polyethylene glycol-induced DNA uptake of protoplasts. Regeneration of protoplasts was accomplished on a hypertonic agar medium containing sodium succinate and yeast extract. The spectinomycin and streptomycin resistance plasmid pCG4, originally from Corynebacterium glutamicum T250, could transform various glutamate-producing bacteria such as C. glutamicum, Corynebacterium herculis, Brevibacterium flavum, and Microbacterium ammoniaphilum. The plasmid was structurally unchanged and stably maintained in new hosts. The transformation frequency of most competent protoplasts with pCG4 DNA isolated from primary transformants was high (ca. 10(6) transformants per microgram of covalently closed circular DNA) but was still two orders of magnitude below the frequency of transfection with modified DNA of the bacteriophage phi CGI. The difference was ascribed to the involvement of regeneration in transformation.

Lysine overproducer mutants with an altered dihydrodipicolinate synthase from protoplast culture of Nicotiana sylvestris (Spegazzini and Comes).

Abstract: Two S-(2-aminoethyl)L-cysteine (AEC) resistant lines were isolated by screening mutagenized protoplasts from diploid N. sylvestris plants. Both lines accumulated free lysine at levels 10 to 20-fold higher than in controls. Lysine overproduction and AEC-resistance were also expressed in plants regenerated from the variant cultures. A feedback insensitive form of dihydrodipicolinate synthase (DHPS), the pathway specific control enzyme for lysine synthesis, was detected in callus cultures and leaf extracts from the resistant lines. Aspartate kinase (AK), the other key enzyme in the regulation of lysine biosynthesis, was unaltered in the mutants. Crosses with wild type plants indicated that the mutation conferring insensitivity to feedback in DHPS, with as result overproduction of lysine and resistance to AEC, was inherited as a single dominant nuclear gene.

Plant regeneration from suspension culture and mesophyll protoplasts of Medicago sativa L.

Abstract: A system was established for achieving plant regeneration from mesophyll protoplasts and cotyledon-derived cell suspension cultures of alfalfa, Medicago sativa L. Peeled leaflets or cells from 6-day-old cell suspensions were incubated in an enzyme mixture containing 1% Driselase, 1% Rhozyme, 0.1% Cellulase and 72 gl-1 mannitol at pH 5.8 for 2–16 h to liberate protoplasts. A complex Kao medium supported cell division and colony formation, whereas a high auxin/low cytokinin treatment on Schenk and Hildebrandt medium followed by culture on growth regulator-free Blaydes or Linsmaier and Skoog medium resulted in somatic embryo formation. Of the three varieties tested. Citation, Answer and Regen S, the latter two produced embryos from which plants could be regenerated.

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.

Abstract: The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.

Protoplast fusion, genetic engineering in higher plants

Abstract: 1 Introduction.- 2 Techniques of Parasexual Hybridization.- 2.1 Culture Techniques.- 2.1.1 Culture of Isolated Plant Protoplasts.- 2.1.2 Culture of Single Cells and Protoplasts.- 2.2 Isolated Protoplast Stage and Plant Cell Engineering.- 2.2.1 Protoplast Fusion.- 2.2.2 Cell Reconstruction Involving Fusion of Subprotoplasts.- 2.2.3 Cell Modification Through Uptake of Isolated Cell Organelles by Plant Protoplasts.- 2.3 Problems of Genetic Variability.- 2.3.1 Genetic Variability Associated with in Vitro Manipulation of Plant Cells.- 2.3.2 Genetic Changes in Cells Induced During the Stage of Isolated Protoplasts.- 3 Protoplast Fusion and Parasexual Hybridization of Higher Plants.- 3.1 Perspectives.- 3.1.1 Terminology.- 3.1.2 History of Parasexual Hybridization of Higher Plants.- 3.2 Methods of Screening for Parasexual Hybrids.- 3.2.1 Introduction.- 3.2.2 Genetic Complementation.- 3.2.3 Physiological Complementation.- 3.2.4 Restoration of Growth Capacity in Hybrids Upon Fusion of Inactivated Cells.- 3.2.5 Mechanical Isolation.- 3.2.6 Physical Enrichment.- 3.3 Frequency of Formation of Parasexual Hybrids.- 3.4 Protoplast Fusion and the Bypassing of Sexual Barriers.- 3.5 Analysis of the Parasexual Hybridization Process and the Genetic Composition of Plant Forms Arising from Protoplast Fusion.- 3.5.1 General Guidelines.- 3.5.2 Genetic Methods of Analysis.- 3.5.2.1 Hybrid Crosses.- 3.5.2.2 Cloning.- 3.5.2.3 Cytogenetic Studies.- 3.5.2.4 Cytophotometric Measurement of Nuclear DNA Content.- 3.5.3 Biochemical Analysis.- 3.5.3.1 Multiple Molecular Forms of Enzymes.- 3.5.3.2 Analysis of Fraction I Protein.- 3.5.3.3 Restriction Endonuclease Analysis of Chloroplast and Mitochondrial DNA.- 3.5.3.4 Molecular Hybridization of Nucleic Acids.- 3.5.3.5 Analysis of Low Molecular Weight Substances.- 4 Transmission Genetics of Parasexual Hybridization in Closely Related Crosses.- 4.1 General Considerations.- 4.1.1 Perspectives and Philosophy of Analysis of Transmission Genetics.- 4.1.2 Initial Experiments on Transmission Genetics of Parasexual Hybrids.- 4.1.3 Hybridization of More Than Two Parental Cells.- 4.2 Fate of Extranuclear Genetic Determinants.- 4.2.1 Perspectives.- 4.2.2 Protoplast Fusion and the Formation of Cytoplasmic Heterozygotes.- 4.2.3 Mitotic Segregation of Plasmagenes and the Sorting Out of Parental Genotypes.- 4.2.4 Recombination of Plasmagenes.- 4.3 Fate of Nuclear Genes in the Process of Somatic Hybridization.- 4.3.1 Segregation of Nuclei.- 4.3.2 Chromosome Sets of Hybrid Cells.- 4.4 Transmission Genetics in Hybridization Systems Utilizing Parental Cell Inactivation.- 4.4.1 Consideration of Animal and Bacterial Systems.- 4.4.2 Treatment with Iodoacetate.- 4.4.3 Irradiation.- 4.5 Genetic Diversity of Hybrid Plants Obtained by Protoplast Fusion.- 5 Protoplast Fusion and Hybridization of Distantly Related Plant Species.- 5.1 Introduction.- 5.2 Initial Stages of Culture of Hybrid Cells of Distantly Related Plant Species.- 5.3 Interfamily Cell Hybrids of Higher Plants.- 5.4 Intertribal Hybrids of Higher Plants.- 5.4.1 Hybridization of Cruciferae Species.- 5.4.2 Hybridization of Distantly Related Members of the Solanaceae and Other Families.- 5.4.3 General Conclusions of Results Obtained with Intertribal Hybrids of Higher Plants.- 5.5 Intergeneric Hybrids.- 5.6 Influence of Physical and Chemical Factors on the Fate of Genetic Material in Distantly Related Species Combinations.- 5.6.1 Introduction.- 5.6.2 Irradiation and the Induction of Genetic Asymmetry.- 5.6.3 Induction of Mitotic Chromosome Segregation by Chemical Agents.- 6 Use of Somatic Hybridization.- 6.1 Genetic Analysis Using Parasexual Hybridization.- 6.1.1 Introduction and Perspective.- 6.1.2 Analysis of the Nature of Inherited Traits.- 6.1.3 Analysis of Nuclear Genes.- 6.1.3.1 Resistance to 5-Methyltryptophan.- 6.1.3.2 Resistance to S-(2-Aminoethyl)-L-Cysteine.- 6.1.3.3 Resistance to Cycloheximide.- 6.1.3.4 Resistance to Azetidine-2-Carboxylate.- 6.1.3.5 Additional Complementation Studies.- 6.1.4 Cosegregation of Extranuclear Genes.- 6.1.5 Analysis of Mitotic Cycle Mechanisms.- 6.1.6 Analysis of the Mechanisms of Differentiation and Morphogenesis.- 6.2 Practical Use of Somatic Cell Hybridization.- 6.2.1 Introduction.- 6.2.2 Bypassing of Incompatibility in Interspecific Hybridization.- 6.2.3 Reconstruction of Cytoplasmic Genomes.- 6.2.4 Transfer of Cytoplasmic Genomes and Cytoplasmic Male Sterility.- 7 Conclusion.- References.

The behavior of the plasma membrane following osmotic contraction of isolated protoplasts: Implications in freezing injury

Abstract: Following osmotic contraction of isolated rye protoplast (Secale cereale L. cv. Puma) that results in nearly a 50% reduction in volume, the plasma membrane was smooth, with no folding or pleating. Instead, deletion of plasma membrane occurred and numerous cytoplasmic vesicles were observed. As a result, the area of the plasma membrane was reduced by approximately 40%. Thin sections revealed that the cytoplasmic vesicles were membrane bound and not merely voids in the cytoplasm. High resolution video microscopy revealed the extent of vesiculation showing large clusters of cytoplasmic vesicles following osmotic contraction. Labeling the plasma membrane with fluorescein-Con-A prior to hypertonic contraction suggested that the cytoplasmic vesicles were derived from the plasma membrane. Freeze-fracture particle density on both the protoplasmic (PFp) and exoplasmic face (EFp) of the plasma membrane remained unchanged following contraction, which is consistent with a unit-membrane deletion into cytoplasmic vesicles. Upon partial re-expansion of the protoplasts, thin sections showed that the vesicles remained in the cytoplasm. These results using osmotic manipulation confirm earlier observations of isolated protoplasts at the light microscope level. Upon contraction plasma membrane is deleted into cytoplasmic vesicles, which are not readily reincorporated into the plasma membrane upon expansion. Lysis occurs before the original volume and surface area are regained.

Intergeneric nuclear gene transfer between somatically and sexually incompatible plants through asymmetric protoplast fusion

Abstract: A stable intergeneric transfer of nuclear genes from Physalis minima into the genome of Datura innoxia has been achieved through asymmetric protoplast fusion. No hybrid plants could be obtained from these species either by traditional methods of sexual breeding or by somatic hybridization via fusion of protoplasts containing complete nuclear genomes. The incompatibility barriers were bypassed by the fusion of highly X-irradiated (LD100) wild-type Physalis with nuclear albino mutant Datura protoplasts. In this intergeneric reconstruction, 1.15% of the total heterokaryons restored the chlorophyll synthesis in their regenerants. Two representative transformed lines, TRL-A and TRL-D, were further characterized, showing 0.43–0.78 pg. additional nuclear DNA (4.45–8.07% nuclear DNA of P. minima). Since chromosomes of the species of Datura and Physalis were distinguishable, the mitotic complement of the transformed regenerants showed only 3 and 1 chromosomes of the donor in tetraploid (2n=48) and octoploid (2n=96) genomes of the recipient, respectively. The introduction and expression of limited genes of Physalis in Datura have also been confirmed by the allelic expression of various isoenzymes. Such stable gene transfer via asymmetric fusion of protoplasts has been discussed in relation to its application in the genetic manipulation of plants.

High frequency of fusion induced in freely suspended protoplast mixtures by polyethylene glycol and dimethylsulfoxide at high pH.

Abstract: Fusion of freely suspended protoplast mixtures (hypocotyl protoplasts of Brassica napus mixed with mesophyll protoplasts of either B campestris or Nicotiana plumbaginifolia) was induced by a solution containing 10% polyethylene glycol, 10% dimethyl-sulfoxide and 01M glycine-NaOH buffer (pH 100) The fusion products represented 15 to 17 percent of the surviving cells More than 50% of the fusion products divided within two days after fusion, indicating that the fusion procedure did not significantly affect the viability of fused cells The fusion products were not bound to the surface of the fusion vessel, so they could be isolated with a micropipette immediately after fusion

Determination of Compartmented Metabolite Pools by a Combination of Rapid Fractionation of Oat Mesophyll Protoplasts and Enzymic Cycling

Abstract: In vivo pool sizes of a range of metabolites have been determined in subcellular fractions of darkened and illuminated mesophyll protoplasts of Avena sativa L. These estimations were made by combining a method of rapid protoplast fractionation with enzymic cycling techniques. Results are given for reduced and oxidized pyridine nucleotides, triose phosphates, 3-phosphoglycerate, inorganic phosphate, aspartate, malate, oxaloacetate, glutamate, 2-oxoglutarate, and citrate, from chloroplasts, mitochondria, and a fraction representing the remainder of the protoplast. The results indicate distinct differences of compartmented levels of certain metabolites between darkened and illuminated protoplasts.

Flow sorting and culture of plant protoplasts

Abstract: Conditions have been established for the rapid flow analysis of leaf protoplasts of Nicotiana tabacum L. cv. Xanthi using a flow cytometer-cell sorter. A procedure based upon chlorophyll autofluorescence was devised to permit the systematic evaluation of flow conditions in order to identify those under which protoplast damage was minimized. These conditions were employed for the flow sorting of protoplasts, following which it was possible to regenerate the sorted protoplasts into complete plants. The application of flow sorting is discussed for the rapid identification and selection of somatic hybrids produced by protoplast fusion.

Dimethyl sulfoxide can initiate cell divisions of arrested callus protoplasts by promoting cortical microtubule assembly

Abstract: A serious problem in the technology of plant cell culture is that isolated protoplasts from many species are reluctant to divide. We have succeeded in inducing consecutive divisions in a “naturally” arrested system—i.e., protoplasts from a hibiscus cell line, which do not divide under standard conditions—and in an artificially arrested system—i.e., colchicine-inhibited callus protoplasts of Nicotiana glutinosa, which do readily divide in the absence of colchicine. In both cases, the reinstallation of a net of cortical microtubules, which had been affected either by colchicine or by the protoplast isolation procedure, resulted in continuous divisions of the formerly arrested protoplasts. Several compounds known to support microtubule assembly in vitro were tested for their ability to promote microtubule assembly in vivo. Best results were obtained by addition of dimethyl sulfoxide to the culture medium. Unlimited amounts of callus could be produced with the dimethyl sulfoxide method from protoplasts which never developed a single callus in control experiments.

Novel alloplasmic Nicotiana plants by "donor-recipient" protoplast fusion: cybrids having N. tabacum or N. sylvestris nuclear genomes and either or both plastomes and chondriomes from alien species.

Abstract: The “donor-recipient” fusion method was utilized to investigate interspecific transfer of organelles and organelle-controlled traits from donor to recipient Nicotiana species To follow organelle transfer and sorting-out under conditions which will be either selective or non-selective for chloroplast transfer we used, respectively, albino (N tabacum VBW) or normally pigmented (N sylvestris) plants as protoplast-recipients N alata, N bigelovii and N undulata served as donors Organelle composition of the cybrid plants was investigated by the analysis of chloroplast DNA restriction pattern, tentoxin sensitivity, chloroplast pigmentation, mitochondrial DNA restriction pattern and alloplasmic male sterility In certain cybrids the analysis of restriction patterns of chloroplast and mitochondrial DNA was augmented by Southern hybridization with the respective organelle-DNA probes A total of 341 fusion-derived plants (from 109 calli) were analysed and 102 of these were found to be cybrids with novel nuclear/organelle compositions The incidence of chloroplast and mitochondrial transfer was strongly facilitated when iodoacetate-treated protoplasts of N tabacum VBW (albino) rather than N sylvestris (normal chloroplasts) protoplasts served as recipient Mitochondrial DNA restriction patterns in cybrids commonly differed from those of either fusion partner All fusion combinations resulted in at least some male-sterile cybrids, having either donor or recipient plastomes thus indicating that interspecific “donor-recipient” protoplast fusion is an efficient mean to produce plants with alloplasmic male sterility

In vitro transformation of petunia cells by an improved method of co-cultivation with A. tumefaciens strains

Abstract: A method (termed co-cultivation) for transforming plant cells in vitro with A. tumefaciens strains, which was originally developed by Marton et al. (1978) Nature 277: 129-131, has been modified by the incorporation of a novel feeder plate culture system and been extended to use with petunia protoplasts. Using efficient cell plating and selection conditions for phytohormone-independent growth, large numbers of independent transformed calli can be obtained efficiently (∼10(-1)) and in less than 3 weeks following protoplast isolation. Southern hybridization analysis has confirmed that the majority of the resulting in vitro transformants contain a single copy of full length T-DNA.The high efficiency of this procedure allows simple screening to identify plant cells transformed by Ti plasmids attenuated by deletion of internal T-DNA regions. Results are presented that demonstrate the co-cultivation method can be used in conjunction with short term assays for monitoring plant gene expression.

Production and Regeneration of Lactobacillus casei Protoplasts

Abstract: Methods for the production and regeneration of Lactobacillus casei protoplasts are described Protoplasts of L casei strains were obtained by treatment with mutanolysin or with mutanolysin and lysozyme together in a protoplast formation buffer containing 002 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 70), 1 mM MgCl(2), 05% gelatin, and 03 M raffinose Cells were regenerated on a complex medium supplemented with bovine serum albumin, MgCl(2), CaCl(2), gelatin, and raffinose Lengthy digestion with lytic enzymes inhibited the capacity of protoplasts to regenerate The optimum conditions of protoplast formation varied from strain to strain Using predetermined optimal conditions it was possible to prepare protoplasts of several L casei strains and regenerate them with 10 to 40% efficiency The methods were applicable to other species of lactobacilli as well

Resistance, germination, and permeability correlates of Bacillus megaterium spores successively divested of integument layers.

Abstract: A variant strain that produced spores lacking exosporium was isolated from a culture of Bacillus megaterium QM-B1551. Two additional spore morphotypes were obtained from the parent and variant strains by chemical removal of the complex of coat and outer membrane. Among the four morphotype spores, heat resistance did not correlate with total water content, wet density, refractive index, or dipicolinate or cation content, but did correlate with the volume ratio of protoplast to protoplast plus cortex. The divestment of integument layers exterior to the cortex had little influence on heat resistance. Moreover, the divestment did not change the response of either the parent or the variant spores to various germination-initiating agents, except for making the spores susceptible to germination by lysozyme. The primary permeability barrier to glucose for the intact parent and variant spores was found to be the outer membrane, whereas the barrier for the divested spores was the inner membrane.

A rapid inexpensive method for the isolation of restrictable mitochondrial DNA from various plant sources.

Abstract: A simplified method for the isolation of mitochondrial DNA (mtDNA) of several plant species from either coleoptile or tissue cultured cells is described. The procedure does not require gradient ultracentrifugation or organic solvent extractions (such as phenol, chloroform, ether, etc.). Protoplast isolation is not required for the release of organelles from cell suspension cultured cells. The entire procedure can be performed in a single day and employs differential low speed centrifugations for isolation of mitochondria and differential precipitations for the recovery of restrictable DNA.

Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer

Abstract: The Staphylococcus aureus plasmid pC194 which codes for resistance to chloramphenicol was introduced into six Bacillus thuringiensis strains representing five varieties by protoplast transformation. Six other varieties could not be transformed. pC194 could be identified in transformed strains as autonomous plasmid. The transformed clones contained in addition a new extrachromosomal element of somewhat lower electrophoretic mobility hybridizing with pC194, and pC194 in multimeric forms. pC194 was also transferred from one B. thuringiensis variety to another and from Bacillus thuringiensis to Bacillus subtilis and vice versa by a conjugation-like process, requiring close cell-to-cell contact.

Protoplast Fusion of Trichoderma reesei, Using Immature Conidia

Abstract: Protoplast fusion of strains derived from Trichoderma reesei QM9414 and QM9136 and the segregation of the resulting fusants were studied. Combinations of protoplasts prepared from young conidia with double amino acid requirements, one of which was a common requirement and the other uncommon, were fused in the presence of polyethylene glycol 6000. Fusants were selected as regenerant colonies requiring only the commonly deficient amino acid. The frequency of fusion was 0.9 x 10 to 4.0 x 10 for the starting conidia and 3.0 x 10 to 4.9 x 10 for the regenerated protoplasts, which was significantly higher than the expected reversion frequencies by mutation. Conidia generated on the fusant colonies showed diverse phenotypes, i.e., parental types (40 to 80%) and nonparental types (20 to 60%). Colonies developed from single conidia of the nonparental phenotype contained special spots called "knobs" that have a higher density of mycelia. The phenotype of the knobs was again varied among prototrophs, parental types, and recombinant types; and their traits were inherited stably. The phenotype of the mycelia in the nonknob part was essentially the same as that of the original conidia and again formed knobs in colonies upon transfer of a piece of mycelia to a fresh medium. The conidial DNA content of the knob clone was almost the same as that of the parents, but that of the fusants was 1.2 to 2.0 times higher than that of the parents. From these results, we conclude that knobs are the segregants from the fusants. One knob clone showed twice the carboxymethyl cellulose hydrolyzing activity of the parents, suggesting the possibility of breeding T. reesei cells by the protoplast fusion technique.

Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension.

Abstract: A method for fusion of protoplasts bearing amplified plasmids and human diploid fibroblasts or other cell types in suspension is described. Transient expression of plasmid-encoded proteins occurs in up to 50% of the human cells, as demonstrated for simian virus 40 T antigen by immunofluorescence and the Escherichia coli xanthine-guanine phosphoribosyl transferase by autoradiography. In contrast, frequencies of stable transformants were similar to those obtained by the CaPO4 coprecipitation technique. However, experiments with both methods involving the recombinant pRSVneo (in which the Rous sarcoma virus long terminal repeat regulates expression of the antibiotic-inactivating aminoglycoside phosphotransferase) revealed a much higher frequency of colonies in G418 selective medium with constructions in which the early region of simian virus 40 DNA was present as well. We propose a role for the simian virus 40 T antigen in enhancing stable transformation in this system.

Large scale isolation of maternally inherited lincomycin resistance mutations, in diploid Nicotiana plumbaginifolia protoplast cultures

Abstract: Lincomycin-resistant clones were isolated in diploid protoplast cultures of Nicotiana plumbaginifolia. Selection of the resistant clones was based on the ability of resistant calli to green in the presence of the antibiotic (1,000 mg l-1). Sensitive colonies formed white calli under the same conditions. In the absence of mutagenic treatment the frequency of the resistant clones was 1.0×10-4. This frequency could be increased up to 5.8×10-4 and 7.2×10-4 by treatment with 0.1 mM and 0.3 mM N-ethyl-N-nitrosourea (NEU), respectively.

Generation of heteroplastidic Nicotiana cybrids by protoplast fusion: analysis for plastid recombinant types.

Abstract: Protoplasts of a mutant line of Nicotiana tabacum having a maternally-transmitted chlorophyll deficiency were fused with protoplasts of two alloplasmic-male-sterile Nicotiana lines by the “donor-recipient” technique. In both fusion experiments variegated plantlets were regenerated which were shown to contain cytoplasms of mixed chloroplast nature. This confirms that with the “donor-recipient” method one can obtain mixed cytoplasms of genetically different chloroplasts. We present a convenient system to assay for genetic recombination between chloroplasts by combining use of several cytoplasmic markers: vis. chlorophyll pigmentation, chloroplast DNA restriction patterns, tentoxin resistance and male sterility. Within the limits of the experiment no recombinant types were recovered.

Transmission genetics of the somatic hybridization process in Nicotiana : 1. Hybrids and cybrids among the regenerates from cloned protoplast fusion products.

Abstract: Callus protoplasts of a Nicotiana tabacum chlorophyll-deficient mutant were fused with mesophyll protoplasts from one of following five sources: 4 cmsanalogs of tobacco bearing the cytoplasms of N. plumbaginifolia, N. suaveolens, N. repanda, and N. undulata, respectively, as well as wild species N. glauca. In another series of experiments, callus protoplasts from the chlorophyll-deficient genome Su/Su mutant of tobacco were fused with mesophyll protoplasts of the wild species N. glauca and those of a plastome chlorophyll-deficient tobacco mutant. The screening of hybrids consisted of visual identification followed by mechanical isolation and cloning of heteroplasmic fusion products in microdroplets of nutrient medium. Studies of regenerated plants included the analyses of gross morphology of plants, leaf and flower morphology, analysis of chromosome size and morphology and chromosome numbers, studies of multiple molecular forms of esterase and amylase, analysis of chloroplast DNA restriction patterns and analyses of chlorophyll-deficiency controlled by Su and P (-) genes. The study of progeny of 41 clones representing all species' combinations demonstrated that regenarants of most (63%) clones from intraspecific (for nuclear genes) combinations were cybrid forms, whereas in the case of the fusion N. tabacum + N. glauca, the true nuclear hybrids prevailed and the proportion of cybrids did not exceed 26%. Clones regenerating both hybrid and cybrid plants from the same fusion product were also found.

Somatic hybridisation using a double mutant of nicotiana tabacum

Abstract: Leaf mesophyll protoplasts of a nitrate reductase deficient, streptomycin resistant double mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type N. rustica. Hybrid colonies were selected for nitrate reductase proficiency and streptomycin resistance. Green, actively proliferating colonies were recovered in the selection medium. Eleven of twenty green colonies, transferred to regeneration medium, produced plants. Regenerated plants were analysed for their vegetative and floral characteristics, Fraction 1 protein polypeptide composition and leaf esterases. All the eleven regenerants were nuclear somatic hybrids possessing the chloroplast of the N. tabacum parent. By developing a double mutant with a negative (auxotrophic) and a positive (resistance) selection marker in a species of interest, this species can be hybridised by somatic cell fusion with wild type species lacking selectable markers.

Wound signals in plants: A systemic plant wound signal alters plasma membrane integrity.

Abstract: Within 4 hr after wounding the lower leaves of young potato and tomato plants, a rapid and remarkable change is induced in the cells of upper undamaged leaves that results in extensive lysis of protoplasts during their isolation. Protoplast yields from unwounded upper leaves, 4 hr after wounding a lower leaf by crushing with a hemostat, decreased 25% below yields from leaves of unwounded plants. From 8 to >20 hr after wounding, protoplast yields were less than half of those from control plants. Multiple woundings decreased yields even further, as did chewing of the lower leaves by tobacco hornworms over a period of several minutes. In addition, within 4 hr of excising young tomato plants at their base with a razor blade, a 90% decrease in leaf protoplast yields was recorded. The major loss of protoplasts induced by wounding was primarily due to an increased cell lysis during protoplast isolation. Cell lysis was apparently due to a weakened cell membrane, because newly recovered protoplasts released from leaves of wounded plants were extremely fragile and exhibited 70% lysis during low speed centrifugation, compared to 20% lysis of protoplasts recovered from control plants. We conclude that a signal is released by wounding that is rapidly transmitted or transported through the plants to induce a profound change in the leaf cell membranes that renders them fragile during protoplast isolation. It is proposed that this signal may play a role in inducing cellular changes in the plant cells as part of their responses to environmental stress such as pest attacks.