Showing papers on "Protoplast published in 1975"
TL;DR: Polyethylene glycol is very effective in producing hybrids capable of indefinite multiplication even in cases, such as early passage human skin fibroblasts and lymphocytes, known to be highly recalcitrant to other treatments.
Abstract: Polyethylene glycol (PEG) is known to promote fusion of plant protoplasts. Various adaptations of this treatment to mammalian, including human, cell cultures are reported here. PEG is very effective in producing hybrids capable of indefinite multiplication even in cases, such as early passage human skin fibroblasts and lymphocytes, known to be highly recalcitrant to other treatments.
516 citations
TL;DR: Intact plant vacuoles were prepared in large numbers from protoplasts of mature leaves, flower petals, stems, pedicels, filaments, styles, and young fruits to isolate chloroplasts with a high degree of integrity and excellent photochemical activity.
Abstract: Intact plant vacuoles were prepared in large numbers (10/sup 6/) from protoplasts of mature leaves, flower petals, stems, pedicels, filaments, styles, and young fruits. Treatment of protoplasts with 0.2 molar K/sub 2/HPO/sub 4/-HCl, pH 8, with slow stirring resulted in gentle osmotic rupture of the protoplasts and release of intact vacuoles. Particulate components of the protoplast, less the vacuole, were largely shed as an aggregate, which was removed by filtration. Vacuoles were recovered from the filtrate by low-speed centrifugation. The general procedure was also used to isolate chloroplasts with a high degree of integrity and excellent photochemical activity. (auth)
146 citations
TL;DR: Protoplasts were isolated from mesophyll cells of axenic shoot cultures derived from haploid and diploid plants of Petunia hybrida to establish optimal plating efficiencies and the haploid chromosome numbers in 10 plants grown from isolated protoplasts proved their origin from haploids.
134 citations
TL;DR: In experiments where tissue slices containing intact cells are supplied with [14C]sugar nucleotide in vitro, alkali-insoluble beta-1,4-glucan is synthesized at rates which greatly exceeded those obtained using isolated membrane preparations.
Abstract: Membrane preparations from growing regions of 8-day old Pisum sativum epicotyls contain multiple beta-1,4-glucan (cellulose) synthetase activities (UDP- or GDP-glucose: beta-1,4-glucan-glucosyl transferase), and the levels of some of these are influenced by treatments with the growth hormone, indoleacetic acid (IAA). When membranes from control epicotyl segments (zero time) are fractionated by isopycnic sedimentation in sucrose density gradients, all of the synthetase activities are associated mainly with Golgi membrane (density 1.55 g/cm3). After decapitation and treatment of epicotyls with IAA, synthetases also appear in a smooth vesicle fraction (density 1.11 g/cm3) which is rich in endoplasmic reticulum (ER) marker enzyme. Major fractions of these synthetases are not recovered in association with plasma membrane or washed cell walls. When [14-C]sucrose is supplied in vivo to segments +/- IAA, radioactive cellulose is deposited only in the wall. Cellulose or cellodextrin precursors do not accumulate in those membranes in which synthetase activities are recovered in vitro. In experiments where tissue slices containing intact cells are supplied with [14C]sugar nucleotide in vitro, alkali-insoluble beta-1,4-glucan is synthesized (presumably outside the protoplast) at rates which greatly exceeded (20-30 times) those obtained using isolated membrane preparations. Progressive disruption of cell structure results in increasing losses of this high activity. These results are consistent with the interpretation that Golgi and ER-associated synthetases are not themselves loci for cellulose synthesis in vivo, but represent enzymes in transit to sites of action at the wall:protoplast omterface. There they operate only if integrity of cellular organization is maintained.
104 citations
TL;DR: As cowpeas belong to a plant family other than tobacco, the new protoplasts allow comparative infection studies with plant viruses, and Poly-l-ornithine was not essential for CPMV infection of the cowpea mesophyll protoplast, but it had a stimulating effect.
89 citations
TL;DR: Induction and growth of callus from seeds, stems and pieces of leaf of Arabidopsis thaliana were successful on a Gamborg B 5 medium and on a modified B 5medium and the morphogenetic potential appeared to depend on the age of the callus in culture.
82 citations
TL;DR: In the presence of MgSO4 as osmotic stabilizer, nucleated protoplasts of Schizophyllum commune developed a large vacuole and could be isolated on the basis of their low buoyant density, suggesting that the synthesis of R-glucan is required for the initiation of hyphal morphogenesis.
Abstract: In the presence of MgSO4 as osmotic stabilizer, nucleated protoplasts of Schizophyllum commune developed a large vacuole and could be isolated on the basis of their low buoyant density. All these protoplasts were capable of wall regeneration and about 50 percent reverted to the hyphal mode of growth in liquid medium. The kinetics of the formation of three main cell-wall components, S-glucan (α-1,3-glucan), R-glucan (β-1,3, β-1,6-glucan) and chitin were studied from the onset of regeneration. S-glucan and chitin accumulation as well as RNA and protein synthesis started simultaneously after a short lag, but R-glucan formation was delayed. The reversion to hyphal tubes only began after several hours of rapid R-glucan synthesis. Cycloheximide (0.5 μg/ml), inhibiting protein synthesis by 98% inhibited the formation of R-glucan and the reversion to hyphal growth but the formation of chitin and S-glucan did start and continued seemingly unimpaired for several hours. This indicates that the enzymes responsible for the synthesis of S-glucan and chitin remained intact during protoplast preparation. Polyoxin D inhibited both the synthesis of chitin and R-glucan and also the reversion to hyphal growth. However, the synthesis of S-glucan was not suppressed. These inhibitor studies as well as the kinetics of R-glucan formation during normal regeneration suggest that the synthesis of R-glucan is required for the initiation of hyphal morphogenesis.
72 citations
TL;DR: The procedure was found to be a simplified alternative to those previously described for tobacco protoplast regeneration and should permit studies related to the influence of differing osmoticum levels on a variety of cell functions.
Abstract: A method is described for the isolation of large numbers of tobacco (Nicotiana tabacum L. cv. Xanthi-nc) mesophyll cell protoplasts under relatively low external osmotic conditions. The procedure utilized 0.2 m sucrose as the primary osmoticum and a mixture of 0.5% macerozyme, 4% cellulase, and 2% polyvinylpyrrolidone, pH 5.4. The viability of resultant protoplasts was confirmed through regeneration of fertile plants. Plating and regeneration studies revealed, however, that qualitative and quantitative modifications in plating and differentiation media were necessary for protoplasts prepared in this manner. Over-all, the procedure was found to be a simplified alternative to those previously described for tobacco protoplast regeneration. In addition, the system should permit studies related to the influence of differing osmoticum levels on a variety of cell functions.
72 citations
TL;DR: Protoplasts were isolated from primary leaves of cowpeas by a technique that makes stripping of the leaf epidermis unnecessary and enzymatic digestion released more than five times as many protoplasts from treated, as compared to untreated, leaves and in less than one-third the digestion time.
57 citations
TL;DR: In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C(4) mesophyll cells.
Abstract: The use of mesophyll protoplast extracts from various C 4 species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C 4 photosynthesis. Conditions regulating the formation of the C 4 acids were studied with protoplast extracts from Digitaria sanguinalis , an NADP-malic enzyme C 4 species, Eleusineindica , an NAD-malic enzyme C 4 species, and Urochloa panicoides , a phosphoenolpyruvate (PEP) carboxykinase C 4 species. Light-dependent induction of CO 2 fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and α-ketoglutarate, or 3-phosphoglycerate induced high rates of CO 2 fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C 4 mesophyll cells. Induction by pyruvate or alanine and α-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO 2 fixation was not. Several differences between these species representing the three C 4 groups were observed. Substrate induction of CO 2 fixation in mesophyll protoplast extracts of D. sanguinalis gave malate as a major product; only by an apparent exchange reaction with cold aspartate did substantial label appear in aspartate (up to 53% of labeled products). In contrast, aspartate was a major product when alanine and α-ketoglutarate served as inducing substrates with E. indica (up to 57%) and U. panicoides (up to 86%). With induction by pyruvate or 3-phosphoglycerate, mesophyll preparations of U. panicoides and E. indica were less effective in forming malate (up to 31% of products) than D. sanguinalis (up to 87% of products). After 2 seconds of whole leaf 14 CO 2 fixation, malate was the major labeled product (57%) with D. sanguinalis , whereas with E. indica and U. panicoides aspartate was the predominant product (73% and 76%, respectively). With mesophyll protoplast extracts of D. sanguinalis , aspartate inhibited CO 2 fixation (about 50% at 0.6 mm), while malate was relatively uninhibitory at comparable concentrations. CO 2 fixation by mesophyll protoplast extracts of E. indica was inhibited by malate (about 50% at 0.6 mm), while aspartate was relatively uninhibitory. With mesophyll preparations of U. panicoides , malate or aspartate (2 mm) caused only slight inhibition of CO 2 fixation. The regulation of aspartate and malate synthesis in C 4 mesophyll cells is discussed relative to initial products of photosynthesis in C 4 species in vivo and species differences in the mechanisms of C 4 photosynthesis.
51 citations
TL;DR: In the cells as a whole there were two types of change; first, a shock phase consequent upon protoplast isolation and second, a phase of very rapid dedifferentiation to a meristematic state.
Abstract: Mesophyll protoplasts isolated by treating tobacco leaves with macerozyme and cellulase were cultured in a modifiedMurashige, andSkoog medium (1962) containing 1% agar. Changes occurring in the cells during isolation and culture were followed by light and electron microscopy. During protoplast preparation, pseudocrystals appeared in the chloroplasts and spherical portions of the chloroplast stroma became surrounded by membranes and cut out into the cytoplasm. During culture, the chloroplasts lost three quarters of their volume, thylacoids were reduced and pseudocrystals disappeared. The cytoplasm at first became vacuolated and poor in ribosomes but, by the third day of culture, it was enriched in membranes and polyribosomes, indicating high metabolic activity. In the cells as a whole there were two types of change; first, a shock phase consequent upon protoplast isolation and second, a phase of very rapid dedifferentiation to a meristematic state.
TL;DR: Undifferentiated callus derived from asparagus protoplast cultures has been used for studies on organogenesis and Adenine has been found to be effective, together with a cytokinin, in promoting the formation of somat ic embryoids in this tissue.
Abstract: Undifferentiated callus derived from asparagus protoplast cultures has been used for studies on organogenesis. Root and shoot formation has been obtained with different hormonal balances. Adenine has been found to be effective, together with a cytokinin, in promoting the formation of somat ic embryoids in this tissue.
TL;DR: Differences in the growth requirements of isolated protoplasts and regenerated cells of two species, Petunia hybrida and Parthenocissus tricuspidata crown gall, were sufficiently consistent to develop a selection procedure, aimed at preferentially recovering potential somatic hybrid material, following protoplast fusion and subsequent culture.
TL;DR: The results suggest that CO(2) fixation in C(4) mesophyll cells is dependent on chloroplasts and extrachloroplastic phosphoenolpyruvate carboxylase, which provides further evidence for carbon fixation through the beta-carboxylation pathway.
Abstract: Mesophyll cells, protoplasts, and protoplast extracts of Digitaria sanguinalis were used for comparative studies of light-dependent CO(2) fixation. CO(2) fixation was low without the addition of organic substrates. Pyruvate, oxaloacetate, and 3-phosphoglycerate induced relatively low rates (10 to 90 mumoles/mg chlorophyll.hr) of CO(2) fixation when added separately. However, a highly synergistic relationship was found between pyruvate + oxaloacetate and pyruvate + 3-phosphoglycerate for inducing light-dependent CO(2) fixation in the mesophyll preparations. Highest rates of CO(2) fixation were obtained with protoplast extracts. Pyruvate, in combination with oxaloacetate or 3-phosphoglycerate induced light-dependent rates from 150 to 380 mumoles of CO(2) fixed/mg chlorophyll.hr which are equivalent to or exceed reported rates of whole leaf photosynthesis in C(4) species. Concentrations of various substrates required to give half-maximum velocities of CO(2) fixation were determined, with the protoplast extracts generally saturating at the lowest substrate concentrations. Chloroplasts separated from protoplast extracts showed little capacity for CO(2) fixation. The results suggest that CO(2) fixation in C(4) mesophyll cells is dependent on chloroplasts and extrachloroplastic phosphoenolpyruvate carboxylase.The stimulation of pyruvate-induced CO(2) fixation by oxaloacetate and 3-phosphoglycerate is thought to be due to induction of noncyclic electron transport which generates ATP for the conversion of pyruvate to phosphoenolpyruvate by pyruvate Pi dikinase. The primary products of the substrate-induced CO(2) fixation were oxaloacetate and malate, which provides further evidence for carbon fixation through the beta-carboxylation pathway. High rates of light-dependent CO(2) fixation with a significant percentage of (14)C fixed into malate suggest an efficient operation of both photosystems I and II.The substrate inductions are discussed with respect to the proposed role of the mesophyll cell in C(4) photosynthesis, and schemes suggesting the stoichiometry of energy requirements for photosynthetic carbon metabolism in C(4) mesophyll cells are presented.
TL;DR: It is suggested that the activator protein accelerates a normal process of transport (leakage) of autolysin molecules into the periplasmic space, which would then unmask deoxyribonucleic acid binding sites located on the plasma membrane.
Abstract: Treatment of pneumococci with activator (a protein that induces bacterial "competence" to absorb deoxyribonucleic acid molecules and undergo genetic transformation) can cause either protoplast formation or leakage of intracellular components to the medium depending on postincubation conditions. The leaked intracellular components include nucleoside phosphates, beta-galactosidase, deoxyribonuclease, autolysin, and hemolysin. Leakage and protoplast formation are induced by the electrophoretically pure activator, and these phenomena require the same conditions as induction of competence for genetic transformation, namely, genetic capacity for competence, protein synthesis, incorporation of choline, and the optimal pH for activation. It is suggested that the activator protein accelerates a normal process of transport (leakage) of autolysin molecules into the periplasmic space. The activity of these autolysin molecules from within would then unmask deoxyribonucleic acid binding sites located on the plasma membrane.
TL;DR: In vitro culture of somatic cells of higher plants allows the plant breeder to apply the techniques of microbial genetics to specific breeding problems in crop plants, but many techniques still require additional refinement before they will become generally applicable in a plant breeding situation.
TL;DR: Protoplasts were isolated enzymatically from leaves of an inbred line of Petunia parodii and underwent cell wall regeneration, followed by repeated cell division to produce callus.
TL;DR: The distinctive isoperoxidases which reacted to infection were the same as those responsive to mechanical injury, confirming that the enzyme reaction to infection results from a nonspecific response to injury.
Abstract: Leaves and pith of Turkish, Wisconsin 38, and Samsun NN tobacco (Nicotiana tabacum) varieties, which differ in their sensitivity to tobacco mosaic virus, showed the same qualitative isoperoxidase patterns and a similar distribution of distinctive isoperoxidases between the cell protoplast and wall-free, ionically, and covalently bound fractions. No changes in the qualitative isoenzyme spectrum were found in relation to age, mechanical injury, or leaf infection with tobacco mosaic virus. The distinctive isoperoxidases which reacted to infection were the same as those responsive to mechanical injury, confirming that the enzyme reaction to infection results from a nonspecific response to injury. The increase in peroxidase activity in response to infection or mechanical injury, or both, was greater in young tissue than in the older ones. The great increase in Samsun NN leaves and no increase in those of the two other varieties in response to infection may be due to differences in the degree to which the pathogen affected processes controlling the nonspecific peroxidase reaction to injury. Peroxidase development in the infected Samsun NN leaves was due to isoenzymes which form the wall-bound fraction in very young tissues, and to those which increase in activity with aging in the protoplast and wall-free fractions. In mechanically injured tissue, only the first group of isoenzymes increased in activity. In Samsun NN plants, the increased peroxidase activity in upper intact leaves above the infected ones was only due to isoenzymes whose activity increases with both normal and virus-accelerated senescence. Peroxidase reaction to challenge inoculation in these leaves was the same whether the lower ones were intact, infected and/or mechanically injured. Thus, the induced systemic resistance to tobacco mosaic virus may be due to other than peroxidase factors.In infected tissues, peroxidase was detected in the endoplasmic reticulum, Golgi apparatus, vacuole, cell wall, and intercellular spaces. The Golgi vesicles were often localized near the tonoplast and plasmalemma, fusing with membranes and secreting their contents. The possible "rejuvenating" effects of injury on synthesis and transport of distinctive isoperoxidases are discussed.
TL;DR: Protoplasts of the moss Physcomitrella patens were isolated by enzymatic degradation of cell walls under the combined influence of two polygalacturonases, resulting in cell wall regeneration and development to typical haploid plants.
TL;DR: Forespores and protoplasts showed osmometric behaviour in solutions of non-permanent solutes consistent with the presence of an intact permeability barrier in these structures.
Abstract: Measurement of certain membrane-bound enzymic activities was used to study the orientation of the outer membrane of the double-membraned forespore of Bacillus megaterium KM. 2. Adenosine triphosphatase, NADH dehydrogenase and L-malate intact protoplasts, but were readily detected in intact stage II or IV forespores, consistent with reversed polarity of the outer forespore membrane relative to the mother-cell plasma membrane. 3. Measurement of NADH oxidase activity revealed that intact stage III forespores had the same high affinity for NADH as protoplast membrane preparations and protoplast lystates, consistent with ready access of NADH to oxidation sites on the outer forespores membrane. 4. Forespores and protoplasts showed osmometric behaviour in solutions of non-permanent solutes consistent with the presence of an intact permeability barrier in these structures.
TL;DR: The results are consistent with the proposal that oxygen inhibits C 3 photosynthesis by competing with CO 2 in the RuDP carboxylase/oxygenase system and the oxygen enhancement of C 4 mesophyll photosynthesis is proposed to be due to pseudocyclic electron flow supplying additional ATP for the CO 2 fixation process.
TL;DR: Cell-wall enzymes were assayed by the difference between enzyme activities in the whole cell and the protoplast and indicated that peroxidase and not malate dehydrogenase was located in the wall, while only two anodic isoenzymes of per oxidase were present in the cell wall.
Abstract: Cell-wall enzymes were assayed by the difference between enzyme activities in the whole cell and the protoplast. Both peroxidase (85.2%) and acid phosphatase (21.9%) were located in the wall. However, malate dehydrogenase was found only in the protoplast. A study of the time-course of the release of peroxidase and malate dehydrogenase into the incubation medium from cells either treated with cellulase or untreated, also indicated that peroxidase and not malate dehydrogenase was located in the wall. Only two anodic isoenzymes of peroxidase were present in the cell wall. These were more negatively charged than those of horseradish peroxidase.
TL;DR: A method is described which utilizes low density plating of tobacco mesophyll protoplast over x-irradiated feeder protoplasts to obtain rapid regeneration of plantlets from small cell colonies.
TL;DR: Comparison of the frequency of multiple marker transformations to single marker events as a function of DNA dilution conclusively proves that these markers originated from the same continuous strand of DNA.
Abstract: The transformation of Bacillus subtilis by homologous deoxyribonucleic acid (DNA) made available by gently lysing a stable L-form or protoplast suspension was 3 to 10-fold more efficient than DNA isolated by conventional procedures. This increased transformation was not influenced by digestion with pronase, trypsin, or ribonuclease. Preincubation of isolated DNA with L-form lysates did not increase the transformation efficiency above that achieved with untreated, isolated DNA. In addition to displaying a higher efficiency of transformation, the DNA found in these gently prepared lysates was also able to co-transform heretofore unlinked markers at frequencies in excess of those found by congression. Comparison of the frequency of multiple marker transformations to single marker events as a function of DNA dilution conclusively proves that these markers originated from the same continuous strand of DNA.
TL;DR: Gel electrophoretic analysis of the RNA from both cotyledon and first leaf protoplasts showed that both protoplast types incorporated either (14C)- or (3H)-uridine into ribosomal RNA species.
Abstract: Aspects of the metabolism of RNA have been studied in enzymatically isolated protoplasts from cotyledon and first leaf mesophyll tissue of two cultivars of cucumber. The first leaf mesophyll protoplasts incorporated (3H)-uridine into ribosomal RNA at a constant rate for up to 25 hr in a simple salts medium and for up to 45 hr in a growth medium. Pulse-chase labelling experiments on such preparations showed a rapid dilution of the intracellular (3H)-uridine pool(s) and a high metabolic rate in the cells in one cultivar but not in another. Gel electrophoretic analysis of the RNA from both cotyledon and first leaf protoplasts showed that both protoplast types incorporated either (14C)- or (3H)-uridine into ribosomal RNA species. Incorporation of (3H)-uridine into chloroplasts RNA was minimal in cotyledon protoplasts, but significant in leaf protoplasts. Greater incorporation into the chloroplast RNA species could be achieved by longer pulses. Synthesis of all of the ribosomal RNA species was sensitive to actinomycin D at 10 and 25 mug/ml concentrations in all protoplasts tested.
TL;DR: Membraneospholipid was found to be hydrolyzed presumably by an intracellular phospholipase C, and diglyceride-rich membrane vesicles were released from the cells during protoplast formation in Bacillus cereus Bacillus subtilis, Micrococcus lysodeikticus, and Staphylococcus aureus.
Abstract: Membrane phospholipid was found to be hydrolyzed presumably by an intracellular phospholipase C, and diglyceride-rich membrane vesicles were released from the cells during protoplast formation in Bacillus cereus Bacillus subtilis, Micrococcus lysodeikticus, and Staphylococcus aureus. The released membranes consisted mainly of small vesicles of 50 to 100 nm in diameter. They have a lower density than that of protoplast membranes in all the bacteria tested in the present study.
TL;DR: The best conditions found for infection of tobacco mesophyll protoplasts with alfalfa mosaic virus (AMV) were pH 5.2, 2.5 µg/ml virus and 1 µG/ml poly-l-ornithine.
Abstract: Summary
The best conditions found for infection of tobacco mesophyll protoplasts with alfalfa mosaic virus (AMV) were pH 5.2, 2.5 µg/ml virus and 1 µg/ml poly-l-ornithine. Newly synthesized virus could be detected 15 h after inoculation and the virus concentration reached an estimated 106 to 107 particles per infected protoplast 2 days after inoculation.
TL;DR: Protoplasts of Streptococcus faecalis ATCC 9790 synthesized soluble PG fragments in amounts which were of the same order of magnitude as that expected for insoluble PG produced by an equivalent amount of intact streptococci.
Abstract: Protoplasts of Streptococcus faecalis ATCC 9790 were produced with the aid of lysozyme, and the ability of these bodies to synthesize soluble, peptide cross-linked peptidoglycan (PG) fragments was examined. Lysozyme digests of PG isolated using gel filtration from the supernatant medium of protoplasts grown in the presence of [14C]acetate and L-[3H]lysine contained small amounts of PG having KD expected for peptide cross-linked dimers and trimers. Addition of benzyl penicillin (300 mug/ml) to growing protoplast cultures did not affect the net amount of PG fragments synthesized but resulted in inhibition of synthesis of dimer and trimer fractions by 27 and 59%, respectively. Failure of penicillin to completely inhibit the accumulation of the dimer fraction was attributed to the presence of atypical forms of dimer. In fact, the supernatant medium of penicillin-treated cultures did not contain detectable amounts of typical peptide cross-linked dimer. The degree of peptide cross-linkage of protoplast PG was at most only 13% of that found in walls isolated from intact streptococci. The relative amounts of monomers, dimers, and trimers synthesized during early and late stages of protoplast growth was approximately the same. Protoplasts synthesized soluble PG fragments in amounts which were of the same order of magnitude as that expected for insoluble PG produced by an equivalent amount of intact streptococci.
TL;DR: The formation of protoplasts of the fission yeasts after the combined application of snail enzymes and Trichoderma viride enzymes in an osmotic stabilizer was studied by light and electron microscopy and the effect of the enzymes used leads during 30 min to the formation of 100% protoplast population.
Abstract: The formation of protoplasts of the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces versatilis after the combined application of snail enzymes and Trichoderma viride enzymes in an osmotic stabilizer (0.4M KCl, pH 5.5) was studied by light and electron microscopy. The effect of the enzymes used leads during 30 min to the formation of 100% protoplast population. Using electron microscopy no original walls or wall remnants were detected in the suspension of protoplasts. Protoplasts are viable and in liquid nutrient medium they regenerate cell walls and revert into normal cells. Such a protoplast population may be useful for biochemical study of protoplast metabolism by quantitative methods as well as for the chemical study of regenerating cell walls.
TL;DR: Protoplasts obtained from cell suspensions of an anthocyanin synthesizing strain ofDaucus carota cv.
Abstract: Protoplasts obtained from cell suspensions of an anthocyanin synthesizing strain ofDaucus carota cv. “Rote Riesen”, cultured on a modified Murashige and Skoog medium (MS) have been induced to regenerate cell walls, and divide repeatedly to form masses of callus. Techniques have been substantially refined, and optimal conditions for the isolation of protoplasts have been established. An optimal protoplast yield of 80–90% was obtained by treating the cells with 1.5% cellulase (pH 5.0) for 4.5 hours in a gently shaking water-bath maintained at 33 °C. The protoplasts when plated in the agar-solidified medium regenerated cell walls within 2 days, and first division was observed within 3 days. In addition, they showed irregular elongation, budding and the formation of sub-protoplasts; another phenomenon was the formation of a chain of buds, which either separated from each other or formed a “coenocytic”, tube-like structure. On the agar medium which contained 0.425 M sorbitol, growth slowed down or stopped after 3–4 weeks. In order to obtain further growth, it was necessary to transfer small pieces of agar containing protoplasts on to the top of a fresh medium containing lower amounts (0.2 M) of sorbitol. The protoplasts continued to divide to form cell colonies, and finally masses of callus after 4–5 weeks. In a liquid medium (B5) the protoplasts reacted similarly, they regenerated walls within 2 days and after 2 weeks anthocyanin containing callus was formed. In both media embryo-formation occurred after 6 weeks of culture. By combining the polyethylene glycol and the high pH fusion techniques intergeneric fusion was achieved between carrot protoplasts and mesophyll protoplasts from haploidNicotiana tabacum cv. “Badischer Burley”. The fused products could be readily identified and isolated by using the difference in colour as the visual markers. Conditions for the culture of these fused protoplasts are being worked out.