Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

A method for high-frequency intergeneric fusion of plant protoplasts

Summary Protoplasts of Vicia hajastana Grossh. obtained from suspension-culture cells and Pisum sativum L. obtained from leaves adhered tightly to each other in concentrated solutions of high-molecular-weight polyethylene glycol (PEG). The adhesion occurred non-specifically between the free protoplasts from the same species as well as from the different species and genus. It was enhanced by enrichment of the PEG solution with calcium. Very few heteroplasmic fusions occurred during the period when the protoplasts were incubated in the PEG solution. However, many heterokaryons (up to 10%) were formed soon after the PEG solution was diluted out. The same phenomena were also observed in protoplasts from suspension-culture cells of Glycine max L. and from leaves of Hordeum vulgare L. Vicia and soybean protoplasts obtained from cultured cells regenerated cell walls and underwent sustained cell division after such treatment. Some Vicia-pea heterokaryons divided once. Over 10% of the soybean-barley hybrids divided in 7 days. Some divided 4–5 times and formed small clusters of cells in 10 days. The hybrids were recognizable because they contained chloroplasts from the leaf protoplast and exhibited morphological characters typical of the chlorophyll-less cells. None of the protoplasts from pea and barley leaves, either with or without PEG treatment, underwent cell division during the period of observation. The mechanism of adhesion and fusion of the protoplasts has been discussed.National Research Council (Canada) No. 13732.     

Inhibitory Effect of Glycine on the Production of Amylase and Proteinase by Bacillus subtilis

Cytological effects of glycine on Bacillus subtilis var. amyloliquefaciens were compared between the cells of the glycine-sensitive parent and resistant mutant. Glycine induced disruption of the protoplasts which had been prepared by treating the glycine-sensitive cells with lysozyme. This effect of glycine was almost completely prevented by preincubating the protoplasts with spermine. The protoplasts prepared from the resistant cells were markedly stable in the presence of glycine. In this mutant, neither cell lysis nor cessation of the enzyme production by glycine occurred, contrary to the results obtained with the glycine-sensitive parent. Between both type of cells little difference could be observed in the metabolic activity for glycine, but free amino acid content was higher in the glycine-resistant cells than in the parent ones.     

Transformation of protoplasts and intact cells from slowly growing embryogenic callus of wheat (Triticum aestivum L.)

A procedure for culturing protoplasts from slowly growing embryogenic calli of wheat was developed. The procedure was dependent on the ability to isolate large numbers of culturable protoplasts from slowly growing embryogenic callus. Approximately 68% of the isolated protoplasts divided, and 22% formed colonies; of the latter, 67% continued to proliferate. Plating efficiency was reduced when protoplasts were transformed by polythylene glycol, electroporation, and/or Agrobacterium. Intact cells were also directly transformed by electroporation. Direct electroporation of the Agrobacterium binary vector into intact cells resulted in a significant increase of GUS activity over the control.     

Protoplasts formation inFusarium species

Formation of protoplasts from four species ofFusarium genus is described. Protoplasts were isolated from mycelium by enzymatic digestion of the cell wall in the presence of an osmotic stabilizer. The results obtained differed between the studied species. Best yields of protoplasts were obtained fromF. moniliforme (90 % cells as protoplasts).     

ELECTRON MICROSCOPIC OBSERVATIONS OF CULTURED CELLS AND PROTOPLASTS OF AMMI VISNAGA

Protoplasts were prepared from cultured cells of Ammi visnaga (Umbelliferae) by enzymatic digestion of the cell walls and examined microscopically. Staining of fresh protoplasts with Calcofluor and silver hexamine demonstrated the apparent absence of wall material. Protoplasts contained more cell organelles than the whole cells, particularly endoplasmic reticulum and associated polysomes. The plasmalemma of most protoplasts appeared smooth; some protoplasts were connected by structures resembling plasmodesmata. Multinucleates resulting from fusion were frequently observed.     

Polyethylene Glycol-induced Uptake of Bacteria into Yeast Protoplasts

Cells of the nitrogen-fixing bacterium Azotobacter vinelandii and the unicellular cyanobacterium Anacystis nidulans were introduced into protoplasts of Saccharomyces cerevisiae by the polyethylene glycol (PEG) method. Factors influencing the uptake frequency were examined, and experimental conditions were established for maximizing the uptake frequency. Under optimal conditions, each protoplast took-up a few bacterial cells. Electron-microscopic studies showed the localization of integrated bacterial cells in membrane-bound vesicles of the cytoplasm or large vacuoles. The protoplasts at the intermediate stages of uptake revealed two major mechanisms of uptake: (a) “endocytosis” by a single protoplast and (b) “cell fusion” between two or more protoplasts. Some bacterial cells disintegrated during the subsequent incubation period through a heterophagy-like process.     

Isolation,culture, and regeneration of plants from potato protoplasts

A technique is described for the routine isolation of protoplasts from storage parenchyma cells of potato tubers grown in vitro. The protoplasts typically contained many starch grains. On culture, most of the starch grains were metabolised during the first 7 days, after which the cells began to divide. Following further culture, protoplast-derived colonies and calli were obtained, from which shoots and intact plants were regenerated. Cytological study of regenerated plants showed that the majority were octaploid or aneuploid at the octaploid level. This aspect is compared with plants regenerated from mesophyll protoplasts of potato. The use of tuber protoplasts for studies on tissue-specific transient gene expression of chimeric gene constructs, following their introduction into the protoplasts by electroporation, is discussed, together with the uses of tuber protoplasts in fundamental physiological and biochemical studies.     

Transformation of protoplasts of nontransformableBacillus subtilis mutants by plasmid pUB 110 DNA

Protoplasts rather than intact cells of nontransformableB. subtilis mutants were transformed by plasmid pUB 110 DNA. Transformability of protoplasts of the NT mutants indicates that the mechanism of uptake of the donor DNA by protoplasts differs from that by competent intact cells.     

Optical Resolution and Biological Activities of α-Ionylideneacetic Acid.

Nuclease activity associated with cells and protoplasts was analyzed by agarose gel electrophoresis. Datura innoxia protoplasts were found to possess a high exonuclease activity. On the other hand, Datura innoxia cells had an endonuclease activity, but no apparent exonuclease. The exonucleases from the protoplasts were active at pH 5 and 6, but not at pH 9. Endonuclease activity from the cells was also inhibited at pH 9. Cultured cells of Daucus carota, Glycine max, Pisum sativum and Vicia hajastana had endonuclease activity, but did not exhibit exonuclease activity. Nicotiana suaveolens cells had both types of nuclease activity. On the other hand, cells from cereals such as Triticum monococcum, Oryza sativa, and Zea mays had active exonuclease activity.     

Fusion between interphase and mitotic plant protoplasts : Induction of premature chromosome condensation

Morphological changes in interphase nuclei were cytologically studied in heterophasic dinucleate cells formed by the fusion of mitotic and interphase plant protoplasts. Mitotic protoplasts were isolated from a partially synchronized suspension culture of wheat (Triticum monococcum). The mitotic cells were accumulated by colchicine after release of hydroxyurea block. Treatment of protoplast populations with polyethylene glycol-dimethyl sulphoxide solution resulted in metaphase-interphase fusion. Three hours after fusion, the appearance of chromosomes with single chromatid as well as of fragmented, pulverized chromatin in heterophasic cells indicated the induction of premature chromosome condensation (PCC) in somatic wheat cells. Condensation in interphase nuclei of mitotically inactive rice protoplasts was also detected after fusion with mitotic wheat protoplasts.     

Interactions of Cowpea Mosaic Virus with Cowpea Protoplasts1)

The capability of cowpea mosaic virus to attach to and infect protoplasts of immune, hypersensitive, and susceptible cowpea (Vigna unguiculata) lines was examined by inoculating protoplasts with either purified virus or radioiodinated purified virus ¹²⁵I-CPMV. Systems were used in which plants were immune and protoplasts susceptible, plants were immune and protoplasts resistant, and plants and protoplasts were susceptible to CPMV. No differences were observed in the attachment of ¹²⁵I-CPMV to resistant and susceptible protoplasts. Polycations, proteins, or virus particles were added to the inoculation medium to neutralize potential nonspecific interactions between cells and virus particles. The various additives induced quantitative differences in binding of virus particles to protoplasts.     

Electron microscopic observations of cell regeneration from cultured protoplasts ofAmmi visnaga

Summary Protoplasts ofAmmi visnaga initiated cell wall formation within 2 days in culture; after 13 days the new cells were enclosed by a cell wall similar to the walls on the original cultured cells. Budding occurred in protoplasts with little or no detectable cell wall. No evidence was obtained for direct participation of any organelle in cell wall formation. The cytoplasm of regenerating cells contained numerous organelles and appeared typical of actively growing plant cells; they were easily distinguished from degenerate cells and protoplasts. While coated vesicles were common, spiny vesicles occurred in only a few cells. Sustained cell division yielded multicellular aggregates. Multinucleate protoplasts, formed by spontaneous fusion, did not divide; some of them contained annulate lamellae with few pore complexes.Supported by the National Research Council of Canada, Grant A6304.     

Protoplast-like structures formation from two species of enterobacteriaceae by fosfomycin treatment

A procedure for protoplasts formation from Escherichia coli and Serratia marcescens by treatment with fosfomycin alone is described. This method gives high and low yields of stable protoplasts from E. coli and S. marcescens respectively. In the last case numerous spheroplasts were obtained. Electron micrographs of intact cells, protoplasts and spheroplasts are shown.     

Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis

The transient expression of foreign genes in the protoplasts of Porphyrayezoensis was examined using three recombinant vectors, pYez-Rub-GUS, pYez-Rub-GFP and pYez-Rub-LUC, which were constructed with the promoter sequence of the ribulose-bisphosphate-carboxylase / oxygenase (Rubisco) gene as a promoter and the bacterial β-glucuronidase (GUS), mutant of green fluorescent protein (S65T-GFP) and firefly luciferase (LUC) genes, respectively, as reporter genes. When the pYez-Rub-GUS was introduced into protoplasts by electroporation, cells stained dark blue by indigotin were observed after the histochemical GUS assay. GUS activity was also detected by quantitative enzyme assays with a chemiluminescent substrate. When the pYez-Rub-GFP was electroporated into protoplasts, the expression of GFP could be detected in vivo observations with fluorescence microscopy. However, the rates of gene expression cells to the total number of cells were different between the GUS and GFP genes. LUC activity was also detected by assay with a chemiluminescent substrate after the introduction of pYez-Rub-LUC into protoplasts, although the activity levels were considerably lower. Relatively high expression rates of introduced GUS genes were observed 3 to 5 days after electroporation. These results show that the promoter sequence of the chloroplast Rubisco gene functions as a promoter of foreign gene expression and that transient expression occurred in protoplasts of P. yezoensis after the introduction of foreign genes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Actin distribution in somatic embryos and embryogenic protoplasts of white spruce (Picea glauca)

Summary Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and multinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in G₁, and suspensor cells showed signs of DNA degradation.     

Plant regeneration from protoplasts of Sphacelaria (Phaeophyceae)

Protoplast were isolated from a filamentous brown alga, Sphacelaria sp. (Sphacelariales, Phaeophyta), using alginate-lyases extracted from marine molluscs, and commercial pectinase and cellulase. Yields were about 4000 protoplasts per gram of fresh tissue. Different types of protoplasts, originating from apical, subapical, nodal and internodal cells, could be readily identified based on their size and pigmentation. Apical cells produced a higher percentage of protoplasts (approx. 2%), compared with other cell types. All apical-cell protoplasts regenerated into new thalli and most other types of protoplasts divided at least once in culture, but did not develop further.     

Kinetics of β-glucan and chitin formation by cells and protoplasts of the yeastSaccharomyces cerevisiae

Intact cells and protoplasts of the yeastSaccharomyces cerevisiae were grown in liquid medium with radioactive glucose as the sole carbon source, and the kinetics of radioactivity incorporation into β-glucan and chitin fractions were measured and compared. While the synthesis of β-glucan by protoplasts started early after their being suspended in the growth medium, the onset of chitin formation was delayed about 3 h and, unlike β-glucan, its formation depended on synthesis of undisturbed protein. In the intact cells, the ratio of β-glucan to chitin was constantly around 12 during growth; while in protoplasts this ratio steadily decreased in the course of cultivation and reached the value of 1.1 after 16h, which can be ascribed to the higher rate of chitin formation by protoplasts in comparison with normal cells. The deproteinized polysaccharide nets formed on the surface of protoplasts that had been incubated in the presence and in the absence of cycloheximide did not differ substantially in their morphology. The only observed difference was the presence of granular material in the samples from control protoplasts grown in the absence of cycloheximide.     

Application of nurse culture for plant regeneration from protoplasts of Lilium japonicum thunb

Summary The regeneration of lily protoplasts isolated from suspension cells of Lilium japonicum was achieved by using the nurse culture method. The protoplasts divided only under the nurse culture application. The divided protoplasts grew into colonies and developed into visible calluses on a medium containing picloram. After the calluses were transferred to a hormone-free medium, plantlets formed from them. The highest frequency of plant regeneration was obtained on a medium containing 1 μM gibberellin 4 (GA₄). The cleaved amplified polymorphie sequences (CAPS) method was used to confirm that the regenerants were not plants that had escaped from nurse cells. We were able to transplant the plantlets to soil in pots without acclimatization, and they showed normal growth.