Fusion of Agrobacterium and E. coli spheroplasts with Nicotiana tabacum protoplasts — Direct gene transfer from microorganism to higher plant

Spheroplasts of Agrobacterium tumefaciens strains and E. coli were fused with protoplasts of Nicotiana tabacum. Fusion products were cultured in the presence of antibiotics to eliminate remaining bacterial spheroplasts. On hormone free medium, tobacco protoplasts treated with wild type Agrobacterium-strains formed colonies with an average frequency of 10^–4. Opine synthesis was detected in the tissues. Some calli derived from protoplasts treated with A. tumefaciens C58C1pRi15834 formed typical hairy roots. Kanamycin resistant calli were obtained after fusion with A. tumefaciens containing pLGVTi23 neo (frequency=10^–3). Fusion of E. coli spheroplasts containing a virulent pTiB6S3::RP4 co-integrate with tobacco protoplasts yielded two hormone independent growing calli producing octopine out of 10⁵ microcalli.Abbreviations PEG Polyethylene glycol - PVA Polyvinyl alcohol     

Endocytic uptake of Escherichia coli spheroplasts by Neurospora crassa slime cells

Summary Interaction of Escherichia coli spheroplasts with Neurospora crassa slime cells was examined by transmission electron microscopy after treatment with polyvinyl alcohol followed by dilution with the high pH-high Ca buffer. Bacterial spheroplasts were found either adhering to the flat surface, associating with the invaginating surface, or residing within the intracellular vesicle of fungal protoplasts. In addition, bacterial spheroplasts free of the surrounding vesicles and those in the course of breakdown were observed in the fungal cytoplasm. It was concluded that Escherichia coli spheroplasts are taken up by Neurospora crassa protoplasts almost exclusively via endocytosis. This is the first cytological evidence for the endocytic activity of fungal cells.     

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

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 0.1M glycine-NaOH buffer (pH 10.0). 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.Abbreviations PEG polyethylene glycol - DMSO dimethylsulfoxide     

Protoplast formation and cell wall regeneration inClostridium thermocellum

Summary A protocol was developed for the production ofClostridium thermocellum protoplasts and the regeneration of protoplasts into vegetative cells. Protoplasts were prepared by exposure of whole cells to lysozyme (125 ug/ml for 10 min. at 45 C). Protoplasts plated on regeneration agar reverted to walled cells with a frequency of up to approximately 0.3 % of input cells.     

The interaction betweenE. coli spheroplasts and plant protoplasts: A proposed procedure to deliver foreign genes into plant cells

Summary E. coli spheroplasts can be used to deliver DNA vectors into plant protoplasts. The use of fluorescent dyes showed that 25–100% of the protoplast population was associated with 1–9 spheroplasts following incubation with several fusogens. Electron microscopy demonstrated spheroplasts attached to protoplasts via a plasma membrane protrusion after high pH/Ca²⁺ treatment, but PEG-high pH/Ca²⁺ promoted endocytosis of spheroplasts into a plasma membrane bounded vesicle. Ultrastructural profiles showed that fusion between spheroplasts and protoplasts did not occur. Immunofluorescence studies detectedE. coli antigens associated with tobacco protoplasts, and after fusogen treatment the antigens were dispersed within the peripheral cytoplasm. The elimination of residual contaminatingE. coli cells from protoplasts was achieved by lysozyme and antibiotic treatment, thus allowing DNA vector assessment in axenic culture.     

Interspecific hybridization between Brassica juncea and B. spinescens through protoplast fusion

Hypocotyl derived protoplasts of B. juncea cv. RLM-198 were fused with mesophyll protoplasts of B. spinescens using polyethylene glycol to produce interspecific hybrids. Fusion products could be microscopically identified by characteristics of the protoplasts of both parents in the hybrid cells; they are colourless and vacuolated like the hypocotyl protoplasts and possess chloroplasts of the mesophyll protoplasts. The heterokaryotic fusion frequency was around 5%. However, the frequency of calli regenerating hybrid shoots was more than 10% of the regenerating calli. Putative somatic hybrids had morphological features characteristic of both the parents. Twelve plants analysed cytologically, possessed 52 chromosomes (26_II) at meiosis representing the complete genomes of B. juncea (18_II) and B. spinescens (8_II). For esterase isozymes, the hybrids had bands of Doth the parents. Hybrid nature of some of the plants was confirmed by their close resemblance to B. juncea, chromosome number and isozyme bands of B. spinescens as in Rsp-19. Somatic hybrids had rudimentary, non-dehiscent anthers and completely sterile pollen. However, on back crossing with B. juncea, 10 out of 12 plants produced seeds and about 100 plants were realized.Abbreviations PEG Polyethylene glycol     

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.     

E. coli spheroplast-mediated transfer of cloned cauliflower mosaic virus DNA into plant protoplasts

Summary Saishin (Brassica chinensis L.) mesophyll protoplasts and E. coli spheroplasts harbouring hybrid plasmid with tandemly dimerized cauliflower mosaic virus DNA were mixed in ratios of 1:1,000 and incubated for 20 min at 30° C in the presence of 20% polyvinyl alcohol. Subsequently, protoplasts/spheroplasts mixture was washed with high pH-high Ca buffer. After 3 days of culture, 8% of Saishin protoplasts were transfected as monitored by immunofluorescence technique. When plant protoplasts and bacterial spheroplasts were mixed in ratios of 1:100 or 1:2,000, 1% or 5% of protoplasts were transfected, respectively.     

Root hair protoplasts ofLotus corniculatus L. (birdsfoot trefoil) express their totipotency

Treatment of the roots of 24–48 h old seedlings of the forage legumeLotus corniculatus with 1.0% Cellulase YC, and 0.1% Pectolyase Y-23 in 4.2% mannitol solution released protoplasts from the tips of root hairs within 30–40 sec of enzyme incubation. Roots from approximately 1000 seedlings yielded 1.7×10⁵ protoplasts. Ten percent of protoplasts divided to form cell colonies when cultured at 1.0×10⁵ ml^–1 in droplets of KM8P medium with 0.6% Sea Plaque agarose. Colonies formed callus on UM agar medium; protoplast-derived tissues produced shoots on B5 medium containing 0.05 mg 1^–1 of BAP. Regenerated plants were phenotypically and cytologically normal (2n=2x=24±2), and produced nitrogen fixing root nodules following inoculation withRhizobium. These results confirm the totipotency of protoplasts isolated from specialised epidermal cells of seedling roots ofLotus corniculatus.     

Reversion of protoplasts and spheroplasts in the yeast Nadsonia elongata

Summary The time rate of regeneration of the cell wall and reversion of protoplasts of the yeast Nadsonia elongata to cells of normal shape and size has been compared with the capability for regeneration of spheroplasts of this yeast. Nearly all protoplasts in a given culture were able to regenerate new walls and had usually reverted to cells of normal appearance by the 30th h of cultivation. Spheroplasts required only half this time to do this. These results can be interpreted as evidence that regeneration of a wall by protoplasts does not depend upon the presence of a cell wall primer, because the proportion of reverting protoplasts (which lack wall remnants) was the same as that of reverting spheroplasts (which possess them). The presence of wall remnants in spheroplasts appears to have merely an accelerating effect on the formation of a new wall and on subsequent reversion of the spheroplasts to complete cells of normal shape and size.     

Intra- and interspecific gametosomatic hybridisation within the genus Petunia

Following PEG and high pH induced fusion, intraspecific gametosomatic hybrid plants (pollen tetrad protoplasts of a normal purple flowered variety of P. hybrida fused with cell suspension protoplasts of a nuclear albino mutant of the variety Blue Lace) and interspecific gametosomatic hybrid plants (tetrad protoplasts (as above) fused with cell suspension protoplasts of a nuclear albino mutant of P. parviflora) were recovered. Hybrid plants of both combinations possessed an intermediate vegetative and floral morphology with chromosome numbers of 2n=3x=21 and 2n=3x=25 respectively. Hybrid cells were in both systems identified as green colonies against an albino background as a result of complementation to chlorophyll proficiency. Pollen tetrad protoplasts did not divide. The production of such plants at the intra- and interspecific level in Petunia has shown that the concept of gametosomatic hybridisation can be extended to genera other than Nicotiana. An alternative selection strategy is available to that as used earlier for Nicotiana.     

Transformation of Vinca protoplasts mediated by Agrobacterium spheroplasts

Summary Vinca rosea protoplasts and Agrobacterium tumefaciens spheroplasts harboring octopine-type Ti plasmid were mixed and treated with polyethylene glycol or polyvinyl alcohol, which facilitated the introduction of spheroplasts into plant protoplasts. After the protoplasts had been kept at 40° C for 4 days, bacteria were found to be completely eliminated from the medium. Among treated protoplasts 1–2 per 1,000 formed colonies on the Murashige and Skoog medium (1962) lacking hormones. When the colonies were isolated and subcultured, they could be maintained as clones. Octopine, an amino acid specific to crown gall, was detected in half of these clones. The phenotypic features of these putative transformants were compared but did not show any coincidental tendencies in relation to color, hardness, form, growth rate, or octopine production. The significance of this system in transformation of higher plant cells is discussed.     

A Comparison of Methods for Plasmid Delivery into Plant Protoplasts

Three methods of plasmid delivery to mesophyll protoplasts ofNicotiana tabacum cv. Xanthi have been evaluated. Specifically;a) chemically stimulated uptake of isolated plasmid, b) deliveryof plasmid encapsulated in liposomes, and c) fusion of plasmid-containingspheroplasts, were combined with divalent cation (Ca²⁺ and Mg²⁺)or polyalcohol [polyethylene glycol (PEG) and polyvinyl alcohol(PVA)] treatments. The quantity and quality of plasmid associatedwith intact protoplasts, was assessed by DNA-DNA blot hybridisationanalysis, following stringent washing to separate intact protoplastsfrom non-viable protoplasts and debris. Treatments which increasedassociation of plasmid with protoplasts decreased protoplastviability. Optimum association of plasmid with protoplasts,in the context of acceptable loss of viability, was achievedwhen protoplasts were interacted with either naked plasmid orliposomeencapsulated DNA in the presence of 15% w/v PEG 6000,or with Escherichia coli spheroplasts containing chloramphenicol-amplifiedplasmid in the presence of 25% w/v PEG 6000. Divalent cationsdid not stimulate significant plasmid delivery without unacceptableloss of protoplast viability. Strategies to further increasethe efficiency of plasmid delivery are discussed. (Received June 21, 1984; Accepted August 20, 1984)     

Immobilization of plant protoplasts: Viability studies

Protoplasts of Daucus carota Ca68 and Catharanthus roseus have been immobilized by entrapment in gelforming polysaccharides (kappa-carrageenan, agarose and alginate). Uniform spherical beads of carrageenan and agarose containing the protoplasts have been prepared by utilizing an inert hydrophobic phase (vegetable oil). The entrapped protoplasts are viable and stabilized towards osmotic shock by the polymeric backbone. Standard methods have been used to study the viability and integrity of the entrapped protoplasts. Upon incubation in a relatively simple medium the immobilized protoplasts show a much higher viability after 14 days as compared to free protoplasts under the same conditions. The viability of D. carota protoplasts has also been monitored by an enzyme activity present in the cells (digitoxigenin 58-hydroxylase).     

Cell division and differentiation in protoplasts from cell cultures of Glycine species and leaf tissue of soybean

Protoplasts were isolated from cell cultures of G. soja and G. tabacina, respectively. The isolation procedure employed Percoll for the separation and concentration of protoplasts. The cultured protoplasts formed cells which developed into embryo-like structures. Protoplasts also were isolated from leaf tissue of soybean cv. Williams 82. Upon culture, the protoplasts regenerated cell walls and divided to form cell cultures.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid BA|Benzyladenine - BA Benzyladenine     

Examination of genome stability in cultured Lycopersicon

The genetic stability of plants regenerated from either mesophyll protoplasts or leaf slices of the F1 hybrid between Lycopersicon esculentum and L. pennellii was assayed by comparing the ploidy level, leaf morphology and isozyme patterns of the regenerants with their somatic parents. Regenerants from protoplasts were predominantly tetraploid, regenerants from leaf slices were predominantly diploid; both classes of regenerants had isozyme patterns identical to those of the parent plant. Callus was analyzed that grew up from cultures containing fused protoplasts from either irradiated or untreated protoplasts of L. esculentum and L. pennellii. The L. pennellii cell line used was 18 months old and could no longer regenerate. Out of 75 calli scored at 3 isozyme loci, 51 were heterozygous at only one or two of the loci. Irradiation of the two parental lines was not necessary to produce fusion products exhibiting asymmetric expression of parental genes.Abbreviations Got-2 glutamate oxaloacetate transaminase-2 - Pgi-1 phosphoglucoisomerase-1 - Pgm-2 phosphoglucomutase-2     

Further evidence for the transformation ofVinca rosea protoplasts byAgrobacterium tumefaciens spheroplasts

Protoplasts ofVinca rosea were transformed by spheroplasts ofAgrobacterium tumefaciens harboring nopalinetype Ti plasmids according to the procedure of Hasezawa et al. (1981). These transformants frequently differentiated tracheids, but further differentiation to teratomata has not so far been observed. Transformation was confirmed by the improved detection of nopaline synthase, where the sensitivity and specificity of the enzyme reaction was increased by employing¹⁴C--ketoglutaric acid and³H-arginine as substrates. The nopaline synthase activity was identified by the comigration of these two radioisotopes in the cnromatogram. Furthermore, the T-DNA structure of one of these transformants was examined by Southern hybridization according to Thomashow et al. (1980) and compared with that ofVinca rosea crown gall.Abbreviations PEG Polyethylene glycol - PVA polyvinyl alcohol - TLC thin-layer chromatography     

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl₂.2H₂O produced an average of 4.5 × 10⁶ protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10⁵ protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - FDA fluorescein diacetate NRCC No. 27937     

Cell wall synthesis and salt (saline) sensitivity in cultured colt cherry (Prunus avium × pseudocerasus) protoplasts

In order to investigate the cellular basis of salt tolerance, Colt cherry (Prunus avium ×pseudocerasus) protoplasts from mesophyll tissues and root cell suspension cultures were cultured in the presence of NaCl, KCl or Na₂SO₄, at normalities of 25, 50, 100 or 200 mN for each salt and with or without 2,6-dichlorobenzonitrile, an inhibitor of cell wall synthesis. Results showed that the acquisition of salt tolerance was concomitant with the onset of cell wall regeneration, with protoplasts exhibiting a greater salt tolerance than cells.Abbreviations DBN 2,6-dichlorobenzonitrile - FDA fluorescein diacetate     

Callus culture as substrate for the production of specific cell wall degrading enzymes for derived protoplast isolation

Callus culture of spruce (Picea excelsa LINK) appears to be a suitable substrate for the fungusTrichoderma reesei to produce an efficient extracellular lytic system for protoplast isolation. In comparison with Onozuka R-10 cellulase, a yield of protoplasts from the spruce callus 2·5 higher was obtained. Another testea commercial cellulase DK was less efficient. The addition of Macerozyme R–10 significantly enhanced release of protoplasts within all tested enzyme preparations. No difference in the viability of protoplasts has been observed.