Immunological methods for the agglutination of protoplasts from cell suspension cultures of different genera

Summary Protoplasts from cell suspension cultures of Vicia hajastana Grossh., soybean (Glycine max L.) and brome grass (Bromus inermis Leyss.) were tightly agglutinated by immune sera prepared against them in rabbits. After incubation, the aggregated protoplasts became adpressed over a considerable area of their surface. Antibody prepared against Vicia protoplasts agglutinated both Vicia and soybean protoplasts alone, as well as a mixture of the two. Soybean and bromegrass antibody likewise cross-reacted with and agglutinated Vicia protoplasts. The heterologous reactions were nearly as strong as, and in some cases stronger than, the homologous. When sheep anti-rabbit globulin was reacted with a mixture of the protoplasts previously coated with homologous antibody, agglutination occurred much more quickly and the aggregates could not be dispersed without physical damage. Carbol-fuchsin staining of nuclei showed that Vicia and soybean protoplasts were randomly mixed in the aggregate. The protoplasts were viable and underwent division after the antibody treatment. The immune serum, which presumably contained complement, lysed the protoplasts unless it was heat-treated prior to use.Issued as NRCC No. 13168.     

A new protoplast culture system in Daucus carota L. and its applications for mutant selection and transformation

Petiole protoplasts from in vitro-grown carrot plants are a very good alternative to traditionally obtained protoplasts from suspension cultures. High plating and regeneration efficiencies were obtained in most of the breeding lines that were tested. The embedding of the protoplasts in alginate was crucial for initiating cell division and further development. Several streptomycin resistant and chlorophyll-deficient plant lines were selected for using the petiole protoplast system. Maternally inherited streptomycin resistance was demonstrated by sexual crosses. Protoplast fusion of several chlorophyll-deficient lines did not result in complementation, indicating the cytoplasmic nature of the mutations. Petiole protoplasts were used for direct transformation with plasmid DNA pNUNV containing NPTII as a selectable marker. High transformation frequencies (up to 1%) were obtained after PEG treatment of the protoplasts. Kanamycin resistance was shown to be inherited as a single dominant nuclear trait.     

Regeneration of somatic hybrid plants formed between Lycopersicon esculentum and L. pennellii

Summary Somatic hybrid plants have been regenerated following polyethylene glycol mediated fusion of leaf mesophyll protoplasts from tomato and protoplasts from Lycopersicon pennellii callus. Three different cultivars of tomato were used as sources of protoplasts: Early Girl, Manapal, and UC82B. Fusions were performed between protoplasts of these tomato cultivars and protoplasts of L. pennellii, and between protoplasts of the cultivars and protoplasts of L. pennellii that had been exposed to 3 or 6 krads of gamma radiation. Somatic hybrid plants were identified on the basis of heterozygous isozyme banding patterns, and leaf and flower morphology. Somatic hybrid plants were regenerated following fusion of tomato protoplasts with either untreated or irradiated L. pennellii protoplasts. All were heterozygous for isozyme loci on five different chromosomes. Regenerated somatic hybrids showed inheritance of either or both parental chloroplast genomes, but predominantly the L. pennellii mitochondrial genome. The regenerated somatic hybrid plants exhibited reduced fertility, less than 20% viable pollen. A total of 34 somatic hybrid calli were identified. Of these, 21 regenerated shoots, and 7 produced seed following manual pollinations.     

Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture

A continuous micropropagation was established from protoplasts of thegreen alga Enteromorpha intestinalis. The effects of two differentcrude enzymes and the osmolarity at different concentrations of the enzymesolution on algal protoplast yields were tested. The optimal enzymecomposition for cell wall digestion and protoplast viability was 2%cellulase R 10 Onozuka and 2% Aplysie with 0.5 m mannitol. Largenumbers of Enteromorpha protoplasts were released (10.0 × 10⁶protoplasts from 1 g fresh thalli) and settled on a rangeof substrata. Regeneration of the protoplasts followed the normal patternfor this species. Conditions for pure cultures and efficient systems offloating supports with nets were determined to optimise the product qualityof plantlets of Enteromorpha. A promising storage process has beendeveloped which involves including protoplasts in beads of alginic acid gel.Plants regenerated from protoplasts may also be used as seedstock tofacilitate propagation for macroalgal culture.     

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.     

Nontranslation of foreign genetic information for fraction 1 protein under circumstances favorable for direct transfer ofNicotiana gossei isolated chloroplasts intoN. Tabacum protoplasts

Summary The nuclei and cytoplasm ofN. gossei andN. tabacum are compatible to the extent that reciprocal, interspecific F₁ hybrids can be produced by conventional breeding techniques. Conditions were established in which manyN. gossei isolated chloroplasts could be seen by phase and fluorescence microscopy to adhere to 40% of the population of protoplasts obtained from white tissue of variegatedN. tabacum plants and to remain attached after washing the protoplasts. Chloroplasts also could be seen to enter the interior of the protoplasts. After treating albino protoplasts withN. gossei chloroplasts, the protoplasts were subjected to further conditions whereby 65 calluses containing shoots developed. TwentyN. tabacum protoplasts not treated with foreign chloroplasts also produced calluses with shoots to serve as a control. All calluses developed chlorophyll irrespective of whether or not the albino protoplasts had been treated with isolatedN. gossei chloroplasts. The Fraction 1 protein ofN. tabacum has a different electrophoretic mobility from the protein ofN. gossei or anN. gossei xN. tabacum F₁ hybrid. The Fraction 1 protein large subunit is coded by chloroplast DNA, whereas the small subunit is coded by nuclear DNA. Fraction 1 protein was isolated from the variegated shoots of the 65 calluses obtained after treating albino protoplasts with foreign chloroplasts. Immunoelectrophoresis demonstrated the protein from each callus to have a mobility identical toN. tabacum protein. Therefore, under circumstances highly favorable for the direct transfer ofN. gossei isolated chloroplasts (and possibly nuclei also) intoN. tabacum protoplasts, no evidence was obtained to suggest that genetic information contained in the isolated foreign organelles was being translated into the polypeptides of either the large or small subunits of Fraction 1 protein contained in newly differentiated leaves derived from the protoplasts. Supported by Research Grant PCM-75-07368 from the National Science Foundation.     

Wirkung von Virushemmstoffen auf die Infektion von Tabakprotoplasten mit dem Tabakmosaik-Virus

Effects of virus inhibitors on the infection of tobacco protoplasts with tobacco mosaic virus Yeast extract inhibits the infection of Nicotiana glutinosa plants with tobacco mosaic virus (TMV), whereas in N. sandérae yeast extract is not effective. This phenomena was compared with the effect of yeast extract on protoplasts, and on the infection of protoplasts of both tobacco species with TMV. Additionally, skim milk and ribonuclease were included in the experiments as further inhibitors of early stages of virus infection. It was examined whether these inhibitors damage non-inoculated protoplasts (a), and whether they affect virus infections in protoplasts as they do in cells of intact plants (b). To investigate protoplast damage by the inhibitors, conductivity measurements of protoplast suspensions containing inhibitors, and the ability of protoplasts for cell wall regeneration after treatment with the inhibitors, were used. Inhibitor concentrations which prevent virus infections in plants did not damage the protoplasts. The inhibitor effect on the course of infection was investigated by protoplast treatments before, during and after inoculation with TMV, and by addition of the substances to the culture medium. Measurements of virus content in protoplasts after cultivation revealed different results for the three inhibitors, however, there was no difference in the response of protoplasts from the two tobacco species to yeast extract. It is concluded that there are principal differences between the inhibition of plant and protoplast infections. Therefore, it is unlikely that protoplasts are a useful system for the mode of action studies on inhibitors of early stages of virus infection in plants.     

Valine resistant plants derived from mutated haploid and diploid protoplasts of Nicotiana sylvestris and N. tabacum

Summary Protoplasts were derived from haploid and diploid Nicotiana sylvestris and N. tabacum. Exposure of the protoplasts to mutagenic doses of ultraviolet (U.V.) radiation prior to two selection rounds in the presence of 4 mM (or 5 mM) and 8 mM of valine, respectively, was required to obtain cell lines with persistent valine resistance. Such lines were obtained from haploid and diploid N. sylvestris protoplasts as well as from haploid protoplasts of N. tabacum but not from (1.8 × 10⁷) diploid N. tabacum protoplasts. The ratio between number of verified valine-resistant cell lines and the initial number of U.V. exposed protoplasts enabled the estimation of the following order of mutation frequency: haploid N. sylvestris > haploid N. tabacum > diploid N. sylvestris. Plants which retained the valine resistance and transmitted it to their sexual progeny were derived from the resistant cell lines.     

Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

Summary Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.Abbreviations PEG Polyethylene glycol - DAPI 4,6-diaminido-2-phenylindole - 2,4-D 2,4-dichlorophenoxyacetic acid     

Fusion characteristics of plant protoplasts in electric fields

The electrical parameters important in the fusion of plant protoplasts aligned dielectrophoretically in high-frequency alternating electric fields have been established. Protoplasts were aligned in an alternating electric field between two relatively distant (1 mm) electrodes, by dielectrophoresis induced by field inhomogeneities caused by the protoplasts themselves. This arrangement allowed ease of manipulations, large throughput and low loss of protoplasts. In analytical experiments, sufficiently large samples could be used to study pulse duration-fusion response relations at different pulse voltages for protoplasts of different species, tissues and size (mesophyll protoplasts of Solanum brevidens, Triticum aestivum, Hordeum vulgare; suspension-culture protoplasts of Nicotiana sylvestris, N. rustica, Datura innoxia and S. brevidens; root-tip protoplasts of Vicia faba, hypocotyl protoplasts of Brassica napus). The percentage of aligned protoplasts that fused increased with increasing pulse parameters (pulse duration; voltage) above a threshold that was dependant on pulse voltage. The maximum fusion values obtained depended on a number of factors including protoplast origin, size and chain length. Leaf mesophyll protoplasts fused much more readily than suspension-culture protoplasts. For both types, there was a correlation of size with fusion yield: large protoplasts tended to fuse more readily than small protoplasts. In short chains (five protoplasts), fusion frequency was lower, but the proportion of one-to-one products was greater than in long chains (ten protoplasts). In formation by electrofusion of heterokaryons between mesophyll and suspension-culture protoplasts, the fusion-frequency response curves reflected those of homofusion of mesophyll protoplasts rather than suspension-culture protoplasts. There was no apparent limitation to the fusion of the smallest mesophyll protoplast with the largest suspension-culture protoplasts. Based on these observations, it is possible to direct fusion towards a higher frequency of one-to-one (mesophyll/suspension) products by incorporating low densities of mesophyll protoplasts in high densities of suspensionculture protoplasts and by using a short fusion pulse. The viability of fusion products, assessed by staining with fluorescein diacetate, was not impaired by standard fusion conditions. On a preparative scale, heterokaryons (S. brevidens mesophyll-N. sylvestris or D. innoxia suspension-culture) were produced by electrofusion and cultured in liquid or embedded in agar, and were capable of wall formation, division and growth. It is concluded that the electrode arrangement described is more suitable for carrying out directed fusions of plant protoplasts than that employing closer electrodes.     

Preparation of protoplasts from Laminaria japonica using native and recombinant abalone alginate lyases

Laminaria japonica protoplasts were released with high yields using the abalone alginate lyase HdAly in combination with a cellulase and chelating agents. Addition of EDTA at concentrations higher than 10 mM to Laminaria thalli which had been preincubated with HdAly and Cellulase Onozuka, dramatically improved the yield of protoplasts. EDTA was far more effective than EGTA, indicating that chelating divalent metal ions such as Mg²⁺ and Sr²⁺ in addition to Ca²⁺ is a key factor for high-yield production of Laminaria protoplasts. Protoplasts had a mean diameter of 27 μm, suggesting that most protoplasts were derived from cortical cells rather than epidermal layer cells. Recombinant HdAly (rHdAly) was produced from a cDNA clone in the Sf9 insect cell expression system. rHdAly had substantially the same enzymatic properties and protoplast-producing ability as did native HdAly. The optimal conditions for high yield production of protoplasts from Laminaria using native and recombinant HdAlys were investigated.     

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.     

Biosynthesis of oleanolic acid glycosides in protoplasts isolated from Calendula officinalis L. roots

Many medicinal plants contain oleanane saponins in roots, however, only scarce data on their biosynthesis in this organ are available so far, including our previous results concerning Calendula officinalis plant. Thus, the purpose of the present work was to confirm the presumable biosynthetic pathway of oleanolic acid glycosides in roots of young C. officinalis plants. First of all, the effective method of isolation of protoplasts from C. officinalis roots was established. Then, isolated root protoplasts were supplied with radioactive precursors, [2-¹⁴C] mevalonate (MVA) and [3-³H] oleanolic acid (OL) and their transformations were studied with comparison to results obtained with excised roots. The penetration of both precursors into protoplasts was more rapid and effective than in the case of excised roots. The labeling of sterols and OL during the incubation with MVA showed that the isoprenoid pathway leading to triterpenoids was operative in excised roots as well as isolated root protoplasts. Moreover, the transformations of OL into two series of its glycosides, i.e. glucosides and glucuronides were investigated. It has been shown that both series of OL glycosides are synthesized in isolated root protoplasts in the same way as in excised roots of young marigold plants.     

Evacuolation and enucleation of mesophyll protoplasts in self-generating percoll gradients

Mesophyll protoplasts from Brassica oleracea, B. napus, Nicotiana tobaccum and Solanum tuberosum were isolated and subjected to uttracentrifugation at 65000g for 30 min in percoll solutions containing various strengths of salt and osmotic stabilizing agents. After centrifugation, the self-generated percoll gradients were evaluated for their effectiveness in protoplast evacuolation and enucleation. The vacuoles, cell debris, evacuolated protoplasts and enucleated protoplasts were separated. Factors that affected evacuolation and enucleation in the percoll gradients were described. Mesophyll protoplasts produced by epidermis peeling and short enzyme incubation periods were more easily evacuolated and enucleated than those produced by leaf-slicing and long incubation periods. Lower centrifugal force at 25000g for 80 min was also successful in evacuolating and enucleating the mesophyll protoplasts. A green band that contained nearly pure evacuolated protoplasts, of which 45% were enucleated protoplasts, was obtained from the self-generated percoll gradient. Rhodamine 123 staining of mitochondria indicated that the evacuolated protoplasts were metabolically active and were capable of regenerating the vacuole and cell wall. Cell divisions were also observed when the evacuolated protoplasts were cultured.     

Electrofusion of protoplasts from celery (Apium graveolens L.) with protoplasts from the filamentous fungus Aspergillus nidulans

A method was developed for electrofusion of higher-plant protoplasts from celery and protoplasts from the filamentous fungus Aspergillus nidulans. Initially, methods for the fusion of protoplasts from ecch species were determined individually and, subsequently, electrical parameters for fusion between the species were determined. Pronase-E treatment and the presence of calcium ions markedly increased celery protoplast stability under the electrical conditions required and increased fusion frequency with A. nidulans protoplasts. A reduction in protoplast viability was observed after electrofusion but the majority of the protoplasts remained viable over a 24-h incubation period. A small decline in protoplast respiration rate occurred during incubation but those celery protoplasts fused with A. nidulans protoplasts showed elevated respiration rates for 3 h after electrofusion.Abbreviations AC alternating current - DC direct current     

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).     

The production of fertile,triploid somatic hybrid plants (Nicotiana glutinosa (n) + N. tabacum (2n)) via gametic: somatic protoplast fusion

Summary The fusion of gametic protoplasts with somatic protoplasts giving rise to gametosomatic hybrid plants was investigated. Gametosomatic hybrid plants were regenerated following the fusion of nitrate reductase deficient (Nr^–) Nicotiana tabacum Nia-130 leaf mesophyll protoplasts with N. glutinosa tetrad protoplasts. The resulting plants were confirmed as hybrids, based on leaf and floral morphology, chromosome number, leaf esterase and leaf callus peroxidase zymograms and Fraction-1-protein analysis. The five gametosomatic hybrid plants had the expected pentaploid, but functionally triploid chromosome number of 3n=5x=60. The relevance of triploid gametosomatic hybrids in facilitating limited gene transfer, is discussed. The utilisation of tetrads as a generally available source of haploid protoplasts for fusion studies is proposed.     

Relation between protoplast division,cell-cycle stage and nuclear chromatin structure

Summary Using different sources of protoplasts and two complementary techniques, flow cytometry and image analysis, to study the cell-cycle phases, we sought to define the particular protoplast state associated with the disposition to divide. Both inPetunia and inNicotiana plumbaginifolia, tissues with a higher G2 frequency (from different aged plants) yielded protoplasts capable of increased cell division. InSorghum, the age of the plant does not modify the proportion of G2 nuclei in leaf protoplasts, and we used root protoplasts to increase G2 frequencies. InHelianthus annuus, leaf protoplasts did not divide; however, hypocotyl protoplast preparations with relatively high 4C DNA frequencies do divide. Moreover, image analysis of chromatin structure indicated that leaf nuclei were in the G0 phase, unlike those from hypocotyls which were in G1. A high frequency of protoplasts with G2 nuclei appears to be correlated with the ability of a given preparation to undergo division; conversely, the differentiated G0 state is not conducive to division.     

Protoplast fusion ofCatharanthus roseus cells by electrofusion of chemically-agglutinated protoplasts

Mesophyll derived protoplasts ofCatharanthus roseus cv. Little pinkie were fused with protoplasts derived from an habituated cell line ofC. roseus. Polyethylene glycol was used as agglutinating agent while fusions were induced by square pulses. Best results were obtained by fusing protoplasts from primary leaves with those from three-day-old cell cultures. Adding calcium ions considerably enhanced heterofusion rate. Good cell viabilities indicated that this fusion process was not cytotoxic. The heterofusion frequency was up to 10% or more. Most of the heterokaryons were able to regenerate their cell walls and underwent division. Communicated by J. TUPY     

Presence of a plant cell wall is not required for transformation of Nicotiana by Agrobacterium tumefaciens

Agrobacterium has been used to transform zero to six-day-old cell wall nonregenerating (CWNR) and cell wall regenerating (CWR) leaf protoplasts of tobacco. Transformed cells were selected by phoytohormone autotrophic growth and were verified by detection of the presence of lysopine dehydrogenase. Transformation frequencies in CWNR protoplasts were at least as high as those in CWR protoplasts, indicating that a plant cell wall is not required for the process of crown gall tumorigenesis. Transformation frequencies were highest in two-day-old protoplasts. This age coincides with the onset of DNA synthesis and the first mitosis within the cell populations. We suggest that the initiation of cell cycle activity may be important for the transformation process.