Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Studies on isolated starch-containing (Vicia faba) and starch-deficient (Allium cepa) guard cell protoplasts

Guard cell protoplasts from starch-containing Vicia faba and starch-deficient Allium cepa stomata were isolated, stabilized and recovered with an efficiency — in relation to the potential yield — of approx. 62% and 77%, respectively. In vitro, guard cell protoplasts (GCP) respond to abscisic acid and fusicoccin by respectively contracting and swelling, that is, decreasing or increasing in diameter by about 15% and more in comparison to the control. This in vitro response correlates with, but is more than 4 times as rapid as, the in vivo response of the stomata. Among the advantages presented by working with isolated GCPs are: greater sensitivity in response; freedom from influences of cuticular ridges, cell walls, subsidiary cells, and epidermal cells; and direct and parallel comparisons of starch-containing and starch-deficient GCP systems.Abbrecviations ABA abscisic acid - FC fusicoccin - ECP, MCP, and GCP epidermal, mesophyll, and guard cell protoplasts, respectively - PPV packed protoplast volume     

An efficient protocol for plant regeneration from protoplasts of the moss <Emphasis Type="Italic">Atrichum undulatum P. Beauv in vitro</Emphasis>

A system for plant regeneration from protoplasts of the moss, Atrichum undulatum (Hedw.) P. Beauv. in vitro, is first reported. Viable protoplasts were isolated at about 9 × 10⁵ protoplasts g⁻¹ fresh weight from 10 to 18 days protonemata. For regeneration of protoplasts, viable protoplasts were cultured in liquid–solid medium containing surface liquid medium MS (0.4 M mannitol) and subnatant solid medium Benecke (0.3 M mannitol) at 20 °C under a 16-h photoperiod white light after 12 h preculture in darkness at 20 °C. The great majority of protoplasts follow a regenerative sequence: formation of asymmetric cells in 2–3 days; division of the asymmetric cells to 2–3 cells in 4–5 days, and further develop to produce a new chloronemal filament in 15 days. Juvenile gametophyte can be visible in 20 days. The plating ratio of cell cluster regenerated from protoplasts reaches up to 45%. Transient expression experiments indicate the electroporation uptake of DNA is possible.     

Subcellular localization of ubiquitin in plant protoplasts and the function of ubiquitin in selective degradation of outer-wall plasmodesmata in regenerating protoplasts

Immunocytochemical localizations in Vicia faba L. protoplasts and cultures of regenerating Solanum nigrum L. protoplasts support former observations that in plant cells ubiquitin occurs within the cytoplasm, the nucleus, the chloroplasts and at the plasmalemma, but not within the vacuole or the cell wall. Immunoresponses were also observed within mitochondria and associated with the endoplasmic reticulum, which is in accordance with previous findings on animal cells. Moreover, the tonoplast membrane system was found to be labelled. For regenerating S. nigrum protoplasts, evidence is given that ubiquitin plays a role in selective degradation even of whole subcellular structures. Most of the discontinuous plasmodesmata formed in the newly deposited outer cell walls during the early stages of culture disappear later on, except for those near the periphery of division walls or of non-division walls, which are probably used for the formation of continuous cell connections during further culture. Outer-wall plasmodesmata which are destined to disappear show high immunoreactivity to ubiquitin antibody, but no conspicuous immunolabelling was observed with the remaining plasmodesmata. Thus, the selective disintegration of whole plasmodesmatal structures is obviously regulated by ubiquitination of plasmodesmatal proteins. A model for the mechanism of degradation of outer-wall plasmodesmata during extension growth of the cell wall is presented.Dedicated to Professor Dr. Andreas Sievers on the occasion of his retirementThis work was supported by grants to R. K. (Deutsche Forschungsgemeinschaft) and to M. S. (Bennigsen-Foerder Preis des Landes Nordrhein-Westfalen). We thank Dipl.— Biol. Kirsten Leineweber for help with the V. faba protoplast isolation and Dr. Olaf Parge, Institut für Psychologie und Sozialforschung, Kiel, Germany, for giving assistance with the statistical analysis.     

Enhancement of transformation rates in higher plants by low-dose irradiation: Are DNA repair systems involved in the incorporation of exogenous DNA into the plant genome?

Irradiation (X-ray; 5–15 Gy) of protoplasts treated with plasmid-DNA and PEG yielded higher transformation rates in comparison to non-irradiated protoplasts transformed by the same method. This could be demonstrated for four plant species. The irradiation doses used did not affect the total number of colonies regenerated without selection pressure, but resulted in 3–6-fold enhancement of hygromycin- or kanamycin-resistant colonies. Plant regeneration frequencies of transformed colonies derived from irradiated and non-irradiated protoplasts were similar in tobacco as well as in Petunia. Higher integration rates of foreign DNA as a consequence of an increased recombination machinery in irradiated cells may be responsible for the enhancement of the number of stably transformed colonies.     

Plant regeneration from protoplasts of a commercial Asian long-grain javanica rice

A reproducible plant regeneration system has been developed for protoplasts from embryogenic cell suspension cultures of the commercial Asian long-grain javanica rice, Oryza sativa cv. Azucena. Protoplasts were isolated routinely from cell suspensions with yields of 5.5–12.0 × 10⁶ g^-1 fresh weight. A membrane filter nurse-culture method was adopted and was essential to support sustained mitotic division of protoplast-derived cells, leading to cell colony formation. The protoplast plating efficiency was higher when suspension cells of Lolium multiflorum, rather than those of the japonica rice O. sativa L. cv. Taipei 309, were employed as nurse cells. A two-step shoot regeneration procedure, in which protoplast-derived calli were cultured initially on medium semi-solidified with 1% (w/v) agarose followed by culture on medium containing 0.4% (w/v) agarose, induced plant regeneration from protoplast-derived calli. Fifteen percent of protoplast-derived tissues regenerated shoots; tissues not subjected to this treatment failed to develop shoots.     

Development and Characterization of a Nonmorphogenetic Cell Line of Wheat (Triticum aestivum)

This study was conducted to compare characteristics of a wheat (Triticum aestivum L.) cell line to those of the maize (Zea mays L.) black Mexican sweet (BMS) cell line and to compare protoplasts isolated from suspension cells of these cell lines. The wheat cell line was established from immature-embryo derived callus of the experimental line ‘ND7532’ and was conditioned for growth in suspension culture. For both cell lines, measurements of packed cell volume (PCV), fresh weight (FW), and dry weight (DW) were taken at 3 day intervals from suspension cultures. Measurements of FW of calluses cultured from suspension cells of both cell lines were taken at 6 day intervals. The morphogenetic potential of the wheat ND7532 cell line was tested in both callus and suspension cultures using media promoting regeneration and/or organogenesis. Growth rates of ND7532 cells in suspension culture were comparable to those of BMS cells. However, relative growth rates of calluses recovered from ND7532 suspension cells were slower than those of calluses recovered from BMS suspension cells. The ND7532 cell line has very limited morphogenetic potential and has been maintained as rapidly growing callus tissue for 11 years. Yields of protoplasts from suspension cells of the two cell lines were comparable, though ND7532 protoplasts were typically smaller. The wheat cell line has is now designated ND7532-NM (nonmorphogenetic) and is available for cellular and molecular biology research.     

Distribution of alginate oligomers in Catharanthus roseus and Wasabia japonica cells cultured in a medium containing alginate oligomers

Distribution of alginate oligomers (AO) which are endogenous elicitor-like substances, in cultured plant cells were investigated by using AO conjugated with monopotassium 7-amino-1,3-naphthalenedisulfonate (ANDS). When AO-ANDS was added at 0.5 g l^–1 to the Catharanthus roseus cell culture, it adhered to the cells as observed by fluorescence microscopy. Using protoplasts of C. roseus, AO-ANDS was found not only in the cell walls but also in the cell membrane and cytoplasm. When C. roseus was cultivated in a medium containing oligo-galacturonic acids, as an endogenous elicitor, this was also found in the cell wall, cell membrane and cytoplasm of C. roseus cells. Similar results were also obtained with Wasabia japonica cells.     

Flow cytometric analysis of protein content in Taxus protoplasts and single cells as compared to aggregated suspension cultures

Plant suspension cultures are highly aggregated, preventing the direct application of flow cytometry for the study of population dynamics. The utility of single cells to accurately represent aggregated suspension cultures was tested through the analysis of total protein content. Specifically, protein content of two Taxus cuspidata suspension culture lines was studied using the Bradford assay for aggregated suspension cultures, and flow cytometry with fluorescein isothiocyanate staining for protoplasts and single cells. Taxus protein levels were measured at 75–160 mg per gram dry weight via the Bradford assay. Aggregated suspension cultures, protoplasts, and single cells predicted the same trend of protein content over the culture period (21 days). Normalized protein content of isolated single cells was statistically equivalent to aggregated suspensions for both cell lines. However, normalized protein content of isolated protoplasts showed significant differences from aggregated suspensions for one of the two cell lines. Elicitation with methyl jasmonate (MJ) is commonly utilized to increase paclitaxel accumulation in suspension cultures, and therefore the effect of MJ elicitation on protein content in aggregated suspensions, isolated single cells and protoplasts was assessed. Aggregated suspension cultures, protoplasts, and single cells did not show any change in total protein content following elicitation with MJ at 200 M on day 7. This study illustrates the usefulness of flow cytometry for obtaining culture population information and the value of using intact single cells for the study of plant metabolism.     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Soft material-based microculture system having air permeable cover sheet for the protoplast culture of Nicotiana tabacum

In plant cell culture, the delivery of nutrition and gas (mainly oxygen) to the cells is the most important factor for viability. In this paper, we propose a polydimethylsiloxane (PDMS)-based microculture system that is designed to have good aeration. PDMS is known to have excellent air permeability, and through the experimental method, we investigated the relation between the degree of air delivery and the thickness of the PDMS sheet covering the culture chamber. We determined the proper thickness of the cover sheet, and cultured protoplasts of Nicotiana tabacum in a culture chamber covered with a PDMS sheet having thickness of 400 μm. The cells were successfully divided, and lived well inside the culture chamber for 10 days. In addition, protoplasts were cultured inside the culture chambers covered with the cover glass and the PDMS sheet, respectively, and the microcolonies were formed well inside the PDMS covered chamber after 10 days.     

Molecular weight distribution of cellulose in primary cell walls

The distribution pattern of the degree of polymerization (DP) of cellulose present in the cell walls of mesophyll- and suspension-cultured cells of tobacco was compared to that of newly synthesized ¹⁴C-labeled cellulose from regenerating tobacco protoplasts and suspension-cultured cells. The cellulose was nitrated, and, after fractionation according to differences in solubility in acetone/water, the DP pattern of labeled or unlabeled cellulose nitrate was determined by viscosity measurements. A low (DP<500) and high DP-fraction (DP>2500) of cellulose were predominant in the cell walls of protoplasts, suspension — cultured cells, and mesophyll cells. The average DP of the high molecular weight fraction of cellulose in the cell walls of mesophyll was higher (DP4,000) than in protoplasts or suspension — cultured cells (DP 2,500-3,000). In all cell walls tested, minor amounts of cellulose molecules with a broad spectrum of a medium DP were present. Pulse — chase experiments with either protoplasts or suspension —cultured cells showed that a large proportion of the low and medium DP-cellulose are a separate class of structural components of the cellulose network. The results are discussed in relation to the organization of cellulose in the primary cell wall.Abbreviations DP degree of polymerisation - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

Protoplasts of Gelidium robustum (Rhodophyta)

Viable protoplasts were isolated from apices of the agarophyte Gelidium robustum (Gardn.) Hollenb. & Abb. using a combination of commercial cell-wall degrading enzymes and extracellular wall-degrading enzymes isolated from a marine bacterium. The protoplasts were approximately 8–15 µm in diameter, liberated mainly from the surface cell layers and from cells at the distal ends of medullary filaments. The bacterial enzyme alone was not sufficient to liberate significant numbers of protoplasts. Maximum yield was 9 × 10⁵ protoplasts/g tissue (wet wt.). Optimum osmolality occurred between 1750–1950 mOs kg^–1; yield and viability were severely diminished at osmolalities less than 1350 mOs kg^–1. Viability, as determined by flurorescein diacetate staining and Evans Blue exclusion 1 hr after removal from the enzyme solution, was approximately 80–95%. Roughly 80% of the cells did not show Calcofluor fluorescence, while 40% stained positively for the presence of sulfated polysaccharides. Cell wall regeneration was observed with inconsistent reproducibility, and no cell division was observed when the protoplasts were placed in culture medium.Dedicated to the memory of Professor Michael Neushul.     

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.     

Influence of primary callus induction conditions on the establishment of barley cell suspensions yielding regenerable protoplasts

Summary With the aim of the development of a culture method for efficient plant regeneration from barley (Hordeum vulgare L.) protoplasts, we examined several culture conditions for primary calli from immature embryos of cvs. Dissa and Igri, which were used for initiation of cell suspensions. Among the primary callus culture conditions tested, growth condition of donor plants had a great impact on these efficiencies; Igri protoplasts derived from embryos of plants grown in a greenhouse gave rise to albino plants and few green shoots while several cell lines originating from embryos of plants grown in a growth chamber (16h light, 12°C) yielded protoplasts developing into green plants. In contrast, cell suspensions were produced at higher frequencies from calli derived from embryos of greenhouse-grown Dissa plants. In Igri, increased levels of 2,4-dichlorophenoxyaceticacid (2,4-D) significantly reduced the efficiency of cell suspension establishment and plant regeneration from protoplasts was achieved only with suspension cells derived from calli induced at the lowest level (2.5 mg/l), while the effect of the 2,4-D concentration was not clear in Dissa. The developmental stage of immature embryos also affected the efficiency of cell suspension establishment, and the optimal embryo size was determined to be approximately 1mm in diameter. These results demonstrate the importance of callus induction conditions for successful barley protoplast culture.     

Division frequency of pea protoplasts in relation to starch accumulation

Division frequency of alginate-embedded pea (Pisum sativum var. Belman) protoplasts derived from embryonic shoot tips was studied quantitatively by image analysis in relation to starch accumulation and protoplast size. Protoplast divisions were observed from day 4 on and the number of protoplasts undergoing division increased in a stepwise manner to 70% the following days. The starch content increased rapidly during the first 3 days of culture prior to the onset of division and resulted a 4.2-fold increase in the intracellular starch area and a 3.0-fold increase (from 27% to 80%) in the number of protoplasts containing starch. Subsequent periods with rapid increases the number of dividing protoplasts were preceded by further starch accumulation. Dividing protoplasts were 33–60% smaller and contained 8–42% less starch than non-dividing protoplasts. However, calculations showed that, in the dividing protoplasts, the relative area covered by starch was 6–12% higher than in non-dividing protoplasts. These data suggest that starch accumulation precedes division of pea protoplasts.     

The hydraulic conductivity as a criterion for the membrane integrity of protoplasts fused by an electric field pulse

The hydraulic conductivity of the membrane, Lp, of fused plant protoplasts was measured and compared to that for unfused cells, in order to identify possible changes in membrane properties resulting from the fusion process. Fusion was achieved by an electric field pulse which induced breakdown in the membranes of protoplasts in close contact. Close membrane contact was established by dielectrophoresis. In some experiments pronase was added during field application; pronase stabilizes protoplasts against high field pulses and long exposure times to the field. The Lp-values were obtained from the shrinking and swelling kinetics in response to osmotic stress. The Lp-values of fused mesophyll cell protoplasts of Avena sativa L. and of mesophyll and guard cell protoplasts of Vicia faba L. were found to be 1.9±0.9·10^-6, 3.2±2.2·10^-6, and 0.8±0.7·10^-6 cm·bar^-1·s^-1, respectively. Within the limits of error, no changes in the Lp-values of fused protoplasts could be detected in comparison to unfused protoplasts. The Lp-values are in the range of those reported for walled cells of higher plants, as revealed by the pressure probe.Abbreviations GCP guard cell protoplast - Lp hydraulic conductivity - MCP mesophyll cell protoplast     

Isolation and culture of protoplasts from cotyledons of Pinus coulteri D. Don.

A protocol was developed for the isolation and culture of protoplasts from the cotyledons of seedlings of Pinus coulteri D. Don. Incubation of cotyledon pieces in a mixture consisting of cellulase Onozuka R10 2%, Pectolyase Y-23 0.1%, mannitol 10%, CaCl₂ 500 mg/l and other macro and micro-nutrients yielded viable protoplasts. After 24 hours of culture in a complex nutrient medium, the protoplasts regenerated new cell walls and the first divisions were observed within 7–10 days. Small cell colonies were formed within 15–20 days, but these started to accumulate phenolics and no further growth of the colonies was observed.     

Studies on graft unions

Summary De novo formation of cytoplasmic cell connections are studied at the graft interface of 5 day old in vitro heterografts ofVicia faba onHelianthus annuus. Continuous and half plasmodesmata, both branched and unbranched, are described at various stages of development in non-division walls between unlike and like dedifferentiated callus cells. In apical portions of protruding callus cells and in the contact zone between opposing cells extremely thin wall parts with a striking ER/plasmalemma contact are observed. During subsequent thickening of the modified wall parts cytoplasmic strands enclosing constricted ER cisternae are entrapped within the newly deposited wall material. These cytoplasmic strands represent half plasmodesmata which—in case of fusion with corresponding structures of adjoining cells across the loosened wall matrix — form continuous cell connections. Golgi vesicles secreting wall material are involved in the process of forming half and continuous plasmodesmata, thus following the same mechanism of plasmodesmata development as described for isolated protoplasts in cell cultures. The findings suggest the existence of a unifying mechanism of secondary formation of plasmodesmata showing far-reaching similarities with the establishment of primary cell connections.     

Accumulation of acyclic polyols and trehalose as related to growth form and carbohydrate source in the dimorphic fungi Mucor rouxii and Candida albicans

Yeast (Y) and hyphal (H) cells of Mucor rouxii and Candida albicans were cultivated in liquid media containing different carbon nutrient sources (glucose, fructose, ribose), and their free acyclic polyol and trehalose contents determined using capillary gas liquid chromatography (TMS- and OAc-derivatization). Irrespective of growth form and C-source, the fraction of the water-soluble neutral components of the cellular mass of the cultures — highly homogeneous with regard to the respective cell form produced — contained glycerol, ribitol and arabitol, in addition to trehalose. The polyols contributed 0.5–2% to the biomass of M. rouxii and 1.5–6% to that of C. albicans; the values for trehalose ranged from 0.2–11% in the former and 1–3.5% in the latter species. Mucor contained higher amounts of ribitol and arabitol in H cells and larger quantities of trehalose and glycerol in Y cells. In Candida, too, hyphae always exhibited higher ribitol contents, whereas arabitol attained higher levels in yeasts under almost any conditions — regardless of the type of medium (synthetic vs. complex), stage of culture (early vs. late log-phase) and strain used. Glycerol concentration was not correlated with the growth form; trehalose contents tended to be higher in Y cells. Taking into account the facts that C. albicans and certain Mucor species are agents of opportunistic infections and are invasive mainly in the filamentous form, and that the prospective hosts do not accumulate either of these carbohydrates, the possibility is considered of using trehalose- and polyol-metabolizing enzymes as targets for designing antifungal drugs.