Influence of the composition of the fusion medium on the yield of electrofused yeast hybrids

Abstract Electrofusion between cells of yeast strains with different genetic markers in isotonic sorbitol solutions leads to high yields of hybrids when 0.1 mM Ca²⁺ and 0.5 mM Mg²⁺ salts are aded. On average, 1000–2000 hybrids are obtained when electrofusion is performed (in a helical chamber) compared to a yield of about 40–120 in the absence of these bivalent cations. A further increase in yield can be achieved by the addition of 1 mg/ml albumin, which results in up to 4000 hybrids per experimental run. The entire fusion process leads to very reproducible results in the presence of these substances.     

Production of male sterile interspecific somatic hybrids between transgenic N. tabacum (Bar) and N. rotundifolia (NPT II) and their identification by aflp analysis

Summary A protoplast fusion experiment was carried out aiming to obtain somatic hybrid plants of transgenic Nicotiana tabacum (bar) (+) N. rotundifolia (npt II). The bialaphos resistance marker (bar) was introduced into N. tabacum via Agrobacterium tumefaciens using vector pGV1500 carrying the bar gene phosphinothricin acetyltransferase. N. rotundifolia (npt II) was recovered after direct gene transformation of protoplasts by the pGP6 plasmid carrying the npt II gene for neomycin phosphotransferase. Both plasmids possessed 35S CaMV promotors. Hybrid selection was based on dual bialaphos— kanamycin resistance. Amplified fragment length polymorphism (AFLP) analysis of regenerated plants showed the presence of species-specific bands for both parents, which confirmed their hybrid nature. N. tabacum (bar) (+) N. rotundifolia (npt II) hybrids exhibited a great diversity in morphology. Fertile hybrids which possessed N. tabacum or N. rotundifolia morphology were recovered. Flow cytometric analysis revealed that the N. tabacum- and N. rotundifolio-like hybrids had nuclear DNA contents near that of N. tabacum (9.40±0.24pg) or N. rotundifolia (5.29±0.36 pg), respectively, and were highly asymmetric. Other hybrids combined traits from the two species at various levels—N. tabacum habit or branched, similar to N. rotundifolia. Their leaves varied in shape. The flowers of the hybrid plants were of N. tabacum or N. rotundifolia type, or had N. rotundifolia dimensions, pink with N. tabacum corolla or white with curly fused petals. All were self-sterile or male sterile. The nuclear DNA content varied from 8.90±0.30 to 19.57±0.33 pg. The data from the morphological and cytological analysis provided vidence that parental chromosome elimination in the hybrid clones was spontaneous and not species-specific and that diploidization of the tobacco genome might have occurred in some clones during in vitro culture. This reflects the genomic incompatibility between the two species.     

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins

Giant protoplasts of Saccharomyces cerevisiae of 10-35 µm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and “patchability” of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 ± 0.07 µF/cm²) and conductance (23-44 µS/cm²) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.     

Development of baking yeast from Nigerian palm-wine yeasts

Two local strains of Saccharomyces cerevisiae, Nk, and Nk₂, showed leavening activities of 103% and 102% of that of a commercial yeast strain on wheat flour and of 114% and 113% on composite dough with 40% (w/v) maize substitution. Yeast fusion products Nk/Ng, Nk/Nk₁ and Nk/Nk₂ showed activities of 104% to 113% on wheat flour and 111% to 131% on the composite dough. These advantages were maintained in the yeasts' baking properties and organoleptic qualities. The fusion products also showed enhanced osmotic tolerance, as indicated by good growth in 3%, 6% and 10% (w/v) NaCl and 50% (w/v) glucose. Viability of the fusion products, preserved by drying at 30 to 35°C, dropped to between 62% and 72% after 3 months.A.O. Ejiofor was and N. Okafor and E.N. Ugwueze are with the Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, P.M.B. 5025, Awka, Nigeria. A.J. Ejiofor is now with the Abteilung Biovertahrenstechnik, Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany     

Expanding the Symbiodinium (Dinophyceae,Suessiales) Toolkit Through Protoplast Technology

Dinoflagellates within the genus Symbiodinium are photosymbionts of many tropical reef invertebrates, including corals, making them central to the health of coral reefs. Symbiodinium have therefore gained significant research attention, though studies have been constrained by technical limitations. In particular, the generation of viable cells with their cell walls removed (termed protoplasts) has enabled a wide range of experimental techniques for bacteria, fungi, plants, and algae such as ultrastructure studies, virus infection studies, patch clamping, genetic transformation, and protoplast fusion. However, previous studies have struggled to remove the cell walls from armored dinoflagellates, potentially due to the internal placement of their cell walls. Here, we produce the first Symbiodinium protoplasts from three genetically and physiologically distinct strains via incubation with cellulase and osmotic agents. Digestion of the cell walls was verified by a lack of Calcofluor White fluorescence signal and by cell swelling in hypotonic culture medium. Fused protoplasts were also observed, motivating future investigation into intra‐ and inter‐specific somatic hybridization of Symbiodinium. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Generation of Symbiodinium protoplasts opens exciting, new avenues for researching these crucial symbiotic dinoflagellates, including genetic modification.     

A note onStreptomyces No. 6, a chitosanase-producing actinomycete

This communication reports the deposit ofStreptomyces No. 6 in the National Collection of Industrial Bacteria, Aberdeen, AB2 1RY, Scotland, UK (Accession number 13037) and gives some references on its use in lysing cell walls of fungi in the Mucorales leading to protoplast formation. Its use in biotechnological fields is also referred to.     

The effect of external Ca^2＋ and Ca^2＋—channel modulators on red—light—induced swelling of protoplasts of Phaseolus radiatus L.

Red-light-induced swelling of the protoplasts isolated from hypocotyl of etiolated mung bean(Phaseolus radiatusL.)was observed only when Ca^2 ions were present in the medium.The optimal CaCl2 concentration was 250μM,Swlling response declined when Ca^2 was supplied into the medium after red light irradiation.The Ca^2 -chelator EGTA eliminated the red-light-induced swelling and 45Ca^2 accumulation in the protoplasts.In conltrast,A23187,a Ca^2 -ionophore,could mimic the effect of red light in darkness.These results indicate that Ca^2 may play a role in light signal transduction.In addition,swelling response was prevented by TFP and CPZ(both are CaM antagonists),implying the involvement of CaM in red-light-induced and Ca^2 -dependent protoplast swelling.     

DEVELOPMENT OF PROTOPLASTS FROM HOLDFASTS AND VEGETATIVE THALLI OF MONOSTROMA LATISSIMUM (CHLOROPHYTA, MONOSTROMATACAE) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate the protoplasts of the green alga Monostroma latissimum Wittrock and subsequently induce them to form algal filaments to act as an algal "seed" stock. Protoplasts of the alga were isolated enzymatically with 4% cellulase Onozuka R-10 and 2% Macerozyme R-10. The highest number of protoplasts was obtained on a 50-rpm shaker with 1.2 M of sorbitol after 6 h of incubation, with a yield of 9 × 106 protoplasts·g−1 of fresh thallus (including holdfast). Protoplasts from both holdfasts and erect thalli usually began to form new cell walls within 5 h after isolation and began to divide from day 6 to day 9 in PES medium; cell clusters, filaments, and/or tubular thalli were formed from day 14 to day 18. For algae collected in March, about 60% of protoplasts isolated from vegetative thalli regenerated to form tubular thalli, and about 45% of protoplasts isolated from holdfasts regenerated to form filaments. However, for algae collected in May, about 1% of protoplasts isolated from vegetative thalli developed directly to form tubular thalli, and 59% of protoplasts regenerated to form cell clusters without the ability to differentiate, whereas protoplasts isolated from holdfasts failed to develop. Regenerated filaments were kept in an incubator for more than 3 years at 24° C under the low irradiance of 66μmol photons·m−2·s−1. After this time, they retained the ability to develop to form tubular thalli under irradiance of 166 and 300 μmol photons·m−2·s−1 at 18°–30° C. Subsequently, these tubular thalli can develop to form leafy thalli after being cultivated at high irradiance of 300 μmol photons·m−2·s−1 and at 18°–22° C. Therefore, the filaments could serve as"seed" stock for algal mass culture.     

雷公藤悬浮细胞原生质体的制备及瞬时转化体系的建立

为探索药用植物雷公藤(Tripterygium wilfordii)悬浮细胞原生质体提取的最优条件,并建立雷公藤原生质体瞬时转化体系,以雷公藤悬浮细胞为材料,对酶解液配比、酶解时间、甘露醇浓度及处理转速进行考察。用PEG介导的瞬时转化法将外源基因转化到雷公藤原生质体中。结果表明,以雷公藤悬浮细胞为材料提取原生质体的最佳条件是酶液配比为2.0%纤维素酶+0.5%果胶酶+0.5%离析酶,甘露醇浓度为0.6 mol·L–1,酶解10小时,处理转速为67×g;用PEG介导法将含有编码GFP的植物表达载体转化雷公藤悬浮细胞原生质体,激光共聚焦扫描显微镜下细胞显示绿色荧光。通过实验筛选得到雷公藤悬浮细胞原生质体的最佳提取条件,建立了雷公藤悬浮细胞原生质体的瞬时转化体系,为进一步开展雷公藤功能基因及合成生物学研究奠定了基础。     

Modified culture conditions for increased viability and cell wall synthesis in grapevine (Vitis vinifera L. cv. Sultanina) leaf protoplasts

Studies were undertaken to determine optimum conditions for grapevine protoplast culture. Highest viability was obtained at 25° in the dark, and at initial pH values of 5.2 to 7.2, in the presence of 1 or 2% (w/v) sucrose and 0.6 or 0.7 M mannitol or osmolality between 729 and 930 mOsmol kg^-1. Optimum plant growth regulators were 6-BAP/NAA at 2.3×10^-6/10–15×10^-6 M, respectively. ³⁵S-Methionine incorporation into de novo synthesized proteins for protoplasts of Nicotiana tabacum cv Xanthi (a readily regenerating species) and for the recalcitrant grapevine was studied. In tobacco the rate of protein synthesis showed 3 maxima which coincided with the distinct stages of naked protoplasts, cell wall reconstitution and induction of cell division. In grapevine protoplasts the first peak exceeded the second one, whereas no peak corresponding to the induction of cell division was observed.