A simple method for large-scale electrofusion and culture of plant protoplasts

A simple method is described for the electrofusion of plant protoplasts. Protoplasts were aggregated in a radio-frequency field (10 V RMS, 0.5 MHz) for 15-30 s with an inter-electrode distance of J mm. They were then fused with a 300-V DC pulse. The protoplasts were able to divide after this treatment. A trans-ferrable electrode permitted electrofusion of l-ml volumes of culture in standard tissue-culture dishes in about 20 s.     

A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO₄), and a protectant (CaCl₂ 2H₂O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.     

A simple method for isolation,liquid culture,transformation and regeneration of Arabidopsis thaliana protoplasts

Summary An efficient technique was developed for the isolation, culture, transformation and regeneration of protoplasts derived from auxin conditioned Arabidopsis root cultures. On an average 30% of root protoplasts underwent cell division in liquid culture and formed somatic embryolike structures which regenerated to plants without embedding in Ca²⁺-alginate. The protoplast protocol was applicable to different landraces of Arabidopsis thaliana (L.) Heynh., such as RLD, Columbia or C24. PEG-mediated DNA uptake into protoplasts using different uidA reporter gene constructs yielded transient gene expression in over 25% of treated cells indicating that root-derived protoplasts are suitable recipients for transformation.Abbreviations BA 6-benzylaminopurine - 2,4D 2,4dichlorophenoxyacetic acid - IAA indole-3-acetic acid - ISA indole-3buryric acid - IPAR 6-(,-dimethylallylamino)purine riboside - NAA naphthaleneacetic acid - uidA ß-glucuronidase gene - GUS ß-glucuronidase enzyme - CaMV Cauliflower Mosaic Virus - nos nopaline synthase - MES 2[N-morpholino]ethane-sulfonicacid - PEG polyethylene glycol - X-gluc 5bromo-4-chloro-3-indolyl glucuronide - MUG 4-methyl umbelliferyl glucuronide - MU 4-methylumbelliferone     

An alginate-layer technique for culture of Brassica oleracea L. protoplasts

Ten accessions belonging to the Brassica oleracea subspecies alba and rubra, and to B. oleracea var. sabauda were used in this study. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants. The influence of selected factors on the yield, viability, and mitotic activity of protoplasts immobilized in calcium alginate layers was investigated. The efficiency of protoplast isolation from hypocotyls was lower (0.7 ± 0.1 × 10⁶ ml⁻¹) than for protoplasts isolated from leaf mesophyll tissue (2 ± 0.1 × 10⁶ ml⁻¹). High (70–90%) viabilities of immobilized protoplasts were recorded, independent of the explant sources. The highest proportion of protoplasts undergoing divisions was noted for cv. Reball F1, both from mesophyll (29.8 ± 2.2%) and hypocotyl (17.5 ± 0.3%) tissues. Developed colonies of callus tissue were subjected to regeneration and as a result plants from six accessions were obtained.     

A rapid, simple method for nuclei isolation from plant protoplasts

A rapid, simple method for nuclei isolation and purification from soybean (Glycine max L. Merr.) protoplasts is described. The isolated nuclei exhibited active amino acid incorporation and RNA synthesis, but DNA synthesis was not detectable. Analysis by CsCl density gradient centrifugation showed that DNA isolated from nuclei had a single band, while DNA isolated from protoplasts consisted of three bands comprised of nuclear DNA, mitochondrial DNA, and chloroplast DNA.     

A simple,versatile feeder layer system for Brassica oleracea protoplast culture

Protoplasts from cauliflower (Brassica oleracea ssp. botrytis) and broccoli (ssp. italica) leaves and hypocotyls were successfully cultured on membrane filters over a feeder layer of cells from a B. campestris suspension culture. Cells from rice, tomato and tobacco suspensions were not as effective as the B. campestris cells. Plants were recovered from protoplasts of previously recalcitrant Brassica genotypes. Protoplasts cultured in low numbers (10–100) on the feeder layer divided and formed colonies capable of plant regeneration, as did fused protoplasts.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - PCV packed cell volume     

A simple method for the isolation of intact mesophyll protoplasts from cereal plants

Intact mesophyll protoplasts from cereal plants were easilyprepared by incubating leaves with the abaxial epidermis peeledoff at 20–25?C for 2–3 hr in 0.6 M mannitol containing1% cellulase at pH 5.6. From one gram (fresh weight) of leaves1.5–6?10⁶ protoplasts, more than 90% of which were morphologicallyintact, could be obtained. Protoplasts isolated from wheat,oat, corn and barley were efficiently infected with brome mosaicvirus (BMV), and supported viral multiplication. (Received June 21, 1977; )     

A simple plant and tissue culture growth chamber for the regeneration of tobacco mesophyll protoplasts

The isolation and regeneration of tobacco mesophyll protoplasts from fully developed leaves, an important methodological step in plant genetic engineering as well as in plant cell biology and physiology, has been proven unreliable to the extent that it has become a significant setback to basic research. This unfortunate situation is primarily due to the suboptimal physiological state of greenhouse-grown protoplast donor plants. A technically simple and inexpensive method, based on the utilization of commercial Phototron units, is described for the production of suitable tobacco donor plants. Furthermore, a modified version of such a culture unit can be used to regenerate plants from protoplast-derived calli.     

A method for the microinjection and culture of protoplasts at very low densities

A method has been developed which allows the recovery of calli from a high proportion of individual, injected, mesophyll protoplasts of Nicotiana tabacum c.v. Xanthi. A small drop of low melting point agarose is used both to hold protoplasts during microinjection and for their subsequent culture in feeder dishes. The feeder dishes consist of "beads" of protoplasts at a high density set in agarose to "feed" the infected protoplasts across a liquid medium.The method has been used successfully both with normal protoplasts and protoplasts from which the vacuole has been removed.Abbreviations NT medium Nagata-Takebe medium (Nagata and Takebe, 1971) - MS medium Murashige-Skoog medium (Murashige and Skoog, 1962) - NAA 1-Naphthaleneacetic acid - BAP 6-Benzylaminopurine - LMP agarose low melting point agarose     

A simple method for estimating intactness of spinach leaf protoplasts by glycolate oxidase assay

A method was developed for the quantitative analysis of intactness of spinach leaf protoplasts using glycolate oxidase activity as an index. Since glycolate does not penetrate into protoplasts at neutral pH, the increase of O₂ consumption by the addition of glycolate to protoplast suspension was due to the glycolate oxidase activity released from damaged protoplasts. The proportion of damaged protoplasts in the whole preparation was calculated from the ratio of released and total glycolate oxidase activity. Freshly prepared spinach leaf protoplasts were found to be 80 to 90% intact as estimated by the method. The effect of osmolarity on the respiratory activities of spinach leaf protoplasts was also examined by applying the same principle.     

A simple method for hyaluronic acid quantification in culture broth

The carbazole assay has been used for determination of the percentage of hyaluronic acid in biological fluids. However, it is difficult to measure the concentration of hyaluronic acid in culture broth because glucose and polysaccharides remaining after cultures can react with sulfuric acid and carbazole. The glucose and polysaccharide remnants must be completely removed in order to get the correct value for hyaluronic acid. The turbidity assay, another method for estimating the concentration of hyaluronic acid, is based on the formation of insoluble complexes between hyaluronic acid and cetyltrimethylammonium bromide. This method is very easy and fast compared with the carbazole assay. Because concentrations of hyaluronic acid measured by the turbidity assay were ranged around 100% of those measured by the carbazole assay, the content of hyaluronic acid in culture broth can be determined by the turbidity assay. The turbidity method also has the advantage of being safer than the carbazole assay.     

A simple method for hemoglobin measurement in cell culture system.

A simple method of hemoglobin analysis in a cell culture system is described. Hemoglobins synthesized in cell cultures are labeled with radioactive amino acids. The cell extract containing radiolabeled hemoglobin is mixed with A, F, S, C, hemoglobin markers and separated by cellulose acetate electrophoresis. Individual bands of hemoglobin are cut from the gel and analyzed for radioactivity. This method is especially useful for determination of newly synthesized minute amount of hemoglobin in cell extracts that are difficult to visualize by staining procedure.     

A simple endpoint dilution method for evaluating serum used for cell culture

A simple endpoint dilution method for evaluating foetal calf serum quality is described. The test uses a series of doubling dilutions of cells on microtitre trays with the test sera added to replicate dilution series. After five to six days of incubation the cells are stained with crystal violet and the end points read macroscopically. The cell growth-promoting property of serum may be expressed as a reciprocal of the cell dilution resulting in an approximately 50% coverage of cells.     

Increased totipotency of protoplasts from Brassica oleracea plants previously regenerated in tissue culture

Factors affecting the division of cells derived from leaf and cotyledon protoplasts from Brassica oleracea L. var. italica (Green Comet hybrid broccoli) were examined to optimize conditions for plant regeneration and to determine whether there was a genetic basis for improved regeneration from protoplasts derived from plants previously regenerated from tissue cultures [15]. When leaf protoplasts from different plants grown from hybrid seed were isolated and cultured simultaneously, division efficiencies of 1–95% were obtained. Cells from some plants showed high division efficiencies in consecutive experiments while cells from other plants had consistently low division rates. More plants from hybrid seed gave high division efficiencies when cotyledon protoplasts were used. However, cotyledon or leaf protoplasts from selfed progeny of regenerated plants produced more vigorous calli and more shoots than protoplasts from hybrid seed. These results suggest that there may be a genetic component to the increased totipotency of Brassica oleracea protoplasts.     

A method for isolation of protoplasts from dermatophytes

A method has been developed to isolate protoplasts from dermatophytes using Novozym 234. A simple technique of flotation in MgSO4 has been adapted to separate protoplasts from incubation mixture. Electron microscopic studies confirmed the absence of cell wall material on these protoplasts. The recovery of DNA from protoplasts was higher than from mycelia.     

A simple culture method for epithelial stem cells derived from human hair follicle

The challenge arises among researchers when hair follicle stem cells (HFSCs) derived from a human hair follicle remain poorly expanded in defined culture medium. In this study, we isolated the HFSCs and they became confluent after 10 days of cultivation. Comparing the viability of HFSCs cultured in defined keratinocytes serum free medium (KSFM) in a coated plate and CnT07 medium in an uncoated plate, the number of HFSCs cultured in CnT07 was significantly higher at days 2, 4, 6 and 8 (P=0.004). The population doubling time of HFSCs was 21.48±0.44 hours in non-coated plates with CnT07 and 30.73±0.75 hours in coated plates with KSFM. Our primary HFSC cultures were positive for CD200 and K15 with brownish color. Flow cytometry analysis showed that the percentage of HFSCs expressing CD200 and K15 were 65.20±3.16 and 72.07±6.62 respectively. After reaching 100% confluence, the HFSCs were differentiated into an epidermal layer in vitro using CnT02-3D defined media. HFSCs were differentiated into an epidermal layer after 2 weeks of induction. Involucrin- and K6-positive cells were detected in the differentiated epidermal layer. This method is a simple technique for HFSC isolation and has a lower cost of processing and labor, and it represents a promising tool for skin tissue engineering.