Method For Isolation Of Protoplasts Research Articles

Preparation of leaf protoplasts from Populus (Populus × xiaohei T. S. Hwang et Liang) and establishment of transient expression system

Poplar, as a typical woody plant, is an ideal raw material for the production of lignocellulose biofuel. However, the longer life cycle is not conducive to the rapid identification of poplar genes. At present, protoplasts have been used for gene function identification and high-throughput analysis in many model plants. In this paper, a simplified and efficient protoplast isolation and transient expression system of Populus (Populus × xiaohei T. S. Hwang et Liang) is described. Firstly, we proposed an efficient enzyme hydrolysis method for isolating protoplasts from leaves of Populus × xiaohei. Secondly, we optimized the conditions of protoplast transformation mediated by PEG, and established an efficient transient expression system of protoplasts of Populus × xiaohei. Finally, the subcellular localization of three identified Dof transcription factors (PnDof19, PnDof20 and PnDof30) was also observed in the nucleus by using this scheme, which proved that the method was feasible. In general, this efficient method of protoplast isolation and transformation can be used for the study of protein subcellular localization and can be applied to other fields of molecular biology, such as protein interaction, gene activation and so on.

Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera

BackgroundCamellia oleifera (C. oleifera) is a woody edible oil crop of great economic importance. Because of the lack of modern biotechnology research, C. oleifera faces huge challenges in both breeding and basic research. The protoplast and transient transformation system plays an important role in biological breeding, plant regeneration and somatic cell fusion. The objective of this present study was to develop a highly efficient protocol for isolating and purifying mesophyll protoplasts and transient transformation of C. oleifera. Several critical factors for mesophyll protoplast isolation from C. oleifera, including starting material (leaf age), pretreatment, enzymatic treatment (type of enzyme, concentration and digestion time), osmotic pressure and purification were optimized. Then the factors affecting the transient transformation rate of mesophyll protoplasts such as PEG molecular weights, PEG4000 concentration, plasmid concentration and incubation time were explored.ResultsThe in vitro grown seedlings of C. oleifera ‘Huashuo’ were treated in the dark for 24 h, then the 1st to 2nd true leaves were picked and vacuumed at − 0.07 MPa for 20 min. The maximum yield (3.5 × 107/g·FW) and viability (90.9%) of protoplast were reached when the 1st to 2nd true leaves were digested in the enzymatic solution containing1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10 and 0.25% (w/v) Snailase and 0.4 M mannitol for 10 h. Moreover, the protoplast isolation method was also applicable to the other two cultivars, the protoplast yield for ‘TXP14’ and ‘DP47’ was 1.1 × 107/g·FW and 2.6 × 107/g·FW, the protoplast viability for ‘TXP14’ and ‘DP47’ was 90.0% and 88.2%. The purification effect was the best when using W buffer as a cleaning agent by centrifugal precipitation. The maximum transfection efficiency (70.6%) was obtained with the incubation of the protoplasts with 15 µg plasmid and 40% PEG4000 for 20 min.ConclusionIn summary, a simple and efficient system for isolation and transient transformation of C. oleifera mesophyll protoplast is proposed, which is of great significance in various aspects of C. oleifera research, including the study of somatic cell fusion, genome editing, protein function, signal transduction, transcriptional regulation and multi-omics analyses.

Single-cell transcriptome atlas reveals developmental trajectories and a novel metabolic pathway of catechin esters in tea leaves.

The tea plant is an economically important woody beverage crop. The unique taste of tea is evoked by certain metabolites, especially catechin esters, whereas their precise formation mechanism in different cell types remains unclear. Here, a fast protoplast isolation method was established and the transcriptional profiles of 16 977 single cells from 1st and 3rd leaves were investigated. We first identified 79 marker genes based on six isolated tissues and constructed a transcriptome atlas, mapped developmental trajectories and further delineated the distribution of different cell types during leaf differentiation and genes associated with cell fate transformation. Interestingly, eight differently expressed genes were found to co-exist at four branch points. Genes involved in the biosynthesis of certain metabolites showed cell- and development-specific characteristics. An unexpected catechin ester glycosyltransferase was characterized for the first time in plants by a gene co-expression network in mesophyll cells. Thus, the first single-cell transcriptional landscape in woody crop leave was reported and a novel metabolism pathway of catechin esters in plants was discovered.

Influence of change in ROS production in plant cells on resistance of agricultural plants to oxidative stress

The paper considers theoretical data on the mechanisms of the formation of reactive oxygen species (ROS) and the transduction of stress signals in plant cells. The negative effects of ROS with excessive generation are described. A method of protoplast isolation and a fluorescent microscopy were selected for the experiments. Also, in this work, a comparative analysis of the production of ROS during hypoxia and reaeration for agricultural plants that are differently resistant to the described stress is carried out. Based on the results of the experimental part of the work, conclusions were drawn and the practical application of this study was described.

Development of protoplast isolation method in some cultivars of Ficus carica as crucial stage for implementation of single-cell RNA-seq technology in plant investigation

Development of protoplast isolation method in some cultivars of <i>Ficus carica</i> as crucial stage for implementation of single-cell RNA-seq technology in plant investigation

Optimized protoplast isolation and establishment of transient gene expression system for the Antarctic flowering plant Colobanthus quitensis (Kunth) Bartl.

Colobanthus quitensis is one of two terrestrial plants that grow in the maritime Antarctic. Despite its important ecological niche in extreme environments, the molecular mechanisms of its adaptation and tolerance have not been elucidated due to difficulties with genetic or transgenic approaches. For this reason, in many other plant species mesophyll protoplasts as a versatile cell-based system have been developed and used to analyze the biological functions of genes of interest. Here we report an optimized method of protoplast isolation from C. quitensis leaves. The main parameters evaluated to reach the highest protoplast yield were the use of a cell wall-degrading enzyme, an osmotic stabilizer, and different pH conditions. Moreover, transient expression and subcellular localization of proteins were validated by an immunoblot assay and a confocal microscopy, respectively, using C. quitensis protoplasts. Therefore, these results suggest that protoplasts can provide a useful cell-based system to facilitate the molecular, biochemical, and cellular characterizations of C. quitensis genes. C. quitensis protoplasts can provide a physiologically relevant cell system to facilitate the molecular, biochemical, and cellular characterization of C. quitensis genes.

Efficient isolation of protoplasts from freesia callus and its application in transient expression assays

Freesia hybrida is one of the most famous cut flower variety domesticated from wild species in Freesia genus, distinguished for its colorful flower colors and sweet scents. It has potential to be a model plant to unravel the molecular basis of plant specialized metabolites biosynthesis in family Iridaceae, which are crucial to genetically modify ornamental traits such as flower color and flower scents. However, the lengthy life cycle propagated from tissue culture and extremely low transformation rate severely decelerate functional characterization of genes through transgenic method in this iridaceous plant. Using calluses induced from young flower buds from one popular cultivar of Freesia hybrida, ‘Red River®’, an efficient protoplast isolation method was established and optimized, which was further employed in the gene transient expression system. Results demonstrated that the newly explored transient system was versatile and could be applied in numerous aspects, such as protein immunoblotting, enzyme activity, protein subcellular localization, protein–protein interaction, protein–DNA interaction and regulatory gene transregulation property assays. Moreover, calluses induced from another cultivar of Freesia hybrida, ‘Ambiance’ could also be used to isolate protoplasts for gene transient transfection analysis, indicating the wide applicability of this system. Results in this study provided new methods for the elucidation of gene function in Freesia in vivo, which might also be inspirational in other species with long life cycles or hardly to be transformed, such as plants in family Iridaceae. Freesia protoplast isolation and transfection methods were established and illustrated the utilization in protein immunoblotting, enzyme activity, subcellular localization, protein-protein or protein-DNA interaction, and investigation of transactivation capacities of regulators.

Simple protoplast isolation system for gene expression and protein interaction studies in pineapple (Ananas comosus L.)

BackgroundAn efficient transformation protocol is a primary requisite to study and utilize the genetic potential of any plant species. A quick transformation system is also crucial for the functional analysis of genes along with the study of proteins and their interactions in vivo. Presently, however, quick and effective transformation systems are still lacking for many plant species including pineapple. This has limited the full exploration of the genetic repository of pineapple as well as the study of its genes, protein localization and protein interactions.ResultsTo address the above limitations, we have developed an efficient system for protoplast isolation and subcellular localization of desired proteins using pineapple plants derived from tissue culture. A cocktail of 1.5% (W/V) Cellulase R-10 and 0.5% (W/V) Macerozyme R-10 resulted in 51% viable protoplasts with 3 h digestion. Compared to previously reported protocols, our protoplast isolation method is markedly faster (saving 4.5 h), requires only a small quantity of tissue sample (1 g of leaves) and has high yield (6.5 × 105). The quality of the isolated protoplasts was verified using organelle localization in protoplasts with different organelle markers. Additionally, colocalization analysis of two pineapple Mg2+ transporter genes in pineapple protoplasts was consistent with the results in a tobacco transient expression system, confirming that the protoplast isolation method can be used to study subcellular localization. Further findings showed that the system is also suitable for protein–protein interaction studies.ConclusionBased on our findings, the presently described method is an efficient and effective strategy for pineapple protoplast isolation and transformation; it is convenient and time saving and provides a greater platform for transformation studies.

A Universal Method of Protoplast Isolation and Transient Expression in Plants

A Universal Method of Protoplast Isolation and Transient Expression in Plants

ПОЛУЧЕНИЕ ПРОТОПЛАСТОВ ОБЛЕПИХИ ( HIPPOPHAE RHAMNOIDES L.)

The aim of research was to study the effect of a method of protoplast isolation on Hippophae rhamnoides cell viability. The main objectives were the release of protoplasts H. rhamnoides from callus tissue by a combination of mechanical and enzymatic effects, as well as evaluation of the general state of isolated cells after isolation and purification. The objects of studies were mesophyll cells and leaf mesophyll explants introduced in culture in vitro. It is assumed that considerable damage of cells in the process of mechanical-enzymatic treatment is not only a result of mechanical impact, but also an effect of the release of metabolic products (polyphenolic components) of cells and their decay. Also protoplasts are dedifferentiated and simplify the process of differentiation in various breeding and biotechnological experiments. As a result of the work, an optimized method of H. rhamnoides protoplast isolation with decreased levels of cell damage is set up. It is revealed that important factors for high yield of viable protoplasts are: proper concentration of D-mannite as an osmoticum; proper concentration of cellulase R10 and macerozime R10; use of sodium tiosulfate as an antioxidante; proper time of incubation of leaf explants in medium with enzymes.

Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays.

Background Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function.Methodology/Principal FindingsWe optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events.Conclusions/SignificanceThis study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.

Protoplast preparation from Laminaria japonica with recombinant alginate lyase and cellulase.

Functional recombinant abalone alginate lyase (rHdAly) and β-1,4-endoglucanase (rHdEG66) were expressed as secreted proteins with baculoviral expression systems. The specific activity of each recombinant enzyme, 2,490 and 18.2 U/mg for rHdAly and rHdEG66, respectively, was comparable to its native form at 30°C. Purified rHdAly and rHdEG66 showed the highest specific activity both at 35°C and optimum pH 8.7 and 5.9, respectively. These properties were also comparable to those of the native enzymes. Protoplast isolation was attempted from Laminaria japonica using both rHdAly and rHdEG66. When L. japonica blades were incubated in artificial seawater containing rHdAly and rHdEG66, very low numbers of protoplasts (<1 × 10(3) protoplasts/g fresh weight) resulted. However, using blades pretreated with proteinase K, the protoplast was increased up to 5 × 10(6) protoplasts/g fresh weight. Since the average diameter of isolated protoplasts was 11.6 μm, these cells were mostly derived from the epidermal layer rather than the cortical layer. Our results suggest that at least three enzymes, alginate lyase, cellulase, and protease, are essential for effective protoplast isolation from L. japonica. The protoplast isolation method in this study is more useful than earlier methods because it preferentially yielded protoplasts of the epidermal layer, which are known to be able to be regenerated.

Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method

BackgroundProtoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using Arabidopsis mesophyll protoplasts (TEAMP), is currently commonly used for studies of subcellular protein localization, promoter activity, and in vivo protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments.ResultsIn this report, we present a new procedure that we call the Tape-Arabidopsis Sandwich. This is a simple and fast mesophyll protoplast isolation method. Two kinds of tape (Time tape adhered to the upper epidermis and 3 M Magic tape to the lower epidermis) are used to make a "Tape-Arabidopsis Sandwich". The Time tape supports the top side of the leaf during manipulation, while tearing off the 3 M Magic tape allows easy removal of the lower epidermal layer and exposes mesophyll cells to cell wall digesting enzymes when the leaf is later incubated in an enzyme solution. The protoplasts released into solution are collected and washed for further use. For TEAMP, plasmids carrying a gene expression cassette for a fluorescent protein can be successfully delivered into protoplasts isolated from mature leaves grown under optimal conditions. Alternatively, these protoplasts may be used for bimolecular fluorescence complementation (BiFC) to investigate protein-protein interactions in vivo, or for Western blot analysis. A significant advantage of this protocol over the current method is that it allows the generation of protoplasts in less than 1 hr, and allows TEAMP transfection to be carried out within 2 hr.ConclusionThe protoplasts generated by this new Tape-Arabidopsis Sandwich method are suitable for the same range of research applications as those that use the current method, but require less operator skill, equipment and time.

Synergism in replication of cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) RNA in orchid protoplasts.

An in vitro orchid protoplast isolation method to study replication kinetics of CymMV and ORSV was developed. This method allows the isolation of viable and raphid-free petal protoplasts from an orchid hybrid, Dendrobium Sonia (Dendrobium Caesar x Dendrobium Tomie Drake). The optimum field strength for both viral RNA to achieve good efficiency of electroporation was 750 V/cm and the optimum viral RNA concentration required was 1 microgram and 4 micrograms per 2 x 10(6) protoplasts for CymMV and ORSV, respectively. Autoradiographs of Northern blots depicting the viral genomic and subgenomic RNA in the extracts, referred to as the "Replication Footprint Profiles" (RFP) of specific CymMV/ORSV virus were prepared at different time intervals. Viral RNA synthesis reached a maximum at 18 h for CymMV and 24 h for ORSV. When CymMV and ORSV viral RNA were electroporated into the protoplasts simultaneously, detection signals of both the positive and negative strand viral RNA increased as compared to the singly infected protoplasts. Thus, synergism in replication of CymMV and ORSV was observed in orchid protoplasts.

Transcription efficiency and hormone-responsiveness of ?-amylase gene promoters in an improved transient expression system from barley aleurone protoplasts

An improved method for isolation of protoplasts from barley aleurone layers was developed which utilizes KC1 instead of mannitol as the osmoticum. Protoplasts prepared by this method were shown to be hormone-responsive even after polyethyleneglycol-mediated DNA uptake. Both yield and viability were increased by about 50% and 6%, respectively, compared to the method we had used previously. These protoplasts were used in a transient expression system to compare the relative transcription rates of promoters from barley genes encoding high-pI and low-pI α-amylases. The protoplasts were used also to identify the hormone-responsive elements and other enhancer elements in barley α-amylase gene promoters which have not been studied previously. Promoter fragments from two high-pI and one low-pI α-amylase genomic clones and deletions derived from them were fused to a promoterless vector containing the coding region of bacterial chloramphenicol acetyltransferase (CAT) gene. Deletion studies of promoters from two barley high-pI α-amylase genes, gRAmy152 and gRAmy56, indicate that the sequences CTTTTG, TAACAAA, and TATCCAC which are conserved in several α-amylase genes must act in concert to confer hormone-responsiveness to these two promoters as has been found for other α-amylase genes. Removal of any one of these three regions causes a severe reduction in overall level of expression and GA-responsiveness. Additional sequences present both upstream and downstream of these three conserved elements also enhance hormone-responsiveness of the reporter genes. For the low-pI α-amylase gene gKAmy 155 promoter, the presence of ACTTGACCAT-CACC (Opaque 2S-like element), a pyrimidine-rich sequence, and TAACAGA alone is not adequate for GA-induced gene expression as has been observed previously for another barley low-pI α-amylase gene Amy32b. They have to work cooperatively with other element(s) located between positions −256 and −197 of the gKAmy 155 promoter to compose an effective GA response complex. ABA- and GA-responsive elements appear to be coincident or close to each other in both high- and low-pI α-amylase genes.

Isolation of Protoplasts from the Coccal Green Alga Eremosphaera viridis De Bary for Patch‐clamp Measurements

AbstractA method for enzymatic isolation of protoplasts from the unicellular green alga Eremosphaera viridis for patch‐clamp measurements is described. Viable protoplasts with “patch‐clean” plasma membranes could only be isolated when combining high enzyme concentrations and long incubation times.In whole‐cell recordings the protoplasts exhibited electrical properties similar to those measured in intact cells. Taken together with the protoplasts' ability for rapid deplasmolysis after transfer into hypotonic solution, this indicates the viability of the isolated protoplasts.

CMV-RESISTANT CULTIVAR SELECTION BY ENZYME-LIKED IMMUNOSORBENT ASSAY AND PROTOPLAST ISOLATION METHOD IN CUCUMBER.

This experiment was carried out to select resistant cultivar to CMV in cucumber using Elisa-test and protoplast isolation method. Twenty domestic cultivars or lines and 8 European cultivars were ested for resistance by Elisa test. Among the domestic cultivars, DADAKI group was found to be susceptible and CHEONGJANG group resistant. Among all the cultivars and lines tested, a European cultivar, DALIBOR and Janghyungheukjinju Korean line were found to be highly resistant. When comparing for the protoplast yield depending upon the positions of seedlings (cotyledon, young leaf and hypocotyl), the highest protoplast yield could be obtained from cotyledons in macerozyme 0.5% + cellulose 2.0%. Protoplast yields in susceptible cultivars were higher than those from resistant cultivars. Differences in cell wall thickness between susceptible and resistant cultivars were observed.

Isolation of protoplasts from developing barley endosperm: a tool for transient expression studies

We have developed a method for the routine isolation of protoplasts from developing starchy endosperm of barley (Hordeum vulgare L.). Preplasmolysis of the intact endosperms, a low concentration of hydrolytic enzymes and gravity sedimentation before any centrifugation step, were crucial factors for a good preparation. Best yields were obtained early after pollination (8-13 days) or with mutants with low starch content. Transient expression of a reporter gene under the control of the 35S promoter, after polyethyleneglycol transfection of endosperm protoplasts, was of the same order as that found in coleoptile derived protoplasts. No significant difference in expression was found for a given tissue between cv. Bomi and its mutant Risø 1508.

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 Non-Enzymatic Method for the Isolation of Protoplasts From Callus of Saintpaulia ionantha (African Violet)

Summary The in vitro culture of Saintpaulia petiole cross-sections on a solid medium containing relatively high levels of auxin, produced callus cells with thin walls. Slight teasing of the callus tissue with dissecting needles released high yields of small densely cytoplasmic protoplasts which were stable in the osmotic pressure of standard tissue culture media. This resulting non-enzymatic protoplast isolation method is dependent on the auxin 2,4-dichlorophenoxyacetic acid which partially inhibits cell wall synthesis without preventing cytokinesis in proliferating Saintpaulia callus stock tissue, and suggests a basic approach which may be applicable to a wide range of plant species. These non-enzymatically isolated Saintpaulia protoplasts provide a suitable experimental system for various physiological studies including assessment of the toxicity of cell wall degrading enzymes.