Use Of Protoplasts Research Articles

Colour change in potato (Solanum tuberosum L.) tubers by disruption of the anthocyanin pathway via ribonucleoprotein complex delivery of the CRISPR/Cas9 system

Potato is an important part of the traditional Norwegian diet, and the crop faces several challenges with respect to pests and diseases, as well as the increasingly challenging changes in climate. Genome editing may provide tools to improve the resilience of Norwegian potato cultivars to new climate challenges. We have altered the skin colour of two potato cultivars, ‘Desirée’ and ‘Nansen’ from red to yellow, as a proof-of-concept for the use of CRISPR/Cas9 in a Norwegian cultivar. Our method has involved the use of protoplasts and we have grown the regenerants for three successive clonal tuber generations to evaluate the stability of the edited plants over time and under varying temperature conditions in contained rooms in a greenhouse. We found that the protoplast method is well suited to achieving CRISPR/Cas9 applications. The results show that the yellow skin is consistent over the three generations of tuber propagation. We found some suspected somaclonal variation in the protoplast regenerants. Some of the variation which we observed under high temperatures (up to nearly 40ºC) during the second growth cycle, disappeared when cultivated under lower temperatures in the third cultivation cycle.

Preparing Arabidopsis thaliana root protoplasts for cryo electron tomography.

The use of protoplasts in plant biology has become a convenient tool for the application of transient gene expression. This model system has allowed the study of plant responses to biotic and abiotic stresses, protein location and trafficking, cell wall dynamics, and single-cell transcriptomics, among others. Although well-established protocols for isolating protoplasts from different plant tissues are available, they have never been used for studying plant cells using cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET). Here we describe a workflow to prepare root protoplasts from Arabidopsis thaliana plants for cryo-ET. The process includes protoplast isolation and vitrification on EM grids, and cryo-focused ion beam milling (cryo-FIB), with the aim of tilt series acquisition. The whole workflow, from growing the plants to the acquisition of the tilt series, may take a few months. Our protocol provides a novel application to use plant protoplasts as a tool for cryo-ET.

Plant protoplasts in the age of synthetic biology.

Protoplasts, which are plant cells with their cell walls removed, have been used for decades in plant research and have been instrumental in genetic transformation and the study of various aspects of plant physiology and genetics. With the advent of synthetic biology, these individualized plant cells are fundamental to accelerate the "design-build-test-learn" cycle, which is relatively slow in plant research. Despite their potential, challenges remain to expanding the use of protoplasts in synthetic biology. The capacity of individual protoplasts to hybridize to form new varieties, and to regenerate from single cells, creating individuals with new features is underexplored. The main objective of this review is to discuss the use of protoplasts in plant synthetic biology and to highlight the challenges to exploiting protoplast technologies in this new "age of synthetic biology".

An improved protocol for protoplast production, culture, and whole plant regeneration of the commercial brown seaweed Undaria pinnatifida

Protoplasts (i.e., living plant cells devoid of cell wall) represent an important biotechnological tool with utility for the conduction of in vitro studies and crop improvement in economic cultivars. Undaria pinnatifida is an annual brown kelp commonly used in Asian cuisine and several industries because of its biological properties. It is one of the major seaweed cultivars used worldwide. This study establishes a simple and effective protocol for protoplast isolation, culture, and whole plant regeneration of U. pinnatifida . The results showed that high protoplast yields (2–4 × 10 7 protoplast g −1 of fresh weight) were obtained using the commercially available enzymes cellulase “Onozuka” RS (1 %) and alginate lyase (4 U mL −1 ) without any pre-treatment and with a short incubation time (2–4 h). The isolation protocol described herein presented with more effective results with meristematic explants and was superior to the protocols reported for U. pinnatifida and other brown algae. Additionally, the enzymatic mixture could be reused up to six times. Whole plant regeneration was accomplished after 3 months in culture at 20 °C using two culture media, and a mixture of antibiotics (penicillin G, streptomycin, and chloramphenicol) was added once at the beginning of the culture. Our regeneration protocol was less laborious and more cost-effective, exhibiting final plating efficiency higher than previously reported and demonstrating the potential to be used to produce up to 3 × 10 4 sporophytes g −1 of explant. The established protocol can be easily used for genetic manipulation, protoplast fusion, or micropropagation, thus enabling the expansion of the use of protoplasts in algae. • Single-step production of protoplasts from the edible seaweed Undaria pinnatifida using commercial enzymes • The reusability of the enzyme shows that the process is economically viable. • Antibiotics and temperature are key factors for protoplast survival, division and normal regeneration. • Regeneration protocol is simpler and more effective than previously reported.

Strain Improvement and Strain Maintenance Revisited. The Use of Actinoplanes teichomyceticus ATCC 31121 Protoplasts in the Identification of Candidates for Enhanced Teicoplanin Production.

Multicellular cooperation in actinomycetes is a division of labor-based beneficial trait where phenotypically specialized clonal subpopulations, or genetically distinct lineages, perform complementary tasks. The division of labor improves the access to nutrients and optimizes reproductive and vegetative tasks while reducing the costly production of secondary metabolites and/or of secreted enzymes. In this study, we took advantage of the possibility to isolate genetically distinct lineages deriving from the division of labor, for the isolation of heterogeneous teicoplanin producer phenotypes from Actinoplanes teichomyceticus ATCC 31121. In order to efficiently separate phenotypes and associated genomes, we produced and regenerated protoplasts. This approach turned out to be a rapid and effective strain improvement method, as it allowed the identification of those phenotypes in the population that produced higher teicoplanin amounts. Interestingly, a heterogeneous teicoplanin complex productivity pattern was also identified among the clones. This study suggests that strain improvement and strain maintenance should be integrated with the use of protoplasts as a strategy to unravel the hidden industrial potential of vegetative mycelium.

Protoplast Isolation and Shoot Regeneration from Protoplast-Derived Callus of Petunia hybrida Cv. Mirage Rose.

Despite the increasing use of protoplasts in plant biotechnology research, shoot regeneration from protoplasts remains challenging. In this study, we investigated the factors involved in protoplast isolation, callus induction, and shoot regeneration in Petunia hybrida cv. Mirage Rose. The following conditions were found to be most optimal for protoplast yield and viability: 0.6 M mannitol, 2.0% cellulase, and 6 h digestion time. A plating density of 10 × 104 protoplasts/mL under osmoticum condition (0.58 M mannitol) showed high microcolony viability in liquid culture. The Kao and Michayluk medium was found to be appropriate for callus proliferation from microcalli under a 16-h light photoperiod. Calli cultured in Murashige and Skoog medium containing 1.0 mg/L 6-benzylaminopurine and 0.2 mg/L 3-indole butyric acid showed the highest shoot regeneration frequency and number of shoots obtained per explant. Random amplification of polymorphic DNA analysis showed that the protoplast-derived shoots exhibited the same banding patterns as those of donor plants. Collectively, these findings can contribute to solving problems encountered in protoplast isolation and shoot regeneration in other petunia cultivars and related species. As the protocol developed by us is highly reproducible, it can be applied in biotechnology research on P. hybrida cv. Mirage Rose.

Development of Model Systems for Plant Rhabdovirus Research.

This chapter reviews the discoveries and initial characterizations (1930-1990) of three plant rhabdoviruses, sonchus yellow net virus, potato yellow dwarf virus, and lettuce necrotic yellows virus, that have become model systems for research on this group of enveloped negative-strand RNA plant viruses. We have used our personal perspectives to review the early historical studies of these viruses, the important technologies and tools, such as density gradient centrifugation, that were developed during the research, and to highlight the eminent scientists involved in these discoveries. Early studies on sites of virus replication, virion structure, physicochemical composition, and the use of protoplasts and vector insect cell culture for virus research are discussed, and differences between the nuclear and cytoplasmic lifestyles of plant rhabdoviruses are contrasted. Finally, we briefly summarize the genome organization and more recent developments culminating in the development of a reverse genetics system for plant negative-strand RNA viruses.

Expression of potato virus S gRNA fragments in transgenic potato plants triggers RNA-interference against related and unrelated viruses

This chapter reviews the discoveries and initial characterizations (1930–1990) of three plant rhabdoviruses, sonchus yellow net virus, potato yellow dwarf virus, and lettuce necrotic yellows virus, that have become model systems for research on this group of enveloped negative-strand RNA plant viruses. We have used our personal perspectives to review the early historical studies of these viruses, the important technologies and tools, such as density gradient centrifugation, that were developed during the research, and to highlight the eminent scientists involved in these discoveries. Early studies on sites of virus replication, virion structure, physicochemical composition, and the use of protoplasts and vector insect cell culture for virus research are discussed, and differences between the nuclear and cytoplasmic lifestyles of plant rhabdoviruses are contrasted. Finally, we briefly summarize the genome organization and more recent developments culminating in the development of a reverse genetics system for plant negative-strand RNA viruses.

Transient Secretory Enzyme Expression in Leaf Protoplasts to Characterize SNARE Functional Classes in Conventional and Unconventional Secretion.

Despite a long case history, the use of protoplasts in cell biology research still divides scientists but their weaknesses can be exploited as strengths. Transient expression in protoplasts can saturate protein-protein interactions very efficiently, inhibiting the process of interest more efficiently than other approaches at gene expression level. The method described here consists of an assay providing a functional characterization of SNARE proteins in a heterogeneous population of cells, by the comparison of native and dominant negative mutant forms. In particular, it allows for discriminating between t-SNARE and i-SNARE functional classes.

Arabidopsis Regenerating Protoplast: A Powerful Model System for Combining the Proteomics of Cell Wall Proteins and the Visualization of Cell Wall Dynamics.

The development of a range of sub-proteomic approaches to the plant cell wall has identified many of the cell wall proteins. However, it remains difficult to elucidate the precise biological role of each protein and the cell wall dynamics driven by their actions. The plant protoplast provides an excellent means not only for characterizing cell wall proteins, but also for visualizing the dynamics of cell wall regeneration, during which cell wall proteins are secreted. It therefore offers a unique opportunity to investigate the de novo construction process of the cell wall. This review deals with sub-proteomic approaches to the plant cell wall through the use of protoplasts, a methodology that will provide the basis for further exploration of cell wall proteins and cell wall dynamics.

Dissection of miRNA Pathways Using Arabidopsis Mesophyll Protoplasts

MicroRNAs (miRNAs) control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.

Dissection of miRNA pathways using Arabidopsis mesophyll protoplasts.

microRNAs (miRNAs) control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this paper, we describe a transient expression system in Arabidopsis mesophyll protoplasts that is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, either by overexpression or by the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.

A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes

Transient gene expression assays using protoplasts have been frequently used in high-throughput screening and functional characterization of plant genes. In barley, however, very few studies have explored the use of protoplasts isolated from green tissues. In this study, a reliable and efficient transient gene expression system has been established using barley green tissue protoplasts. Due to the importance of osmolarity in maintaining protoplast viability and transfection efficiency, different mannitol concentrations were tested to determine the optimal osmolarity suitable for barley protoplast preparation. The method and conditions were also described for efficient isolation of protoplasts from barley leaf and stem tissues and transient expression of exogenous gene constructs. This transient expression system has been successfully demonstrated for protein immunoblot analysis, subcellular protein localization and quantitative analysis of gene expression. Furthermore, a simplified method has been described to quickly evaluate microRNAs for post-transcriptional regulation of their target genes in barley protoplasts.

Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection.

BackgroundGenetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants.Methodology/Principal FindingsWe recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation.Conclusions/SignificanceWe have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.

A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes

BackgroundPlant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. In rice, protoplasts are commonly prepared from suspension cultured cells or etiolated seedlings, but only a few studies have explored the use of protoplasts from rice green tissue.ResultsHere, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP-tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts.ConclusionsWe show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes.

From protoplasts to gene clusters

Advances in protoplast technology underpinned many crucial developments in our understanding of the molecular biology of filamentous fungi. This review follows one of these developments, namely the discovery and analysis of difuran toxin gene clusters. Our understanding of the biosynthetic pathway of the agriculturally important toxin, aflatoxin, has been dramatically enhanced by the use of protoplasts and protoplast-based gene transformation methods. Since the identification of the first pathway genes by complementation of mutants with transforming DNA, transformation has continued to play a critical role in the elucidation of gene function and regulation. But despite the wealth of knowledge accumulated so far some fundamental questions remain to be answered. How did these gene clusters evolve? What is the biological role of aflatoxin? The discovery of homologues of aflatoxin genes in other fungal species such as the pine needle pathogen Dothistroma septosporum may help to shed some light on these questions.

Use of protoplasts from paired heterogenic bacterial species to detect tin contaminants: Prospects for biosensor development

Two different bacteria gave different respiratory responses to the test analytes, tributyl tin (TBT) and cadmium as expressed by positive sigmoid responses by Halomonas sp. (slope, +1.71 [TBT]; +1.76 [Cd]) and negative sigmoid responses by Bacillus pumilis (slope, -1.06 [TBT]; -0.59 [Cd]). The EC50 values determined from Hill plots for the response of Halomonas sp. to the TBT and Cd were 1 and 8.5 mM, respectively, which were lower by a factor of 10 than the corresponding values for B. pumilis. With protoplasts of B. pumilis there was a major shift in the signal from sigmoid negative to positive with TBT (+1.35) but not Cd (-0.5), while the signals with the remaining protoplast-analyte combinations remained unchanged. For all four protoplast-analyte combinations the EC50 values were in the order of 10-100-fold lower than those for their whole cell counterparts. When other analytes were tested the protoplasts gave a similar response to tin as for TBT, but detected copper and 2,4-dichlorophenol with similar signal profiles to Cd and with lower sensitivity. The difference in signal and higher sensitivity of the two species protoplast system towards TBT/tin compared to the other analytes tested, suggests that it may feasible to develop this approach for the detection of tin residues.

Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation

A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool for molecular genetic analysis of interesting Bacillus strains, which are hard to transform by conventional methods.

Biolistic mediated site-specific integration in rice

Cre-lox mediated site-specific integration in tobacco or Arabidopsis used polyethylene glycol or Agrobacterium, respectively, to deliver the integrating DNA. The polyethylene glycol method is inconvenient since it requires the use of protoplasts. The Agrobacterium method is inefficient as the single-stranded T-DNA is not a substrate for Cre-lox recombination. In this study, we tested the biolistic method for the site-specific insertion of DNA into the rice genome. Two target callus lines, each harboring a single genomic lox target, were generated by Agrobacterium-mediated transformation. The target callus lines were subjected to a second round of transformation by particle bombardment with a construct designed to excise the plasmid backbone from the integrating DNA, followed by the recombination of the integrating DNA into the genomic lox target. Site-specific integration was obtained from both target callus lines. Three integrant plants were regenerated from one target line and were found to have a precise copy of the integrating DNA at the target site, although only one plant has the integrating DNA as the sole copy in the genome. Site-specific integration through the biolistic delivery of DNA can be considered for other plants that are transformable via particle bombardment.

Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5'-monophosphate decarboxylase.

A homologous transformation system for the opportunistic fungal pathogen Aspergillus fumigatus was developed. It is based on the A. fumigatus pyrG gene, encoding orotidine 5'-monophosphate decarboxylase, which was cloned and sequenced. Transformation of both Aspergillus (Emericella) nidulans and A. fumigatus pyrG mutant strains by the use of protoplasts or electroporation established the functionality of the cloned gene. DNA sequencing of the A. fumigatus pyrG1 mutant allele revealed that it encodes a truncated, non-functional, PyrG protein. Transformation of an A. fumigatus pyrG1 mutant with a plasmid carrying the novel pyrG2 allele constructed by in vitro mutagenesis yielded prototrophic transformants following recombination between both mutation sites. Analysis of transformants carrying the entire plasmid showed that up to 45% of integration had occurred at the pyrG locus. This provides a tool to target defined genetic constructs at a specific locus in the A. fumigatus genome in order to study gene regulation and function.