Plant Protoplasts Research Articles

High-Efficient Transient Gene Transfection for Transcriptional Activity Assay Using Rice Protoplasts on Large-Scale Platform.

Transient gene expression in protoplasts offers a straightforward and efficient approach for investigating gene function. Here, I introduce a method that utilizes a 96-well plate format to assess transcriptional activation capacity of transcription factors through transient expression system in rice protoplasts. This method includes plant cultivation, protoplast preparation, transfection, and transactivation assay and allows for the simultaneous measurement and validation of 23 samples with 4 replicates per sample at a time.

Multiscale chromatin dynamics and high entropy in plant iPSC ancestors.

Plant protoplasts provide starting material for of inducing pluripotent cell masses that are competent for tissue regeneration in vitro, analogous to animal induced pluripotent stem cells (iPSCs). Dedifferentiation is associated with large-scale chromatin reorganisation and massive transcriptome reprogramming, characterised by stochastic gene expression. How this cellular variability reflects on chromatin organisation in individual cells and what factors influence chromatin transitions during culturing are largely unknown. Here, we used high-throughput imaging and a custom supervised image analysis protocol extracting over 100 chromatin features of cultured protoplasts. The analysis revealed rapid, multiscale dynamics of chromatin patterns with a trajectory that strongly depended on nutrient availability. Decreased abundance in H1 (linker histones) is hallmark of chromatin transitions. We measured a high heterogeneity of chromatin patterns indicating intrinsic entropy as a hallmark of the initial cultures. We further measured an entropy decline over time, and an antagonistic influence by external and intrinsic factors, such as phytohormones and epigenetic modifiers, respectively. Collectively, our study benchmarks an approach to understand the variability and evolution of chromatin patterns underlying plant cell reprogramming in vitro.

Genome editing of a recalcitrant wine grape genotype by lipofectamine-mediated delivery of CRISPR/Cas9 ribonucleoproteins to protoplasts.

The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene-free grapevine plants, exploiting the lipofectamine-mediated direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo (V. vinifera), an extremely in vitro recalcitrant wine genotypeused to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG-mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.

Ratiometric gibberellin biosensors for the analysis of signaling dynamics and metabolism in plant protoplasts.

Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation-based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)-based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co-expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA-INSENSITIVE, GAI; REPRESSOR-of-ga1-3, RGA; RGA-like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA-oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta.

Pooled effector library screening in protoplasts rapidly identifies novel Avr genes

Crop breeding for durable disease resistance is challenging due to the rapid evolution of pathogen virulence. While progress in resistance (R) gene cloning and stacking has accelerated in recent years1–3, the identification of corresponding avirulence (Avr) genes in many pathogens is hampered by the lack of high-throughput screening options. To address this technology gap, we developed a platform for pooled library screening in plant protoplasts to allow rapid identification of interacting R–Avr pairs. We validated this platform by isolating known and novel Avr genes from wheat stem rust (Puccinia graminis f. sp. tritici) after screening a designed library of putative effectors against individual R genes. Rapid Avr gene identification provides molecular tools to understand and track pathogen virulence evolution via genotype surveillance, which in turn will lead to optimized R gene stacking and deployment strategies. This platform should be broadly applicable to many crop pathogens and could potentially be adapted for screening genes involved in other protoplast-selectable traits.

Enabling High-throughput Transgene Expression Studies Using Automated Liquid Handling for Etiolated Maize Leaf Protoplasts.

The field of plant biotechnology has witnessed remarkable advancements in recent years, revolutionizing the ability to manipulate and engineer plants for various purposes. However, as research in this field increases in diversity and becomes increasingly sophisticated, the need for early, efficient, dependable, and high-throughput transient screening solutions to narrow down strategies proceeding to stable transformation is more apparent. One method that has re-emerged in recent years is the utilization of plant protoplast, for which methods of isolation and transfection are available in numerous species, tissues, and developmental stages. This work describes a simple automated protocol for the randomized preparation of plasmid within a 96-well plate, a method for the isolation of etiolated maize leaf protoplast, and an automated transfection procedure. The adoption of automated solutions in plant biotechnology, exemplified by these novel liquid handling protocols for plant protoplast transfection, represents a significant advancement over manual methods. By leveraging automation, researchers can easily overcome the limitations of traditional methods, enhance efficiency, and accelerate scientific progress.

A high-efficient protoplast transient system for screening gene editing elements in Salvia miltiorrhiza.

A high-efficiency protoplast transient system was devised to screen genome editing elements in Salvia miltiorrhiza. Medicinal plants with high-value pharmaceutical ingredients have attracted research attention due to their beneficial effects on human health. Cell wall-free protoplasts of plants can be used to evaluate the efficiency of genome editing mutagenesis. The capabilities of gene editing in medicinal plants remain to be fully explored owing to their complex genetic background and shortfall of suitable transformation. Here, we took the Salvia miltiorrhiza as a representative example for developing a method to screen favorable gene editing elements with high editing efficiency in medical plants by a PEG-mediated protoplast transformation. Results indicated that using the endogenous SmU6.1 of S. miltiorrhiza to drive sgRNA and the plant codon-optimized Cas9 driven by the promoter SlEF1α can enhance the efficiency of editing. In summary, we uncover an efficacious transient method for screening editing elements and shed new light on increasing gene editing efficiency in medicinal plants.

Protoplast Isolation, Culture, and Regeneration in Common and Tartary Buckwheat.

Techniques based on the use of plant protoplasts are a convenient model for better understanding and observing developmental changes in the cells. The establishment of research tools based on protoplasts consists of many steps needed for optimization. Here, we describe the culture of morphogenic callus (MC)- and hypocotyl-derived protoplasts of common (Fagopyrum esculentum Moench) and Tartary (F. tataricum (L.) Gaertn.) buckwheat. Protoplasts embedding in agarose matrix and application of plant hormones, including phytosulfokine (PSK), enable the development of protoplast cultures and plant regeneration.

Studying Secretory Protein Synthesis in Nicotiana benthamiana Protoplasts.

Transient gene expression in plant protoplasts facilitates the analysis of hybrid genes in a fast and reproducible manner. The technique is particularly powerful when studying basic conserved biochemical processes including de novo protein synthesis, modification, assembly, transport, and turnover. Unlike individual plants, protoplast suspensions can be divided into almost identical aliquots, allowing the analysis of independent variables with uncertainties restricted to minor pipetting errors/variations. Using the examples of protein secretion and ER retention, we describe the most advanced working practice of routinely preparing, electroporating, and analyzing Nicotiana benthamiana protoplasts. A single batch of electroporation-competent protoplasts permits up to 30 individual transfections. This is ideal to assess the influence of independent variables, such as point mutations, deletions or fusions, or the influence of a co-expressed effector gene in dose-response studies.

Phytosulfokine peptide library: chemical synthesis and biological evaluation on protoplast regeneration

A focussed library of 12 phytosulfokine analogues are synthesized and evaluated as artificial plant peptide hormones to boost regeneration of plant protoplasts.

Plant Extracellular Vesicles: Current Landscape and Future Directions.

Plant cells secrete membrane-enclosed micrometer- and nanometer-sized vesicles that, similarly to the extracellular vesicles (EVs) released by mammalian or bacterial cells, carry a complex molecular cargo of proteins, nucleic acids, lipids, and primary and secondary metabolites. While it is technically complicated to isolate EVs from whole plants or their tissues, in vitro plant cell cultures provide excellent model systems for their study. Plant EVs have been isolated from the conditioned culture media of plant cell, pollen, hairy root, and protoplast cultures, and recent studies have gathered important structural and biological data that provide a framework to decipher their physiological roles and unveil previously unacknowledged links to their diverse biological functions. The primary function of plant EVs seems to be in the secretion that underlies cell growth and morphogenesis, cell wall composition, and cell-cell communication processes. Besides their physiological functions, plant EVs may participate in defence mechanisms against different plant pathogens, including fungi, viruses, and bacteria. Whereas edible and medicinal-plant-derived nanovesicles isolated from homogenised plant materials ex vivo are widely studied and exploited, today, plant EV research is still in its infancy. This review, for the first time, highlights the different in vitro sources that have been used to isolate plant EVs, together with the structural and biological studies that investigate the molecular cargo, and pinpoints the possible role of plant EVs as mediators in plant-pathogen interactions, which may contribute to opening up new scenarios for agricultural applications, biotechnology, and innovative strategies for plant disease management.

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.

Engineering and characterization of carbohydrate-binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts.

Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nkon ) and desorption (koff ) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.

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".

Polyploid Induction and Karyotype Analysis of Dendrobium officinale

Dendrobium officinale Kimura et Migo is an orchid with both medicinal and edible values and a high economic value. The wild resources of D. officinale are in an endangered state. Compared with the wild D. officinale, cultivated D. officinale exhibits inferior quality and a low content of medicinal components. Polyploid induction is a conventional breeding tool for genome doubling of species, which can effectively increase the total amount of plant components to improve the medicinal efficacy of D. officinale. In this study, D. officinale tetraploids were generated by treating the protocorms with colchicine. Morphological observations showed that tetraploids exhibited decreased plant size and leaf shape index and increased stem diameter. Cytological observations showed that the polyploid plants had larger stomata and a lower number of stomata per unit area compared with normal plants. The highest stomata variation of 30.00% was observed when the plant was treated with 0.3% colchicine for 24 h. Chromosomal observations showed that treatment of plants with 0.2% colchicine for 48 h resulted in the highest tetraploid induction rate of 10.00%. A total of 10 tetraploids were successfully obtained by inducing plant protoplasts with colchicine. The number of diploid D. officinale chromosomes was 38 with a base number of 19, and the karyotype formula was 2n = 2x = 38 = 24m + 14sm with a karyotype asymmetry coefficient of 60.59%, belonging to type 2B. The number of tetraploid D. officinale chromosomes was 76 with a base number of 19, and the karyotype formula was 2n = 4x = 76 = 58m + 18sm with a karyotype asymmetry coefficient of 60.04%, belonging to type 2B. This study determined the optimal mutagenesis treatment based on the chromosome observation results, investigated the relationship between the phenotype and ploidy level, and generated the polyploid germplasm of D. officinale, thereby laying the foundation for the breeding of new D. officinale cultivars enriched with compounds of medicinal value.

Synthetic Promoter Screening Using Poplar Mesophyll Protoplast Transformation.

Plant protoplasts are useful to study both transcriptional regulation and protein subcellular localization in rapid screens. Protoplast transformation can be used in automated platforms for design-build-test cycles of plant promoters, including synthetic promoters. A notable application of protoplasts comes from recent successes in dissecting synthetic promoter activity with poplar mesophyll protoplasts. For this purpose, we constructed plasmids with TurboGFP driven by a synthetic promoter together with TurboRFP constitutively controlled by a 35S promoter, to monitor transformation efficiency, allowing versatile screening of high numbers of cells by monitoring green fluorescent protein expression in transformed protoplasts. Herein, we introduce a protocol for poplar mesophyll protoplast isolation followed by protoplast transformation and image analysis for the selection of valuable synthetic promoters. Graphical overview.

Single-Cell Confinement Methods to Study Plant Cytoskeleton.

Progress in cytoskeletal research in animal systems has been accompanied by the development of single-cell systems (e.g., fibroblasts in culture). Single-cell systems exist for plant research, but the presence of a cell wall hinders the possibility to relate cytoskeleton dynamics to changes in cell shape or in mechanical stress pattern. Here we present two protocols to confine wall-less plant protoplasts in microwells with defined geometries. Either protocol might be more or less adapted to the question at hand. For instance, when using microwells made of agarose, the composition of the well can be easily modified to analyze the impact of biochemical cues. When using microwells in a stiff polymer (NOA73), protoplasts can be pressurized, and the wall of the well can be coated with cell wall components. Using both protocols, we could analyze microtubule and actin dynamics in vivo while also revealing the relative contribution of geometry and stress in their self-organization.

A high-efficiency PEG-Ca2+-mediated transient transformation system for broccoli protoplasts.

Transient transformation of plant protoplasts is an important method for studying gene function, subcellular localization and plant morphological development. In this study, an efficient transient transformation system was established by optimizing the plasmid concentration, PEG4000 mass concentration and genotype selection, key factors that affect transformation efficiency. Meanwhile, an efficient and universal broccoli protoplast isolation system was established. Using 0.5% (w/v) cellulase R-10 and 0.1% (w/v) pectolyase Y-23 to hydrolyze broccoli cotyledons of three different genotypes for 3h, the yield was more than 5×106/mL/g, and the viability was more than 95%, sufficient to meet the high standards for protoplasts to be used in various experiments. The average transformation efficiency of the two plasmid vectors PHG-eGFP and CP507-YFP in broccoli B1 protoplasts were 61.4% and 41.7%, respectively. Using this system, we successfully performed subcellular localization of the products of three target genes (the clubroot resistance gene CRa and two key genes regulated by glucosinolates, Bol029100 and Bol031350).The results showed that the products of all three genes were localized in the nucleus. The high-efficiency transient transformation system for broccoli protoplasts constructed in this study makes it possible to reliably acquire high-viability protoplasts in high yield. This research provides important technical support for international frontier research fields such as single-cell sequencing, spatial transcriptomics, plant somatic hybridization, gene function analysis and subcellular localization.

The TARGET System: Rapid Identification of Direct Targets of Transcription Factors by Gene Regulation in Plant Cells.

The TARGET system allows for the rapid identification of direct regulated gene targets of transcription factors (TFs). It employs the transient transformation of plant protoplasts with inducible nuclear entry of the TF and subsequent transcriptomic and/or ChIP-seq analysis. The ability to separate direct TF-target gene regulatory interactions from indirect downstream responses and the significantly shorter amount of time required to perform the assay, compared to the generation of transgenics, make this plant cell-based approach a valuable tool for a higher throughput approach to identify the genome-wide targets of multiple TFs, to build validated transcriptional networks in plants. Here, we describe the use of the TARGET system in Arabidopsis seedling root protoplasts to map the gene regulatory network downstream of transcription factors-of-interest.

Polish Contribution to Global Research on Somatic Embryogenesis

AbstractSomatic embryogenesis (SE) is a complex process that begins with regaining totipotency in some somatic cells, proceeds through embryo development and maturation, and ends with the formation of a whole plant. Since the first publications on SE in 1958, this regeneration process has been applied to the in vitro propagation of many plant species and has led to the development of some specific model systems. SE has been used to expand our understanding of the cytomorphological, physiological, biochemical, and genetic processes that govern the earliest developmental events in the life of plants. This paper summarizes the achievements of Polish research groups working on SE systems established for several plants (gentians, the tree fernCyathea delgadiiSternb., and conifers) and three model species (Arabidopsis thaliana,Medicagosativa, andM. truncatula). SE systems have used a broad spectrum of experimental approaches involving genomic tools (transcriptomics, proteomics, and chromatin analyses), physiological methods which focus on phytohormones, and cytological techniques. Studies on the experimental models ofA. thalianaandMedicagospp. have resulted in the identification of new genetic and epigenetic elements of the complex regulatory network controlling embryogenic induction in plant somatic cells. The protocol developed for ferns has provided a unique and simple system for cytological analysis of early SE events that occur in a single cell of initial explants. Gentian embryogenic suspension cultures have successfully been used in broad biotechnological applications, including plant transformation, protoplast isolation, culture, and fusion. Systems described for coniferous species effectively produced many vigorous somatic seedlings and cost-efficient storage of genotypes during clonal field-testing. The research undertaken by Polish scientists has resulted in developing experimental systems that have enabled significant advances in SE knowledge.