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Methods in Molecular Biology (Clifton,... 2022Wheat is one of the major staple crops around the world. A transient expression system is crucial for gene functional studies in wheat as stable transfection is still...
Wheat is one of the major staple crops around the world. A transient expression system is crucial for gene functional studies in wheat as stable transfection is still difficult in most cultivars. Protoplasts could serve as a versatile transient expression tool in wheat research. Here, we describe protocols for wheat protoplast isolation and transfection that are enabled by cellulase R-10 and macerozyme R-10 containing enzymatic solution and polyethylene glycol-mediated method, respectively. In addition, we show an example of efficiency evaluation of the emerging base editors in wheat protoplasts. These protocols are of wide use in both conventional gene functional analysis and reagent functionality evaluation of genome editing in wheat.
Topics: Gene Editing; Protoplasts; Transfection; Triticum
PubMed: 35258830
DOI: 10.1007/978-1-0716-2164-6_10 -
Methods in Molecular Biology (Clifton,... 2022Protoplast, a plant cell without cell wall, can be readily transfected by exogenous macromolecules (DNA, RNA, protein) and therefore offer a versatile single cell-based...
Protoplast, a plant cell without cell wall, can be readily transfected by exogenous macromolecules (DNA, RNA, protein) and therefore offer a versatile single cell-based functional analysis system to rapidly assess these exogenous macromolecules' functions. Properly prepared Arabidopsis leaf mesophyll protoplasts exhibit similar responses as intact plants to diverse abiotic and biotic stress signals as well as different hormones and nutrients, based on well-established reporter and marker gene assays. The protoplast transient expression system has been proven to be a vital and reliable tool for elucidation of the activities of transcription factors and protein kinases, protein subcellular localization and trafficking, protein-protein interaction, and protein stabilities in various signal transduction pathways. Moreover, protoplast also offers a platform for single cell-based plant regeneration, gene silencing, and genome editing. Healthy protoplasts isolated from plant tissues and the high transfection efficiency are key steps for successful use of the protoplast transient expression system. In this chapter, we describe the detailed methods of the protoplast transient expression system in Arabidopsis, including plant material preparation, high-quality maxi-plasmid DNA extraction, non-stressed protoplast isolation, highly efficient PEG-calcium transfection of plasmid DNA, and protoplast culture and harvest. We also provide several examples of gene functional analysis using this protoplast transient expression system.
Topics: Arabidopsis; Plant Leaves; Protoplasts; Signal Transduction; Transfection
PubMed: 35258823
DOI: 10.1007/978-1-0716-2164-6_3 -
Methods in Molecular Biology (Clifton,... 2022Protoplast-based transient gene expression platforms can be used to study a range of questions concerning gene regulation. Crucial to the success of these studies is the...
Protoplast-based transient gene expression platforms can be used to study a range of questions concerning gene regulation. Crucial to the success of these studies is the isolation of large quantities of healthy protoplasts from the tissue of interest. Herein, we describe protocols for isolating and transfecting maize mesophyll protoplasts for gene expression studies. The isolation protocol yields approximately 1.8-1.9 × 10 protoplasts with 80-90% viability from 6 g of etiolated leaf tissue, and the polyethylene glycol-mediated transfection protocol results in 55-58% transfection efficiency. The transfection protocol describes the use of a dual-expression vector that carries the coding sequence for two fluorescent proteins (FPs), one driven by a constitutive promoter for normalization for transfection efficiency and the other driven by the construct of interest. The use of a dual-FP expression vector eliminates the need for co-transfection and separate steps for enzymatic/substrate processing as required for luciferase-based assays. These protocols have been tested on leaf tissue from the maize genotypes B73 and PHR03 and, as written, can be completed in 24 h.
Topics: Plant Leaves; Protoplasts; Transfection; Zea mays
PubMed: 35258827
DOI: 10.1007/978-1-0716-2164-6_7 -
Methods in Molecular Biology (Clifton,... 2022Protoplast production with the moss Physcomitrium (Physcomitrella) patens has a long and successful history. As a tool, it has not only been the base of reverse genetic...
Protoplast production with the moss Physcomitrium (Physcomitrella) patens has a long and successful history. As a tool, it has not only been the base of reverse genetic studies covering research fields as diverse as development, metabolism, or gene network regulation but also allowed its development as a bioengineering platform for protein production. We present here a standardized protocol for protoplast production from Physcomitrium (Physcomitrella) patens protonemata. Additionally, we detail procedures for their transfection, their plating for optimal regeneration, and three alternative selection approaches. To improve the consistency of protoplast regeneration, we describe a new option for protoplast embedding. The use of an alginate matrix to regenerate moss protoplast alleviates the use of warm agarized medium. Thus, it optimizes transformed protoplast survival without any morphological detrimental effect or impact on transfection efficiency.
Topics: Bryopsida; Protoplasts; Transfection
PubMed: 35258821
DOI: 10.1007/978-1-0716-2164-6_1 -
Methods in Molecular Biology (Clifton,... 2022The protocol outlined in this chapter describes a detailed procedure for protoplast isolation and transformation using polyethylene glycol (PEG)-mediated transfection...
The protocol outlined in this chapter describes a detailed procedure for protoplast isolation and transformation using polyethylene glycol (PEG)-mediated transfection and DNA microinjection, highlighting also the critical steps associated with the method. Briefly, we will describe the efficient isolation of protoplasts from 3-month-old suspension calli collected at 14 days after cultured. Digestion of the calli with an optimal composition of enzyme solution yielded over 2 × 10 protoplasts/mL with the viability of more than 80%. The concentrations of DNA, PEG, and magnesium chloride and application of heat shock treatment are the crucial determinants for efficient PEG-mediated transfection. Using the optimal PEG transfection conditions, a transfection efficiency of more than 20% could be obtained. At the same time, protoplasts embedded in alginate layer cultured for 3 days and injected with 100 ng/μL of total DNA solution are the optimal factors for microinjection. We successfully regenerated the injected protoplasts to calli expressing green fluorescent protein (GFP) signals when cultured in optimal medium and cultivation procedures.
Topics: DNA; Microinjections; Polyethylene Glycols; Protoplasts; Transfection
PubMed: 35258834
DOI: 10.1007/978-1-0716-2164-6_14 -
Biotechnology Journal Dec 2021The dairy yeast Kluyveromyces marxianus is a promising cell factory for producing bioethanol and heterologous proteins, as well as a robust synthetic biology platform...
The dairy yeast Kluyveromyces marxianus is a promising cell factory for producing bioethanol and heterologous proteins, as well as a robust synthetic biology platform host, due to its safe status and beneficial traits, including fast growth and thermotolerance. However, the lack of high-efficiency transformation methods hampers the fundamental research and industrial application of this yeast. Protoplast transformation is one of the most commonly used fungal transformation methods, but it yet remains unexplored in K. marxianus. Here, we established the protoplast transformation method of K. marxianus for the first time. A series of parameters on the transformation efficiency were optimized: cells were collected in the late-log phase and treated with zymolyase for protoplasting; the transformation was performed at 0 °C with carrier DNA, CaCl , and PEG; after transformation, protoplasts were recovered in a solid regeneration medium containing 3-4% agar and 0.8 m sorbitol. By using the optimized method, plasmids of 10, 24, and 58 kb were successfully transformed into K. marxianus. The highest efficiency reached 1.8 × 10 transformants per μg DNA, which is 18-fold higher than the lithium acetate method. This protoplast transformation method will promote the genetic engineering of K. marxianus that requires high-efficiency transformation or the introduction of large DNA fragments.
Topics: Genetic Engineering; Kluyveromyces; Protoplasts; Synthetic Biology
PubMed: 34554645
DOI: 10.1002/biot.202100122 -
Methods in Molecular Biology (Clifton,... 2022Protoplasts are plant cells that have had their cell walls removed, which allows for a variety of cellular manipulations that are not possible within the context of...
Protoplasts are plant cells that have had their cell walls removed, which allows for a variety of cellular manipulations that are not possible within the context of intact plant tissue. Unfortunately, the removal of cell walls is not trivial and can be sensitive to cell type and cell differentiation state. Here, we describe a modified protoplasting protocol that improves isolation of viable protoplasts from the seedling maize shoot apex.
Topics: Cell Wall; Meristem; Plant Shoots; Protoplasts; Seedlings; Zea mays
PubMed: 35258829
DOI: 10.1007/978-1-0716-2164-6_9 -
Methods in Molecular Biology (Clifton,... 2019Efficient plant protoplast production from cell suspension cultures, leaf, and stem tissue allows for single-cell plant biology. Since protoplasts do not have cell...
Efficient plant protoplast production from cell suspension cultures, leaf, and stem tissue allows for single-cell plant biology. Since protoplasts do not have cell walls, they can be readily transformed to enable rapid assessment of regulatory elements, synthetic constructs, gene expression, and more recently genome-editing tools and approaches. Historically, enzymatic cell wall digestion has been both expensive and laborious. Protoplast production, transformation, and analysis of fluorescence have recently been automated using an integrated robotic system. Here we describe its use for bulk protoplast isolation, counting, transformation, and analysis at very low cost for high-throughput experiments.
Topics: Protoplasts; Robotics; Nicotiana; Transformation, Genetic
PubMed: 30610649
DOI: 10.1007/978-1-4939-8991-1_26 -
Biotechnology and Bioengineering Jun 2020A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome...
A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing.
Topics: Alginates; Biocompatible Materials; Cell Division; Cells, Immobilized; Protoplasts; Saccharomyces cerevisiae; Saccharomycetales
PubMed: 32100874
DOI: 10.1002/bit.27318 -
Methods in Molecular Biology (Clifton,... 2022Protoplast transfection is widely used in plant research to rapidly evaluate RNA degradation, reporter assay, gene expression, subcellular localization, and...
Protoplast transfection is widely used in plant research to rapidly evaluate RNA degradation, reporter assay, gene expression, subcellular localization, and protein-protein interactions. In order to successfully use protoplast transfection with the newly emerging clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein editing platform, high yield of protoplasts, stable transfection efficiency, and reliable regeneration protocols are necessary. The Nicotiana tabacum transient protoplast transfection and regeneration system can effectively obtain target gene mutations in regenerated plants without transgenes and is thus a very attractive technique for evaluating gene editing reagents using CRISPR/Cas-based systems. Here, we describe in detail sterilized seed germination, culture conditions, isolation of Nicotiana tabacum protoplasts from tissue culture explants, construction of a vector containing the Cas protein and sgRNA cassette, highly efficient polyethylene glycol-calcium transient transfection of plasmids delivered into protoplasts, evaluation of mutagenesis efficiency and genotype analysis from protoplasts and regenerated plants, and the regeneration conditions to obtain CRISPR-edited plants from single protoplasts.
Topics: CRISPR-Cas Systems; Gene Editing; Mutagenesis; Protoplasts; Nicotiana
PubMed: 35258824
DOI: 10.1007/978-1-0716-2164-6_4