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Frontiers in Plant Science 2019Somatic embryogenesis (SE) in not only one of the most promising techniques for mass propagation of selected trees, but also is a valuable tool for basic research... (Review)
Review
Somatic embryogenesis (SE) in not only one of the most promising techniques for mass propagation of selected trees, but also is a valuable tool for basic research studies in cell biology and genetic engineering, and it allows the long-term conservation of genetic resources by cryopreservation techniques. This review reports the most recent progress in SE, protoplast culture, and cryopreservation of four important Japanese pines ( var. , and ). Induction of embryogenic tissues (ET), embryogenic culture maintenance/proliferation, production of somatic embryos, germination, and conversion to plants are described focusing on the protocols most commonly reported for plant production in species through to SE.
PubMed: 30745904
DOI: 10.3389/fpls.2019.00031 -
Frontiers in Genome Editing 2021The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global... (Review)
Review
The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global population. However, the delivery and access of gene-editing tools to the host genome and subsequent recovery of successfully edited plants form significant bottlenecks in the application of new plant breeding technologies. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. Hence, it is of importance to develop and improve multiple strategies for delivery and regeneration in order to be able to approach each application from various angles. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. Here, we will discuss the use of protoplast regeneration in the application of new plant breeding technologies and review pertinent literature on successful protoplast regeneration.
PubMed: 34713266
DOI: 10.3389/fgeed.2021.734951 -
Plant & Cell Physiology Nov 2021Many plant processes occur in the context of and in interaction with a surrounding matrix such as soil (e.g. root growth and root-microbe interactions) or surrounding... (Review)
Review
Many plant processes occur in the context of and in interaction with a surrounding matrix such as soil (e.g. root growth and root-microbe interactions) or surrounding tissues (e.g. pollen tube growth through the pistil), making it difficult to study them with high-resolution optical microscopy. Over the past decade, microfabrication techniques have been developed to produce experimental systems that allow researchers to examine cell behavior in microstructured environments that mimic geometrical, physical and/or chemical aspects of the natural growth matrices and that cannot be generated using traditional agar plate assays. These microfabricated environments offer considerable design flexibility as well as the transparency required for high-resolution, light-based microscopy. In addition, microfluidic platforms have been used for various types of bioassays, including cellular force assays, chemoattraction assays and electrotropism assays. Here, we review the recent use of microfluidic devices to study plant cells and organs, including plant roots, root hairs, moss protonemata and pollen tubes. The increasing adoption of microfabrication techniques by the plant science community may transform our approaches to investigating how individual plant cells sense and respond to changes in the physical and chemical environment.
Topics: Biological Assay; Bryophyta; Imaging, Three-Dimensional; Microfluidic Analytical Techniques; Plant Cells; Plant Roots; Pollen Tube; Protoplasts
PubMed: 34027549
DOI: 10.1093/pcp/pcab067 -
Microbial Cell Factories Oct 2017Filamentous fungi have been of great interest because of their excellent ability as cell factories to manufacture useful products for human beings. The development of... (Review)
Review
Filamentous fungi have been of great interest because of their excellent ability as cell factories to manufacture useful products for human beings. The development of genetic transformation techniques is a precondition that enables scientists to target and modify genes efficiently and may reveal the function of target genes. The method to deliver foreign nucleic acid into cells is the sticking point for fungal genome modification. Up to date, there are some general methods of genetic transformation for fungi, including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation. This article reviews basic protocols and principles of these transformation methods, as well as their advantages and disadvantages.
Topics: Agrobacterium; Biolistics; Electroporation; Fungi; Gene Transfer Techniques; Genetic Techniques; Genome, Fungal; Protoplasts; Transformation, Genetic
PubMed: 28974205
DOI: 10.1186/s12934-017-0785-7 -
International Journal of Molecular... Nov 2023Protoplasts, derived from plant cells, exhibit remarkable totipotency and hold significant value across a wide spectrum of biological and biotechnological applications.... (Review)
Review
Protoplasts, derived from plant cells, exhibit remarkable totipotency and hold significant value across a wide spectrum of biological and biotechnological applications. These versatile applications encompass protein subcellular localization and interaction analysis, gene expression regulation, functional characterization, gene editing techniques, and single-cell sequencing. Protoplasts' usability stems from their inherent accessibility and their ability to efficiently incorporate exogenous genes. In this review, we provide a comprehensive overview, including details on isolation procedures and influencing factors, purification and viability assessment methodologies, and the utilization of the protoplast transient expression system. The aim is to provide a comprehensive overview of current applications and offer valuable insights into protoplast isolation and the establishment of transient expression systems in a diverse range of plant species, thereby serving as a valuable resource for the plant science community.
Topics: Protoplasts; Plants; Biotechnology; Gene Editing
PubMed: 38069215
DOI: 10.3390/ijms242316892 -
PloS One 2022A high yield of isolated protoplast and reliable regeneration system are prerequisite for successful somatic hybridization and genome editing research. However,...
A high yield of isolated protoplast and reliable regeneration system are prerequisite for successful somatic hybridization and genome editing research. However, reproducible plant regeneration from protoplasts remains a bottleneck for many crops, including cassava. We evaluated several factors that influence isolation of viable protoplasts form leaf mesophyll, induction of embryogenic calli, and regeneration of plants in three cassava cultivars; Muchericheri, TMS60444 and Karibuni. A relatively higher protoplast yield was obtained with enzyme mixture containing 5 g/L Macerozyme and 10 g/L cellulase. Muchericheri recorded relatively higher protoplast yield of 20.50±0.50×106 whereas TMS60444 (10.25±0.25×106) had the least protoplast yield in 10 g/L cellulase and 4 g/L cellulase. Freshly isolated protoplast cells were plated on callus induction medium (CIM) solid medium containing MS basal salt, 60 g/L D-glucose, 30 g/L sucrose, B5 vitamins, 100 mg/L myo-inositol, 0.5 mg/L copper sulphate, 100 mg/L casein hydrolysate, 4.55 g/L mannitol, 0.1 g/L MES, 10 mg/L picloram and 3 g/L gelrite to induce protoplast growth and development. The three cultivars reached colony formation but no further development was observed in this culture method. Protoplast growth and development was further evaluated in suspension culture using varying cell densities (1, 2 and 3× 105 p/mL). Development with highest number of minicalli was observed in cell density of 3× 105 p/mL. Minicalli obtained were cultured on CIM supplemented with 10mg/L picloram. Callus induction was observed in all cell densities with the cultivars. Highest somatic embryogenesis was observed in 2× 105 p/ml while no somatic embryogenesis was observed in cell density of 1×105 p/mL. Somatic embryos were matured in EMM medium supplemented with 1 mg/L BAP, 0.02 mg/L NAA and 1.5 mg/L GA3 then germinated in hormone free medium for plant regeneration. This protocol which used simple mixture of commercial enzymes is highly reproducible and can be applied in biotechnology research on cassava.
Topics: Manihot; Protoplasts; Picloram; Vegetables; Plant Leaves; Cellulase; Callosities; Regeneration
PubMed: 36454974
DOI: 10.1371/journal.pone.0278717 -
Plant Cell Reports Sep 2022We have established a DNA-free genome editing method via ribonucleoprotein-based CRISPR/Cas9 in cultivated tomato and obtained mutant plants regenerated from transfected...
We have established a DNA-free genome editing method via ribonucleoprotein-based CRISPR/Cas9 in cultivated tomato and obtained mutant plants regenerated from transfected protoplasts with a high mutation rate. The application of genome editing as a research and breeding method has provided many possibilities to improve traits in many crops in recent years. In cultivated tomato (Solanum lycopersicum), so far only stable Agrobacterium-mediated transformation carrying CRISPR/Cas9 reagents has been established. Shoot regeneration from transfected protoplasts is the major bottleneck in the application of DNA-free genome editing via ribonucleoprotein-based CRISPR/Cas9 method in cultivated tomato. In this study, we report the implementation of a transgene-free breeding method for cultivated tomato by CRISPR/Cas9 technology, including the optimization of protoplast isolation and overcoming the obstacle in shoot regeneration from transfected protoplasts. We have identified that the shoot regeneration medium containing 0.1 mg/L IAA and 0.75 mg/L zeatin was the best hormone combination with a regeneration rate of up to 21.3%. We have successfully obtained regenerated plants with a high mutation rate four months after protoplast isolation and transfection. Out of 110 regenerated M plants obtained, 35 (31.8%) were mutated targeting both SP and SP5G genes simultaneously and the editing efficiency was up to 60% in at least one allele in either SP or SP5G genes.
Topics: CRISPR-Cas Systems; Gene Editing; Solanum lycopersicum; Plant Breeding; Protoplasts; Ribonucleoproteins
PubMed: 35773498
DOI: 10.1007/s00299-022-02893-8 -
International Journal of Molecular... Mar 2022As one of the pioneer crops widely planted in saline-alkaline areas, provides daily necessities, including natural fiber, vegetable proteins, and edible oils. However,...
As one of the pioneer crops widely planted in saline-alkaline areas, provides daily necessities, including natural fiber, vegetable proteins, and edible oils. However, cotton fiber yield and quality are highly influenced by salt stress. Therefore, elucidating the molecular mechanisms of cotton in response to salinity stress is importance to breed new cultivars with high tolerance. In this study, we first developed a method for single-cell RNA-seq based on isolating protoplast from cotton root tips; then, we studied the impact of salinity stress on gene expression profiling and their dynamic changes using the developed high-efficiency method for protoplast dissociation suitable for single-cell RNA-seq. A total of 3391 and 2826 differentially expressed genes (DEGs) were identified in salt-treated samples before and after protoplast dissociation, respectively, which were enriched into several molecular components, including response to stimulus, response to stress, and cellular macromolecule metabolic process by gene ontology (GO) analysis. Plant hormone signal transduction, phenylpropanoid biosynthesis, and MAPK signaling pathway were found to be enriched via Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Twenty-two and nine salinity-responsive DEGs participated in plant hormone signaling and MAPK signaling in roots, before and after protoplast dissociation, respectively; six upregulated DEGs were involved in ABA signaling transduction, namely, , , , , , and which indicates their potential functions on plants adapting to salt stress. Additionally, 384 and 257 transcription factors (TFs) were differentially expressed in salt-stress roots before and after protoplast dissociation, respectively, of which significantly up-regulated TFs mainly belonged to the AP2/ERF-ERF family, which implied their potential roles responding to salt stress. These results not only provide novel insights to reveal the regulatory networks in plant's root response to salt stress, but also lay the solid foundation for further exploration on cellular heterogeneity by single-cell transcriptome sequencing.
Topics: Gene Expression Profiling; Gene Expression Regulation, Plant; Gossypium; Plant Breeding; Plant Growth Regulators; Protoplasts; Salt Stress; Stress, Physiological; Transcriptome
PubMed: 35269989
DOI: 10.3390/ijms23052845 -
Biology Aug 2020Despite the increasing use of protoplasts in plant biotechnology research, shoot regeneration from protoplasts remains challenging. In this study, we investigated the...
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 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 × 10 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 cv. Mirage Rose.
PubMed: 32824283
DOI: 10.3390/biology9080228 -
Plant Methods Dec 2022Camellia 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...
BACKGROUND
Camellia 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.
RESULTS
The 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 × 10/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 × 10/g·FW and 2.6 × 10/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.
CONCLUSION
In 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.
PubMed: 36550558
DOI: 10.1186/s13007-022-00972-1