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BMC Biotechnology Mar 2017Cassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel...
Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz).
BACKGROUND
Cassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced.
METHODS
Fully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization.
RESULTS
In this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 10 protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H/monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system.
CONCLUSION
We optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.
Topics: Gene Expression Profiling; Genetic Enhancement; High-Throughput Screening Assays; Manihot; Mesophyll Cells; Plant Proteins; Polyethylene Glycols; Protein Engineering; Protoplasts
PubMed: 28292294
DOI: 10.1186/s12896-017-0349-2 -
International Journal of Molecular... Mar 2020Protoplast systems have been proven powerful tools in modern plant biology. However, successful preparation of abundant viable protoplasts remains a challenge for...
Protoplast systems have been proven powerful tools in modern plant biology. However, successful preparation of abundant viable protoplasts remains a challenge for orchids. Herein, we established an efficient protoplast isolation protocol from orchid petals through optimization of enzymatic conditions. It requires optimal D-mannitol concentration (0.5 M), enzyme concentration (1.2 % (w/v) cellulose and 0.6 % (w/v) macerozyme) and digestion time (6 h). With this protocol, the highest yield (3.50 × 10/g fresh weight of orchid tissue) and viability (94.21%) of protoplasts were obtained from flower petals of . In addition, we achieved high transfection efficiency (80%) through the optimization of factors affecting polyethylene glycol (PEG)-mediated protoplast transfection including incubation time, final PEG4000 concentration and plasmid DNA amount. This highly efficient protoplast-based transient expression system (PTES) was further used for protein subcellular localization, bimolecular fluorescence complementation (BiFC) assay and gene regulation studies of flowering related genes in orchids. Taken together, our protoplast isolation and transfection protocol is highly efficient, stable and time-saving. It can be used for gene function and molecular analyses in orchids and other economically important monocot crops.
Topics: Cell Separation; Flowers; Gene Expression Regulation, Plant; Orchidaceae; Plant Proteins; Protein Binding; Protoplasts
PubMed: 32218171
DOI: 10.3390/ijms21072264 -
International Journal of Molecular... Sep 2022Protoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem...
Protoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem cells, but their regeneration ability decreases or becomes lost altogether with extension of the culture period. In this study, we isolated and cultured EC-derived protoplasts (EC-pts) from carrots and compared them with non-EC-derived protoplasts (NEC-pts) with respect to their totipotency. The protoplast isolation conditions were optimized, and the EC-pts and NEC-pts were characterized by their cell size and types. Both types of protoplasts were then embedded using the alginate layer (TAL) method, and the resulting EC-pt-TALs and NEC-pt-TALs were cultured for further regeneration. The expression of the EC-specific genes SERK1, WUS, BBM, LEC1, and DRN was analyzed to confirm whether EC identity was maintained after protoplast isolation. The protoplast isolation efficiency for EC-pts was 2.4-fold higher than for NEC-pts (3.5 × 106 protoplasts·g−1 FW). In the EC-pt group, protoplasts < 20 µm accounted for 58% of the total protoplasts, whereas in the NEC-pt group, small protoplasts accounted for only 26%. In protoplast culture, the number of protoplasts that divided was 2.6-fold higher for EC-pts than for NEC-pts (7.7 × 104 protoplasts·g−1 FW), with a high number of plants regenerated for EC-pt-TALs, whereas no plants were induced by NEC-pt-TAL. Five times more plants were regenerated from EC-pts than from ECs. Regarding the expression of EC-specific genes, WUS and SERK1 expression increased 12-fold, and LEC1 and BBM expression increased 3.6−6.4-fold in isolated protoplasts compared with ECs prior to protoplast isolation (control). These results reveal that the protoplast isolation process did not affect the embryogenic cell identity; rather, it increased the plant regeneration rate, confirming that EC-derived protoplast culture may be an efficient system for increasing the regeneration ability of old EC cultures through the elimination of old and inactivate cells. EC-derived protoplasts may also represent an efficient single-cell system for application in new breeding technologies such as genome editing.
Topics: Alginates; Daucus carota; Plant Breeding; Protoplasts; Totipotent Stem Cells
PubMed: 36232857
DOI: 10.3390/ijms231911556 -
Plant Physiology Dec 2001Plant protoplasts show physiological perceptions and responses to hormones, metabolites, environmental cues, and pathogen-derived elicitors, similar to cell-autonomous... (Review)
Review
Plant protoplasts show physiological perceptions and responses to hormones, metabolites, environmental cues, and pathogen-derived elicitors, similar to cell-autonomous responses in intact tissues and plants. The development of defined protoplast transient expression systems for high-throughput screening and systematic characterization of gene functions has greatly contributed to elucidating plant signal transduction pathways, in combination with genetic, genomic, and transgenic approaches.
Topics: Arabidopsis; Gene Expression Regulation, Plant; Plant Leaves; Plants, Genetically Modified; Protoplasts; Signal Transduction; Zea mays
PubMed: 11743090
DOI: No ID Found -
International Journal of Molecular... Mar 2021The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling...
The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice () protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct , consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with , , , or alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.
Topics: Cytokinins; Gene Expression Regulation, Plant; Genes, Plant; Oryza; Plant Growth Regulators; Plant Proteins; Plant Shoots; Promoter Regions, Genetic; Protoplasts; Signal Transduction
PubMed: 33807467
DOI: 10.3390/ijms22073647 -
Cellular and Molecular Life Sciences :... Dec 2016Efforts to reduce the global burden of bacterial disease and contend with escalating bacterial resistance are spurring innovation in antibacterial drug and biocide... (Review)
Review
Efforts to reduce the global burden of bacterial disease and contend with escalating bacterial resistance are spurring innovation in antibacterial drug and biocide development and related technologies such as photodynamic therapy and photochemical disinfection. Elucidation of the mechanism of action of these new agents and processes can greatly facilitate their development, but it is a complex endeavour. One strategy that has been popular for many years, and which is garnering increasing interest due to recent technological advances in microscopy and a deeper understanding of the molecular events involved, is the examination of treated bacteria for changes to their morphology and ultrastructure. In this review, we take a critical look at this approach. Variables affecting antibacterial-induced alterations are discussed first. These include characteristics of the test organism (e.g. cell wall structure) and incubation conditions (e.g. growth medium osmolarity). The main body of the review then describes the different alterations that can occur. Micrographs depicting these alterations are presented, together with information on agents that induce the change, and the sequence of molecular events that lead to the change. We close by highlighting those morphological and ultrastructural changes which are consistently induced by agents sharing the same mechanism (e.g. spheroplast formation by peptidoglycan synthesis inhibitors) and explaining how changes that are induced by multiple antibacterial classes (e.g. filamentation by DNA synthesis inhibitors, FtsZ disruptors, and other types of agent) can still yield useful mechanistic information. Lastly, recommendations are made regarding future study design and execution.
Topics: Anti-Bacterial Agents; Bacteria; Cell Wall; Organelles; Peptidoglycan; Protoplasts
PubMed: 27392605
DOI: 10.1007/s00018-016-2302-2 -
Journal of Applied Microbiology Feb 2018To establish a proper protoplast-preparation route from enriched motile flagellates and nonmotile resting cells of Haematococcus pluvialis.
AIMS
To establish a proper protoplast-preparation route from enriched motile flagellates and nonmotile resting cells of Haematococcus pluvialis.
METHODS AND RESULTS
Through cultivations in two mixotrophic media, enriched Haematococcus flagellates and resting cells were respectively produced and applied in enzymatic protoplast preparations. Great differences of enzymatic sensitivity and osmotic-lability were identified between them. Flagellates showed the same osmotic-lability as protoplasts and the extracellular matrix-removing rate was applied for an evaluation of protoplast-releasing. During the treatment of flagellates, an addition of more than 0·2 mmol l Ca was found to be essential for maintenance of high cellular viability. More than 80% cellular viability and a 90% protoplast-releasing rate were obtained simultaneously after 2-3 h treatment of 0·06% proteinase K in 0·05 mol l Tris-HCl (pH7·8) buffer with 0·2 mmol l CaCl and 0·2 mol l sorbitol/mannitol. For resting cells, a treatment of both 0·12% proteinase K and a combination of 2% cellulase + 1% snailase could function similarly in order to degrade the cellulosic cell wall, while the protoplast yield was limited to about 40%, due to the existence of an undegradable secondary wall in the mature resting cell.
CONCLUSION
Proteinase K was efficient for protoplast-releasing from either flagellates or resting cells. Due to the great difference of enzymatic sensitivity and osmotic-lability between flagellates and resting cells, it was necessary to select a different enzymatic treating process based upon the main cell type in the culture. A better protoplast-preparing efficiency was obtained from Haematococcus cells when flagellates were in the majority.
SIGNIFICANCE AND IMPACT OF THE STUDY
The protoplast preparation of H. pluvialis was firstly established based on two main cell types of H. pluvialis, motile flagellates and nonmotile resting cells. Increase of flagellate stability and viable protoplast-preparing efficiency through addition of Ca in enzymatic solution was firstly reported.
Topics: Biocatalysis; Cell Survival; Cellulase; Chlorophyta; Endopeptidase K; Osmosis; Protoplasts
PubMed: 29154453
DOI: 10.1111/jam.13643 -
The CRISPR Journal Oct 2021Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving CRISPR and Cas proteins rely on low...
Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving CRISPR and Cas proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification, or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50% in and 13.6% in rapid cycling without antibiotic selection. Using a 60 nt donor containing 27 nt in each homologous arm, 6/22 regenerated plants showed targeted insertions, and one contained a precise insertion of a 6 bp III site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants.
Topics: Costs and Cost Analysis; Gene Editing; Gene Targeting; Genome, Plant; Mutagenesis, Insertional; Protoplasts; Nicotiana
PubMed: 34569819
DOI: 10.1089/crispr.2021.0045 -
Plant Physiology and Biochemistry : PPB Sep 2023Somatic cell fusion is a process that transfers cytoplasmic and nuclear genes to create new germplasm resources. But our limited understanding of the physiological and...
BACKGROUND
Somatic cell fusion is a process that transfers cytoplasmic and nuclear genes to create new germplasm resources. But our limited understanding of the physiological and molecular mechanisms that shape protoplast responses to fusion.
METHOD
We employed flow cytometry, cytology, proteomics, and gene expression analysis to examine the sugarcane (Saccharum spp.) protoplast fusion.
RESULTS
Flow cytometry analysis revealed the fusion rate of protoplasts was 1.95%, the FSC value and SSC of heterozygous cells was 1.17-1.47 times higher than that of protoplasts. The protoplasts viability decreased and the MDA increased after fusion. During fusion, the cell membranes were perforated to different degrees, nuclear activity was weakened, while microtubules depolymerized and formed several short rod like structures in the protoplasts. The most abundant proteins during fusion were mainly involved in RNA processing and modification, cell cycle control, cell division, chromosome partition, nuclear structure, extracellular structures, and nucleotide transport and metabolism. Moreover, the expression of key regeneration genes, such as WUS, GAUT, CESA, PSK, Aux/IAA, Cdc2, Cyclin D3, Cyclin A, and Cyclin B, was significantly altered following fusion.
PURPOSE AND SIGNIFICANCE
Overall, our findings provide a theoretical basis that increases our knowledge of the mechanisms underlying protoplast fusion.
Topics: Protoplasts; Saccharum; Flow Cytometry; Proteomics; Cytoplasm
PubMed: 37619268
DOI: 10.1016/j.plaphy.2023.107918 -
BMC Plant Biology Jun 2024Angelica Gigas (Purple parsnip) is an important medicinal plant that is cultivated and utilized in Korea, Japan, and China. It contains bioactive substances especially...
Response surface methodology mediated optimization of phytosulfokine and plant growth regulators for enhanced protoplast division, callus induction, and somatic embryogenesis in Angelica Gigas Nakai.
BACKGROUND
Angelica Gigas (Purple parsnip) is an important medicinal plant that is cultivated and utilized in Korea, Japan, and China. It contains bioactive substances especially coumarins with anti-inflammatory, anti-platelet aggregation, anti-cancer, anti-diabetic, antimicrobial, anti-obesity, anti-oxidant, immunomodulatory, and neuroprotective properties. This medicinal crop can be genetically improved, and the metabolites can be obtained by embryonic stem cells. In this context, we established the protoplast-to-plant regeneration methodology in Angelica gigas.
RESULTS
In the present investigation, we isolated the protoplast from the embryogenic callus by applying methods that we have developed earlier and established protoplast cultures using Murashige and Skoog (MS) liquid medium and by embedding the protoplast in thin alginate layer (TAL) methods. We supplemented the culture medium with growth regulators namely 2,4-dichlorophenoxyaceticacid (2,4-D, 0, 0.75, 1.5 mg L), kinetin (KN, 0, 0.5, and 1.0 mg L) and phytosulfokine (PSK, 0, 50, 100 nM) to induce protoplast division, microcolony formation, and embryogenic callus regeneration. We applied central composite design (CCD) and response surface methodology (RSM) for the optimization of 2,4-D, KN, and PSK levels during protoplast division, micro-callus formation, and induction of embryogenic callus stages. The results revealed that 0.04 mg L 2,4-D + 0.5 mg L KN + 2 nM PSK, 0.5 mg L 2,4-D + 0.9 mg L KN and 90 nM PSK, and 1.5 mg L 2,4-D and 1 mg L KN were optimum for protoplast division, micro-callus formation and induction embryogenic callus. MS basal semi-solid medium without growth regulators was good for the development of embryos and plant regeneration.
CONCLUSIONS
This study demonstrated successful protoplast culture, protoplast division, micro-callus formation, induction embryogenic callus, somatic embryogenesis, and plant regeneration in A. gigas. The methodologies developed here are quite useful for the genetic improvement of this important medicinal plant.
Topics: Angelica; Plant Growth Regulators; Plant Somatic Embryogenesis Techniques; Protoplasts; Cell Division
PubMed: 38858674
DOI: 10.1186/s12870-024-05243-w