-
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 -
Plants (Basel, Switzerland) Dec 2023Plant cells secrete membrane-enclosed micrometer- and nanometer-sized vesicles that, similarly to the extracellular vesicles (EVs) released by mammalian or bacterial... (Review)
Review
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.
PubMed: 38140468
DOI: 10.3390/plants12244141 -
Nature Communications Jul 2023Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a destructive citrus disease worldwide. Generating disease-resistant cultivars is the most effective,...
Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a destructive citrus disease worldwide. Generating disease-resistant cultivars is the most effective, environmentally friendly and economic approach for disease control. However, citrus traditional breeding is lengthy and laborious. Here, we develop transgene-free canker-resistant Citrus sinensis lines in the T0 generation within 10 months through transformation of embryogenic protoplasts with Cas12a/crRNA ribonucleoprotein to edit the canker susceptibility gene CsLOB1. Among the 39 regenerated lines, 38 are biallelic/homozygous mutants, demonstrating a 97.4% biallelic/homozygous mutation rate. No off-target mutations are detected in the edited lines. Canker resistance of the cslob1-edited lines results from both abolishing canker symptoms and inhibiting Xcc growth. The transgene-free canker-resistant C. sinensis lines have received regulatory approval by USDA APHIS and are exempted from EPA regulation. This study provides a sustainable and efficient citrus canker control solution and presents an efficient transgene-free genome-editing strategy for citrus and other crops.
Topics: Citrus sinensis; RNA, Guide, CRISPR-Cas Systems; CRISPR-Cas Systems; Disease Resistance; Plant Breeding; Citrus; Xanthomonas; Plant Diseases
PubMed: 37402755
DOI: 10.1038/s41467-023-39714-9 -
Biology Methods & Protocols 2024Protoplast regeneration has become a key platform for genetic and genome engineering. However, we lack reliable and reproducible methods for efficient protoplast... (Review)
Review
Protoplast regeneration has become a key platform for genetic and genome engineering. However, we lack reliable and reproducible methods for efficient protoplast regeneration for tomato () cultivars. Here, we optimized cell and tissue culture methods for protoplast isolation, microcallus proliferation, shoot regeneration, and plantlet establishment of the tomato cultivar Micro-Tom. A thin layer of alginate was applied to protoplasts isolated from third to fourth true leaves and cultured at an optimal density of 1 × 10 protoplasts/ml. We determined the optimal culture media for protoplast proliferation, callus formation, shoot regeneration, and root regeneration. Regenerated plantlets exhibited morphologically normal growth and sexual reproduction. The entire regeneration process, from protoplasts to flowering plants, was accomplished within 5 months. The optimized protoplast regeneration platform enables biotechnological applications, such as genome engineering, as well as basic research on plant regeneration in species.
PubMed: 38414647
DOI: 10.1093/biomethods/bpae008 -
Plants (Basel, Switzerland) Jul 2023In the modern world we are constantly bombarded by environmental and natural stimuli that can result in oxidative stress. Antioxidant molecules and enzymes help the...
In the modern world we are constantly bombarded by environmental and natural stimuli that can result in oxidative stress. Antioxidant molecules and enzymes help the human body scavenge reactive oxygen species and prevent oxidative damage. Most organisms possess intrinsic antioxidant activity, but also benefit from the consumption of antioxidants from their diet. Leafy green vegetables such as spinach are a well-researched rich source of dietary antioxidant molecules. However, plant cell walls are difficult to digest for many individuals and the bio-accessibility of nutrients and antioxidants from these sources can be limited by the degree of digestion and assimilation. Through a specific enzymatic process, Solarplast contains organic spinach protoplasts without the cell wall, which may facilitate higher yield and efficacy of beneficial antioxidant molecules. In this study, analytical techniques coupled to in vitro bioassays were used to determine the potential antioxidant activity of Solarplast and determine its antioxidant enzymatic capabilities. Solarplast demonstrated superior antioxidant activity when compared to frozen spinach leaves in TOC, FRAP and TEAC antioxidant assays. Several antioxidant enzymes were also increased in Solarplast, when compared to frozen spinach. As a functional readout, Solarplast attenuated hydrogen peroxide-, ethanol- and acetaminophen-induced increases in oxidative stress and cytotoxicity in both intestinal (HT-29) and liver (HepG2) cell lines. These findings suggest that Solarplast may represent a non-GMO, plant-based food supplement to help reduce oxidative stress in the human body.
PubMed: 37514292
DOI: 10.3390/plants12142678 -
Frontiers in Plant Science 2023Single-cell and spatial transcriptomics have diverted researchers' attention from the multicellular level to the single-cell level and spatial information. Single-cell... (Review)
Review
Single-cell and spatial transcriptomics have diverted researchers' attention from the multicellular level to the single-cell level and spatial information. Single-cell transcriptomes provide insights into the transcriptome at the single-cell level, whereas spatial transcriptomes help preserve spatial information. Although these two omics technologies are helpful and mature, further research is needed to ensure their widespread applicability in plant studies. Reviewing recent research on plant single-cell or spatial transcriptomics, we compared the different experimental methods used in various plants. The limitations and challenges are clear for both single-cell and spatial transcriptomic analyses, such as the lack of applicability, spatial information, or high resolution. Subsequently, we put forth further applications, such as cross-species analysis of roots at the single-cell level and the idea that single-cell transcriptome analysis needs to be combined with other omics analyses to achieve superiority over individual omics analyses. Overall, the results of this review suggest that combining single-cell transcriptomics, spatial transcriptomics, and spatial element distribution can provide a promising research direction, particularly for plant research.
PubMed: 37636094
DOI: 10.3389/fpls.2023.1185377 -
Plants (Basel, Switzerland) Dec 2023The species, a group of plants native to Australia, have been historically valued for their pharmacological properties and have played a significant role in traditional...
The species, a group of plants native to Australia, have been historically valued for their pharmacological properties and have played a significant role in traditional medicine and pharmaceutical research. Persistent efforts are underway to enhance the efficacy of the active ingredient scopolamine, employing both conventional breeding methods and advanced biotechnology tools. The primary objective of this research was to establish a highly efficient method for isolating mesophyll protoplasts and facilitating their regeneration, thereby laying a robust foundation for the application of various advanced plant biotechnology tools in the pursuit of genetic enhancement. The mesophyll protoplast isolation process was developed for hybrid × with careful optimisation of the following parameters: leaf strip size; incubation conditions; physical treatment; and enzyme concentration. The optimal parameters were combined in each individual step; the best enzyme concentration was determined to be 2% (/) cellulysin and 0.5% (/) macerase. Protoplast yield was found to be greatly affected by the enzyme concentrations. The isolated protoplasts were cultured at a density of 0.5 × 10 to best sustain the highest cell division (33.2%) and a microcalli induction frequency of 17.9%. After 40 days of culture in a modified KM8P medium at 25 °C in darkness, visible microcalli were transferred to a solidified Murashige and Skoog (MS) medium with 1 mg L 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction under a 16 h photoperiod. After 30 days of culture, compact organogenic calli were transferred into a solid MS medium with 6-benzylaminopurine (BA) alone or thidiazuron (TDZ) alone or in combination with BA or naphthalene acetic acid (NAA) for shoot regeneration. The maximum shoot regeneration frequency (63.3%) was observed in the medium with 1.5 mg L TDZ alone. For the first time, a reliable protoplast isolation and regeneration system from mesophyll cells was established for with high protoplast viability, successful microcalli formation, and intact plant regeneration. This innovation will significantly contribute towards the genetic enhancement of the species.
PubMed: 38202348
DOI: 10.3390/plants13010040 -
Microbiology and Molecular Biology... Jun 2023The quest for bacterial survival is exemplified by spores formed by some members. They turn up everywhere one looks, and their ubiquity reflects adaptations to the... (Review)
Review
The quest for bacterial survival is exemplified by spores formed by some members. They turn up everywhere one looks, and their ubiquity reflects adaptations to the stresses bacteria face. Spores are impactful in public health, food safety, and biowarfare. Heat resistance is the hallmark of spores and is countered principally by a mineralized gel-like protoplast, termed the spore core, with reduced water which minimizes macromolecular movement/denaturation/aggregation. Dry heat, however, introduces mutations into spore DNA. Spores have countermeasures to extreme conditions that are multifactorial, but the fact that spore DNA is in a crystalline-like nucleoid in the spore core, likely due to DNA saturation with small acid-soluble spore proteins (SASPs), suggests that reduced macromolecular motion is also critical in spore dry heat resistance. SASPs are also central in the radiation resistance characteristic of spores, where the contributions of four spore features-SASP; Ca, with pyridine-2,6-dicarboxylic acid (CaDPA); photoproduct lyase; and low water content-minimize DNA damage. Notably, the spore environment steers UV photochemistry toward a product that germinated spores can repair without significant mutagenesis. This resistance extends to chemicals and macromolecules that could damage spores. Macromolecules are excluded by the spore coat which impedes the passage of moieties of ≥10 kDa. Additionally, damaging chemicals may be degraded or neutralized by coat enzymes/proteins. However, the principal protective mechanism here is the inner membrane, a compressed structure lacking lipid fluidity and presenting a barrier to the diffusion of chemicals into the spore core; SASP saturation of DNA also protects against genotoxic chemicals. Spores are also resistant to other stresses, including high pressure and abrasion. Regardless, overarching mechanisms associated with resistance seem to revolve around reduced molecular motion, a fine balance between rigidity and flexibility, and perhaps efficient repair.
Topics: Spores, Bacterial; Ultraviolet Rays; DNA Damage; Mutation; Water; Bacillus subtilis; Bacterial Proteins; DNA, Bacterial
PubMed: 36927044
DOI: 10.1128/mmbr.00080-22 -
Cell Surface (Amsterdam, Netherlands) Dec 2023Across all kingdoms of life, cells secrete an extracellular polymer mesh that in turn feeds back onto them. This entails physical connections between the plasma membrane... (Review)
Review
Across all kingdoms of life, cells secrete an extracellular polymer mesh that in turn feeds back onto them. This entails physical connections between the plasma membrane and the polymer mesh. In plant cells, one connection stands out: the Hechtian strand which, during plasmolysis, reflects the existence of a physical link between the plasma membrane of the retracting protoplast and the cell wall. The Hechtian strand is part of a larger structure, which we call the Hechtian structure, that comprises the Hechtian strand, the Hechtian reticulum and the Hechtian attachment sites. Although it has been observed for more than 100 years, its molecular composition and biological functions remain ill-described. A comprehensive characterization of the Hechtian structure is a critical step towards understanding this plasma membrane-cell wall connection and its relevance in cell signaling. This short review intends to highlight the main features of the Hechtian structure, in order to provide a clear framework for future research in this under-explored and promising field.
PubMed: 38024561
DOI: 10.1016/j.tcsw.2023.100115 -
International Journal of Molecular... Jul 2023Mangoes ( L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been...
Mangoes ( L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system. This study optimized the conditions of the protoplast isolation and transformation system, which can provide a lot of help in the gene expression regulation study of mango. The most beneficial protoplast isolation conditions were 150 mg/mL of cellulase R-10 and 180 mg/mL of macerozyme R-10 in the digestion solution at pH 5.6 and 12 h of digestion time. The 0.16 M and 0.08 M mannitol in wash solution (WI) and suspension for counting (MMG), respectively, were optimal for the protoplast isolation yield. The isolated leaf protoplasts (~5.4 × 10 cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaCl, from which 84.38% of the protoplasts were transformed. About 0.08 M and 0.12 M of mannitol concentration in MMG and transfection solutions, respectively, were optimal for protoplast viability. Under the florescence signal, GFP was seen in the transformed protoplasts. This showed that the target gene was successfully induced into the protoplast and that it can be transcribed and translated. Experimental results in this paper show that our high-efficiency protoplast isolation and PEG-mediated transformation protocols can provide excellent new methods for creating a rapid and effective tool for the molecular mechanism study of mangoes.
Topics: Mangifera; Protoplasts; Plant Leaves; Transfection
PubMed: 37569360
DOI: 10.3390/ijms241511984