Preparation Of 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.

An Efficient Fluorescence-Activated Protoplast Sorting (FAPS) and Regeneration Protocol for Canola (Brassica napus).

Protoplast sorting and purification methods are powerful tools enabling the enrichment of cellular subpopulations for basic and applied studies in plant sciences. Fluorescence-activated protoplast sorting (FAPS) is an efficient method to isolate specific protoplast populations based on innate features (size and autofluorescence) or expression of fluorescent proteins. FAPS-based methods have recently been deployed in single-cell purification for single-cell RNA sequencing-based transcriptional profiling studies. Protoplast sorting methods integrated with the ability to culture and recover whole plants add value to functional genomics and gene editing applications. Enriching cells expressing nucleases linked to fluorescent proteins can maximize knockout or knockin editing efficiencies and minimize toxic and off-target effects. Here, we report the protocol for protoplast preparation, sterile cell sorting, culture, and downstream regeneration of plants from canola protoplasts. This protocol can be successfully applied to all totipotent protoplast methods that can regenerate into whole plants. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Preparation of transfected canola protoplasts for sorting Basic Protocol 2: Fluorescence-activated protoplast sorting Basic Protocol 3: Bead culture of sorted protoplasts and recovery of plantlets.

Fusiform nanoparticle boosts efficient genetic transformation in Sclerotinia sclerotiorum

BackgroundSclerotinia sclerotiorum is a highly destructive phytopathogenic fungus that poses a significant threat to a wide array of crops. The current constraints in genetic manipulation techniques impede a thorough comprehension of its pathogenic mechanisms and the development of effective control strategies.ResultsHerein, we present a highly efficient genetic transformation system for S. sclerotiorum, leveraging the use of fusiform nanoparticles, which are synthesized with FeCl3 and 2,6-diaminopyrimidine (DAP). These nanoparticles, with an average longitude length of 59.00 nm and a positively charged surface, facilitate the direct delivery of exogenous DNA into the mycelial cells of S. sclerotiorum, as well as successful integration with stable expression. Notably, this system circumvents fungal protoplast preparation and tedious recovery processes, streamlining the transformation process considerably. Furthermore, we successfully employed this system to generate S. sclerotiorum strains with silenced oxaloacetate acetylhydrolase-encoding gene Ss-oah1.ConclusionsOur findings demonstrate the feasibility of using nanoparticle-mediated delivery as a rapid and reliable tool for genetic modification in S. sclerotiorum. Given its simplicity and high efficiency, it has the potential to significantly propel genetic research in filamentous fungi, offering new avenues for elucidating the intricacies of pathogenicity and developing innovative disease management strategies.Graphical

Development of a Robust Saccharomyces cerevisiae Strain for Efficient Co-Fermentation of Mixed Sugars and Enhanced Inhibitor Tolerance through Protoplast Fusion.

The economical and efficient commercial production of second-generation bioethanol requires fermentation microorganisms capable of entirely and rapidly utilizing all sugars in lignocellulosic hydrolysates. In this study, we developed a recombinant Saccharomyces cerevisiae strain, BLH510, through protoplast fusion and metabolic engineering to enhance its ability to co-ferment glucose, xylose, cellobiose, and xylooligosaccharides while tolerating various inhibitors commonly found in lignocellulosic hydrolysates. The parental strains, LF1 and BLN26, were selected for their superior glucose/xylose co-fermentation capabilities and inhibitor tolerance, respectively. The fusion strain BLH510 demonstrated efficient utilization of mixed sugars and high ethanol yield under oxygen-limited conditions. Under low inoculum conditions, strain BLH510 could completely consume all four kinds of sugars in the medium within 84 h. The fermentation produced 33.96 g/L ethanol, achieving 84.3% of the theoretical ethanol yield. Despite the challenging presence of mixed inhibitors, BLH510 successfully metabolized all four sugars above after 120 h of fermentation, producing approximately 30 g/L ethanol and reaching 83% of the theoretical yield. Also, strain BLH510 exhibited increased intracellular trehalose content, particularly under conditions with mixed inhibitors, where the intracellular trehalose reached 239.3 mg/g yeast biomass. This elevated trehalose content contributes to the enhanced stress tolerance of BLH510. The study also optimized conditions for protoplast preparation and fusion, balancing high preparation efficiency and satisfactory regeneration efficiency. The results indicate that BLH510 is a promising candidate for industrial second-generation bioethanol production from lignocellulosic biomass, offering improved performance under challenging fermentation conditions. Our work demonstrates the potential of combining protoplast fusion and metabolic engineering to develop superior S. cerevisiae strains for lignocellulosic bioethanol production. This approach can also be extended to develop robust microbial platforms for producing a wide array of lignocellulosic biomass-based biochemicals.

Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi.

Iterative metabolic engineering of Fusarium fujikuroi has traditionally been hampered by its low homologous recombination efficiency and scarcity of genetic markers. Thus, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas9) system has emerged as a promising tool for precise genome editing in this organism. Some integrated CRISPR/Cas9 strategies have been used to engineer F. fujikuroi to improve GA3 production capabilities, but low editing efficiency and possible genomic instability became the major obstacle. Herein, we developed a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in F. fujikuroi. This system, based on an autonomously replicating sequence, demonstrated the capability of a single plasmid harboring all editing components to achieve 100%, 75%, and 37.5% editing efficiency for single, double, and triple gene targets, respectively. Remarkably, even with a reduction in homologous arms to 50bp, we achieved a 12.5% gene editing efficiency. By employing this system, we successfully achieved multicopy integration of the truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase gene (tHMGR), leading to enhanced GA3 production. A key advantage of our plasmid-based gene editing approach was the ability to recycle selective markers through a simplified protoplast preparation and recovery process, which eliminated the need for additional genetic markers. These findings demonstrated that the single-plasmid CRISPR/Cas9 system enables rapid and precise multiple gene deletions/integrations, laying a solid foundation for future metabolic engineering efforts aimed at industrial GA3 production.

Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system.

The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.

Optimization of preparation conditions for Salsola laricifolia protoplasts using response surface methodology and artificial neural network modeling

BackgroundSalsola laricifolia is a typical C3–C4 typical desert plant, belonging to the family Amaranthaceae. An efficient single-cell system is crucial to study the gene function of this plant. In this study, we optimized the experimental conditions by using Box-Behnken experimental design and Response Surface Methodology (RSM)-Artificial Neural Network (ANN) model based on the previous studies.ResultsAmong the 17 experiment groups designed by Box-Behnken experimental design, the maximum yield (1.566 × 106/100 mg) and the maximum number of viable cells (1.367 × 106/100 mg) were obtained in group 12, and the maximum viability (90.81%) was obtained in group 5. Based on these results, both the RSM and ANN models were employed for evaluating the impact of experimental factors. By RSM model, cellulase R-10 content was the most influential factor on protoplast yield, followed by macerozyme R-10 content and mannitol concentration. For protoplast viability, the macerozyme R-10 content had the highest influence, followed by cellulase R-10 content and mannitol concentration. The RSM model performed better than the ANN model in predicting yield and viability. However, the ANN model showed significant improvement in predicting the number of viable cells. After comprehensive evaluation of the protoplast yield, the viability and number of viable cells, the optimal results was predicted by ANN yield model and tested. The amount of protoplast yield was 1.550 × 106/100 mg, with viability of 90.65% and the number of viable cells of 1.405 × 106/100 mg. The corresponding conditions were 1.98% cellulase R-10, 1.00% macerozyme R-10, and 0.50 mol L−1 mannitol. Using the obtained protoplasts, the reference genes (18SrRNA, β-actin and EF1-α) were screened for expression, and transformed with PEG-mediated pBI121-SaNADP-ME2-GFP plasmid vector. There was no significant difference in the expression of β-actin and EF1-α before and after treatment, suggesting that they can be used as internal reference genes in protoplast experiments. And SaNADP-ME2 localized in chloroplasts.ConclusionThe current study validated and evaluated the effectiveness and results of RSM and ANN in optimizing the conditions for protoplast preparation using S. laricifolia as materials. These two methods can be used independently of experimental materials, making them suitable for isolating protoplasts from other plant materials. The selection of the number of viable cells as an evaluation index for protoplast experiments is based on its ability to consider both protoplast yield and viability. The findings of this study provide an efficient single-cell system for future genetic experiments in S. laricifolia and can serve as a reference method for preparing protoplasts from other materials.

Enhanced Production of Sisomicin in Micromonospora inyoensis by Protoplast Mutagenesis and Fermentation Optimization.

Sisomicin is a broad-spectrum aminoglycoside antibiotic and is the precursor of netilmicin and plazomicin. However, the fermentation level of sisomicin is still low compared with other antibiotics, which restricts the application of sisomicin and its derivatives. In this study, to improve sisomicin production, breeding of high-yielding sisomicin strains was conducted with chemical mutagenesis using Micromonospora inyoensis OG-1 (titer, 1042 U·mL-1) as the starting strain. Protoplast preparation was conducted under optimal conditions, and protoplast mutagenesis was performed with a suitable concentration of diethyl sulfate. Subsequently, a high-yielding and genetically stable strain (H6-32) was obtained by screening, with a sisomicin titer of 1486 U·mL-1 (an increase of 42.6%). Finally, carbon and nitrogen sources were optimized to further improve sisomicin production, and a sisomicin titer of 1780 U·mL-1 was ultimately obtained by controlling the dissolved oxygen level at 30% in a 5-L fermenter, which to the best of our knowledge is the highest reported titer ever achieved by fermentation. Comparative genome analysis showed that a total of 13 genes in the genome of the mutant strain H6-32 were mutated compared to the original strain. This study not only provides a reference for further breeding of high-yielding strains and fermentation optimization, but also enhances our understanding of sisomicin production.

High-quality genome assembly and genetic transformation system of Lasiodiplodia theobromae strain LTTK16-3, a fungal pathogen of Chinese hickory.

Lasiodiplodia theobromae, as one of the causative agents associated with Chinese hickory trunk cankers, has caused huge economic losses to the Chinese hickory industry. Although the biological characteristics of this pathogen and the occurrence pattern of this disease have been well studied, few studies have addressed the related mechanisms due to the poor molecular and genetic study basis of this fungus. In this study, we sequenced and assembled L. theobromae strain LTTK16-3, isolated from a Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Phylogenetic analysis and comparative genomics analysis presented crucial cues in the prediction of LTTK16-3, which shared similar regulatory mechanisms of transcription, DNA replication, and DNA damage response with the other four Chinese hickory trunk canker-associated Botryosphaeria strains including, Botryosphaeria dothidea, Botryosphaeria fabicerciana, Botryosphaeria qingyuanensis, and Botryosphaeria corticis. Moreover, it contained 18 strain-specific protein clusters (not conserved in the other L. theobromae strains, AM2As and CITRA15), with potential roles in specific host-pathogen interactions during the Chinese hickory infection. Additionally, an efficient system for L. theobromae protoplast preparation and polyethylene glycol (PEG) -mediated genetic transformation was firstly established as the foundation for its future mechanisms study. Collectively, the high-quality genome data and the efficient transformation system of L. theobromae here set up the possibility of targeted molecular improvements for Chinese hickory canker control.IMPORTANCEFungi with disparate genomic features are physiologically diverse, possessing species-specific survival strategies and environmental adaptation mechanisms. The high-quality genome data and related molecular genetic studies are the basis for revealing the mechanisms behind the physiological traits that are responsible for their environmental fitness. In this study, we sequenced and assembled the LTTK16-3 strain, the genome of Lasiodiplodia theobromae first obtained from a diseased Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Further phylogenetic analysis and comparative genomics analysis provide crucial cues in the prediction of the proteins with potential roles in specific host-pathogen interactions during the Chinese hickory infection. An efficient PEG-mediated genetic transformation system of L. theobromae was established as the foundation for the future mechanisms exploration. The above genetic information and tools set up valuable clues to study L. theobromae pathogenesis and assist in Chinese hickory canker control.

Optimization of preparation and transformation of protoplasts from Populus simonii × P. nigra leaves and subcellular localization of the major latex protein 328 (MLP328)

BackgroundPopulus simonii × P. nigra is an ideal material for studying the molecular mechanisms of woody plants. In recent years, research on Populus simonii × P. nigra has increasingly focused on the application of transgenic technology to improve salt tolerance. However, the rapid characterization of gene functions has been hampered by the long growth cycle and exceedingly poor transformation efficiency. Protoplasts are an important tool for plant gene engineering, which can assist with challenging genetic transformation and the protracted growth cycle of Populus simonii × P. nigra. This study established an optimized system for the preparation and transformation of protoplasts from Populus simonii × P. nigra leaves, making genetic research on Populus simonii × P. nigra faster and more convenient. Major Latex Protein (MLP) family genes play a crucial role in plant salt stress response. In the previous study, we discovered that PsnMLP328 can be induced by salt treatment, which suggested that this gene may be involved in response to salt stress. Protein localization is a suggestion for its function. Therefore, we conducted subcellular localization analysis using protoplasts of Populus simonii × P. nigra to study the function of the PsnMLP328 gene preliminarily.ResultsThis study established an optimized system for the preparation and transformation of Populus simonii × P. nigra protoplasts. The research results indicate that the optimal separation scheme for the protoplasts of Populus simonii × P. nigra leaves included 2.5% cellulase R-10, 0.6% macerozyme R-10, 0.3% pectolyase Y-23, and 0.8 M mannitol. After enzymatic digestion for 5 h, the yield of obtained protoplasts could reach up to 2 × 107 protoplasts/gFW, with a high viability of 98%. We carried out the subcellular localization analysis based on the optimized transient transformation system, and the results indicated that the MLP328 protein is localized in the nucleus and cytoplasm; thereby proving the effectiveness of the transformation system.ConclusionIn summary, this study successfully established an efficient system for preparing and transforming leaf protoplasts of Populus simonii × P. nigra, laying the foundation for future research on gene function and expression of Populus simonii × P. nigra.

Construction of a Mycoviral Infectious Clone for Reverse Genetics in Botrytis cinerea.

The most important advances in our understanding of the viral life cycle, such as genome replication, packaging, transmission, and host interactions, have been made via the development of viral infectious full-length clones. Here, we describe the detailed protocols for the construction of an infectious clone derived from Botrytis virus F (BVF), a mycoflexivirus infecting the plant pathogenic fungus Botrytis cinerea, the determination of the complete sequence of the cloned mycovirus, the preparation of fungal protoplasts, and the transfection of protoplasts using transcripts derived from the BVF infectious clone.

An efficient targeted gene deletion approach for Cochliobolus heterostrophus using Agrobacterium tumefaciens-mediated transformation

Cochliobolus heterostrophus is a plant pathogenic fungus of southern corn leaf blight, which has been regarded as a model necrotrophic plant pathogen. Many methods have been developed to knock out targeted genes in C. heterostrophus, of which the most widely-used one is protoplast-mediated transformation. However, there are several problems of this method associated with protoplast preparation, DNA product, time consumption, or high cost. In this study, a highly efficient target gene deletion approach in C. heterostrophus was established and optimized, based on Agrobacterium tumefaciens-mediated transformation (ATMT); the transformation efficiency of this approach was 85–88 transformants per 105 conidia, and the homologous recombination efficiency was approximately 68.3%. Furthermore, six gene knockout mutants of C. heterostrophus were obtained using this ATMT method. The phenotypes of this fungus altered in the mutant strains, and the virulence of the mutants significantly reduced compared to of the wild type strain. Taken together, this ATMT system established in this study can be used as a genetic manipulation tool for C. heterostrophus, to better understand the functions of genes and its relation to virulence.

Preparation of leaf protoplasts from Populus (Populus × xiaohei T. S. Hwang et Liang) and establishment of transient expression system

Poplar, as a typical woody plant, is an ideal raw material for the production of lignocellulose biofuel. However, the longer life cycle is not conducive to the rapid identification of poplar genes. At present, protoplasts have been used for gene function identification and high-throughput analysis in many model plants. In this paper, a simplified and efficient protoplast isolation and transient expression system of Populus (Populus × xiaohei T. S. Hwang et Liang) is described. Firstly, we proposed an efficient enzyme hydrolysis method for isolating protoplasts from leaves of Populus × xiaohei. Secondly, we optimized the conditions of protoplast transformation mediated by PEG, and established an efficient transient expression system of protoplasts of Populus × xiaohei. Finally, the subcellular localization of three identified Dof transcription factors (PnDof19, PnDof20 and PnDof30) was also observed in the nucleus by using this scheme, which proved that the method was feasible. In general, this efficient method of protoplast isolation and transformation can be used for the study of protein subcellular localization and can be applied to other fields of molecular biology, such as protein interaction, gene activation and so on.

Establishment of protoplast preparation protocol and genetic transformation system for Sclerotiophoma versabilis

Establishment of protoplast preparation protocol and genetic transformation system for Sclerotiophoma versabilis

Optimization of Protoplast Preparation System from Leaves and Establishment of a Transient Transformation System in Apium graveolens

Protoplast culture and transformation technology offer a novel method for developing new plant varieties. Nonetheless, the effective preparation of protoplasts and transformation technology specific to celery has yet to be achieved. This study utilized celery seedling leaves as the primary materials to examine the key factors influencing protoplast isolation. The aim was to prepare leaf protoplasts with a high yield and of high quality and subsequently conduct transient gene transformation and expression. The findings indicated that the most effective procedure for isolating and purifying protoplasts was enzymatic digestion using an enzyme solution consisting of 2.0% cellulase, 0.1% pectolase, and 0.6 M mannitol for a duration of 8 h. Subsequently, the protoplasts were filtered through a 400-mesh sieve and purified through centrifugation at 200× g. Within this system, the overall protoplast yield was exceptionally high, reaching a viability rate of up to 95%. The transient transformation system yielded a maximum transformation efficiency of approximately 53%, as evaluated using the green fluorescent protein (GFP) as a reporter gene. The parameters of the transient transformation system were as follows: a protoplast concentration of 5 × 105 cells·mL−1, exogenous DNA concentration of 500 μg·mL−1, final concentration of PEG4000 at 40%, and transformation duration of 15 min. The transient transformation system was also utilized to further analyze the protein localization characteristics of the celery transcription factor AgMYB80. The findings indicated that AgMYB80 predominantly localizes in the nucleus, thereby confirming the reliability and effectiveness of the transient transformation system. This study successfully established an efficient system for isolating, purifying, and transforming celery protoplasts, and will serve as a basis for future studies on molecular biology and gene function.

Development of lead (Pb) tolerant strain by protoplast technology and their remediation.

The lead is poisonous metal and because of its chemical nature it acts as an environmental contaminant through the water or soil and it becomes toxic to humans. The toxicity of Pb occurs as a change in the conformation of nucleic acid and protein, inhibition of enzyme activity, disruption of membrane function and oxidative phosphorylation. For protoplast preparation, the removal of the cell wall and protoplast formation obtained by specific lytic enzyme. In cytoplasmic membrane, the envelope of bacteria consists of overlying cell wall. From hypertonic environment, the complete cell wall removal occurs due to which it maintains the osmotic integrity of the cell and produces the protoplast. In current work, protoplasts were produced by specific lytic enzyme (lysozyme and macerozyme), chemo fused (with the help of Polyethylene Glycol) and regenerated from strains Staphylococcus sp. and Bacillus sp. The fused protoplast was spherical in shape observed under microscopy. Colonies were screened on specific medium supplemented with Pb (Concentration at the rate of 1.5mM). One resistant colony (MICBT-1) was selected and further examined and applied for the transformation of Pb in the broth medium. The strain removed 98% of Pb at 1mM concentration. Next, sucrose containing medium was best which gives maximum protoplast regeneration. From various organisms, fusion technique has been used to combine the genes to create the strains having desired properties. This is a significant technique for engineering of bacterial strains for advantageous applied properties. Further MICBT-1 applied in artificially contaminated soil and removed maximally in exchangeable fraction (remains up to 0.05 mM). An efficient bioremediating agent for lead transformation from soil and water is expected to ease the ever-increasing problem. Further, it is needful to obtain new strain with the help of protoplast technology which can reduce the pollutant. This lead tolerant strain can be applied for bioremediation purposes in the Pb contaminated soil and water environment.

Highly efficient endosperm and pericarp protoplast preparation system for transient transformation of endosperm-related genes in wheat

Highly efficient endosperm and pericarp protoplast preparation system for transient transformation of endosperm-related genes in wheat

Development of a protoplast isolation system for functional gene expression and characterization using petals of Camellia Oleifera

Protoplasts preparation and purification have been frequently used in plant genetics and breeding studies, whereas application of protoplasts in woody plants is still in its infancy. Although transient gene expression using purified protoplasts is well-documented and widely used in model plants and agriculture crops, no instance of either stable transformation or transient gene expression in the woody plant Camellia Oleifera has as of yet been reported. Here, we developed a protoplast preparation and purification method using C. oleifera petals by optimizing osmotic condition with D-mannitol and polysaccharide-degrading enzyme concentrations for petal cell wall digestion, to reach a high efficiency of protoplast productivity and viability. The achieved protoplasts yield was approximately 1.42 × 107 cells per gram of petal material and the viability of protoplasts was up to 89%. In addition, we explored influencing factors of protoplast transformation, including concentrations of PEG4000 and plasmid DNA. The transformation efficiency of 81% could be reached under the optimized condition. This protoplast isolation and transient expression system were deployed to further identify the functional regulation of C. oleifera related genes and the subcellular distribution of their encoded products. In summary, the protoplast isolation and transient expression system we established using oil-tea tree petals is an efficient, versatile and time-saving system, being suitable for gene function characterization and molecular mechanism analysis.

Single-cell transcriptome atlas reveals spatiotemporal developmental trajectories in the basal roots of moso bamboo (Phyllostachys edulis).

Roots are essential for plant growth and development. Bamboo is a large Poaceae perennial with 1642 species worldwide. However, little is known about the transcriptional atlas that underpins root cell-type differentiation. Here, we set up a modified protocol for protoplast preparation and report single-cell transcriptomes of 14 279 filtered single cells derived from the basal root tips of moso bamboo. We identified four cell types and defined new cell-type-specific marker genes for the basal root. We reconstructed the developmental trajectories of the root cap, epidermis, and ground tissues and elucidated critical factors regulating cell fate determination. According to in situ hybridization and pseudotime trajectory analysis, the root cap and epidermis originated from a common initial cell lineage, revealing the particularity of bamboo basal root development. We further identified key regulatory factors for the differentiation of these cells and indicated divergent root developmental pathways between moso bamboo and rice. Additionally, PheWOX13a and PheWOX13b ectopically expressed in Arabidopsis inhibited primary root and lateral root growth and regulated the growth and development of the root cap, which was different from WOX13 orthologs in Arabidopsis. Taken together, our results offer an important resource for investigating the mechanism of root cell differentiation and root system architecture in perennial woody species of Bambusoideae.

Nondestructive and Quantitative Analysis of Cell Wall Regeneration in the Medicinal Macrofungus Ganoderma lingzhi by a Membrane-Fusing Fluorescent Probe.

Ganoderma is a prize medicinal macrofungus with a broad range of pharmaceutical values. To date, various attempts have been made to cultivate Ganoderma to improve the production of secondary metabolites with pharmacological activity. Among the adopted techniques, protoplast preparation and regeneration are indispensable. However, the evaluation of protoplasts and regenerated cell walls usually relies on electron microscopy assays, which require time-consuming and destructive sample preparation and merely provide localized information in the selected area. In contrast, fluorescence assays enable sensitive real-time detection and imaging in vivo. They can also be applied to flow cytometry, providing a collective overview of every cell in a sample. However, for macrofungi such as Ganoderma, the fluorescence analysis of protoplasts and regenerated cell walls is difficult owing to the hindrance of the homologous fluorescent protein expression and the lack of an appropriate fluorescence marker. Herein, a specific plasma membrane probe, TAMRA perfluorocarbon nucleic acid probe (TPFN), is proposed for the nondestructive and quantitative fluorescence analysis of cell wall regeneration. Exploiting the perfluorocarbon membrane-anchoring chains, hydrophilic nucleic acid linker, and fluorescent dye TAMRA, the probe is proven to be selective, soluble, and stable, enabling rapid fluorescence detection of a protoplast sample free of transgenic expression or immune staining. Based on the TPFN and flow cytometry techniques, a quantitative approach is constructed to monitor the process of cell wall growth in a fast, quantitative, and high-throughout manner, and the obtained results are consistent with those of conventional electron microscopy. In principle, with slight modifications or integration, the proposed probe and approach can be adapted to the preparation of cell protoplasts, inspection of cell wall integrity under environmental stress, and programmable membrane engineering for cytobiology and physiology research.