Protoplast Transformation System Research Articles

Establishment of an Efficient Protoplast Isolation and Transfection Method for Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of E. ulmoides gene function has been severely hampered by the limitations of genetic transformation methods and breeding cycles. The polyethylene glycol (PEG)-mediated protoplast transformation system is a multifunctional and rapid tool for the analysis of functional genes in vivo, but it has not been established in E. ulmoides. In this study, a large number of highly active protoplasts were isolated from the stems of E. ulmoides seedlings by enzymatic digestion, and green fluorescent protein expression was facilitated using a PEG-mediated method. Optimal enzymatic digestion occurred when the enzyme was digested for 10 h in an enzymatic solution containing 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol. The active protoplast yield under this condition was 1.13 × 106 protoplasts/g fresh weight, and the protoplast activity was as high as 94.84%. This study established the first protoplasm isolation and transient transformation system in hard rubber wood, which lays the foundation for subsequent functional studies of E. ulmoides genes to achieve high-throughput analysis, and provides a reference for future gene function studies of medicinal and woody plants.

Polyethylene glycol–mediated genetic transformation and fluorescent labelling of Colletotrichum sublineola for studying sorghum anthracnose pathogenicity

AbstractAnthracnose, a disease primarily caused by the fungus Colletotrichum sublineola, affects sorghum production in China. However, the infection mechanisms and complex host interactions of C. sublineola remain to be elucidated. Thus, this study aimed to establish and optimize a protoplast transformation system of C. sublineola so as to lay a foundation for further research on the infection mechanisms and complex host interactions of C. sublineola. A genetic transformation system was established and optimized for C. sublineola strain HX‐5‐1 and used to introduce fluorescent reporter genes, namely, green fluorescent protein (GFP) and mCherry. Results showed that the growth of HX‐5‐1 was completely inhibited on a regenerating medium containing 300 μg mL−1 hygromycin B, indicating that hygromycin B acted as a transformant‐selective marker. HX‐5‐1 produced the highest yield of protoplasts when the mycelia were cultured for 60 h and dissolved in a 0.1% lysozyme solution (0.7 M NaCl) for 3 h. Subsequently, a 106 mL−1 protoplast solution transformed using 5 μg of plasmid DNA exhibited the highest transformation efficiency. Laser confocal microscopy showed that GFP and mCherry fluorescent signals were successfully expressed in the mycelia and spores of HX‐5‐1, indicating that they were stably inherited in the transformants and did not affect colony morphology, growth rate, or pathogenicity. In conclusion, we successfully established a genetic transformation system for C. sublineola and employed it to obtain stable fluorescent protein expression. This study may serve as a foundation for understanding the infection process of C. sublineola.

Cotyledon peeling method for passion fruit protoplasts: a versatile cell system for transient gene expression in passion fruit (Passiflora edulis).

Passion fruit (Passiflora edulis) is a perennial evergreen vine that grows mainly in tropical and subtropical regions due to its nutritional, medicinal and ornamental values. However, the molecular biology study of passion fruit is extremely hindered by the lack of an easy and efficient method for transformation. The protoplast transformation system plays a vital role in plant regeneration, gene function analysis and genome editing. Here, we present a new method ('Cotyledon Peeling Method') for simple and efficient passion fruit protoplast isolation using cotyledon as the source tissue. A high yield (2.3 × 107 protoplasts per gram of fresh tissues) and viability (76%) of protoplasts were obtained upon incubation in the enzyme solution [1% (w/v) cellulase R10, 0.25% (w/v) macerozyme R10, 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] for 2 hours. In addition, we achieved high transfection efficiency of 83% via the polyethylene glycol (PEG)-mediated transformation with a green fluorescent protein (GFP)-tagged plasmid upon optimization. The crucial factors affecting transformation efficiency were optimized as follows: 3 μg of plasmid DNA, 5 min transfection time, PEG concentration at 40% and protoplast density of 100 × 104 cells/ml. Furthermore, the established protoplast system was successfully applied for subcellular localization analysis of multiple fluorescent organelle markers and protein-protein interaction study. Taken together, we report a simple and efficient passion fruit protoplast isolation and transformation system, and demonstrate its usage in transient gene expression for the first time in passion fruit. The protoplast system would provide essential support for various passion fruit biology studies, including genome editing, gene function analysis and whole plant regeneration.

Using A Protoplast Transformation System to Enable Functional Studies in Mangifera indica L.

Mangoes (Mangifera indica 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 × 105 cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaCl2, 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.

Direct leaf-peeling method for areca protoplasts: a simple and efficient system for protoplast isolation and transformation in areca palm (Areca catechu)

BackgroundAreca palm (Areca catechu) is a woody perennial plant of both economical and medicinal importance grown in tropical and subtropical climates. Yet, the molecular biology study of areca palm is extremely impeded by its unavailability of a transformation method. An efficient protoplast isolation and transformation system could be highly desirable to overcome this barrier.ResultsHere, we described a simple and efficient method for protoplast isolation and transformation from the perennial plant areca palm. A high yield of protoplasts (2.5 × 107 protoplasts per gram of fresh leaf tissues) was obtained from the fresh light green leaflet from the newly-emerged leaf digested overnight in the enzyme solution [2% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, 0.7 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] by the direct leaf-peeling method. The isolated areca protoplasts maintain viability of 86.6% and have been successfully transformed with a green fluorescent protein (GFP)-tagged plasmid (pGreen0029-GFP, 6.0 kb) via the polyethylene glycol (PEG)-mediated transformation. Moreover, the mannitol concentration (optimal: 0.7 M) was determined as a key factor affecting areca protoplast isolation. We also demonstrated that the optimal density of areca protoplast for efficient transformation was at 1.0–1.5 × 106 cells/ml. With the optimization of transformation parameters, we have achieved a relatively high transformation efficiency of nearly 50%.ConclusionWe have established the first efficient protocol for the high-yield isolation and transformation of areca palm protoplasts. This method shall be applied in various biological studies of areca palm, such as gene function analysis, genome editing, protein trafficking and localization and protein–protein interaction. In addition, the protoplast system offers a great genetic transformation approach for the woody perennial plant-areca palm. Moreover, the established platform may be applied in protoplast isolation and transformation for other important species in the palm family, including oil palm and coconut.

Pectate Lyase Genes Abundantly Expressed During the Infection Regulate Morphological Development of Colletotrichum camelliae and CcPEL16 Is Required for Full Virulence to Tea Plants.

Colletotrichum camelliae is the dominant species causing foliar diseases of tea plants (Camellia sinensis) in China. Transcriptome data and reverse transcription-quantitative PCR (qRT-PCR) analysis have demonstrated that the pectate lyase genes in C. camelliae (CcPELs) were significantly upregulated during infectious development on tea plants (cv. Longjing43). To further evaluate the biological functions of CcPELs, we established a polyethylene glycol (PEG)-mediated protoplast transformation system of C. camelliae and generated targeted deletion mutants of seven CcPELs. Phenotypic assays showed that the genes contribute to mycelial growth, conidiation, and appressorium development. The polypeptides encoded by each CcPEL gene contained a predicted N-terminal signal peptide, and a yeast invertase secretion assay suggested that each CcPEL protein could be secreted. Cell death-suppressive activity assays confirmed that all seven CcPELs did not suppress Bax-induced cell death in tobacco leaf cells. However, deletion of CcPEL16 significantly reduced necrotic lesions on tea leaves. Taken together, these results indicated that CcPELs play essential roles in regulating morphological development, and CcPEL16 is required for full virulence in C. camelliae. IMPORTANCE In this study, we first established a PEG-mediated protoplast transformation system of C. camelliae and used it to investigate the biological functions of seven pectate lyase genes (CcPELs) which were abundantly expressed during infection. The results provided insights into the contributions of pectate lyase to mycelial growth, conidial production, appressorium formation, and the pathogenicity of C. camelliae. We also confirmed the secretory function of CcPEL proteins and their role in suppressing Bax-induced cell death. Overall, this study provides an effective method for generating gene-deletion transformants in C. camelliae and broadens our understanding of pectate lyase in regulating morphological development and pathogenicity.

Functional Characterization of Two Cell Wall Integrity Pathway Components of the MAPK Cascade in Phomopsis longicolla.

The pathogenic fungus Phomopsis longicolla causes numerous plant diseases, such as Phomopsis seed decay, pod and stem blight, and stem canker, which seriously affect the yield and quality of soybean production worldwide. Because of a lack of technology for efficient manipulation of genes for functional genomics, understanding of P. longicolla pathogenesis is limited. Here, we developed an efficient polyethylene glycol-mediated protoplast transformation system in P. longicolla that we used to characterize the functions of two genes involved in the cell wall integrity (CWI) pathway of the mitogen-activated protein kinase (MAPK) cascade, including PlMkk1, which encodes MAPK kinase, and its downstream gene PlSlt2, which encodes MAPK. Both gene knockout mutants ΔPlMkk1 and ΔPlSlt2 displayed a reduced growth rate, fragile aerial hyphae, abnormal polarized growth and pigmentation, defects in sporulation, inadequate CWI, enhanced sensitivity to abiotic stress agents, and significant deficiencies in virulence, although there were some differences in degree. The results suggest that PlMkk1 and PlSlt2 are crucial for a series of growth and development processes as well as pathogenicity. The developed transformation system will be a useful tool for additional gene function research and will aid in the elucidation of the pathogenic mechanisms of P. longicolla. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera

BackgroundCamellia 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.ResultsThe 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 × 107/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 × 107/g·FW and 2.6 × 107/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.ConclusionIn 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.

Establishment of a Protoplasts-Based Transient Expression System in Banana (Musa spp.)

The breeding of triploid banana cultivars with improved traits, such as yield and disease resistance, remains a major challenge for breeders. One reason is that the molecular study and functional gene analysis in bananas fall behind due to the difficulties of its genetic manipulation. The plant protoplast-based transient transformation has been documented and widely used as a versatile and convenient system for functional gene analysis in many plant species. However, an efficient high-quality protoplast isolation and transformation system is still lacking for bananas. Here, we established an efficient protoplast isolation and transformation method for bananas by selecting proper source materials, optimizing conditions for enzymatic hydrolysis and PEG-mediated transfection. We found the best source materials for banana protoplasts’ isolation are young suckers, which give a yield of protoplasts ranging from 2.5 × 106 to 10.1 × 107 g−1 fresh weight after 5 to 6 h of enzymolysis. The yield is sufficient for most assays that have been established in protoplasts-based systems, such as protein subcellular localization and protein interaction assays. Moreover, using the established transient gene expression system in banana protoplasts, we validated the subcellular localization of Arabidopsis VESICLE SORTING RECEPTOR 1 (VSR1) and the protein self-interaction of Arabidopsis CNGC20 on the cell membrane. The results indicated this system works well and could be routinely used for the functional characterization of banana genes.

Construction of protoplast genetic transformation system for Mycena--symbiont of Gastrodia elata

Mycena, a symbiont of Gastrodia elata, promotes seed germination of G. elata and plays a crucial role in the sexual reproduction of G. elata. However, the lack of genetic transformation system of Mycena blocks the research on the interaction mechanism of the two. In order to establish the protoplast transformation system of Mycena, this study analyzed the protoplast enzymatic hydrolysis system, screened the resistance markers and regeneration medium, and explored the transient transformation. After hydrolysis of Mycena hyphae with complexes enzymes for 8 h and centrifugation at 4 000 r·min~(-1), high-concentration and quality protoplast was obtained. The optimum regeneration medium for Mycena was RMV, and the optimum resistance marker was 50 mg·mL~(-1) hygromycin. The pLH-HygB-HuSHXG-GFP-HdSHXG was transformed into the protoplast of Mycena which then expressed GFP. The established protoplast transformation system of Mycena laid a foundation for analyzing the functional genes of Mycena and the molecular mechanism of the symbiosis of Mycena and G. elata.

Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2.

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

Cloning of transcription factor PcFBA-1 in Pogostemon cabin and its interaction with FPPS promoter

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.

An efficient genetic manipulation protocol for dark septate endophyte Falciphora oryzae.

To investigate the protoplast preparation and transformation system of endophytic fungus Falciphora oryzae. F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9M KCl solution and 10mgmL-1 glucanase at 30 °C with shaking at 80rpm for 2-3h. When the protoplasts were plated on a regenerations-agar medium containing 1M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired green fluorescent protein-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots. This study provided a protoplast transformation system of F. oryzae, creating opportunities for future genetic research in other endophytic fungi.

Enhanced polyethylene glycol (PEG)-mediated protoplast transformation system for the phytopathogenic fungus, Ganoderma boninense.

The basidiomycete fungus, Ganoderma boninense, has been identified as the main causal agent of oil palm basal stem rot (BSR) disease which has caused significant economic losses to the industry especially in Malaysia and Indonesia. Various efforts have been initiated to understand the disease and this plant pathogen especially at the molecular level. This is the first study of its kind on the development of a polyethylene glycol (PEG)-mediated protoplast transformation system for G. boninense. Based on the minimal inhibitory concentration study, 60µg/mL and above of hygromycin were effective to completely inhibit G. boninense growth. Approximately 5.145×107cells/mL of protoplasts with the viability of 97.24% was successfully obtained from G. boninense mycelium tissue. The PEG-mediated G. boninense protoplast transformation using 1µg of transformation vector, 25% of PEG solution, 10min of pre-transformation incubation, and 30min of post-transformation incubation has improved the transformation rate as compared with the previous reported protocols for other basidiomycete fungi. Optimization of four transformation parameters has improved the transformation efficiency of G. boninense from an average of 2 to 67 putative transformants. The presence of hygromycin phosphotransferase (hpt) and enhanced green fluorescent protein (eGFP) genes in the putative transformants was detected by PCR and verified by gene sequence analysis. Southern hybridization result further confirmed the integration of hpt gene in G. boninense transformants, and the green fluorescent signal was detected in the G. boninense transformants under the microscopic analysis. The establishment of this transformation system will accelerate the gene function studies of G. boninense especially those genes that may contribute to the pathogenesis of this fungus in oil palm.

Advances allowing feasible pyrG gene editing by a CRISPR-Cas9 system for the edible mushroom Pleurotus eryngii

For decades, the edible mushroom Pleurotus eryngii (P. eryngii) has been cultivated as important raw materials for food and pharmaceutical industries in most of Asian countries, especially in China. Unfortunately, the generation and improvement of new cultivars are very difficult since there are many barriers which have not been solved thoroughly by gene editing tools, even though the CRISPR-Cas9 technique has been widely applied in other species. In this study, we identified the point-mutated variant of the endogenous sdhB gene (cbxr) as a more stable selection marker than hygromycin B resistance gene (hph) in P. eryngii. Furthermore, using a codon-optimized Cas9, a predicted native U6 promoter-guided sgRNA, as well as an optimized protoplast transformation system, a highly efficient pyrG gene editing system was established in P. eryngii, that incorporated varied insertions and deletions (indels) by non-homologous end joining (NHEJ) and homology-directed repair (HDR). Findings for a successful targeted gene editing strategy in the edible mushroom P. eryngii may open a new chapter for the improvement of edible mushroom cultivars.

Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana

BackgroundTo date, CRISPR/Cas9 RNP editing tools have not been applied to the genetic modification of banana. Here, the establishment of a PEG-mediated banana protoplast transformation system makes it possible to build an efficient DNA-free method for a site-directed mutagenesis system.ResultsProtoplasts constitute a versatile platform for transient expression in plant science. In this study, we established a PEG-mediated banana protoplast transformation system. This system was further optimized for successfully delivering CRISPR/Cas9 and CRISPR/Cas12a plasmids and CRISPR/Cas9 ribonucleoproteins (RNPs) for targeted delivery of the PDS gene into banana protoplasts. Specific bands were observed in PCR-Restriction Enzyme Digestion (PCR-RE) assays, and Sanger sequencing of single clones further confirmed the occurrence of indels at target sites. Deep amplicon sequencing results showed that the editing efficiency of the CRISPR/Cas9 system was higher than that of the other two systems.ConclusionsThe PEG-mediated banana protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in banana. The application of the CRISPR/Cas9 RNP system enables the generation of banana plants engineered by DNA-free gene editing.

An Efficient and Economical Protocol for Isolating, Purifying and PEG-Mediated Transient Gene Expression of Chinese Kale Hypocotyl Protoplasts.

In this study, we report the isolation and purification of protoplasts from Chinese kale (Brassica oleracea var. alboglabra) hypocotyls, and their transient gene expression transformation and subcellular localization of BaMYB75 (Bol042409). The upshot is that the vintage protocol included 5-d hypocotyls that were enzymatically hydrolyzed for 8 h in enzyme solution (3.0% cellulase, 0.5% pectolase, and 0.5 M mannitol), and the protoplasts were purified by precipitation. The total yield of protoplasts was 8 × 105 protoplast g−1 fresh weight, and the protoplasts’ viability was 90%. The maximum transformation efficiency obtained by using green fluorescent protein (GFP) as a detection gene was approximately 45% when the polyethylene glycol (PEG)4000 concentration was 40% and transformation time was 20 min. In addition, BaMYB75 was ultimately localized in the nucleus of Chinese kale hypocotyl protoplasts, verifying the validity and reliability of this transient transformation system. An effective and economical hypocotyl protoplast isolation, purification, and transformation system was established for Chinese kale in this study. This effectively avoided interference of chloroplast autofluorescence compared to using mesophyll cells, laying the foundation for future research in the molecular biology of Brassica vegetables.

An efficient mesophyll protoplast isolation, purification and PEG-mediated transient gene expression for subcellular localization in Chinese kale

Abstract Transient gene expression serves as a valuable tool for gene functional study in plants. Here we reported protoplast isolation and purification from the plantlet young leaves of Chinese kale (Brassica oleracae var. alboglabra Bailey), and their transient gene expression with polyethylene glycol (PEG)-mediated transformation and subcellular localization of phytoene desaturase (BaPDS1). The procedures of isolation andtransformation ofmesophyll protoplasts derived from Chinese kale were optimized, and the influencing factors were analyzed. The results showed that the optimal protocol of protoplast isolation and purification was initialized by digestion in enzyme solution (2.0% cellulase, 0.1% pectolase, and 0.6 M mannitol) for 9 h. After filtered through 400 mesh and centrifuged at 179 ×g for purification, the total yield of protoplast reached as high as 6.04 × 107 protoplasts g−1 fresh weight (FW) and the viability of the protoplasts was up to 95%. A maximum transformation efficiency of approximately 30% measured by using green fluorescent protein (GFP) as a detecting gene was obtained when PEG4000 was at a final concentration of 40% and transformation time was set to 15 min. In addition, the subcellular localization of BaPDS1 in Chinese kale was targeted to the chloroplast, confirming the efficiency and reliability of this transient transformation system. Taken together, an efficient protoplast isolation, purification and transformation system in Chinese kale was established in this study, laying a foundation for future research in molecular biology and gene function in Chinese kale and other Brassica vegetables.

Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici.

Take-all, caused by Gaeumannomyces tritici, is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors—temperature, enzyme lysis time, and concentration of the lysing enzyme—were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 107 protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46–54 transformants/μg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici. An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.

Targeted Gene Deletion in Cordyceps militaris Using the Split-Marker Approach.

The macrofungus Cordyceps militaris contains many kinds of bioactive ingredients that are regulated by functional genes, but the functions of many genes in C. militaris are still unknown. In this study, to improve the frequency of homologous integration, a genetic transformation system based on a split-marker approach was developed for the first time in C. militaris to knock out a gene encoding a terpenoid synthase (Tns). The linear and split-marker deletion cassettes were constructed and introduced into C. militaris protoplasts by PEG-mediated transformation. The transformation of split-marker fragments resulted in a higher efficiency of targeted gene disruption than the transformation of linear deletion cassettes did. The color phenotype of the Tns gene deletion mutants was different from that of wild-type C. militaris. Moreover, a PEG-mediated protoplast transformation system was established, and stable genetic transformants were obtained. This method of targeted gene deletion represents an important tool for investigating the role of C. militaris genes.