34 results about "Protoplast" patented technology

The present application relates to the field of plant tissue culture technology, and particularly discloses a lily stem cell abundance ex vivo regulation method. The lily bulb is used as the starting material, stem cell magnitude amplification and regulation culture can produce lily tissue organ materials with high stem cell abundance. The present application provides sterile lily materials with high stem cell abundance, which has the characteristics of strong regenerative ability, high proliferation rate, etc., is the basis for protoplast preparation and high-efficiency regeneration, and is suitable for different lily varieties, and provides technical support for cell engineering breeding, seedling scale breeding, etc.

This application belongs to the fields of microbiology and cell biology, and provides a method for directly preparing germination-promoting fungal protoplasts from orchid protocorms, comprising: (1) physical homogenization; (2) filtration; (3) removal of plant contents and residual cell walls; (4) purification of mycelial clusters; and (5) release and purification of fungal protoplasts. Through the rational design of homogenization and purification steps, this application achieves high-yield, high-purity, and high-vitality preparation of germination-promoting fungal protoplasts, providing excellent materials for orchid gene variety breeding and genetic engineering operations.

This invention discloses a gene editing method and application for *Trichoderma reesei* based on a ribonucleoprotein complex. It aims to overcome the problems of low editing efficiency, easy generation of heterozygotes, and risk of exogenous DNA integration in *Trichoderma reesei* using plasmid-based CRISPR / Cas9 systems. This invention combines a dual-site cleavage strategy guided by double sgRNAs with direct RNP delivery. The method involves: designing two guide RNAs spaced 50-700 bp apart for the target gene, pre-assembling them with Cas9 protein in vitro to form an active RNP complex; delivering this complex along with exogenous donor DNA to *Trichoderma reesei* protoplasts. The exogenous donor DNA contains a left homologous arm, an editing sequence, and a right homologous arm, wherein the left and right homologous arms are homologous to the genomic sequences outside the dual cleavage sites on the target gene determined by the double sgRNAs, respectively; upon entering the cell, the RNP complex introduces dual-site cleavage at the target site, achieving precise editing under the guidance of the exogenous donor DNA.

This invention discloses a stable and efficient method for preparing protoplasts from *Pseudolarix amabilis*. The method directly uses etiolated *Pseudolarix amabilis* seedlings as material to prepare protoplasts, requiring no other aseptic procedures, and the entire process is simple and convenient. Furthermore, by setting different buffer component ratios and a specific mannitol concentration, this invention achieves stable maintenance of the osmotic pressure of *Pseudolarix amabilis* seedling cells during the separation process. Experimental verification shows that the protoplast yield using this method is 1.32 × 10⁻⁶. 5 The protoplasts were prepared at a density of 1 protoplast per ml, with 95% of them intact and undamaged, resulting in significantly improved quality and activity. Furthermore, the enzymatic hydrolysate described in this invention overcomes the difficulty of enzymatic hydrolysis caused by the high degree of leatheriness in *Pseudolarix amabilis* leaf tissue. This method is suitable for widespread application in the field of herbaceous plant biotechnology.

The application discloses a chrysanthemum constitutive high-expression promoter and application thereof in gene editing. The nucleotide sequence of the promoter CmUbi is shown as SEQ ID NO. 4. Through double luciferase reporter experiments and stable genetic transformation experiments, it is proved that the expression activity of the CmUbi promoter in chrysanthemum protoplast and callus is significantly higher than that of commonly used CaMV 35S, AtRPS5a and corn ZmUbi promoters. A CRISPR / Cas9 gene editing vector is constructed by using the promoter, and a key gene of branch of hexaploid chrysanthemum is edited BRC1b , and a plurality of allele simultaneous mutation complete editing strains are successfully obtained, and the number of branches of the mutants is significantly increased. The endogenous high-activity promoter of the chrysanthemum provided by the application effectively solves the problems of low expression activity of an existing heterologous promoter in the chrysanthemum and poor gene editing efficiency of a polyploid, and provides an efficient and adaptive expression regulation element for chrysanthemum molecular breeding and functional genomics research.

The present application belongs to the field of plant biotechnology, and particularly relates to a Korean pine cell fusion method based on chemical induction combined with a magnetic bead method. The method comprises the following steps: S1, preparing first protoplasts and second protoplasts; S2, preparing passivated first protoplasts and passivated second protoplasts; S3, mixing the passivated first protoplasts with the passivated second protoplasts in equal volumes after the passivated first protoplasts are cultured with a sodium silicate aqueous solution to obtain mixed protoplasts; S4, performing electrofusion on the mixed protoplasts in a cell fusion instrument to obtain fusion products; and S5, screening hybrid cells from the fusion products by using a magnetic bead method. The present application adopts a double screening mechanism of chemical passivation and magnetic bead screening, inactivates parent cells by treating with iodoacetamide and rose Bengal B, and then specifically enriches hybrid cells by using the magnetic bead method, so that the acquisition rate and screening accuracy of hybrid cells are significantly improved.

This invention discloses a PEG-mediated genetic transformation method for *A. anthracnose* protoplasts. Through the preparation, transformation, and transformation of *A. anthracnose* protoplasts, and the screening of transformants, the hygromycin B resistance gene fragment was successfully transferred into the *A. anthracnose* genome, achieving successful genetic transformation of *A. anthracnose* protoplasts. More importantly, this invention establishes a new enzymatic digestion system and improves the culture mechanism in protoplast preparation. During the culture and screening of transformants, a more suitable resistance concentration is used, balancing the stringency of screening with the survival of transformants. This improves transformation efficiency, shortens the overall transformation process, and establishes a more efficient and convenient genetic transformation system for *A. anthracnose* protoplasts.

This invention belongs to the field of plant gene editing technology, specifically relating to a bifunctional gene editing system and its application in single-parent maize. The invention provides a bifunctional gene editing system comprising: a fusion protein expression cassette and a guide RNA combined expression cassette; wherein the guide RNA combined expression cassette includes: pegRNA associated with the maize D9 gene; and sgRNA associated with the maize YUC4 and YUC2 genes. Transient expression validation using the bifunctional gene editing system in maize protoplasts revealed that the system achieves a precise editing efficiency of 3.5% at the G571V site of the D9 gene, a knockout efficiency of 40%-65% for the YUC4 gene, and a knockout efficiency of 37%-54% for the YUC2 gene. This demonstrates that the technical solution of this application can simultaneously and efficiently achieve precise point mutation and gene knockout within the same cell, thus enabling its application in the synergistic improvement of lodging resistance in maize.

The application discloses application of a LtCel61A0 (Lasiodiplodia theobromae glycoside hydrolase family 61) gene in regulating growth, reproduction, pathogenicity and adversity resistance of Lasiodiplodia theobromae. The LtCel61A0 gene is derived from the L. theobromae, a knockdown or overexpression vector of the LtCel61A0 gene of the L. theobromae is constructed, and the vector is introduced into protoplast of the L. theobromae to obtain a knockdown or overexpression strain. It is found through experiments that the LtCel61A0 gene is significantly affected by the knockdown or overexpression, has an effect on the adversity resistance of the L. theobromae, and significantly reduces the pathogenicity of the L. theobromae to tea tree leaves. Therefore, the LtCel61A0 can be used as a fungicide target and a key protein of a pathogenic mechanism of a disease, is used for developing a medicament of a disease caused by the L. theobromae and cultivating a new variety of a disease-resistant plant, and has a wide application prospect in the prevention and treatment of plant pathogenic fungal diseases.

The application discloses a mutant strain of Grifola frondosa for improving yields of mycelium, protein and triterpenes and application thereof, and belongs to the technical field of microorganisms. The mutant strain of Grifola frondosa has a preservation number of CGMCC No. 42006. The mutant strain is obtained by using diethyl sulfate mutagenesis technology to treat protoplasts of Grifola frondosa, screening excellent strains of Grifola frondosa, continuously culturing the strains obtained through preliminary screening, and finally obtaining the mutant strain of Grifola frondosa with obviously improved yields of mycelium dry weight, intracellular protein and triterpenes. The mutant strain has good genetic stability and is suitable for being used as a strain for industrialized production of mycelium, triterpenes and protein of Grifola frondosa, thereby providing technical support for promoting development of the Grifola frondosa industry.

The application relates to the field of DNA nanotechnology, and discloses a method for sorting protoplasts by using DNA tetrahedral framework nucleic acid specific anchoring, as follows: cholesterol functionalized DNA tetrahedral framework nucleic acid is synthesized through a PCR reaction; high-activity protoplasts are obtained through enzymolysis; the DNA tetrahedral framework nucleic acid is uniformly mixed with an MMG buffer solution and then added to a protoplast resuspension solution for incubation; and the DNA tetrahedral framework nucleic acid is specifically anchored on the surface of the protoplasts to realize the sorting of the protoplasts. The DNA tetrahedral framework nucleic acid is functionalized with a cholesterol group by using the above method, the DNA tetrahedral framework nucleic acid is specifically anchored in protoplasts with stronger division ability, and the sorting of the protoplasts is successfully realized; the method is simple in operation, remarkable in effect, and low in cost, and successfully breaks through the lack of a sorting method in the current protoplast research, thereby providing a new direction and thought for protoplast research.

The invention concerns a method for producing a shoot of a plant comprising germline progenitor cells of a recalcitrant plant. The germline progenitor cells may be modified to comprise a mutation in a sequence of interest. The invention further pertains to plants obtainable by the method of the invention, wherein the plant preferably comprises at least the L2-meristem layer of the recalcitrant plant.

The application discloses a kit and a method for extracting nuclei in plant fruit kernels. The kit comprises a lysis solution, a washing solution and a purification solution. The lysis solution comprises 0.04%-0.4% w / v Triton X100, 0.2-0.4 mM spermine, 0.5-1 mM spermidine, 0.4-1.6 U / muL RNase inhibitor, 1-2.56 mM DTT, 10-15 mM NaCl, 1X PBS and 200-350 mM sucrose. The washing solution comprises 2% w / v BSA, 1.25-2.5 mM DTT and 0.2-0.4 U / muL RNase inhibitor. The purification solution comprises methanol and at least two Percoll solutions with different concentrations. The kit with specific components and the optimized extraction method can quickly obtain a high-purity nucleus suspension without extracting protoplasts, effectively remove impurities and secondary metabolites, reduce the risk of RNA degradation, and are simple to operate and low in cost.

This invention belongs to the field of gene editing technology for edible and medicinal fungi, and specifically relates to an antibiotic-free gene editing system based on a green fluorescent strain of *Pleurotus ostreatus*, its construction method, and its application. This invention constructs a *Pleurotus ostreatus* strain that stably expresses green fluorescent protein, and modulates the green fluorescent gene... egfp Editing was performed, and in the absence of resistance screening, with egfp sgRNAs were designed targeting the gene, and after in vitro cleavage verification, they were used to perform PEG-mediated protoplast transformation on green fluorescent strains. 113 potential transformants were randomly selected on regeneration medium for initial screening, and 11 transformants were obtained through genomic PCR verification. egfp The edited strain achieved an editing efficiency of 9.73%, and the edited strain lost its green fluorescence. The green fluorescent strain and gene editing system provided in this application lay the foundation for research on the gene function of *Pleurotus ostreatus*, provide technical support for the cultivation of superior varieties, and have significant practical implications for promoting safe breeding and high-quality development of the edible fungi industry.

The application provides a kind of preparation of red radish protoplast enzymatic hydrolysate and the preparation method and application of red radish protoplast, belong to the technical field of plant cytology.The enzymatic hydrolysate includes:1.0~1.5w / v% cellulase, 0.3~0.5w / v% dissociation enzyme and 0.3~0.5w / v% hemicellulase.The cellulase has the effect of degrading cellulose in plant cell wall, dissociation enzyme is used with cellulase, which is beneficial to separate plant tissue into single cell, and the main effect of hemicellulase is to degrade hemicellulose in plant cell wall.The enzymatic hydrolysate can synergistically promote the effect of red radish tissue cell wall lysis through the comprehensive effect of three enzymes, can better and faster destroy cell wall skeleton, is more conducive to the preparation of red radish protoplast and further maximally release protoplast, is conducive to improving the yield of protoplast, and can also keep the protoplast with high activity.

The present disclosure concerns a method of obtaining a composition having yeast proteins derived from yeast protoplasts. The present disclosure also includes compositions comprising yeast proteins derived from yeast protoplasts, edible products derived from said composition as well as usages thereof.

The application discloses a method for separating and purifying waxberry protoplasts. The method mainly comprises the following steps: culturing and obtaining sterile seedlings or cluster seedlings of the waxberry, and selecting tender new leaves at the top of the second to third leaves; cutting the leaves into strips and then placing the leaves in a permeation liquid for pretreatment; then placing the leaves in an enzymolysis liquid containing cellulase R-10, dissociation enzyme R-10 and pectinase Y-23, and performing enzymolysis under constant temperature, darkness and shaking conditions; and finally filtering through a cell sieve, centrifuging and resuspending to obtain the waxberry protoplasts. The yield of the protoplasts obtained by using the method is as high as 3.56x10 6 The application realizes efficient separation and purification of the waxberry protoplasts for the first time, and provides a key technical basis for molecular breeding and genetic improvement of the waxberry.

The application discloses application of a LtEgID9 (Lasiodiplodia theobromae glycoside hydrolase family 61) gene in regulation of pathogenicity and stress resistance of Lasiodiplodia theobromae. The LtEgID9 gene is derived from L. theobromae, a knockdown vector of the LtEgID9 gene of L. theobromae is constructed, and the vector is introduced into protoplasts of L. theobromae to obtain a knockdown strain. It is found through experiments that the knockdown of the LtEgID9 gene can have an effect on the stress resistance of L. theobromae without affecting the growth of L. theobromae, and significantly reduce the pathogenicity of L. theobromae to tea tree leaves. Therefore, the LtEgID9 can be used as a fungicide target and a key protein of a pathogenic mechanism of a disease, for development of a medicament for the disease caused by L. theobromae infection and cultivation of a new variety of disease-resistant plants, and has a wide application prospect in the prevention and treatment of plant pathogenic fungal diseases.

This invention generally pertains to means and methods for cultivation of plant protoplasts. Specifically, the invention pertains to a method for cultivating plant protoplasts, wherein the method at least comprises (a) providing an aqueous solution comprising plant protoplasts and a hydrogel-forming pectin, (b) causing the pectin to form a hydrogel comprising the plant protoplasts and (c) contacting the hydrogel comprising the plant protoplast with a first culture medium. The invention further pertains to a pectin hydrogel comprising plant protoplasts and a pectin hydrogel comprising clonal microcalli.

The application discloses a kind of based on needle and tender stem's Pinus massoniana protoplast preparation and its transient genetic transformation method, belong to plant cell biotechnology field.The application is by optimizing enzyme solution formula, using the enzyme solution containing 1%-3% cellulase and 0.1%-0.5% pectinase is handled Pinus massoniana seedling needle leaf or tender stem explant, successfully obtain high yield, high activity protoplast.Based on this, the application further establishes PEG-mediated transient transformation system: needle leaf protoplast is incubated in 30%PEG, 0.4M mannitol, 100mM CaCl2 Condition is incubated for 15 minutes, and transformation effect is best;And tender stem protoplast is incubated in 15%PEG, 0.6M mannitol, 50mM CaCl2 Condition is incubated for 45 minutes, and reach the highest genetic transformation efficiency.This application provides a kind of efficient, stable and with tissue adaptation's Pinus massoniana gene function research technical scheme.

The application belongs to the field of cell biology and particularly relates to a high-efficiency separation method of Angelica dahurica protoplasts. The method comprises the following steps: S1, culture of A. dahurica: selecting A. dahurica containing more than 3 basal leaves for dark culture, and the dark culture time is 20-60 days; S2, selection of protoplast material: selecting the yellowing leaf of A. dahurica treated in step S1 as the protoplast material; S3, separation of protoplasts: performing enzymolysis on the yellowing leaf obtained in step S2, wherein the enzymolysis solution used in the enzymolysis contains 1.3-1.7% cellulase, 0.6-0.8% macerozyme and 0.35-0.45 mol / L mannitol. The A. dahurica protoplasts obtained by the separation method provided in the application have the characteristics of high yield, cell integrity, few impurities and high gene transformation rate.

The invention discloses a preparation method of tomato leaf vacuole-free cytoplasm. The method comprises the following steps: cutting tomato leaves, putting the cut tomato leaves into an enzymolysis buffer solution containing cellulase, macerozyme, mannitol, potassium chloride, MES, calcium chloride, beta-mercaptoethanol and fetal calf serum, and carrying out vacuumizing enzymolysis to obtain protoplast; removing vacuoles through density gradient centrifugation, and collecting tiny protoplasts; and performing staged homogenate cracking and centrifugal purification to obtain the tomato leaf vacuole-free cytoplasm. The vacuole-free cytoplasm prepared by the invention has dsRNA cleavage activity, and can be used for tomato virus in-vitro research and plant cell-free system research and development.

The application discloses a culture method for propagating Brachythecium populeum, and belongs to the technical field of Brachythecium populeum tissue culture. The application uses sporangium as an explant to propagate Brachythecium populeum, and quickly obtains Brachythecium populeum spores with a high germination rate, establishes a regeneration system of Brachythecium populeum, lays a foundation for large-scale propagation of Brachythecium populeum, shortens the propagation cycle of Brachythecium populeum, and better maintains excellent properties. The application first establishes a method for culturing Brachythecium populeum by using spores, fills the blank of the prior art, and provides a technical basis for propagation, seed preservation, preparation of protoplasts, recombination experiments and gametophyte culture of Brachythecium populeum.

This invention provides a method for isolating cell nuclei from *Pinus tabuliformis* tissue, relating to the field of plant molecular and cell biology. The method uses *Pinus tabuliformis* callus tissue as explants, releases protoplasts using a specific enzymatic hydrolysis solution, lyses them using a specific lysis buffer, and finally filters and centrifuges to obtain *Pinus tabuliformis* cell nuclei. Using this invention, cell nuclei suitable for single-cell and histone modification library construction and sequencing can be rapidly isolated, providing a foundation for research on single-cell nuclear genetic material transcription analysis, tissue-specific analysis, and cell fate determination in gymnosperms, and offering research ideas for the future use of cell nuclei in coniferous genetic engineering.

The invention relates to a method for efficiently preparing and converting immature xylem protoplast of catalpa bungei. The invention particularly relates to an enzyme solution for preparing immature xylem protoplast of catalpa bungei as well as a preparation method and a conversion method of the enzyme solution. The preparation method provided by the invention can realize efficient preparation and conversion of the protoplast.

The application provides a hybrid potato seedling hypocotyl single cell extraction method and application thereof. According to the characteristics of the potato seedling hypocotyl single cell, the application significantly improves the extraction efficiency of protoplast by adjusting the amount of different cellulases in the enzyme solution and combining a slight extrusion mode. The method is simple and fast, does not need to use complex treatment and expensive reagents, and is convenient for popularization and application. In addition, the protoplast extracted by the method can be effectively used for subsequent sequencing and detection, and it is proved that the cell activity extracted by the method is high, and the method has wide application value.

This invention provides a method for efficient isolation of callus protoplasts from Brazilian bananas and its application, belonging to the field of plant cell biology. The method for isolating Brazilian banana protoplasts involves: cutting unexpanded heart leaves of Brazilian bananas into strips and mixing them with an enzymatic hydrolysate at a ratio of 4-5 g: 15 mL for enzymatic hydrolysis. The enzymatic hydrolysate comprises the following components at the following concentrations: cellulase 1-1.5 w / v%, pectinase 0.2-0.4 w / v%, mannitol 0.35-0.45 M, KCl 18-22 mM, MES 18-22 mM, and CaCl₂. 2 8~12 mM, BSA bovine serum albumin 0.08~0.12%. This invention, by improving the enzymatic hydrolysate formulation, significantly enhanced the viability of protoplasts from the Brazilian banana plantation, achieving a protoplast yield of 3.1 × 10⁻⁶. 6 g ‑1 The viability of the protoplasts prepared by this invention is as high as 92%. These protoplasts can be used for subcellular localization and bimolecular fluorescence complementation experiments, laying a solid foundation for gene function research and molecular breeding work in the Musa genus, and also demonstrating good potential feasibility for their application in plant regeneration.

This invention belongs to the field of edible fungi genetic breeding technology, and relates to a Cordyceps militaris strain that does not produce aspergillin, with the preservation number CGMCCNo.40266. This strain does not contain the CCM_02059 gene, CCM_02060 gene, or any of the CCM_02059 to CCM_02060 genes. It also relates to a method for creating this strain through CRISPR / Cas9 gene editing, comprising the following steps: S1, determining and synthesizing the sgRNA nucleotide sequence of the target gene; S2, determining and synthesizing the nucleotide sequences of the upstream and downstream homologous arms of the target gene; S3, ligating the sgRNA nucleotide sequence described in S1 and the homologous arm nucleotide sequences described in S2 into the pAMA1-Cas9-hyg vector to construct a knockout vector; and S4, transforming the knockout vector described in S3 into Cordyceps militaris protoplasts via PEG-mediated transformation to obtain a Cordyceps militaris strain with the target gene knocked out. This invention utilizes CRISPR / Cas9 editing technology to edit the gene cluster of inoculin synthesis in Cordyceps militaris, disrupting the inoculin synthesis pathway and breeding Cordyceps militaris strains that do not produce inoculin and have no foreign gene insertions, which is of great significance to the development of the Cordyceps militaris industry.