997 results about "Protoplast" patented technology

The invention relates to Agrobacterium mediated transformation of moulds comprising species of the fungal sub-divisions Ascomycotina, Basidiomycotina, Deuteromycotina, Mastigomycotina, and Zygomycotina.Examples demonstrate the transformation of Aspergillus awamori (both protoplasts and conidia), Aspergillus nidulans, Aspergillus niger, Colletotrichum gloeosporioides, Fusarium solani pisi, Neurospora crassa, Trichoderma reesei, Pleurotus ostreatus and Agaricus bisporus (all conidia), and Fusarium graminearum (both conidia and rehydrated freeze dried ATCC material).Especially for Aspergillus awamori the transformation frequency is much higher than with conventional mould transformation techniques.It has further been found that not only one expressable gene can be introduced into these moulds, but even multiple copies of such gene, which, moreover, can be targeted e.g. in the chromosomal pyrG locus, as exemplified for A. awamori. These multiple copies can be of a gene encoding a desired, homologous or heterologous, protein.

The invention aims to disclose a method for producing vitamin K2 by utilizing bacillus natto. Protoplast fusion is conducted on bacillus natto BS-53 highly yielding the vitamin K2 and bacillus natto CICC10262 having a high growth speed but a low vitamin K content, and fusants with parent advantages are screened out; obtained high-yield strains having a high growth speed are utilized to produce the vitamin K2 under an optimized fermentation condition; and isopropanol and n-hexane are utilized to extract and separate the vitamin K2. The growth speed of the strains and the yield of the vitamin K2 is increased and an effective method is provided for scale production of the vitamin K2, thereby realizing the aim of the invention.

The invention belongs to the field of plant tissue cultivation, and particularly relates to a method of cutting propagation of a peony immature stem. The method includes the steps of material selection, cutting cultivation, indoor management, oversummer maintenance, overwinter management and strong seedling cultivation. By using low-frequency ultrasonic waves to conduct processing, rooting can be promoted, plant cell division can be remarkably accelerated and induced, the cell growth is stimulated, protein synthesis of protoplast can be accelerated, and the adventitious bud reproducibility can be improved. Due to the fact that intermittent irradiation is adopted, no drastic cavitation effect can be produced, and partial damage to cells caused by the ultrasonic waves can be avoided. Due to the fact that cultivation and rooting are conducted in the dark cultivation process, growth inhibitor is greatly reduced, and rooting is facilitated. The complete and specific method including the steps of oversummer maintenance, auxiliary bud inducing, overwinter management and strong seedling cultication is provided, and therefore application of the peony rapid cutting propagation technology in actual production is promoted, and the method has significance in large-scale production of peony seedlings.

The invention discloses a method for preparing and regenerating a phomopasis asparagi protoplast, which relates to the technical field of fungus protoplast preparation and regeneration in cell engineering. The method comprises the following detailed operation steps of: (1) preparing a phomopasis asparagi conidium suspension; (2) preparing a fresh mycelium; (3) performing enzymolysis on mycelium cell walls; (4) separating the protoplast; and (5) regenerating the protoplast. The method is easy to operate, low in requirements on equipment and short in enzymolysis time and regeneration period, and effectively enhances the efficiency of preparation and regeneration of the protoplast.

The invention discloses a preparation method of a Calibrachoa protoplast. The method comprises the following steps: preprocessing tender leaves in 3-5DEG C clear water under dark conditions for 20-25h; carrying out enzymatic hydrolysis on the preprocessed blades by using an enzymatic hydrolysis solution; adding a CPW washing solution having a same volume with the enzymatic hydrolysis solution, shaking up, filtering by using a 300 mesh cell sieve, centrifuging at a rotating speed of 1100r/min for 2min, and absorbing the obtained supernatant; and adding a CPW washing solution to the obtained precipitate to wash a protoplast, centrifuging at a rotating speed of 1100r/min for 2min, collecting the obtained precipitate, adding a suspension, and suspending to obtain the protoplast with the output of (4.7-5.3)*10<6>/g and the activity of 85%, wherein the activity of 80% of the protoplast is maintained 9h later. The protoplast with high activity, high output and good state is obtained through a separation technology, protoplast culturing and plant regeneration conditions are met, and an experiment material is provided for plant protoplast fusion cultivation of new kinds.

The invention relates to a protoplast mutation breeding method for improving the cordycepin content of cordyceps militaris. The method comprises the following steps of activated culture of bacterial strains, solid culture of mycelia, liquid mycelia culture, mycelia enzymolysis, protoplast nitrosoguanidine (NTG) mutation, protoplast regeneration culture, regenerated bacteria subculture, fermentation culture, filtering, further washing of mycelia, measurement of cordycepin content in the mycelia, preservation of the bacteria strain with high cordycepin content, measurement of the genetic stability and the like. Due to the adoption of the method, the cordycepin content of the cordyceps militaris can be increased.

The invention discloses a process for producing gentamicin B and a cell protoplast fusion technique is adopted to select a new strain which takes the gentamicin B as a main component through breeding producing strains (micromonospora echinospora) of micronomicinin. The process for producing the gentamicin B which is disclosed by the invention has the advantages that the whole production cycle is shortened, the purity of the gentamicin B is increased, the purity of the gentamicin B which is produced is increased from original 85% to present 90%, the production cost is lowered, and the production capacity of the gentamicin B is extremely increased.

According to the invention, the culture time of paddy rice is shortened by adjusting the growing conditions of paddy rice seedlings; a paddy rice green protoplast is prepared by an enzyme method; the preparation method is optimized, so vacuum pumping and the usage of a highly toxic reagent are avoided; and thus the preparation method of the paddy rice green protoplast is simpler and safer. The protoplast prepared by the method of the invention has a high cell survival rate, and a high transformation rate, is applicable to various analysis such as subcellular localization of target gene codingprotein, protein interaction, western blotting and the like, and is especially applicable to instantaneous expression research of light/chloroplast related gene.

The invention a novel and efficient method for protoplast culture comprising the steps of isolating the protoplasts from a cell suspension, mixing the protoplasts with equal volumes of alginate solution, placing 40-50 mul of CaCl2 solution on a glass microslide, placing a mixture of protoplasts and alginate solution on the glass microslide and immediately covering by a glass coverglass, adding CaCl2 solution from the sides of coverglass, sliding down the coverglass towards one side and placing it in a petridish containing protoplast culture medium, sealing the petridishes, incubating it and transferring the extra thin alginate layer with celled colonies to regeneration medium for development of culture.

The invention provides a method for the genetic transformation of agrobacterium tumefaciens mediated sclerotinia sclerotiorum. The method comprises the following steps: mixing and co-culturing the agrobacterium tumefaciens containing binary vectors and the protoplasts of the sclerotinia sclerotiorum; and screening by using antibiotics to obtain transformants, the transformation efficiency of the method reaches 1/5.9*104 protoplasts. By using the protoplasts of the sclerotinia sclerotiorum as the receptor and using the agrobacterium tumefaciens as the mediator, the method of the invention overcomes the problem of the difficult genetic transformation of sclerotinia sclerotiorum, which is resulted from the multinucleate hypha cells and the difficulty in obtaining a large amount of ascospore, and has the advantages of simple method and stable transformation efficiency. Meanwhile, the invention further provides a simple and effective method for preparing the protoplasts of sclerotinia sclerotiorum.

The invention discloses application of a gene FoPDCD5 (Program Cell Death Protein 5) to regulation of pathogenicity of fusarium oxysporum, and belongs to the field of plant genetic engineering. According to the application of the gene FoPDCD5 to regulation of the pathogenicity of the fusarium oxysporum, the gene is knocked out from the fusarium oxysporum by a homologous recombination method, and aknockout mutant [delta]Fopdcd5 is obtained; by constructing a gene complement vector, the gene complement vector is introduced into a [delta]Fopdcd5 protoplast; and by using a method of random insertion, the gene is completed into the knockout mutant, a complement mutant [delta]Fopdcd5-com is obtained. A pathogenicity test shows that pathogenicity of the knockout mutant [delta]Fopdcd5 is significantly reduced; and pathogenicity of the complement mutant [delta]Fopdcd5-com is restored to a wild-type level. The application of the gene FoPDCD5 to regulation of the pathogenicity of the fusarium oxysporum proves that the FoPDCD5 is necessary for production of conidia of the fusarium oxysporum and response to oxidative stress and pathogenicity. The application is helpful to elucidate a pathopoiesia molecular mechanism of the fusarium oxysporum thoroughly, and target genes are provided for development of effective fungicides.

High yield antibiotics producing fungus strain, preparation method and use thereof are provided. The fungus strain is a mutant derived from Glarea lozoyensis, and deposited in CGMCC with the accession number of CGMCC 2933. The preparation method concludes following steps: (a) mixing the culture media of Glarea lozoyensis strain ATCC 20957 with nitrosoguanidine, and obtaining mixture a; (b) mixing lywallzyme with the mixture a, and obtaining protoplasts; (c) regenerating the protoplasts, and obtaining single clones; and (d) culturing the single clones, then obtaining the mutant strain. This fungus strain has stable genetic and producing property, produces little impurities in fermentation, and is suitable to be used in industry.

The invention belongs to the technical field of rape molecular breeding, and specifically relates to a breeding method of cabbage type rape woad oil cytoplasm male sterile line. The invention also relates to a protoplast fusion technology to recombinate the cytoplasm and nuclear genome of cabbage type rape-woad so as to establish cytoplasmic male sterility of recombinant cytoplasm. The breeding process comprises the following steps: obtaining the leaf meat protoplast of cabbage type rape-woad through separation and extraction, obtaining the protoplast fused somatic cell hybrid of cabbage type rape-woad through a cell fusion method; taking the obtained somatic cell hybrid of cabbage type rape-woad and leaf meat protoplast cell hybrid of cabbage type rape-woad as the female parent, taking cabbage type rape as the male parent, carrying out multi-generation backcross, and obtaining cytoplasmic male sterile line with carpelloid stamen; wherein the mitochondrion gene PCR amplification verifies that mitochondrial genome carries out recombination during the fusion process, thus the nucleoplasm becomes uneven, and the sterile character appears.

The invention relates to a separation and culturing method of Sinkiang saussurea protoplast, comprising four steps of culturing of sterile saussurea involucrate seedlings, inducement and differentiation of embryonic callus, separation and purification of plasmogen and culturing of the protoplast which are all applicable to the separation and culturing of the Sinkiang saussurea involucrate protoplast. The invention has simple and easily-operated method and lower requirements on the equipment and culturing conditions; in addition, the enzymolysis method is practical, the conditions for cell wallremoval are mild, and the separated protoplast has high yield and strong activity, and is easy for the cultuing of the saussurea involucrate protoplast and a plant regeneration, thus laying the foundations for the culturing and fusing of the protoplast as well as the germplasm resource innovation of the Sinkiang saussurea involucrate and the research of transgenic saussurea involucrate.

The invention relates to an Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method. The method comprises: pre-cultured Stevia rebaudiana stem apex explant is immersed into an Agrobacterium rhizogenes solution of pRI101-ANDNA plasmid carrying target gene, treatments such as infection, co-culture and sterilization are performed, the hairy root is cultured in a kanamycin-containing screening culture medium, the anti-kanamycin hairy root is formed on the explant through induction, the hairy root is induced to form callus, differentiation of adventitious bud is performed, the adventitious bud continuously grows to form the test-tube plantlet, and a PCR amplification method is adopted to identify the transgene plant carrying out the target gene. With the technical scheme, the target gene can be transformed into the Agrobacterium rhizogenes, such that the steps of separation from the protoplast and culture are eliminated compared with genetic transformation of polyethylene glycol and other chemical methods; and compared with genetic transformation of gene gun and other physical methods, the method of the present invention has characteristics of no requirement of expensive equipment, and simple operation technology.

This invention relates to a method for increasing the conversion efficiency of pollen tube pathway method by using agrobacterium tumefaciens virulence gene. The method comprises: combining or mixing agrobacterium tumefaciens virulence gene and the target gene to be converted (green fluorescent protein gene) in vitro, packaging with liposome, and converting plants, especially cotton by using pollen tube pathway method. The method combines agrobacterium-liposome-pollen tube pathway technique, and has obviously increased protoplast conversion efficiency. Plasmids are not only protected by liposome when entering embryo sacs, but also encapsulated by the toxic protein to be expressed when entering egg cells, which can improve the stability of exogenous DAN in cell conversion process, realize precise cutting in T-DNA region, and increase the conversion efficiency.