32 results about "Subculture (biology)" patented technology

The invention discloses a method for producing a hog cholera C-strain virus by culturing ST Cells in low serum, and belongs to the technical field of veterinary medicine and biology. The method comprises the following steps: S1, preparing a cell subculture and culture solution; S2, domesticating cells to adapt to a low-serum culture medium; S3, domesticating cells to adapt to a low-serum culture environment; S4, breeding a cell virus strain; S5, breeding prepared venom. The method for producing the hog cholera C-strain virus by culturing ST Cells in low serum provided by the invention can remarkably reduce the production cost and can also improve the downstream purifying efficiency, and can quickly and stably expand production scale, so that the quality is easy to balance and stabilize.

The invention discloses a rapid propagation method for performing tissue culture by using a taro stem tip, belonging to the technical field of biology. The method comprises the following steps of: selecting an aseptic taro stem tip as an explant; and performing a series of operating steps such as bud induction, subculture multiplication, rooting culture and the like to form a complete test-tube plantlet. The method comprises treatment methods for culture medium components at each abovementioned stage and induction culture. A seedling with two or more leaves can grow by performing primary culture on the taro stem tip for about 20 days, so that the process of primary culture is accelerated greatly; the growth coefficient is 4.3 on average after one-time secondary culture, secondary culture can be performed for five times generally to culture 1,470 (4.35) seedlings, and two batches can be cultured every year to form 2,940 tissue culture seed tuber seedlings, so that large-scale commercial production of taros is facilitated; and the method is also easy for obtaining regeneration plants with high genetic stability and low aberration rates.

The invention belongs to the technical field of metabolic engineering and synthetic biology, and relates to genetic engineering bacteria producing beta-carotene. The genetic engineering first uses wild-type yeast as host bacteria, and strong promoter-expressed heterologous genes crtYB, crtI, crtE, and a catalytic region (tHMG1) gene of a rate-limiting step HMG1 are separately inserted into a genome of the host bacteria to construct initial recombinant saccharomyces cerevisiae producing beta-carotene; and then sterol acyltransferase genes (ARE1 and ARE2 genes) are overexpressed in the initial recombinant saccharomyces cerevisiae, and genes (PAH1, DPP1 and LPP1 genes) encoding phosphatidic acid phosphatase activity are knocked out to perform chassis microorganism transformation on the recombinant saccharomyces cerevisiae. The obtained recombinant saccharomyces cerevisiae can produce beta-carotene with relatively high yield and relatively low cost, the strain is stable during subculture,and no reverse mutations can appear.

The invention discloses an efficient method for inducing somatic embryos of white-flower camellia oleifera abel Ganwu-2, and relates to methods for inducing the somatic embryos of the white-flower camellia oleifera abel in the technical field of biology. The method comprises the following steps of (1), selecting explants; (2), disinfecting the explants; (3), preparing a callus induction culture medium; (4), performing callus induction and subculture; and (5), performing somatic embryo induction. According to a formula of the culture medium, the mature embryos of the camellia oleifera abel serve as the explants, and a system for inducing the somatic embryos of the white-flower camellia oleifera abel Ganwu-2 is established for the first time; the system for inducing the somatic embryos of the white-flower camellia oleifera abel Ganwu-2 is efficiently established, wherein multiple indexes are higher than those of the same kind of white-flower camellia oleifera abel; and on the basis, a certain foundation is laid for overcoming a genetic transformation system of the white-flower camellia oleifera abel, and the development of camellia oleifera abel breeding career is better promoted.

The invention relates to a silver crucian carp back fin cell line. The silver crucian carp back fin cell line is obtained through primary culture and subculture conducted on the back fin of a silver crucian carp, and a used cell culture fluid is prepared by using an M199 culture medium. The invention further provides an establishing method and application of the silver crucian carp back fin cell line. The silver crucian carp back fin cell line has the advantages that the established silver crucian carp back fin cell line can be subjected to continuous passage, and a large amount of silver crucian carp back fin cell lines can be provided. The silver crucian carp back fin cell line can be directly applied to research on silver crucian carp immunity related function genes and can be applied to a silver crucian carp virus infection experiment, a large amount of manpower and material resources are saved, and effect is better. The silver crucian carp back fin cell line can be subjected to multigelation and can be applied to important research in cell biology, virology, molecular biology, genetics, immunology and the like.

The invention discloses an efficiency and rapid transgenic method for chrysanthemum, belonging to the fields of genetic breeding of the chrysanthemum and molecular biology. The method comprises the following steps: (1) selecting a chrysanthemum stem as an explant, and carrying out pre-culturing on a pre-culturing medium; (2) activating and culturing recombinant expression vector transformed agrobacterium liquid, enriching thalli, and carrying out resuspension by virtue of an MS liquid culture medium, so as to obtain infection liquid; (3) soaking the explant pre-cultured in the step (1) into the infection liquid, and carrying out infection by virtue of a vacuum negative pressure method; and (4) putting the infected explant onto the pre-culturing medium, carrying out co-culturing in a dark condition, carrying out decarboxylation culture on a decarboxylation culture medium, finally, sequentially carrying out selective culture by virtue of a selective culture medium 1 and a selective culture medium 2, when determining that agrobacterium does not outbreak, carrying out subculture on a root culture medium, and carrying out further rooting screening, so as to obtain genetic chrysanthemumplants. Compared with a traditional leaf-disc-method transgenic method for the chrysanthemum, the method has the advantages that the transgenosis time is remarkably shortened, the conversion efficiency of transgenosis of the chrysanthemum is increaed, the workload of science researchers is reduced, a new way is provided for the efficient conversion and gene function analysis of the chrysanthemum,and a new thought is provided for the transgenosis of other plants.

The invention relates to an establishment method of turbot hepatic parenchymal cell line and the turbot hepatic parenchymal cell line. The establishment method of the turbot hepatic parenchymal cell line comprises the following steps: obtaining turbot liver tissue; cutting the liver tissue into tissue blocks, and carrying out washing; carrying out digestion treatment by adopting type 4 collagenase, and carrying out washing again; carrying out digestion treatment on the tissue blocks obtained after the digestion treatment by adopting trypsin again; adding a primary complete medium to terminate digestion to obtain treated tissue blocks; uniformly attaching the treated tissue blocks into a culture bottle, placing the attached culture bottle into an incubator at 23 DEG C for 6 hours; inverting the culture bottle for 6 hours; adding 4 ml of a primary complete culture medium after the culture bottle is upright; performing primary culture; and then, performing subculture. The turbot hepatic parenchymal cell line established by the method can realize continuous passage so as to provide a large number of turbot hepatic cells; and the established stable and sustainable turbot hepatocyte line can be applied to the molecular cell biology research of turbot nutrition metabolism, thereby overcoming defects in the prior art at the cellular level.

The invention provides a method for establishing a genetic transformation system of dendrobium nobile and relates to the technical field of genetic transformation. The method provided by the inventioncomprises the following steps: inoculating an induction culture medium with aseptic seedlings of the dendrobium nobile, so as to obtain calli; inoculating a subculture medium with the calli, so as toobtain subculture calli; transferring a target gene into the subculture calli, so as to obtain infected calli; inoculating a 1/2MS plate culture medium with the infected calli; inoculating a differentiation culture medium with the co-cultured infected calli, so as to obtain clustered buds; inoculating a bud strengthening culture medium with the clustered buds, so as to obtain strengthened buds; and inoculating a rooting and seedling strengthening co-culture medium with the strengthened buds, thereby obtaining transgenic dendrobium nobile. According to the method, the system is established based on a tissue culture rapid-propagation system, and a technical foundation is settled for carrying out genetic improvement on ornamental properties and medicinal characteristics of the dendrobium nobile by using molecular biology ways.

The invention relates to an in-vitro culture, identification and induced differentiation method of sebastes schlegeli myoblasts, and belongs to the field of molecular biology. The cultured sebastes schlegeli myoblast can be subjected to subculture, and is used for basic research on the muscle growth of marine scleroderma; the induced differentiation of myoblasts in vitro can be effectively carriedout, and a basis is provided for the molecular mechanism research of fusing monocytes into polycytes in vivo.

The invention discloses a preparation method of a stem cell factor oral product, and relates to the technical field of biology. The preparation method comprises the following steps of obtaining primary bovine umbilical cord mesenchymal stem cells; carrying out subculture on the primary bovine umbilical cord mesenchymal stem cells; when the P3-P5-generation bovine umbilical cord stem cells are cultured until the cell fusion degree reaches 80-90%, adopting a secretion-promoting culture solution for culture, collecting a supernate after culture is conducted for 48 h, filtering the supernate with a 0.1-micron filter membrane, and performing ultrafiltration and concentration to obtain a concentrated solution, wherein the secretion-promoting culture solution comprises a basic culture solution, and glutamine, noradrenaline, transforming growth factors, glucosamine and astragalus polysaccharide which are added into the basic culture solution; and pre-freezing the concentrated solution, and then carrying out ultralow-temperature freeze drying to obtain freeze-dried powder. According to the invention, the amplification of the umbilical cord mesenchymal stem cells can be effectively improved, the secretion of stem cell factors can be induced, and the obtained product can condition the body from the cellular level, comprehensively improve the body function, improve the sub-health and repair and strengthen the immune system.

The invention belongs to the technical field of plant biology, and mainly provides a populus alba xP .berolinensis hormone autotrophic cell line capable of realizing plant regeneration. A suspension culture system and an adventitious bud differentiation and rooting plant regeneration system are established; callus is induced from populus alba xP .berolinensis pollen in a mononuclear edging stage in an MS culture medium added with 2 mg/L of 2,4-D, 0.5 mg/L of KT, 30g/L of sucrose and 7.5 g/L of agar, and continuous subculture is carried out for 5 times to obtain light yellow callus with loose texture and vigorous growth; the callus still can rapidly grow in an MS culture medium only containing 30 g/L of sucrose and is the hormone autotrophic cell line; after the hormone autotrophic cell line is inoculated into a 150 mL glass triangular flask containing 50 mL of a MS liquid culture medium with 50 g/L of sucrose for suspension culture for 18 days, the growth amount is increased by about 9.2 times; culturing is carried out in an MS culture medium added with 0.05 mg/L of TDZ, 25g/L of sucrose and 7.5 g/L of agar for 3 months, and inducing is carried out to form a large number of adventitious buds; and the adventitious buds are inoculated into a rooting culture medium to be cultured for 30 days, rooting of the adventitious buds is achieved, and regenerated plants are formed.

The invention relates to molecular biology and particularly discloses a gene sequence capable of preventing gradual inactivation of a promoter in subculture and applications of the gene sequence. The nucleotide sequence of the gene sequence is shown as SEQ ID No.1. The gene sequence is used for constructing a carrier and a CHO cell line, can prevent gradual inactivation of the promoter in subculture of CHO cells and can be used for monoclonal antibody production through the CHO cell line to increase the monoclonal antibody yield.

The invention relates to the technical field of biology, in particular to a sunshine rose grape tissue culture and rapid propagation improvement method and device. The explants are disinfected; carrying out primary culture on the disinfected explants to obtain robust bud seedlings; carrying out rooting culture on the robust bud seedlings obtained by primary culture to obtain rooted seedlings; and hardening the rooted seedlings, and putting the seedlings into the ground. The ratio of a primary culture medium to a rooting culture medium is improved, the primary germination rate and the rooting rate are greatly increased, conventional subculture is not carried out, complex procedures and uncontrollable pollution factors of subculture are avoided, and under the condition that production conditions and manual operation are basically consistent, the production efficiency is greatly improved. The conventional tissue culture needs 3 months for producing a batch of healthy nursery stocks (based on 1000 plants), and only 45 days are needed after the method is improved, so that the problems of long time consumption and low efficiency of the existing sunshine rose grape tissue culture and rapid propagation technology are solved.

The invention provides an instantaneous conversion method for duckweed. The instantaneous conversion method for duckweed comprises the following steps: collecting duckweed; carrying out subculture on the collected duckweed on a liquid culture medium; conducting activating and extracting agrobacterium tumefaciens with a fluorescence-labeled target gene; preparing a bacterium dissolving solution containing Silweet-775 and cane sugar; adding agrobacterium with the fluorescence-labeled target gene into the bacterium dissolving solution to completely dissolve thalli in the bacterium dissolving solution, and cracking the agrobacterium to release plasmids; selecting a complete duckweed, pricking a small hole in the back surface of a duckweed leaf by using a needle head, putting the duckweed into the bacterium dissolving solution for soaking and infecting, putting the infected duckweed into a duckweed liquid culture medium, and carrying out dark culture to obtain instantly infected duckweed. The method for instantly infecting the duckweed with the agrobacterium mainly adopts a needle punching method, has the characteristics of easiness in operation, short time, low cost and the like, and can be applied to related theories and application researches such as molecular biology of duckweed, bioreactor construction, fluorescence labeling and the like.

The invention discloses a fig callus culture method. The method comprises the following steps of (1) asepsis treatment of stem segments: cleaning stems with axillary buds of a plant material, removing branches, disinfecting, cleaning, cutting the stem segments short, and inoculating a culture medium with morphological lower ends; (2) culturing the stems of aseptic fig seedlings: culturing under a shading condition, inducing calluses from edge tissues of the stems, cutting off the calluses, putting the calluses into a subculture medium, and culturing until the volume is 1-2 times of the original volume and the color is yellowish white for later use; and (3) screening a optimal culture medium and culture condition. According to the culture method of the 'Mareuyuefen' callus, the induction rate can reach 90%, and a new way is provided for molecular biology and genetic engineering research of the 'Mareuyuefen'.

The invention provides a method for artificially feeding riptortus pedestris in the whole growth period. Feed used in the method is green and tender pods of cowpeas, kidney beans, green beans, mung beans, soybeans and peas. The relative humidity is 20-80%; the illumination period L: D is equal to 8: 16-16: 8; the feeding temperature in the adult stage and the nymph stage is 15-35 DEG C; the feeding temperature in the adult spawning period is 15-35 DEG C; and the feeding temperature in the egg hatching period is 10-35 DEG C. The method provided by the invention can improve various indexes suchas adult duration, adult egg laying amount, egg hatching rate, nymph survival rate, insect body weight and the like. The method is applied to indoor subculture breeding of the riptortus pedestris, anda large number of indoor breeding insect sources can be provided for research in the aspects of biological characteristics, physiological and biochemical characteristics, molecular biology, drug toxicology and the like of the riptortus pedestris.

The invention discloses a Ganoderma applanatum transgenic method, and belongs to the field of biology. The method comprises the following steps: preparing a culture medium comprising a YPG culture medium and an MYG regeneration culture medium, constructing a recombinant eukaryotic expression plasmid vector pCAMBIA1303-pGPD-FIP-gap, suspending purified 1 * 10<7> Ganoderma applanatum protoplast in 250 [mu]L of osmotic pressure stabilizer, adding 5 [mu]g of plasmid pCAMBIA1303-pGPD-FIP-gap, lightly shaking up, then placing on ice, and carrying out ice bath treatment for 10 min; adding 50 [mu]L of a PEG buffer solution, placing on ice, carrying out ice bath treatment for 30 min, adding 1 mL of the PEG buffer solution, uniformly mixing, and standing at 28 DEG C for 20 min, wherein the PEG buffer solution comprises PEG6000, 15mM CaCl2 and 10mM Tris aqueous solution; and carrying out regeneration culture, hygromycin resistance screening and subculture, and then detecting. The result shows that transformants subjected to hygromycin resistance screening twice are positive, the PCR positive detection result shows that the positive transformants can be obtained by 10-60% of the PEG buffer solution, the efficiency is the best when the 30% of the PEG buffer solution is used for carrying out ganoderma applanatum conversion, 1 [mu[g of plasmid can obtain about 110-120 positive transformants, and the positive transformation rate can reach 95%.

The invention discloses a method for separating maternal mesenchymal stem cells from human placenta decidua basalis tissue, which belongs to the technical field of cell biology. The method comprises the following steps: separating maternal mesenchymal stem cells from human placenta decidua basalis tissues, and carrying out subculture, cell harvesting, cryopreservation and cell STR identification to finally obtain a cell product. In the separation and culture process, an improved separation method and an improved culture system are adopted to reduce the damage to cells, improve the separation efficiency, shorten the culture period and obtain high-purity cells with excellent characterization.