121 results about "Transformation efficiency" patented technology

The invention belongs to the technical field of plant biology, and discloses a wild soybean genetic transformation method which comprises the following steps: S1, disinfecting wild soybean seeds; s2, the backs of the disinfected wild soybean seeds are cut once, the cut wild soybean seeds are placed on a culture medium, and imbibition seeds are obtained; s3, seed coats of the wild soybean seeds are removed, growing points of embryo tips are exposed through dissection, and explants are obtained; s4, putting the explant into an agrobacterium infection solution containing target plasmids, and carrying out vacuum infiltration in a vacuum environment; and S5, after releasing vacuum, standing and infecting in an agrobacterium infection solution for 3-16 hours. The glycine soja genetic transformation method can effectively improve the transformation efficiency of glycine soja.

This invention discloses the application of the GhATG18a gene in accelerating callus induction in cotton. The invention constructs a pCAMBIA2300-GhATG18a overexpression vector from the full-length CDS fragment of this gene, then introduces it into Agrobacterium, and transforms cotton using Agrobacterium-mediated transformation to obtain transgenic lines. qRT-PCR results show that, compared with the CCRI24 control material, the expression level of the GhATG18a gene in the overexpression lines OE-GhATG18a-1 and OE-GhATG18a-2 is significantly upregulated. Phenotypic observation and histological sections both show that the OE-GhATG18a line overexpressing this gene accelerates the process of somatic cell dedifferentiation and callus induction in cotton, thereby improving the genetic transformation efficiency of cotton. Therefore, overexpressing this gene and applying it to commercial cotton varieties that are difficult to genetically transform can help accelerate the callus induction process, thereby improving the genetic transformation efficiency and providing technical support for breeding high-yield and stress-resistant high-quality cotton germplasm.

The invention belongs to the technical field of microbial genetic transformation, and particularly relates to a method for high-yield production of pleurotus ostreatus transformants, a genetic transformation system and application of the genetic transformation system. Through optimization of protoplast preparation conditions, the number of the obtained protoplast is 4.95 * 10 < 8 > CFU.mL <-1 >, and the regeneration rate of the protoplast in an OM-YEPS regeneration culture medium is 12.7%. A method for producing high-yield pleurotus ostreatus transformants is researched by using a PEG-mediated protoplast transformation method, a set of efficient and stable genetic transformation and screening method is established, about 200-300 positive transformants with stably expressed exogenous genes are obtained, and the transformation efficiency is 65.38%. The genetic transformation system of the high-yield Pleurotus ostreatus transformant, constructed by the invention, lays a solid technical foundation for subsequent gene function verification, metabolic engineering transformation and molecular breeding with excellent characters, and further accelerates the biological research and industrial application process of Pleurotus ostreatus and even other macro fungi.

This invention discloses a genetic transformation and gene editing method applicable to multiple sesquiterpene species, belonging to the field of plant genetic transformation and gene editing technology. The method uses the cotyledonary nodes of sterile seedlings of the sesquiterpene genus as explants, and introduces an optimized pScEF1α-Cas9 gene editing transformation vector using Agrobacterium infection. Regenerated plants are obtained through recovery culture, selection culture, and rooting culture. The vector contains four gene expression units: pScEF1α:Cas9, pGmU6:sgRNA, pGmUBI:DsRed2, and pCaMV35S:HygR. In vivo screening is further performed using the DsRed2 fluorescent reporter gene, and the gene-edited plants are identified by molecular detection. This method is applicable to common sesquiterpene, stem-nodled sesquiterpene, and spiny sesquiterpene, and has advantages such as strong species versatility, high transformation efficiency, short cycle, visualized screening, and high positive rate, providing efficient technical support for gene function research and genetic improvement of sesquiterpene species.

The present disclosure provides an application method in rice callus differentiation based on an Oryza sativa Leafy Cotyledon 1 (OsLEC1) gene, including the steps of: selecting guide ribonucleic acid (gRNA) target sites; cloning tandem fragments including gRNA; ligating each gRNA fragment; performing polymerase chain reaction (PCR) amplification on a ligation product; performing enzyme digestion on the purified product and a target vector; transforming the ligated vector; performing Agrobacterium-mediated genetic transformation of rice; and screening and identifying transgenetic plants. In the present disclosure, the differentiation of callus directly affects the emergence efficiency of transgenic plants. The knockout of OsLEC1 can promote the differentiation of rice callus, suggesting that OsLEC1 can serve as an important target gene for improving the transformation efficiency of rice and even gramineous crops. OsLEC1 can be used as a starting point to construct various molecular tools to enhance transformation efficiency.

The invention belongs to the technical field of plant cytology, and particularly relates to a method for improving the transformation efficiency of plant protoplast, which comprises the following steps: preparing enzymatic hydrolysate: each liter of enzymatic hydrolysate contains 20 mmol of KCl, 5-20 g of cellulase, 2-6 g of macerozyme, 10 mmol of CaCl2, 1 g of BSA and the balance of water; performing leaf enzymolysis; obtaining a protoplast; preparing a competent protoplast; resuspending the MMG solution; and transforming and incubating the protoplast resuspension by using the target plasmid. According to the method, the high-quality protoplast can be obtained, and when the protoplast is applied to downstream experiments, the conversion efficiency and stability of the experiments can be remarkably improved.

The invention discloses an application of an OsPLT1 gene in promotion of rice callus regeneration. It is found for the first time that overexpression of OsPLT1 can improve the regeneration and transformation efficiency of rice calluses, including rice varieties difficult to differentiate and regenerate. Furthermore, the OsPLT1-rZmG2-rZmW2 is used in a combined manner, and a rice callus specific promoter is used for driving gene segment transformation, so that the transformation efficiency of the rice is further improved.

The invention belongs to the technical field of genetic transformation, and particularly relates to a method for improving apple leaf disc regeneration and genetic transformation efficiency. And culturing the apple leaf disc in the apple leaf disc regeneration culture medium to obtain a leaf disc regeneration adventitious bud. The amino acid sequence of the MdREF1 small peptide is as shown in SEQ ID NO. 1. The apple homologous peptide MdREF1 is identified in an NCBI (National Center of Biotechnology Information) database on the basis of AtPROPEP1-8 and AtPep1-8 sequences of arabidopsis thaliana. The synthetic peptide of the apple MdREF1 is added into a basic regeneration culture medium, so that the leaf disc callus growth amount and the adventitious bud differentiation number can be remarkably increased. Wherein by adding 50nmol / L of MdREF1 peptide, the regeneration frequency can be improved by 1.74 times, the average regeneration bud number can be increased by 2.16 times, and the conversion efficiency can be improved by 3.14 times.

The application discloses a method for improving genetic transformation rate of high-altitude meadow grass, comprising the following steps: (1) taking mature seeds of high-altitude meadow grass as explants to induce callus, and obtaining the callus; (2) adding a surfactant Silwet L-77 with a mass fraction of 3‰ of the bacterial liquid to the agrobacterium bacterial liquid carrying a hygromycin resistance gene, and obtaining a culture bacterial liquid; (3) placing the regenerated plant in a screening culture medium containing 40 mg / L of hygromycin resistance for culture, screening the survived plants, and obtaining the genetically transformed plants. The method can improve the transformation efficiency of the high-altitude meadow grass by about 4.54 times through adding the surfactant in the genetic transformation process and cooperating with the screening of the obtained differentiation culture medium.

The invention belongs to the technical field of synthetic biology and enzyme engineering, and particularly relates to a multi-enzyme in-vivo space co-localization carbon metabolic flux strengthening method, a genetically engineered bacterium and application of the genetically engineered bacterium. According to the invention, a recombinant expression vector is constructed, key enzymes RHLA and RHLB participating in synthesis of a target product are respectively subjected to fusion expression with protein scaffold modules RIAD and RIDD through (G4S) 3 flexible connecting peptide, and are introduced into host chassis bacteria, so that a stable multi-enzyme composite system is formed in cells, the transmission distance of a metabolic intermediate is shortened, and the expression efficiency is improved. Spatial co-localization and concerted catalysis of key catalytic enzymes are realized, and the conversion efficiency of a carbon source to a target product is improved. Compared with the prior art, the genetically engineered bacterium constructed by the invention has the advantages of stable structure, no need of complex regulation, reasonable metabolic flow distribution and the like in the fermentation process, the synthesis efficiency and yield of the target biosurfactant are remarkably improved, and the feasibility of industrial amplification application is enhanced.

The invention discloses a construction method and application of a basic group editor of industrial bacteria of cephalosporium acremonium based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9), and belongs to the field of gene engineering. The cephalosporium acremonium industrial bacterium base editor provided by the invention can be used for specific mutation of a specific site of the DNA level of the cephalosporium acremonium industrial bacterium. The cytosine base editor CBE constructed by the invention is a plasmid comprising a fusion protein A and an sgRNA expression element. The base editor CBE can successfully mediate C.G-T.A, is suitable for the base editor of the high-yield CPC cephalosporium acremonium industrial bacteria, utilizes the protoplast, is high in conversion efficiency, can mediate specific site mutation in the cephalosporium acremonium industrial bacteria, and has application value.

The invention belongs to the technical field of gene engineering, and particularly relates to an efficient electrotransformation method of lactococcus lactis BL19. According to the method disclosed by the invention, the electric shock conversion efficiency of the lactococcus lactis BL19 is remarkably improved, and a foundation is laid for genetic engineering transformation in the future.

The invention discloses a LeRho1 gene expression vector and a method for improving heat resistance and trichoderma resistance of lentinus edodes. The LeRho1 gene expression vector is a recombinant expression vector, a target gene for coding the LeRho1 protein is inserted into the recombinant expression vector, and the amino acid sequence of the LeRho1 protein is shown as SEQ ID NO. 1. The invention finds that the LeRho1 gene has a positive regulation effect on the heat resistance of the shiitake mushroom strain, and the LeUSPA and LeUSPH genes mediate the thermal response of the shiitake mushroom strain through negative regulation. Therefore, according to the method, the LeRho1 gene overexpression vector or LeUSPA and LeUSPH gene silencing vectors are constructed and transferred into the shiitake mushroom strain for genetic transformation, the heat resistance and trichoderma resistance of the shiitake mushroom strain can be remarkably improved, and the transformation efficiency is high.

The invention discloses a method for regulating and controlling efficient conversion of methanol based on (p) ppGpp signal molecules. Specifically, by regulating synthesis and hydrolysis balance of (p) ppGpp signal molecules in microbial cells and combining promoter engineering and nutrition condition optimization, methanol metabolic pathway key gene expression and amino acid metabolic flux are enhanced, and the methanol utilization efficiency and target product synthesis capacity of microorganisms are remarkably improved. The regulation and control mechanism of the (p) ppGpp on methanol conversion under the stress of a low nitrogen source is defined for the first time, efficient conversion of methanol to organic acids such as acetic acid and butyric acid is realized, a brand new strategy is provided for preparation of high-added-value chemicals through methanol bioconversion, and the method has the advantages of being economical, environmentally friendly and high in conversion efficiency and has a wide application prospect.

The invention belongs to the technical field of bacterial plasmid editing, and particularly relates to miniature DNA nuclease as well as a preparation method and application thereof. The preparation method of the micro DNA nucleotidase comprises the following steps: carrying out expression on a p15A-IscB-2. 0 plasmid, so as to obtain the micro DNA nucleotidase. Wherein the nucleotide sequence of the p15A-IscB-2 plasmid is shown as SEQ ID NO.2, and the p15A-IscB-2 plasmid is obtained by performing synonymous mutation on a 5 '-AACNNNNNNGYGC-3' sequence in an IscB gene in p15A-IscB. The p15A-IscB-2 plasmid disclosed by the invention is non-toxic to escherichia coli, can express an IscB protein, and is high in conversion efficiency, so that the p15A-IscB-2 plasmid can be directly used for gene editing of escherichia coli.

This invention relates to a genetic transformation method for Ningxia wolfberry using germinating seeds as explants, belonging to the field of plant genetic transformation technology. The invention mainly includes the following steps: (1) After disinfecting Ningxia wolfberry seeds, inoculate them onto a germination medium, and obtain germinating seeds through vernalization and dark culture treatment; (2) Activate and culture Agrobacterium rhizogenes and Agrobacterium tumefaciens strains carrying target gene expression frames to obtain a bacterial solution for infection; (3) Take the germinating seeds obtained in step (1) and mix them with the bacterial solution obtained in step (2) for infection, then transfer the infected germinating seeds to a co-culture medium or substrate for co-culture; (4) Cultivate the material after co-culture in step (3) to induce root growth, and detect the expression level of the target gene or reporter gene on the roots or plants to obtain transgenic positive plants. This invention has the advantages of simple operation, significantly shortened transformation cycle, and high transformation efficiency.

The present application relates to a method for transforming corn, comprising: the primary callus obtained by the method for inducing corn callus, using the method for obtaining corn suspension cell line to obtain corn suspension cell line; agrobacterium infection corn suspension cell group in the corn suspension cell line. The method for transforming corn of the present application first induces the primary callus from corn proembryo, quickly obtains the suspension cell line, shortens the experimental period to 40-50 days, is simple and convenient to operate, has good repeatability, simultaneously makes the corn genetic transformation not affected by the season, meets the needs of annual stable transformation; then uses agrobacterium infection to transform the corn suspension cell line, and regenerates seedlings again; the transformation efficiency can reach about 30%, the transgenic positive rate can reach about 90%, and the single copy rate is about 50%; since the pressure on corn explant (proembryo) supply is significantly reduced, the human and material resources consumption required for culturing the explant is greatly reduced, and large-scale genetic transformation is realized.

The invention discloses a non-tissue-culture-dependent pearl millet injection transformation method, and belongs to the technical field of plant genetic engineering. The method comprises the following steps: selecting germinated seeds of pearl foxtail millet with the primary bud length of 0.8-1.0 cm as receptors; preparing agrobacterium liquid, and adjusting the concentration to OD600 = 0.5; injecting the bacterial liquid into the embryo along the coleoptile direction of the seed until the bacterial liquid seeps from the end part of the coleoptile; co-culturing the transformed receptor for 24 hours under a dark condition after injection, and transplanting the transformed receptor into a seedling culture pot for cultivation; and finally, carrying out PCR (Polymerase Chain Reaction) screening. By optimizing key parameters such as the receptor development stage, the agrobacterium strain type and the bacterial liquid concentration, efficient genetic transformation of the pearl millet without tissue culture is achieved, and the T0 transformation positive rate reaches 63%. According to the invention, the transformation process is simplified, the transformation period is shortened, the cost is reduced, the transformation efficiency and stability are improved, and an effective technical support is provided for gene function research and molecular breeding of the pearl millet.

The invention provides a method for improving the success rate of a wheat protoplast instantaneous transformation system, which comprises the following steps: S1, fully rinsing a cell culture container for incubation by using a 10% bovine serum albumin solution, so that all structures in the container are infiltrated by the 10% bovine serum albumin solution, and discarding waste liquid after rinsing; and S2, resuspending the wheat protoplast into which the exogenous gene is transferred by using 900-1200mu L of a W5 solution, transferring the wheat protoplast into the cell culture container rinsed in the S1, wrapping the cell culture container by using tin foil paper, and incubating for 12-16 hours at room temperature. Compared with a traditional incubation process, the method has the advantages that the integrity rate and activity of the protoplast are remarkably improved, and the risks of protoplast rupture and death in the incubation process are effectively reduced. According to the system, the instantaneous conversion efficiency can stably reach more than 50%, and the reliability and operability of gene function verification and regulation mechanism research are greatly improved.

The application discloses a construction method of a succinic acid-producing actinobacillus CRISPRi system, and belongs to the technical field of biology. The CRISPRi system provided by the application takes an expression vector pLGZ92M as a skeleton vector, and main acting elements of the CRISPRi system include a cpf1 expression element and a crRNA expression element. A pckA promoter starts expression of a dcpf1 protein, a frd promoter starts expression of crRNA, and the expression is terminated by a terminator rrnB t1 terminator. The CRISPRi system provided by the application can realize inhibition of a target gene by only replacing crRNA for the target gene. The method of the application realizes gene inhibition of succinic acid-producing actinobacillus, and has the advantages of simple operation process and high transformation efficiency.

The invention belongs to the technical field of gene inheritance, and particularly relates to a CDB simple arabian jasmine flower genetic transformation method which comprises the following specific steps: selecting different physiological parts of arabian jasmine flowers as candidate explants, performing scratch pretreatment, setting time gradient for pre-culture, and screening the optimal duration; the method comprises the following steps: screening three agrobacterium adaptive strains, namely AR1193, GV3101 and EHA105, and constructing a vector containing an RUBY visual reporter gene; according to the application, the RUBY reporter gene is adopted, the positive transformant can be directly recognized through red color development under natural light, DNA extraction and PCR detection do not need to be carried out in the early stage, the screening process is simplified, the screening period is shortened, the transformation efficiency can be improved only by lightly scratching an explant with a sterile blade, and complex tissue culture for inducing callus is not needed; the soaking method is adopted for infection, ultrasonic-assisted delivery is matched, operation is convenient and fast, professional high-end equipment is not needed, clear gradients are set for key conditions, optimized parameters can be directly reused, the test exploration cost is reduced, and the method is suitable for being popularized in different laboratories.

This invention discloses a plasmid for gene overexpression in non-pattern *Bacillus laterosporus*. Its leader region contains a methyltransferase expression cassette derived from the *Bacillus laterosporus* i83 genome, enabling stable existence in specific hosts containing the RM system. Based on this, a PidR2 promoter is added to obtain the pMCPidR2 shuttle plasmid. The PidR2 promoter effectively drives target gene expression, the pWV01 replicon ensures plasmid replication in non-pattern hosts, and the chloramphenicol resistance gene is used for positive colony selection. The plasmid contains a TraJ element, allowing it to be introduced into non-pattern microbial hosts via conjugation transfer. This invention also provides a target gene expression strategy in non-pattern microorganisms, achieving high transformation efficiency and high strain yield through conjugation transfer transformation of donor bacteria, conjugation transfer helper bacteria, and recipient bacteria, resulting in stable expression of the target protein. This invention provides an effective vector for efficient and stable gene overexpression in specific non-pattern microorganisms, helping to solve the problem of difficult genetic manipulation of industrial production strains.

The invention discloses a genetic transformation method based on pear somatic embryogenesis, and belongs to the technical field of genetic engineering and molecular breeding. In order to solve the problems that existing pear genetic transformation is low in transformation efficiency, high in chimera rate, long in period, difficult to obtain gene editing regeneration plants and the like, the invention provides two paths: 1, immature zygotic embryos 30-60 days after pear flowering are taken as explants, resistant plants are directly induced through a somatic embryo regeneration path, and the transformation efficiency within 40-60 days reaches 73.91%; secondly, the embryogenic callus with the consistent genetic background is used as an explant for infection, the embryogenic property is maintained and the proliferation efficiency is improved by optimizing a culture medium formula, and a resistant plant with the consistent genetic background can be obtained within 30-50 days. The method overcomes the defect that regeneration plants are difficult to obtain through existing callus transformation, remarkably shortens the breeding period, is high in transformation efficiency, low in chimera rate and controllable in genetic background consistency, and provides technical support for pear functional genomics research and molecular breeding.

The invention relates to the technical field of bioengineering, and discloses a saccharomyces cerevisiae self-replication system and application thereof.The system comprises a recombinant plasmid, and the recombinant plasmid comprises upstream and downstream homologous arms of a saccharomyces cerevisiae delta site, a selection marker TRP1, an autonomous replication sequence ARS310, a G418 resistance gene and a resistance gene promoter; wherein the nucleotide sequence of the autonomous replication sequence ARS310 is as shown in SEQ ID NO. 3; the nucleotide sequence of the G418 resistance gene is as shown in SEQ ID NO. 4; the nucleotide sequence of the selection marker TRP1 is as shown in SEQ ID NO. 5. The invention provides a pressure-driven saccharomyces cerevisiae self-replicating system for high-efficiency expression of foreign protein (target gene), and the expression of the foreign target gene with high conversion efficiency, high stability and high copy number is realized by utilizing natural multiple copies of a delta site, the self-replicating capability of ARS and the screening pressure of a G418 resistance gene.

This invention provides a method for transient transformation of oil palm zygote embryos based on Agrobacterium-mediated transformation, which employs the Agrobacterium-mediated approach and is based on the optimal Agrobacterium concentration (OD) for stable transformation of oil palm. 600 The optimal Agrobacterium infection time for zygotic embryos was determined by measuring the concentration of acetylsuccinone and co-culture time. The transient GUS expression staining rate was compared between zygotic embryos of different oil palm varieties and at different growth stages. The method described in this invention optimizes infection conditions based on stable transformation parameters, clarifies the optimal explant materials and treatment process, and effectively improves transient transformation efficiency and experimental repeatability. This study establishes for the first time an Agrobacterium-mediated transient transformation system for oil palm zygotic embryos and obtains zygotic embryos most suitable for transient transformation, achieving efficient transient expression of exogenous genes in oil palm zygotic embryos. This provides a rapid and low-cost technical means for studying oil palm gene function.

The invention discloses a stable genetic transformation method of floating moss. The genetic transformation method comprises the following steps: (1) crushing thallus tissues of the floating moss, (2) pre-culturing explants, (3) infecting and co-culturing, (4) screening and culturing for the first time, (5) screening and culturing for the second time, (6) enlarging and culturing, and (7) verifying transformation. According to the genetic transformation method, a thallus of floating moss is taken as a starting explant, an agrobacterium-mediated hygromycin and kanamycin screening transformation system of the floating moss is established for the first time, the blank of a floating moss genetic transformation system is filled, and experimental results show that after multi-generation culture, a transformed plant can still stably express a target gene. The method has the advantages of high transformation efficiency and simplicity in operation, and can be used for subsequent genetic transformation research of unknown floating moss genes. The method has a wide application prospect.

The invention provides a PEG / CaCl2 mediated Penicillium citrinum genetic transformation method, and belongs to the technical field of microbial genetic transformation, and the method comprises the following steps: adding a target gene segment into Penicillium citrinum protoplast suspension, and carrying out an ice bath reaction; the target gene segment is obtained by taking pJET-hph-02944 plasmid as a template and carrying out PCR (Polymerase Chain Reaction) amplification by adopting a hygromycin B resistance gene primer pair with gene sequences as shown in SEQ ID No. 1 and SEQ ID No. 2; adding a PTC (Positive Temperature Coefficient) buffer solution into the mixed system, uniformly mixing and standing; continuously adding a TB3 liquid culture medium containing ampicillin, and carrying out recovery culture; uniformly mixing the resuscitated protoplast with a molten TB3 solid culture medium, pouring a plate, and carrying out dark culture for the first time; then covering a TB3 solid culture medium containing hygromycin B and ampicillin, carrying out secondary dark culture to obtain a transformant, and transferring the transformant to a PDA plate containing hygromycin B for screening. According to the method, CaCl2-PEG is adopted for mediated transformation of Penicillium citrinum, the transformation efficiency is higher, and the transformation speed is higher.

The invention provides a method for verifying gene functions based on combination of agrobacterium rhizogenes and a movable element. The provided method comprises the following steps: (1) constructing an expression vector fused by a target gene and a movable element, wherein the target gene is a functional gene to be verified of the plant; (2) transferring the expression vector into agrobacterium rhizogenes to obtain transformed agrobacterium rhizogenes; (3) infecting a plant by utilizing the transformed agrobacterium rhizogenes to obtain a positive plant with hairy roots; (4) determining the function of the target gene in the plant through the expression of the target gene in the upper end tissue of the positive plant and the phenotypic difference between the target gene and the control plant; wherein the movable element is a genetic material capable of transferring the target gene from the hairy root of the positive plant to the upper tissue. The method provided by the invention is simple to operate, high in transformation efficiency and stable and reliable in phenotype, and can be widely applied to plant gene function research.

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 provides an in vitro cultured bezoar, its preparation method, and its application, belonging to the field of traditional Chinese medicine preparation technology. The preparation method of this invention involves obtaining a bile-resistant, high-β-glucuronidase-active *Escherichia coli* strain through bile gradient acclimation, which is then combined with *Enterococcus faecalis* to form a functional bacterial community. This community is then cultured in vitro through a three-stage process of proliferation, transformation, and crystallization, followed by gradient calcium ion-induced crystallization and vacuum freeze-drying to obtain in vitro cultured bezoar. This invention aims to solve the problems of low conversion efficiency, uneven crystallization, insufficient content of effective components, and unstable product quality in existing technologies. The obtained in vitro cultured bezoar product has significantly higher bilirubin and conjugated bile acid contents than the standards of the *Chinese Pharmacopoeia*, and the process is stable, controllable, and suitable for industrialization.