70results about How to "Improve the efficiency of genetic transformation" patented technology

The invention relates to an agrobacterium tumfaciens-mediated transgenic soybean regenerated seedling rooting method, and belongs to the technical fields of soybean tissue culture and transgenic research. Research of soybean cotyledon node and embryo genetic transformation is performed by using an agrobacterium tumfaciens mediating method; a novel transgenic plant rooting method is adopted for the first time; and even if a double-layer rooting culture medium is adopted, the upper layer contains antibiotics and the lower layer does not contain antibiotics, the growth of agrobacterium tumfaciens can be inhibited, adverse influence of the antibiotics on transgenic regenerated plants can be greatly reduced, and the transplanting survival rates of the agrobacterium tumfaciens infected plants reach 90.70 percent and 88.10 percent respectively. The application of the method has significance for improving the agrobacterium tumfaciens-mediated soybean genetic transformation efficiency, and has good reference effect on genetic transformation of other plants.

The invention relates to a method for improving the genetic transfer efficiency of gene-transferred soybean. Wherein, it uses agricillin soybean cotyledon transfer system and germ sharp transfer system to obtain transfer adventitious bud; said adventitious buds without growing root and seed are directly made into graft, via cleft grafting method to be grafted on the soybean seed stock; the graft and the stock via combine period, recover period, fusion period and survival period can improve the gene transfer efficiency of soybean. The grafting can improve the survival rate of plant more than 90%, to shorten the gene transfer period and improve the gene transfer research.

The invention discloses a transgenic method of an agrobacterium-mediated rape field. The method comprises the following steps: eluting an agrobacterium on a YEB solid plate with 1 / 2 MS fluid nutrient media which comprises 5% of sucrose, 0.05-0.1% of Silwet-77, 0.2-0.4% of acetosyringone and 0.0001-0.0002% of 6-BAP by concentration; removing blooming flowers from a main inflorescence and each branched inflorescence and only leaving buds when the buds of a principal branch and part of lateral branches of the rape and a plurality of flowers bloom before the conversion; bundling the inflorescences immediately after soaking in eluted conversion bacterium liquid for 30 seconds; continuously dip-dyeing one inflorescence for 3 times with an interval of 2-3 days; and removing a parchment bag on the inflorescence and the flowers and buds at the top end of the inflorescence. The method has the advantages of simple operation, low cost, high transgenic efficiency and no genotype dependence, thereby having excellent potential application in the aspects of culturing rapes of new species by utilizing the gene engineering means and establishing rape mutant libraries.

The invention provides a Dangshan pear genetic transformation method. The Dangshan pear genetic transformation method comprises the following steps: (1) constructing a genetic transformation receptor; (2) culturing agrobacterium tumefaciens and preparing infection solution; (3) carrying out infection and coculture; (4) carrying out sterilization culture and screening culture; and (5) carrying out resistant callus GUS (glucuronidase) staining identification. The Dangshan pear genetic transformation method has the advantages that Dangshan pear callus is taken as the receptor, NptII is taken as a marker gene, GUS gene is taken as a reporter gene, Dangshan pear genetic transformation is carried out by adopting an agrobacterium EHA105 mediated process for obtaining positive resistant callus, and then redifferentiation is carried out by virtue of the positive resistant callus for obtaining test-tube plantlets; meanwhile, a method for carrying out genetic transformation by firstly inducing resistant callus by virtue of fresh treetop, taken as an explant, grown the same year of Dangshan pear is firstly established, content of phenolic substances in fresh treetops of the Dangshan pear in spring is low, less germs are carried, both inoculation browning rate and contamination rate are low, materials are available, callus induction ratio is high, and demand of genetic transformation can be met.

The invention relates to the field of plant genetic engineering. Specifically, the invention relates to an efficient watermelon genetic transformation system and application. Growth regulation genes such as AtGRF5, TaGRF4, OsGIF1, ZmWUS and ZmBBM are included in watermelon genetic transformation. It is found for the first time that expression of AtGRF5, TaGRF4, OsGIF1, ZmWUS or ZmBBM genes in the watermelon transformation and / or regeneration process can promote the genetic transformation efficiency of watermelon crops, AtGRF5 can significantly improve the genetic transformation efficiency of watermelons by about 80 times, and precious gene resources are provided for genetic breeding. The invention further provides a method for using a fluorescent protein DsRed2 as a genetic transformation screening marker, and genetic transformation screening operation is simplified. When the system is expressed and used in watermelons, the genetic transformation breeding efficiency of the watermelons can be effectively improved, and the breeding cost is reduced.

The invention provides a rubber tree embryoid induction culture medium, an induction method for an embryonic callus and a culture method for regenerating resistant callus plant, and belongs to the technical field of plant tissue culture. The rubber tree embryoid induction culture medium takes an improved MS culture medium as a basic culture medium, and is prepared from Kt, NAA, 6-BA, salicylic acid, spermidine, acid hydrolyzed casein, coconut liquid endosperm coconut water, active carbon, saccharose and phytagel. The somatic embryo induction rate of the embryoid induction culture medium provided by the invention for inducing the embryonic callus of a rubber tree anther somatic embryo is increased by 2.04 to 2.16 times in comparison with that of an anther dedifferentiation callus.

The invention provides a method for improving the hairy root induction efficiency and the genetic transformation efficiency of bupleurum chinense, and application thereof. On the basis of exploration on the conditions of the hairy root agrobacterium strains, the infection bacteria liquid OD value, the co-culture time and the like, the optimum combination condition for improving the infection efficiency of the hairy root agrobacterium is determined; compared with the traditional hairy root induction efficiency being about 10 percent, the hairy root induction efficiency obtained by the technical scheme provided by the invention reaches 70 to 85 percent. The method for improving the hairy root genetic transformation efficiency of the bupleurum chinense provided by the invention has the advantages that the exogenous gene conversion rate being 80 percent or higher can be obtained; better, more efficient, simpler and more convenient selection is provided for the bupleurum chinense functional gene research and / or the industrialization of hairy root metabolites of the bupleurum chinense.

The invention discloses a genetic transformation method for panicum virgatum L., and belongs to the technical field of plant bioengineering. The method comprises disinfection of mature panicum virgatum L. seeds, induction and propagation expanding of type II high-quality embryonic callus, kanamycin screening to obtain kanamycin-resistant callus tissue after agrobacterium infection, kanamycin-resistant callus tissue regeneration and rooting, and finally acquisition of positive transgenic panicum virgatum L. plants. The method is mainly used for solving the problems of large-scale screening and commercialized production of transgenic panicum virgatum L.. Experimental data show that by the adoption of the method, low-cost kanamycin can be used as a transgenic plant resistance screening reagent, the positive transgenic panicum virgatum L. plants are successfully obtained in 4-5 months, genetic transformation efficiency reaches up to 60%, 100 genetic vectors can be transformed by each person every year, and the working efficiency of producing more than 2,000 independent positive transgenic panicum virgatum L. plants is achieved.

The invention relates to an efficient and stable genetic transformation method for strawberries mediated by agrobacterium tumefaciens. The method comprises the steps: preculturing aseptic seedlings of strawberries, activating agrobacteria, infecting, co-culturing, delaying screening, and screening and rooting. According to the efficient and stable genetic transformation method, the genetic transformation steps are optimized, so that the transgenosis efficiency of the strawberries is increased by a large margin; the problems that a traditional agrobacterium-mediated method is low in transformation efficiency, chimeras are easy to generate, the screening is difficult and the workload is great are solved; the genetic transformation efficiency is improved enormously; and meanwhile, the false positive rate of regeneration buds is obviously lowered.

The invention discloses a method for genetic transformation of pineapple somatic embryogenic receptor kinase gene AcSERK1 and application thereof. The genetic transformation method of the invention comprises the following steps: 1) construction of a sense expression vector of pFGC5941-35s-AcSERK1; 2) preparation of callus and callus tolerance test to PPT; 3) preparation of agrobacterium infectionsolution suspended by MS; 4) pre-culture, infection, co-culture and PPT screening culture; 5) PPT resistant seedling rooting and transplanting. Transgenic plants obtained by the genetic transformationmethod of the invention are subjected to PCR and Southern blot and tested by RT-PCR, confirming that the AcSERK1 had been inserted into pineapple genomic DNA and overexpression is occurred in pineapple somatic embryos. Functional validation shows that the number of embryogenic callus particle and somatic embryos obtained by callus which overexpresses the AcSERK1 after somatic embryo induction issignificantly higher than that of wild type, indicating that overexpression of the AcSERK1 has significant promotion effect on formation of pineapple embryogenic cells and somatic embryogenesis.

The invention discloses a method for cultivating regeneration plants of cotton. The method for cultivating target cotton plants, provided by the invention, comprises the following steps of: (1) firstly, removing stem tips, branches and leaves as well as fibrous roots of a target cotton plant, and retaining 1-2 white fibrous roots or basal parts of the fibrous roots to obtain a main body; and secondly, cutting off along a first section, which is close to the root part, of the main body and retaining the first section, provided with the root, of the main body to obtain a plant to be subjected to subculture; (2) culturing the plant to be subjected to subculture in a regeneration plant subculture medium to obtain a regeneration plant subjected to subculture again; and (3) hardening and transplanting the regeneration plant subjected to the subculture again to obtain the transplanted cotton plant, wherein the target cotton plant is the regeneration cotton plant obtained after tissue culture. Proven by experiments, the cultivation method for cultivating the regeneration plants of cotton has the capability of greatly increasing the survival rate of transplanting of the regeneration plants of cotton. The method disclosed by the invention is simple to operate, strong in practicability and is conductive to improving the genetic transformation efficiency of the cotton.

The invention provides an efficient genetic transformation method of agrobacterium-mediated dendrobium candidum. The efficient genetic transformation method comprises the following steps: (1) performing activating culture on agrobacteria; (2) performing a dip dyeing liquid: adding the agrobacteria obtained in the step (1) into an MS liquid culture medium, putting on ice so as to enable the agrobacteria to be completely dissolved in the culture medium, adjusting bacterial liquid by using the MS liquid culture medium till OD600 is 0.4-0.8, then adding 100[mu]mol / L of acetosyringone(AS) and 0.001-0.01% of Triton-100, and uniformly shaking; (3) performing ultrasonic treatment and dip dyeing: under the conditions of 280-320W and 36-45KHz, performing ultrasonic treatment for 1-3min; (4) performing co-culture; (5) screening; (6) performing plant regeneration. A transformation system is complete; the survival rate of protocorms of dendrobium candidum is high; through optimization of the steps and parameters, plants with excellent resistance are obtained through transformation; the transformation rate is high, and the positive transformation rate is as high as 69.7%.

The invention relates to a method for genetic transformation of eucommia ulmoides by injection. According to the method, eucommia ulmoides seedlings germinating for three days are taken, a first trueleaf between two cotyledons is pulled out to serve as a genetic transformation receptor, and agrobacterium resuspensions with different concentrations are injected between the two cotyledons of each eucommia ulmoides seedling. The method comprises the following specific operation steps: (1) preparing the genetic transformation receptor; (2) culturing agrobacterium; (3) preparing the agrobacteriumresuspensions; (4) performing genetic transformation of the eucommia ulmoides; (5) managing a transgenic plant; and (6) identifying the transgenic plant. According to the method, a tissue culture technology is not adopted, besides agrobacterium culture, sterile operation is not needed, and the method has the advantages of being easy to operate, low in cost, capable of converting large-fragment DNA, stable in heredity, low in copy number and the like, and becomes the most widely applied genetic transformation method at present.

The invention belongs to the technical field of kiwi fruit genetic engineering, and particularly relates to a culture medium and a genetic transformation method of Hongyang kiwi fruits. The genetic transformation method sequentially comprises the following steps: agrobacterium tumefaciens culturing, leaf pre-culture, infecting the leaves by the agrobacterium tumefaciens, callus screening and culturing, carrying out bud elongation culture, carrying out bud rooting culture and positive seedlings transplanting; steps of leaf pre-culture, leaf infection by agrobacterium tumefaciens, callus screening and culturing, bud elongation culture and bud rooting culture adopt the corresponding culture medium provided by the invention. According to the method, the genetic transformation time of the kiwifruits can be shortened, the genetic transformation efficiency can be improved, particularly, a culture medium formula for rapidly and efficiently obtaining positive seedlings is found, and the methodplays an important role in shortening the genetic transformation time of the Hongyang kiwi fruits and improving the genetic transformation efficiency.

The invention discloses a method for constructing genetic transformation systems of catalpa bungei. The method includes procedures of screening the critical lethal concentration of hygromycin by the aid of embryogenic calluses of the catalpa bungei; carrying out infection and transformation by the aid of agrobacterium-mediated processes assisted by ultrasonic treatment; detecting resistant regenerated embryogenic calluses and regenerated plants and the like. The embryogenic calluses of the catalpa bungei are used as transformation receptors. The method has the advantages that the embryogenic calluses of the catalpa bungei are used as the receptors by the aid of the method for the first time, accordingly, infection materials are wide in transformation population, and embryogenic cells are high in regeneration capacity; annual genetic transformation requirements can be met by long-term stable subculture storage of the embryogenic calluses; the embryogenic calluses of the catalpa bungei are infected and transformed under the assistance effect of ultrasonic treatment, and accordingly the method is high in transformation efficiency; T-DNA [thymine DNA (deoxyribonucleic acid)] regions of vectors are successfully integrated into genomes of the catalpa bungei by the aid of the method, and specific primers are designed for detecting transgenic catalpa bungei regenerated plants; the method is applicable to molecular genetic improvement and detection on the catalpa bungei, and transgenic catalpa bungei obtained from transformation events can be used as a donor, so that diversified novel transgenic varieties can be obtained.

The invention discloses a construct and an application thereof. The construct comprises a nucleotide sequence shown in SEQ ID NO: 1. The nucleotide sequence shown in SEQ ID NO: 1, namely a blue-green algae ictB gene can be successfully introduced into a receiver plant by using the construct, thus the photosynthetic efficiency of the receiver plant can be remarkably reinforced, and the yield of plants is increased.

The invention discloses an alfalfa genetic inverting method in plant transferring gene technical domain, which comprises the following steps: 1, comprising two methods; evoking callus with alfalfa hypocotyls through floating culture mode; assisting by super sound; sieving body embryo in medium; germinating the body embryo; generating root; getting the transforming plant; 2, using tender true leafof alfalfa as transferring acceptor material; transforming with agricillin medium guild; sieving in traumatic evoked medium; seeding resistance callus in differential medium; evoking out of body embryo; generating root; getting the transforming plant. This invention increases genetic transferring efficiency of alfalfa and shorten genetic transferring period.

The invention discloses a HD-Zip I type transcription factor GmHDL 57 gene with ability of improving salt resistance of leguminous plants. The sequence of the gene is a nucleotide sequence shown as SEQ ID NO:1, and a coded protein sequence is an amino acid sequence shown as SEQ ID NO: 2. The invention further discloses application of the HD-Zip I type transcription factor GmHDL 57 gene with ability of improving salt resistance of leguminous plants in lotus corniculatus. By utilizing an overexpression technique, the salt resistance of the gene in the leguminous plant lotus corniculatus is researched. Results show that compared with control plants, the salt resistance of the plants is obviously enhanced after overexpression, and the gene has great application prospects for widening a planting range of the leguminous plants in saline-alkali soil.

The invention discloses a sterilization method of rape seeds for tissue culture. The sterilization method comprises the following steps: firstly, putting seeds in water for socking for 1 hour, removing seeds floating on the water surface, washing with tap water twice, transferring into a clean bench, sterilizing for 1 minute with 75% alcohol, sterilizing the seeds for 15 minutes with 3% NaClO (keeping a shaking and rotating state in the process), further washing with sterile water 5-6 times, and finally transferring the seeds onto sterile filter paper for airing, wherein the 3% NaClO has the optimal sterilization concentration screened from seed sterilization effects of 0-30% NaClO, and the sterilization time of 15 minutes is the optimal sterilization time screened from 10-30 minutes of seed sterilization with the optimal concentration of 3% NaClO. By adopting the sterilization method, maximum healthy seedlings can be cultured for the production of transgenic oilseed rape, so that thesuccess rate of oilseed rape gene transformation is increased.

The invention provides application of a corn BBM1 gene for improving the plant genetic transformation efficiency. For the first time, the corn BBM1 gene can promote the genetic transformation efficiency of plants such as corn, and precious gene resources are provided for genetic breeding. The gene can improve the genetic transformation efficiency of the transformed corn variety syndrome-31 and thelike, the corn variety B73 and other transformation efficiency is improved, the corn transformation gene limit can be effectively solved, and the corn transformation acceptor varieties are enlarged.

The invention belongs to the technical field of plant genetic engineering, and provides an agrobacterium-mediated genetic transformation method using the mature embryo of sorghum as an explant to induce callus for the disadvantageous that in traditional sorghum genetic transformation systems, material drawing by using young embryos and young ears as explants is seasonally restricted and also for the tedious operation that embryo stripping is needed for other crops in which the mature crops are used as the explants. Sorghum seeds which are germinated and then appear white are utilized as objects for agrobacterium infection and the explants for callusogenesis induction, resistant callus are obtained through screening and cultivation, and then a regenerated and transformed plant is obtained through subculture, differentiation, rooting, and transplantation. The disadvantages that in an existing sorghum genetic transformation system, the material drawing by using the young embryos and youngears as the explants is seasonally restricted is avoided, the tedious operation that the embryo stripping is needed for other crops in which the mature crops are used as the explants is overcome, andthe sorghum genetic transformation efficiency is greatly improved. According to the agrobacterium-mediated genetic transformation method using the mature embryo of sorghum as the explant to induce the callus, references and technical approaches are provided for performing genetic improvement of sorghum by using biotechnology.

The invention belongs to the field of plant genetic engineering, and discloses an EMP gene for influencing differentiation and development of a rice callus and application of EMP gene in differentiation and development of the rice callus. The nucleotide sequence of the EMP gene is shown as SEQ ID NO:1; the EMP gene is related to the differentiation and development of the rice callus, and the regeneration capacity of the rice callus can be quickly detected and identified by detecting the expression quantity of the EMP gene; and moreover, through overexpression of the EMP gene, the differentiation capacity of the rice callus can be improved, the genetic transformation efficiency of rice is improved, and then the transgenosis transformation efficiency of agrobacterium-induced rice is improved, and the application is of great importance in breeding new rice varieties.

The invention discloses a kit comprising serial culture mediums and its application in soybean genetic transformation. The kit comprises recovery culture medium, screening culture medium, growth culture medium, and rooting culture medium. The recovery culture medium comprises 45-55 mg / L of L- asparagine, 45-55 mg / L L-glutamine, 28-32 mg / L EDTA salt, and 11-13 mg / L FeSO4. The screening culture medium contains 45-44 mg / L L-asparagine, 45-55 mg / L L-glutamine, 28-32 mg / L EDTA salt, and 11-13 mg / L FeSO4. The growth culture medium contains 45-55 mg / L L-asparagine, 45-55 mg / L L-glutamine, 28-32 mg / L EDTA salt, and 11-13 mg / L FeSO4. The rooting culture medium contains 45-55 mg / L L-asparagine and 45-55 mg / L L-glutamine. The application of the kit in soybean genetic transformation belongs to the protection scope of this invention. The kit helps improve soybean genetic transformation efficiency and has high application value.

The invention discloses a method for constructing a ramie genetic transformation system, which belongs to the agricultural biological technical field or the plant genetic engineering field. The method mainly comprises following steps: taking a ramie seedling cotyledon as a transformation explant to be cocultured with agrobacterium, then, picking up the cotyledon which is cocultured, transferring the ramie seedling cotyledon on selective medium to eliminate bacteria and to culture selectively, and transferring a resistant plant which is produced in the selective medium in selective elongation medium and selective rooting medium to carry out elongation culture and rooting culture successively until a resistant bud is regenerated into a complete plant. The resistant bud which is obtained is respectively checked through GUS, PCR and Southern hybridization and the result shows that a foreign gene is integrated in a ramie genome. The method of the invention has simple operation, materials are conveniently obtained, the transformation efficiency is high, and the method can be widely used in genetic engineering operations of the ramie.

The present invention belongs to the field of plant transgenic technology, and discloses a method for improving sugarcane genetic transformation efficiency by taking mannose as a screening agent. The method is based on sugarcane young heart leaves-induced yellowish and granular type II embryogenic callus as a transgenic acceptor material, employs a negative pressure assistant and improved agrobacterium tumefaciens mediated method to lead a 6-phosphate mannose isomerase gene into a sugarcane genome, and enables getting transgenic sugarcane plants after screening and differentiating on mannose-containing medium. The method uses a PMI positive selection marker system, requires a short cycle from sugarcane callusing to differentiating into seedlings, is stable in transformation effects, and helps to overcome the defects that a genetic transformation system taking antibiotics and herbicides as negative selection markers is low in transformation efficiency, long time in embryogenic callus induced differentiation, easy in sugarcane callus browning, etc. The transformed callus maintains a high regenerative capacity, and has a transformation rate of 58.97% showed by molecular detection results.

The invention discloses a method for increasing chloroplast genetic transformation efficiency through the genome editing technique. According to the method, chloroplast genome is subjected to fixed shear through the genome editing technique, thereby increasing recombination efficiency and integration efficiency of exogenous gene in the chloroplast genome, and further increasing the chloroplast genetic transformation efficiency.

The invention discloses a white birch transgenosis method, belongs to the field of plant gene engineering and aims at solving the problem that white birch transgenosis is low in efficiency, difficult in sterilization and long in period. The method includes: 1, selecting white birch seeds, soaking, and picking mature seeds which are fully expanded by absorbing, submerged at the bottom of a bottle and germinated completely as transgenosis receptors; 2, sterilizing; 3, preparing engineering bacterium liquid used for infection of the seed receptors; 4, longitudinally cutting the seeds, and dip-dyeing; 5, placing the seeds in a liquid callus induction medium for co-culture; 6, obtaining a transgenosis strain to complete through callus culture, differential culture, subculture and rooting culture. By using the method, culture of a white birch explant is not needed, so that transgenosis period is shortened; a liquid co-culture mode is adopted, so that repeated sterilization steps are omitted, and white birch transgenosis efficiency is improved; conversion efficiency is high, period is short, and the problem that white birch transgenosis is low in efficiency, difficult in sterilization and long in period is solved. The white birch stain is high in transplanting survival rate, and the method is simple to operate and easy to master.

The invention belongs to the technical field of gene engineering, and particularly relates to the application of cell-penetrating peptide TAT2 in transgenic cotton cultivation. The cell-penetrating peptide TAT2 comprises 18 amino acids, and the sequence is shown in SEQ ID NO.1. After cotton pistil fertilization is finished, DNA to be transformed and cell-penetrating peptide TAT2 solution which are evenly mixed are injected into an ovary through a pollen tube to be used for carrying a DNA fragment to be transformed to a cotton genome. Due to the fact that the cell-penetrating peptide can bring a foreign DNA into a fertilization cell directly and smoothly and the foreign DNA can enter a cell nucleus, genetic transformation efficiency is improved remarkably compared with the traditional pollen tube path way; besides, the method does not depend on a cotton tissue culture regeneration system, operation is convenient, expensive instruments are not needed, production cost is low, and therefore a more effective method worth application and popularization can be provided for cotton variety improvement and fundamental research.

The invention relates to the technical field of plant genetic engineering, and provides an agrobacterium-mediated tomato genetic transformation method. The method specifically comprises the followingsteps: obtaining a sterile tomato leaf explant; infecting the leaf explant with agrobacterium tumefaciens; screening the resistance of the explant; carrying out subculture on the explant; rooting culture of the explant; carrying out soil moving culture on the transgenic plant; according to the method, the procedure of genetic transformation is simplified, the whole process is simple, and the transformation period is effectively shortened; ; the heredity of the transgenic offspring tends to be stable; the operation is simple, the expression level of the exogenous gene is obviously improved, andthe conversion efficiency is greatly improved.