147 results about "Rhizobium rhizogenes" patented technology

The invention relates to an agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method, and belongs to the technical fields of molecular biology and genetic engineering. The agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method comprises the following steps of: selecting genotypes of soybean seeds, disinfecting the soybean seeds, and germinating; preparing bacterial liquid, which contains anti-nematode bacillus thuringiensis (Bt) genes, of agrobacterium rhizogene K599; transforming the agrobacterium rhizogenes-mediated soybeans; performing histochemical staining analysis on transgenic roots; and performing polymerase chain reaction (PCR) detection on the transgenic roots. According to the method, an agrobacterium rhizogenes-mediated and vacuum infiltration-assisted transformation method is applied to the soybeans for the first time, and hairy roots of soybean roots are transformed successfully; the disinfected soybean seeds are planted in sterile vermiculite and are germinated, the injury of epidermic meristematic cells is caused at the upper end of hypocotyl, the agrobacterium rhizogene is infected under the vacuum condition, and genetic transformation is performed to obtain the soybean transgenic hairy roots containing the anti-nematode BT genes. By implementing the agrobacterium rhizogenes-mediated and vacuum infiltration-assisted soybean genetic transformation method, the opportunity of the invasion of the agrobacterium rhizogenes can be improved fully, the infection efficiency of the agrobacterium rhizogenes can be improved obviously, and the transformation rate of the soybeans is improved effectively.

The present invention discloses a method using Agrobacterium rhizogenes (Ar) gene to transform Rhodiola sachalinensis A.Bor to achieve a hairy root system for Salidroside production. By preparation of explant and cultivation in Ar bacteria liquid, Rhodiola sachalinensis A.Bor is induced to grow hairy roots by genetic transformation; the PCR testing proves that hairy roots are generated by transformation; a fermenter is used for massive production of hairy roots, and precursor substances and elicitors are added to improve the Salidroside content. The method is applied to produce Salidroside to substitute the drying-up Rhodiola sachalinensis A.Bor resources.

The invention discloses application of a method for smearing and transforming agrobacterium rhizogene-mediated hypocotyl to soybean transformation. The method for smearing and transforming agrobacterium rhizogen-mediated hypocotyl to is applied to soybeans for the first time, and hairy root transformation of soybean roots is successfully carried out. In this invention, the sprouting time of seeds and the concentration of screening agents are optimized, therefore not only increasing the quantity of transgenic hairy roots induced by each soybean explant, but also obviously enhancing the transformation frequency and growth conditions of hairy roots. A screening marker weed killer usually used for soybean transformation is successfully used for obtaining soybean compound plants in aseptic conditions, wherein each soybean compound plant has a transgenic root and a non-transgenic over-ground part and can be used as the ideal material for studying genes relative to the form of root systems, the symbiotic nitrogen fixation of roots and the formation of fungus roots.

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.

The invention relates to a method for producing alkannin by utilizing Arnebia euchroma(Royle)Johnst hairy root. The method includes selection and processing of Arnebia euchroma(Royle)Johnst explant, activation culture of agrobacterium rhizogene, infection of cotyledon explant and induction of hairy root, obtaining of sterile Arnebia euchroma(Royle)Johnst hairy root system and liquid culture of Arnebia euchroma(Royle)Johnst hairy root as well as alkannin production. The used hairy root has the characteristic of rapid and autonomous growth on culture medium without exogenous hormones, thus the method not only can overcome the defects that cell culture grows slowly and hormone is required to be additionally added to maintain the growth thereof when utilizing cell culture to produce alkannin, and the method has the advantages of stable alkannin throughput, simple operation in cultivation, lower cost and no restriction of climate and land natural conditions. Especially the invention provides a stable and continuable novel medicine source for producing alkannin by utilizing hairy root culture method and provides reliable source and material basis for alkannin mass production by applying bioreactor in the future.

The present invention relates to the method of establishing symbiotic relationship between arbuscular nycorrhizal fungus and tomato hairy root. Germinated tomato seedling is first soaked with agrolacterium suspension and inoculated to sucrose-agar culture medium for culture to induce hairy root; the tomato hairy root is then transferred to M culture medium and AM fungi spore is inoculated for dark culture at 25 deg.c; and the germinated hypha invades into root system to establish the symbiotic relationship between arbuscular nycorrhizal fungus and tomato hairy root. The present invention induces tomato hairy root and establishes the symbiotic relationship between AM fungus and tomato hairy root on bacteria-free culture medium. The system of the present invention may be used in the amplification of AM fungus to obtain bacteria-free AM fungus material and in the relevant research of AM fungus under bacteria-free condition.

The invention provides a construction method for a plant root related functional gene research model. The method comprises the following steps of: connecting a plant root related functional gene to a transgenic expression vector pRI101-AN to obtain recombinant plasmid containing a target gene and transferring the recombinant plasmid into agrobacterium rhizogene K599; infecting a part which is 0.2-0.5cm below a cotyledon of a living cucumber seedling germinated for 4 days, and inducing to form a transgenic hairy adventitious root; shearing off a main root below the part where the hairy root isformed when the hairy adventitious root grows to be 5-10cm long; burying the formed hairy adventitious root into soil; and continuously growing for 1-2 months to obtain the plant root related functional gene research model. In the method, by using a cucumber plant with the transgenic hairy adventitious root, a cucumber root related functional candidate gene can be quickly identified, and the identification can be performed without obtaining the transgenic plant, so that an experimental procedure can be greatly reduced, the defect of low transgenic regeneration efficiency is overcome, and quick analysis of functions of the root related gene can be performed on a large scale.

The invention provides a construction method of agrobacterium rhizogenes-mediated transgenic plants, and relates to the technical field of plant genetic engineering. The method comprises the steps offirstly, constructing agrobacterium rhizogenes with a target gene, enabling sterile seedlings of plants to take roots and then planting; when the stem lengths of the planted sterile seedling are morethan 3cm from the roots, injecting the bacterial liquid of the agrobacterium rhizogenes with the target gene into the stems of the sterile seedlings of the plants; after the transgenic hairy roots ofthe plants grow out at the injected parts of the stems, culturing until the lengths of the transgenic hairy roots are greater than 3cm and the number of roots is increased to 10, removing the non-hairy roots and extending the hairy roots into the soil to obtain the transgenic plants with the hairy roots. Compared with the conventional plant transgenic system, the method provided by the invention can omit the plant re-differentiation step of callus cells and overcome the problems of long period and the like which are caused by using the callus cells for genetic transformation. The method provided by the invention requires simple equipment and easy in mastering of operation technology, thus having a broad development and application prospects.

The invention discloses a method for inducting hairy root of glycyrrhizae. The procedure of the method is to co-culture glycyrrhizae cotyledonary node and agrobacterium rhizogenes which contains Ri plasmid and then transfer glycyrrhizae cotyledonary node and agrobacterium rhizogenes to an induction medium for induction cultivation after washing of cephamycin-contained sterile water, then glycyrrhizae cotyledonary node yields glycyrrhizae hairy root. The invention takes glycyrrhizae cotyledonary node as acceptor material for the first time and realizes induction of glycyrrhizae hairy root through the mediating of agrobacterium rhizogenes, thus shortening the growing period of glycyrrhizae hairy root to 4 days and raising the transforming efficiency to more than 96 percent. Furthermore, the active component in the hairy root such as licoflavone is as high as 1.6 percent, which settles a good foundation for producing useful glycyrrhizic acid and licoflavone by using glycyrrhizae hairy root culture.

The invention discloses a method for inducing and quickly propagating hairy roots of tartary buckwheat. The method includes (1), pre-cultivating sterile explants of the tartary buckwheat; (2), infecting the pre-cultivated explants of the tartary buckwheat by the aid of agrobacterium rhizogene infection liquid; (3), co-cultivating the infected explants; (4), transferring the co-cultivated explants into induction cultivation media and carrying out induction cultivation on the co-cultivated explants; (5), carrying out secondary cultivation and amplification cultivation on the hairy roots of the tartary buckwheat. The hairy roots of the tartary buckwheat are generated by means of induction. The method has the advantages that key parameters such as the explant pre-cultivation time, the agrobacterium rhizogene infection liquid concentration, the infection time, the co-cultivation time and the types of agrobacterium rhizogene which can obviously affect the induction efficiency for the hairy roots of the tartary buckwheat are optimized, accordingly, effects of inducing the hairy roots of the tartary buckwheat can be obviously improved, and the hairy roots of the tartary buckwheat can be quickly and efficiently induced; the method is applicable to inducing hairy roots of different varieties of tartary buckwheat, materials are convenient to obtain, the method is high in applicability and induction speed and easy and convenient to implement, heritance is stable, the cost can be saved, and the like.

The invention discloses a cultivation method for inducing scutellaria baicalensis hairy root based on agrobacterium rhizogenes infection, which uses an agrobacterium rhizogenes bacteria liquid to infect root of a scutellaria baicalensis explant and induces the scutellaria baicalensis explant to form a hairy root, the scutellaria baicalensis hairy root presents yellow hairy on a MS solid medium, so that scutelloside can be stably synthesized, an ITS sequencing is performed and the hairy root can be identified as the scutellaria baicalensis hairy root of a scutellaria. Then seed selection is performed for further in a seed selection medium and a cultivation condition can be optimized to select lines which raise the content of active ingredient scutelloside used for hairy root traditional Chinese medicines. Hairy root can be obtained through scutellaria of a scutellaria introduced by agrobacterium rhizogenes, wherein the inductivity can reach 10 - 40%, the obtained scutellaria baicalensis hairy root presents yellow hairy which is capable of stably synthesizing scutelloside. According to the invention, the scutellaria baicalensis hairy root of a scutellaria is cultivated on the optimized medium, the growth is rapid and the biomass can be increased for 23 -35 times in 30 - 35 days, and the cultivation is performed under the dark conditions. The invention has the characteristics of low energy consumption and low cost, which is suitable for industrialization production.

The invention provides a construction method for an economic plant effective root transgene system, and belongs to the technical field of plant transgene. A bacterial liquid containing a target recombinant vector agrobacterium rhizogene is subjected to solid-liquid separation, an obtained thallus containing the target recombinant vector agrobacterium rhizogene is subjected to resuspension, an obtained agrobacterium rhizogene suspension is injected into stems of economic plant seedlings, and 10-12 days after injection is conducted, calluses grow at wounds of the stems of the economic plant seedlings; the seedlings continue to be cultured for 28-35 days, and when the calluses are differentiated into regenerated hairy roots, original hairy roots are cut off. The construction method is applicable to various economic plants, and the regeneration rate of the hairy roots and the regeneration rate of the calluses are high. The construction method provides very rapid and effective gene system function analysis, secondary metabolite engineering and plant stress response studies. According to the constructed root transgene system, only the roots are transgenic roots rather than the whole plants, and the constructed system is a good system for studying signal transduction between the roots and the stems.

The invention discloses a method for increasing content of camptothecin in camptotheca hairy root in the technical field of biology. The method comprises the following steps of: cloning coding frame sequences of CrORCA3 and CrG10H genes from catharanthus roseus; constructing a plant bivalent high-efficiency expression vector including the genes; and genetically transforming the camptotheca into camptotheca hairy root genetically modified by CrORCA3 and CrG10H genes, by using the hair root agrobacterium-mediated technique. The camptothecin content in the obtained genetically-modified camptotheca hairy root is increased obviously. The content of the camptothecin is 5.4 times of the contrast. The content of hydroxycamptothecin is 2.3 times of the contract. The content of the hairy root camptothecin independently genetically-modified by CrORCA3 is increased by 1.5 times of the contract. The content of the hairy root camptothecin independently genetically-modified by CrG10H gene is not increased obviously. The invention provides a method for increasing content of the camptothecin in the camptotheca hairy root, thereby providing a novel method for producing the camptothecin and the hydroxycamptothecin as anti-cancer drugs of important clinical requirements.

The invention discloses a yellow aconite hair root and its acquisition method. The yellow aconite hair root provided by this invention is obtained by selecting yellow aconitic organ as explant materials, performing transfection by hair root agricillin and culturing the hair root. The yellow aconite hair root obtained by this invention can reproduce by fluid culture method, the reproductive speed is quick, the period of production is short (about 40 days or so), and it can not be influenced by the natural condition, which can carry out the intensifited industrial production and can overcome the defects that the speed of reprothropology is slow, the growing cycle is long, the output is low and the hereditary stability is weak in artificial planting of yellow aconite hair root; the medicinal components in hair root-the guanfu base A is of high content, which has wide application foreground and utilization value.

The invention discloses an inducing method of dioscorea alata transgenosis hairy roots, and belongs to the technical field of plant culture and transgenosis. The method includes the steps: 1) explantsterilization; 2) explant culture; 3) infection liquid preparation; 4) hairy root induction and reproduction; 5) hairy root reproduction. According to the method, dioscorea alata stems with leaf axilla serve as explants, an induction culture medium, a differential culture medium and a rooting culture medium are optimized, an integrated plant is acquired through induction, differential and a rooting culture, stems of dioscorea alata aseptic seedlings are infected by agrobacterium rhizogenes K599 (with recombinant plasmid pRI101-AN-gfp) living bodies to obtain the transgenosis hairy roots, PCR (polymerase chain reaction) and fluorescence microscope observation identify whether the hairy roots are transgenosis hairy roots or not, an inducing system of the dioscorea alata transgenosis hairy roots is built, and dioscorea alata is rapidly and effectively bred.

The invention discloses a huperzia serrata hairy root system preparation and cultivation method, and belongs to the biological cell engineering technology. The method comprises the following steps: tender stems of huperzia serrata is adopted for dedifferentiation treatment of an explant to acquire huperzia serrata calluses; the callused and agrobacterium rhizogenes (DL 1968) containing Ri plasmid are co-cultivated and transferred to an induced medium for induced cultivation after residual fungus liquid is extracted by aseptic paper, and then hairy roots grow at the huperzia serrata calluses; and the explant with the hairy roots is placed in an expansion medium for expanded cultivation of the hairy roots after bacteriostatic cultivation. According to the invention, the biological cell engineering technology is utilized to build a huperzia serrata hairy root system cultivation system to prepare a huperzia serrata hairy root system, so that standardized production of huperzia serrata is realized, wild resources are replaced, the ever-increasing market demand for huperzia serrata is relieved, and the problem of dependency on huperzia serrata import is solved. Therefore, the huperzia serrata hairy root system preparation and cultivation method has great significance on industrialized and commercialized development of medicinal plant hairy roots.

A novel process for the remediation of nitrogenous energetic materials such as 1,3,5,-trinitro-1,3,5-triazine (RDX) which can be used in situ on contaminated media is provided. The process comprises the bioremediation by one or more miccroorganisms capable of metabolizing the energetic materials. Examples of such microorganisms include Rhizobium rhizogenes, Burkholderia sp., and Cladosporium cladosporioides (ATCC 66669). Strains of these microorganisms have been deposited. The strain designated A1 has been deposited as Rhizobium rhizogenes BL (ATCC PTA-4110) and the strain designated C8 has been deposited as Burkholderia sp. (ATCC PTA-4111) Additionally, with the addition of a carbon source, such as a sugar, the process can totally degrade the energetic materials in two to three days.

The invention provides a method for inducing the production of tripterygium wilfordii hairy roots by agrobacterium rhizogenes, which comprises the following steps: (1) obtaining aseptic explants; (2) preparing bacterial liquid of agrobacterium rhizogenes; (3) performing agrobacterium rhizogenes infection to induce hairy roots; (4) establishing a hairy root in-vitro culture system, wherein the establishment of the hairy root in-vitro culture system comprises the following steps: culturing hairy roots in a 1/2 MS solid medium containing 500 mg/L cefotaxime sodium under a dark culture condition with a temperature of 27 +/-1 DEG C, performing subculture once every 7 days, reducing the using concentration of cefotaxime sodium gradually during subculture till no bacterium is found, finally putting the completely aseptic hairy roots on the 1/2 MS solid medium for subculture and preservation. During the detection of the produced aseptic tripterygium wilfordii hairy roots, a considerable amount of triptolide is found to be contained in the hairy roots, and the content is significantly higher than the content of triptolide in traditional tripterygium wilfordii wild roots and tissue culture roots; the method has good prospects for industrial application. The production of triptolide by hairy root culture fills the gap of traditional Chinese medicine.

The invention discloses a method for inducing Polygonum ciliinerve to generate hairy roots, which comprises the following steps of: inducing laminae of the Polygonum ciliinerve through an Agrobacterium rhizogenes strain W.T15834 to form the hairy roots, and culturing to obtain a great number of hairy roots of the Polygonum ciliinerve, wherein the laminae of the Polygonum ciliinerve serve as explants. According to the method for inducing the Polygonum ciliinerve to generate the hairy roots, the Polygonum ciliinerve is converted by utilizing Agrobacterium rhizogenes on the basis of establishing a sterile rapid propagation system by utilizing isolated culture, thereby, a great number of hairy roots of the Polygonum ciliinerve can be obtained. The method for inducing and culturing the hairy roots of the Polygonum ciliinerve has the characteristics that hormones autonomously and rapidly grow and differentiate, the proliferation coefficient is high, the inheritance is stable, materials can be conveniently fetched without season limitations, and the culture time is short, and has a positive effect on perfecting the Polygonum ciliinerve transgenic technology, performing mass culture on the hairy roots of the Polygonum ciliinerve and utilizing the biotransformation of secondary metabolic products of the hairy roots of the Polygonum ciliinerve in the later period.

The invention relates to the technical field of bioengineering and discloses a method for improving camptothecin content in hairy roots of the new-type medicine source plant ophiorrhiza pumila of camptothecin. The method disclosed by the invention comprises the following steps of: cloning coding frame sequences of genes of ophiorrhiza japonica strictosidine synthase and geraniol-10-hydroxylase in catharanthus roseus to build a plant bivalent efficient expression vector containing the genes, carrying out genetic transformation on ophiorrhiza pumila via an agrobacterium rhizogenes mediated method to acquire the hairy roots of the ophiorrhiza pumila for transforming CrSTR and CrG10H genes; and inducing and treating high-yield camptothecin strains by adopting plant hormones to acquire high-yield hairy roots with the camptothecin content of 4.703mg/g DW. The MTT (Methyl Thiazolyl Tetrazolium) detection proves that camptothecin crude extract acquired via a transgenosis manner is good in biological activity and the lethality to cancer cells reaches 35.9%. By adopting the method disclosed by the invention, a new medicine source for acquiring the camptothecin is provided and a new method for producing anti-cancer medicine camptothecin in important clinical demand is provided.

The invention belongs to the technical field of biological culture, in particular to a method for improving the tanshinone content in salvia miltiorrhiza hair roots through methyl jasmonic acid treatment. According to the method, agrobacterium rhizogene is firstly used for infecting salvia miltiorrhiza aseptic seedlings to obtain the salvia miltiorrhiza hair roots, then, the sterilization culture of the salvia miltiorrhiza hair roots is carried out, and next, the salvia miltiorrhiza hair roots are selected to be transferred into a liquid culture medium for carrying out suspension culture; and methyl jasmonic acid is added into the salvia miltiorrhiza hair roots in the time of 4 to 6 days before the salvia miltiorrhiza hair root liquid culture harvesting, then, the harvesting is carried out on time, and the tanshinone content in salvia miltiorrhiza hair roots can be greatly improved. The method provided by the invention has the advantages that the tanshinone content in the hair roots can be effectively improved, the operation is simple, the relatively higher front-stage investment required by the utilization of a gene engineering method for improving the tanshinone content is not needed, the production cost of the tanshinone is effectively reduced, and great significance is realized on meeting the large-scale production of the tanshinone.

The invention belongs to the technical field of bioengineering, and provides a method for improving camptothecin content in hairy roots of novel medicine source plant ophiorrhiza pumila of camptothecin. The method comprises the following steps of: cloning coder frame sequences of CrORCA3 and CrG10H genes from catharanthus roseus; constructing a bivalent efficient expression vector of a plant containing such genes; and carrying out genetic transformation to the ophiorrhiza pumila through the agrobacterium rhizogenes mediated transformation method, thus obtaining the hairy roots of the ophiorrhiza pumila containing transformed CrORCA3 and CrG10H genes. The camptothecin content in the obtained hairy roots of the transgenic ophiorrhiza pumila is obviously increased, the camptothecin output in each flask(2.62mg/flask) is averagely increased by 5.52 times in comparison with that in the contrast, and the average content (8.44mg/g DW) is increased by 4.04 times. The total camptothecin output of each flask, induced by plant hormones after culturing one strain for six weeks, is increased by 7.8 times. The preparation method provided by the invention is a novel method for producing an anti-cancer drug that is camptothecin under important clinical demand.