Method for fast obtaining transgenic plant of potato

A potato and genetically modified technology, applied in the field of plant genetic engineering, can solve the problems of large difference in transformation frequency, complex transformation procedures, low transformation frequency, etc., and achieve the effects of good repeatability, simple transformation steps, and cost-saving cultivation

Inactive Publication Date: 2004-08-11
HUAZHONG AGRI UNIV
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  • Abstract
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Problems solved by technology

However, the following problems still exist in the genetic transformation of potato mediated by Agrobacterium to be solved: if the transformation procedure is complicated, that is, through the induction of callus first, then the induction of differentiation, and after the differentiation of shoots, transfer to the rooting medium to induce rooting (De Block M, Theor Appl Genet, 1988, 76: 767-774); genotype restriction, transformation frequency difference between different varieties is large (Stiekema W J etc., Plant Cell Rep, 1988, 7: 47-50; Tavazza R, etc., Plant Sci, 1988, 59: 175-181; Sheerman S and Bevan M W. Plant Cell Rep, 1988, 7: 13-16; Wenzler H et al., Plant Science, 1989, 63: 79-85); transformation frequency is low, most Less than 20% (Ishida B K et al., Plant Cell Rep, 1989, 8: 325-328; Visser R G F et al., Plant Mol Biol, 1989, 12: 329-337); the cycle is longer, generally 5-8 weeks (Beaujean A et al., Journal of Experimental Bot, 1998, 49: 1589-1595; Trujillo C et al., Plant Cell Rep, 2001, 20: 637-641) and aneuploid (such as 47 or 49 chromosomes) transgenic plants Produce (Ooms G etc., Theor Appl Genet, 1987, 73:744-750; Ishida B K, etc., Plant Cell Rep, 1989, 8:325-328) etc.

Method used

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  • Method for fast obtaining transgenic plant of potato
  • Method for fast obtaining transgenic plant of potato
  • Method for fast obtaining transgenic plant of potato

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Agrobacterium LBA4404 containing the expression vector pBSAP was inoculated in LB medium with 50 mg / L kanamycin and 50 mg / L rifampin, and cultured on a shaker at 28°C and 240 r / min until OD 600 is about 0.5. Then it was centrifuged at 5000r / min for 6 minutes, and the pellet was resuspended with MS liquid medium. Under sterile conditions, cut the potato tetraploid cultivar "E Potato No. 3" into small discs with a thickness of 1-2 mm, soak them in the above-mentioned Agrobacterium solution for 10 minutes, take them out and dry them with sterile filter paper The surface bacterial liquid is transferred into the solid differentiation medium numbered SRM3. The composition and proportion of the medium are as follows: MS medium with a large amount of components and trace components, plus 1mg / L IAA, 0.2mg / L GA 3 , 0.5mg / L BA, 2mg / L zeatin, 30g / L sucrose, pH=5.8. Incubate in the dark at 28°C for 2 days. Then they were transferred to the same medium supplemented with 75 mg / L ka...

Embodiment 2

[0041] Agrobacterium LBA4404 containing the expression vector pBSAP was inoculated in LB medium with 50 mg / L kanamycin and 50 mg / L rifampin, and cultured on a shaker at 28°C and 240 r / min until OD 600 is about 0.5. Then it was centrifuged at 5000r / min for 6 minutes, and the pellet was resuspended with MS liquid medium. Under sterile conditions, cut the potato tetraploid cultivar "Gannongshu No. 2" into small discs with a thickness of 1-2mm, soak them in the above-mentioned Agrobacterium solution for 10 minutes, and absorb them with sterile filter paper after taking them out. The dry surface bacterial liquid is transferred into the solid differentiation medium of SRM3 (see example 1) whose composition and proportion, sucrose concentration and pH are the same as those described in Implementation 1. Incubate in the dark at 28°C for 2 days. Then they were transferred to the same medium supplemented with 75 mg / L kanamycin and 400 mg / L cephalosporin, and cultured at a light intens...

Embodiment 3

[0043] Agrobacterium LBA4404 containing the expression vector pBSAP was inoculated in LB medium with 50 mg / L kanamycin and 50 mg / L rifampin, and cultured on a shaker at 28°C and 240 r / min until OD 600 is about 0.5. Then it was centrifuged at 5000r / min for 6 minutes, and the pellet was resuspended with MS liquid medium. Under sterile conditions, cut potato tetraploid cultivar "N552" into small discs with a thickness of 1-2 mm, soak in the above-mentioned Agrobacterium solution for 10 minutes, take it out, and blot the surface bacteria solution with sterile filter paper , into the solid differentiation medium that is numbered SRM3 (see Example 1), the composition and ratio of the medium, sucrose concentration and pH are the same as those described in Implementation 1. Incubate in the dark at 28°C for 2 days. Then they were transferred to the same medium supplemented with 75 mg / L kanamycin and 400 mg / L cephalosporin, and cultured at a light intensity of 2000 lx, a photoperiod o...

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Abstract

A process for quickly culturing transgenic plant of potato includes such steps as agroinfecting the potato explant cultured in test tube, culturing to introduce the target gene to potato receptor, direct culturing in solid differential culture medium to induce resistant buds, culturing in selective rooting culture medium to induco normal root, and verifying by PCR, PCR-southern, or Northern hybridizing method.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a method for rapidly obtaining transgenic potatoes through the mediation of Agrobacterium by using test tube tubers of potato as receptors. Background technique [0002] In my country and the world, potato (Solanum tuberosum L.) is the fourth largest food crop after rice, wheat and corn. Since potato is an autotetraploid vegetative crop, its gene segregation is complicated, the expression frequency of recessive genes is low, the pollen is often infertile, and hybridization is difficult. It takes a long time and is difficult to improve through conventional breeding methods. Big. Therefore, it is of great significance to use genetic transformation technology for genetic improvement of potato. [0003] Before the present invention was made, the genetic transformation of potato was mainly realized through the method mediated by Agrob...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H1/00A01H5/00C12N5/10C12N15/74
Inventor 谢从华柳俊司怀军
Owner HUAZHONG AGRI UNIV
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