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Method of establishing high fescue highly effective agrobacterium mediating conversion system

A technique of Agrobacterium-mediated and establishment method, which is applied in the field of establishment of high-efficiency Agrobacterium-mediated transformation system of tall fescue, which can solve problems such as adhesion, inability of target tissues or cells to carry out bacteria, and inability to produce signal molecules

Active Publication Date: 2007-08-15
深圳市日昇生态科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are two main reasons why monocotyledonous plants are difficult to transform with Agrobacterium: (1) monocotyledonous plants are rarely or completely unable to produce signal molecules that activate the genes in the Vir region of the Ti plasmid; (2) target tissues or cells transformed by monocotyledonous plants cannot Effective bacterial adhesion, no obvious wound response, can not induce dedifferentiation of cells near the wound to form a large number of competent cells, but only those competent cells with strong ability to regenerate and integrate transformation can be transformed into plants
So far, the Agrobacterium-mediated transformation system using tall fescue embryogenic callus as the receptor is still in the stage of laboratory research

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 Acceptor material preparation

[0022] The tall fescue species tested are embryogenic callus induced by Houndog5, Crossfire II, Barlexas, and Millennium, and the callus induction medium refers to MS+9mg / L2, 4-D+0mg / L6-BA+300mg / L hydrolyzed casein; callus subculture medium refers to MS+9mg / L2, 4-D+0.05mg / L6-BA+0.5mg / LKT+50XCu, closed the culture dish with Parafilm, cultured in dark at 26°C. Embryogenic calli aged 3-5 months were picked and pre-cultured for 7 days to be infected.

Embodiment 2

[0023] The determination of embodiment 2 bacteriostatin concentration

[0024] Agrobacterium strain LBA4404 was cultured in YEP liquid medium containing different concentrations of bacteriostatin in the dark at 28°C 200-220rmp, and the OD of the bacterial solution was measured 600 value. After Agrobacterium grows to the logarithmic growth phase, centrifuge the bacterial solution at room temperature 4000rmp for 10 minutes to collect bacteria, dissolve and dilute the bacteria with MS culture medium to make the OD 600 The value is 0.2-0.5, infect the callus for 20 minutes, and shake continuously during the infection process to make the Agrobacterium fully contact with the callus, and vacuumize for 5 minutes. The plant material was taken out, the residual bacterial liquid was blotted dry with filter paper, transferred to callus induction medium containing different concentrations of bacteriostatin, and cultured in the dark at 26°C. Record the pollution situation every day.

Embodiment 3

[0025] Implement 3 donor strain cultures

[0026] Pick a single colony from the plate and inoculate it into bacterial broth supplemented with antibiotics. 28°C, 200-220rmp constant temperature shaker culture to OD 600 0.5-0.8, centrifuge the bacteria solution at room temperature 4000rmp for 10 minutes to collect the bacteria, dissolve and dilute the bacteria with MS culture medium to make the OD 600 The value is about 0.3 for conversion, while adding 100 μmol / L acetosyringone (AS).

[0027] Infection: Pour the bacterial solution into a sterile small beaker filled with embryogenic callus, infect the callus for 20 minutes, shake gently during the infection process to make the bacterial solution fully contact with the callus, and soak in vacuum 5min. Place the infected callus in a petri dish with several sterilized filter papers on the bottom layer, cover the upper layer with a small piece of filter paper again, and apply light pressure with tweezers so that the excess bacteri...

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Abstract

The invention discloses a constituting method of high fescue and high effect agricillin dielectric conductor transferring system, which comprises the following steps: getting germ property traumatic receptor material through high-frequency regenerative system; culturing donor bacterial strain; ascertaining the density of bacteriostatic element; optimizing transforming condition of agricillin; screening and culturing transforming traumatic; regenerating plant; testing molecule of resistance plant; optimizing each condition of bacterium solution strength, disseminating time, disseminating condition, co-culturing time and adding density of bacteriostatic element to provide a transforming system. This invention overcomes the defect of now technology, which constitutes a high effective system.

Description

technical field [0001] The invention relates to genetic engineering, in particular to a method for establishing a high-efficiency agrobacterium-mediated transformation system of tall fescue. Background technique [0002] Tall fescue (Festuca arundinacea Schreb.), also known as reed-like fescue, is a perennial cool-season grass species in temperate regions of the world. It is native to western Europe. It has excellent characteristics such as broad sex, and can be used for both grazing and planting lawns. It is one of the important cultivated forage grasses in Europe and North America, and is listed as the most important turfgrass species along with Bluegrass grass. Introduced in the 1970s in my country, it became an important grass species for establishing artificial grassland and supplementing natural grassland in warm temperate regions of the north. In recent years, the use of tall fescue as a lawn grass has developed rapidly in my country. It has been introduced and cult...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00C12N5/04
Inventor 韩烈保王维飞曾会明齐春晖方文娟
Owner 深圳市日昇生态科技股份有限公司