Construction method of agrobacterium tumefaciens-mediated transgenic plants

A technology of Agrobacterium rhizogenes and transgenic plants, which is applied in the field of plant genetic engineering, can solve problems such as long cycle times, and achieve the effects of simple equipment, no need for redifferentiation, and broad development and application prospects

Active Publication Date: 2019-04-26
BEIJING FORESTRY UNIVERSITY
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] It can be seen that the existing technology mainly uses Agrobacterium rhizogenes to mediate hairy roots, and then induces and differentiates the hairy roots to form new transgenic plants. The method of obtaining transgenic plants by these methods still requires callus Cultivation, differentiation culture, rooting culture and other steps, the cycle is long

Method used

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  • Construction method of agrobacterium tumefaciens-mediated transgenic plants
  • Construction method of agrobacterium tumefaciens-mediated transgenic plants
  • Construction method of agrobacterium tumefaciens-mediated transgenic plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Induction of eGFP gene-transferred apple hairy roots

[0069] (1) Cultivation of aseptic apple seedlings: the aseptic seedlings of the apple variety Gala are provided by the cross-innovation platform of the School of Forestry, Beijing Forestry University. / L sucrose+8g / L agar, pH=5.8, after growing for 10 days at 22~24°C in the subculture medium (such as figure 1 Shown), transfer to MS+0.5mg / L IBA+30g / L sucrose+8g / L agar, pH=5.8 rooting medium, obtain the apple Gala aseptic seedling of rooting after 40 days (such as figure 2 shown).

[0070] (2) Preparation of the transgenic infection liquid: the 35S-eGFP-pROK2 vector was constructed using the Seamless Assembly Cloning Kit of Zhongmei Taihe Company. The constructed vector was transformed into competent cells of Agrobacterium rhizogenes K599 by heat shock method; the positive clone colony was picked, and firstly mixed with 5ml YEP+20mg / L Rif+50mg / LKana liquid medium at 28°C and 180rpm in a shaker. Shake ove...

Embodiment 2

[0078] Example 2 Effects of Different Agrobacterium rhizogenes on the Induction of Apple Gala Hairy Roots

[0079] Table 1 Effects of different Agrobacterium rhizogenes on the induction of hairy roots of apple Gala

[0080]

[0081] Select aseptic seedlings of apple Gala with consistent growth for subculture and rooting. Three kinds of Agrobacterium rhizogenes MSU440, C58C1 and K599 were amplified to OD with YEP+20mg / L Rif liquid medium at 28°C and 180rpm in a shaker 600 =6.0, make the infection bacteria solution. According to the method shown in Example 1, the apple Gala tissue culture seedlings through subculture rooting were injected into the stem, and then the induction of apple Gala hairy roots was observed.

[0082]As shown in Table 1, different Agrobacterium rhizogenes were used to inject the stems of apple Gala. When injected with MSU440, the induction rate of hairy roots was 10%, and when injected with C58C1, the induction rate of hairy roots was 10%. , when K59...

Embodiment 3

[0083] Effect of different concentrations of embodiment 3 Agrobacterium rhizogenes on the induction of apple Gala hairy roots

[0084] Table 2 Effects of different concentrations of Agrobacterium rhizogenes on the induction of apple Gala hairy roots

[0085]

[0086] Select aseptic seedlings of apple Gala with consistent growth for subculture and rooting. Three copies of Agrobacterium rhizogenes K599 were amplified to OD in a shaker at 28°C and 180rpm with YEP+20mg / L Rif liquid medium 600 = 0.2, OD 600 = 0.4, OD 600 =0.6 to make the infection bacteria liquid. According to the method shown in Example 1, the apple Gala tissue culture plantlets through subculture rooting were injected into the stem, and 40 days after the callus was born, the induction status of apple hairy roots was observed.

[0087] As shown in Table 2, apple stems were injected with different concentrations of Agrobacterium rhizogenes, when OD 600 = 0.4, the induction rate of hairy root was the highest...

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Abstract

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.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a method for constructing transgenic plants mediated by Agrobacterium rhizogenes. Background technique [0002] Agrobacterium rhizogenes is a G-Agrobacterium with a broad host range. After Agrobacterium infects plants, it can induce plants to produce a large number of highly branched adventitious roots, commonly known as hairy roots. The hairy roots produced by Agrobacterium rhizogenes infecting plants have the characteristics of fast growth, high degree of differentiation, stable physiology, biochemistry and genetics, and easy operation and control. [0003] In genetic engineering, Agrobacterium rhizogenes infection to form transgenic hairy roots is a fast and efficient transgenic method. In this study, a rapid and efficient apple transgenic system was established through Agrobacterium infection using the aseptic 'Gala' seedling of the same region, the same a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00
CPCA01H4/001A01H4/008C12N15/8205
Inventor 杨清孟冬董碧莹付玉杰牛丽丽宋治华
Owner BEIJING FORESTRY UNIVERSITY
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