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Efficient transient plant transgenic method based on negative pressure and temporary immersion

A technology for instantaneous transformation of plants, applied in the field of plant biotechnology and tissue culture, can solve the problems of complicated operation, time-consuming transgenic system, low infection rate, etc., and achieve simple operation, easy acquisition and high repeatability

Active Publication Date: 2021-03-09
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is to overcome the shortcomings of the transgenic system in the prior art, such as time-consuming, low infection rate, and complicated operation, and provide a method for efficiently infecting and inducing target gene expression in plant leaves in a short time. method

Method used

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  • Efficient transient plant transgenic method based on negative pressure and temporary immersion
  • Efficient transient plant transgenic method based on negative pressure and temporary immersion
  • Efficient transient plant transgenic method based on negative pressure and temporary immersion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Transformation test of pear plants

[0063] 1) Select the OHF333 (Pyrus Communis) group culture seedlings in the vary, and cut the top buds of about 2 cm and transferred to the NN69 solid medium.

[0064] 2) The experiment repeatedly validated its effectiveness twice as a GUS basis, and the experiment was repeated two times, and specifically selected INTGUs having a plant Catalyze intron, constructed into the plant dual expression vector PBI 121. The Agrobacterium Eha105 was selected for the infectious strain, and the PBI121 vector with the reporter was introduced into the Agrobacterium cells using a freezer method.

[0065] Agrobacter with a target vector was inoculated into a liquid LB medium with 50 mg / l kanamycin and 25 mg / l. After 3 days, I was re-vaccined to 50 mg / l kanamycin and 25mg. / L rifetian liquid lb medium is oscillated for 12 hours. 25 ° C, 3000 revolutions were collected 15 minutes to collect cultured Agrobacterium bacteria. Formulated infec...

Embodiment 2

[0073] Example 2 Multi-temporary immersion parameter verification

[0074] This embodiment further verifies the effectiveness of other multiple temporary immersion parameters.

[0075] Based on the same step 1) -2 based on Example 1).

[0076] 3) Under sterile conditions, the OHF333 ohf333 ohf333 in 28-35 days was subsequently cultured, and the 13-day leaves were quickly cut off from the petiole, and the healthy blade was completely carried out to sterile water. After all the blade is prepared, remove from sterile water, after absorbing excess water on the filter paper, quickly immersed in infection with the activated Agrobacterium, the experiment repeated twice, each treatment 120 leaves, place In the vacuum pump, it was maintained for 3 minutes after 3 minutes, and the pressure was quickly released.

[0077] 4) Temporary immersion mode implementation process is as follows:

[0078] A. The blades of the intrinsic infected were allowed to stand for 1 minute on the filter paper, an...

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Abstract

The invention provides a plant transgenic method mediated by agrobacterium. According to the method, exogenous genes are efficiently and instantaneously introduced into plant explants through negativepressure and short-time repeated infection. Compared with a traditional method, the method has the advantages of being short in consumed time, high in efficiency, easy to operate and the like.

Description

Technical field [0001] The present invention relates to plant biological technology and tissue culture, and more particularly to a plant transgenic method that is mediated by Agrobacterium. Background technique [0002] Agrobacterium-mediated transgenic system is a key modern biological means for obtaining transgenic plants to study plant genes, and introduce excellent agronomic traits into the target plant. At present, the mainstream plant transgenic system is usually used as a gene vector carrier, with a plant opal of leaves, pollen, endosperm, and sub-leaf. Generally, the Agrobacterium is infected with an anthracene. And in the infection process, the T-DNA region carrying the exogenous antibiotic and the target gene expression frame is introduced inside the plant cells, which in turn is integrated on the genome. [0003] After the T-DNA is introduced inside the plant cell, the target gene can be transcribed, translated, modified, and finally expressing active proteins using th...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/84
CPCC12N15/8205
Inventor 徐凌飞翟锐
Owner NORTHWEST A & F UNIV