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Method for transforming exogenous gene by coleoptile of germinating seed

A technology for germinating seeds and exogenous genes, applied in the field of plant genetic engineering, can solve the problems of low transformation rate, difficult to achieve large-scale and industrialization, poor repeatability, etc., and achieve the effects of convenient material sampling, good stability and less genetic variation.

Inactive Publication Date: 2011-04-20
LIAONING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its disadvantages are low conversion rate and poor repeatability. It must be carried out on the basis of aseptically operated plant tissue culture, and it is difficult to achieve large-scale and industrialization.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0023] 1. Germination of corn seeds:

[0024] Wash the corn seeds with 2 times the volume of water at 25°C-28°C for 5-10 hours. After the seeds have absorbed enough water, put them in a glass plate for germination and culture. Put 3-5 layers of wet gauze at the bottom of the plate. Seeds are placed on gauze and covered with a petri dish to germinate at room temperature to maintain temperature and humidity.

[0025] 2. Activation culture of Agrobacterium:

[0026] Routine suspension culture of Agrobacterium engineering bacteria with the target gene and herbicide resistance screening gene in YEB medium, centrifuge the bacterial liquid when the OD600 value is 0.5-1.0, pour off the YEB culture medium and resuspend it with N6 culture medium for activation For culturing, adding 100-500 mol / L acetosyringone (3,5methoxy-4-hydroxyacetophenone, AS) can promote the activation of vir gene. When the logarithmic growth phase is reached, the bacterial solution is stored at 4°C until use.

...

Embodiment II

[0032] The specific implementation of embodiment II is substantially similar to embodiment I:

[0033] 1. The specific implementation of embodiment II is the same as step 1 of embodiment I.

[0034] 2. The specific implementation method of embodiment II is the same as step 2 of embodiment I.

[0035] 3. On the basis of the specific implementation steps 1 and 2 of the above-mentioned embodiment II, carry out coleoptile cutting transformation:

[0036] When the seeds germinate to a coleoptile length of 1-3cm, use a double-sided knife to cross-cut the coleoptile 1-3mm above the germ growth point at the base of the coleoptile, expose the growth point and prick the cells at the growth point with a micro-injection needle, and then inject a small amount of 0.1-0.2ml of Agrobacterium in the syringe is dropped on the growth point and in the coleoptile pool. Because the cutting point is slightly higher than the growth point and the coleoptile grows rapidly, the coleoptile pool is form...

Embodiment III

[0039] Embodiment III sets forth the specific implementation mode by taking wheat seed gene transformation as an example, and further illustrates the present invention:

[0040] 1, embodiment III replaces corn seed with wheat seed. The coleoptile length when wheat seeds germinate is generally 2-8cm, so the coleoptile transformation specific implementation is identical with the step 1 of embodiment 1.

[0041] 2. The specific implementation method of embodiment III is the same as step 2 of embodiment 1.

[0042] 3. On the basis of the specific implementation steps 1 and 2 of the above-mentioned embodiment III, when carrying out coleoptile gene transformation, can adopt:

[0043]The coleoptile injection transformation method in step 3 of Example I is carried out or the coleoptile cutting transformation method in step 3 of Example II is used. The specific implementation method is the same as step 3 of Example I or step 3 of Example II.

[0044] 4. The specific implementation me...

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Abstract

The present invention provides a method for transforming exogenous gene by coleoptile of germinating seed, which belongs to the technical field of plant genetic engineering. The invention mainly comprises the following steps: a) germinating seeds in conventional method, carrying out transformation when coleoptiles grow to an appropriate length; b) absorbing the activated and cultivated agrobacterium liquid with a micro-syringe; c) vertically penetrating the syringe needle into the growing points of damaged plumules through the top of coleoptiles, or injecting the bacterium liquid to the growing points after coleoptiles are crosscut with a blade above the growing point of plumules on the base of coleoptiles and conducting transforming and cocultivating; d), spraying herbicide when the seeds grow to seedlings, taking herbicide resistant seedlings as transgenic plants of primary selection because of the gene of herbicide resistant being the marker gene of transformation; e) growing the plants of primary selection in the field and conducting gene identification to the seeds obtained. This invention is characterized by simple, rapid, efficient and large-scale transformation, and can be applied to gramineous plants of which the germinated seeds have coleoptiles.

Description

technical field [0001] The present invention relates to a technology of gene transformation. It uses the coleoptile of germinated seeds as the physiological morphology index of the growth point of the germ growth point, and uses injection or cutting methods to infect and transform the cells of the growth point of the germ growth point with Agrobacterium, realize the introduction of foreign genes into the recipient cells, and obtain transgenic plants. The present invention belongs to the technical field of plant genetic engineering. Background technique [0002] Plant genetic engineering technology is an important way to carry out genetic breeding using modern gene molecular biology theory. However, the low gene conversion rate, poor repeatability, and low efficiency have always been the bottleneck affecting its industrialization and application, and it is also a research hotspot in various countries around the world. The present invention provides a new technique for solvi...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/84
Inventor 王关林方宏筠那杰
Owner LIAONING NORMAL UNIVERSITY
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