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Method for improving cotton transgenosis efficiency

A transgenic cotton technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of low transformation efficiency, restrict the industrial production of transgenic plants, etc. The effect of increasing the degree of trauma

Inactive Publication Date: 2016-01-27
COTTON RES INST SHANXI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the plant transgenic technology based on Agrobacterium transformation currently has the problem of low transformation efficiency. The transformation rate of most plants is about 1%. This problem has also become the main factor limiting the industrial production of transgenic plants.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] S1. After activating the Agrobacterium containing the target gene to the transformed state, centrifuge and resuspend at 6000rpm to obtain the Agrobacterium resuspension;

[0021] S2. Soak the stem tips of the sterile cotton seedlings in the Agrobacterium resuspension, centrifuge at 4800rpm for 5min at 20°C, transfer to a sterile glass container, let stand and perform ultrasonic treatment for 15 minutes; wherein, the ultrasonic output power is 300 The frequency of ultrasonic treatment is 68KHz;

[0022] S3, after finishing the treatment, take out the cotton stem tip after the infection, blot the remaining bacterial liquid with sterile filter paper, and transfer it to a 6-glycosylaminopurine containing concentration of 0.2mg / L, concentration of 0.1mg / L The indole-3-acetic acid of L and the co-cultivation medium of the α-naphthaleneacetic acid of 0.05mg / L carry out co-cultivation;

[0023] S4. After the co-cultivation is over, the infected cotton shoot tips are screened u...

Embodiment 2

[0025] S1. After activating the Agrobacterium containing the target gene to the transformed state, centrifuge and resuspend at 11000rpm to obtain the Agrobacterium resuspension;

[0026] S2. Soak the stem tips of sterile cotton seedlings in the Agrobacterium resuspension, centrifuge at 8200rpm for 10min at 25°C, transfer to a sterile glass container, let stand and perform ultrasonic treatment for 20 minutes; wherein, the ultrasonic output power is 340 The frequency of ultrasonic treatment is 72KHz;

[0027] S3, after finishing the treatment, take out the cotton stem tip after the infection, blot the remaining bacterial liquid with sterile filter paper, and transfer it to a 6-glycosylaminopurine containing concentration of 0.5mg / L, concentration of 0.3mg / L The indole-3-acetic acid of L and the co-cultivation medium of the α-naphthaleneacetic acid of 0.2mg / L carry out co-cultivation;

[0028] S4. After the co-cultivation is over, the infected cotton shoot tips are screened unde...

Embodiment 3

[0030] S1. After activating the Agrobacterium containing the target gene to the transformed state, centrifuge and resuspend under the condition of 8500 to obtain the Agrobacterium resuspension;

[0031] S2. Soak the stem tips of the sterile cotton seedlings in the Agrobacterium resuspension, centrifuge at 6500rpm for 7.5min at 22.5°C, transfer to a sterile glass container, let stand and perform ultrasonic treatment for 17.5 minutes; wherein, the ultrasonic output power is 320W, the frequency of ultrasonic treatment is 70KHz;

[0032] S3, after finishing the treatment, take out the cotton stem tip after the infection, blot the remaining bacterial liquid with sterile filter paper, and transfer it to a 6-glycosylaminopurine containing concentration of 0.35mg / L, concentration of 0.2mg / L The indole-3-acetic acid of L and the co-cultivation medium of the alpha-naphthaleneacetic acid of 0.125mg / L carry out co-cultivation;

[0033] S4. After the co-cultivation is over, the infected c...

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PUM

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Abstract

The invention discloses a method for improving cotton transgenosis efficiency. The method includes the steps: activating agrobacteria containing a target gene into a transformation state, centrifuging under the condition of 6000-11000rpm, and resuspending to obtain an agrobacterium resuspension solution; soaking stem tips of aseptic cotton seedlings into the agrobacterium resuspension solution, transferring to a sterile glass vessel after centrifugal treatment, standing and ultrasonically treating for 15-20min; after treatment is finished, taking out the infected cotton stem tips, using sterile filter paper to suck dry residual bacterial liquid, and transferring to a co-culture medium containing 6-glycosyl aminopurine, indole-3-acetic acid and alpha-naphthylacetic acid to perform co-culture; after co-culture is finished, screening the infected cotton stem tips under a selection pressure. By adoption of the method, transgenosis efficiency can be effectively improved while the boll set rate of transgenic cotton can be effectively increased.

Description

technical field [0001] The invention relates to the field of cotton cultivation, in particular to a method for improving the transgenic efficiency of cotton. Background technique [0002] Significant progress has been made in the genetic improvement of cotton using genetic engineering technology. Cotton shoot tips have the characteristics of direct differentiation and regeneration, once transformed, the formation of leaves and roots can be induced under appropriate culture conditions to obtain transgenic plants. The Agrobacterium-mediated shoot tip transformation method can not only solve the problem of genotype dependence, but also shorten the transformation cycle and improve the survival rate of transplanting. . However, the plant transgenic technology based on Agrobacterium transformation currently has the problem of low transformation efficiency, and the transformation rate of most plants is about 1%. This problem has also become the main factor limiting the industrial...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00
Inventor 马燕斌张树伟王新胜吴霞李燕娥
Owner COTTON RES INST SHANXI ACAD OF AGRI SCI