Method for screening transgenic rape using chanamyn seed soaking method

A technology of kanamycin and transgene, which is applied in the field of screening transgenic rapeseed by kanamycin seed soaking method, can solve the problems of heavy workload, complicated operation, false positive, etc., and achieve fast screening speed, less false positive, and simple method Effect

Inactive Publication Date: 2008-08-06
SICHUAN AGRI UNIV
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Problems solved by technology

Its advantage is that the concentration of kanamycin is relatively stable, and because of less volatilization loss, it only needs to be added once, but this method has the following problems: (1) the workload is large, the operation is complicated, and a large amount of seeds need to be placed in the Screening under bacterial conditions is very heavy; kanamycin belongs to the class of antibiotics and cannot be sterilized by high temperature, so it must be filtered and sterilized when preparing the medium, which is complicated to operate; (2) the survival rate of transplanting is very low , the surviving seedlings after kanamycin resistance screening are generally weaker, and the survival rate of transplanting them in the field is very low; When the root is upturned and separated from the medium, there will be a false positive

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0017] The screening of transgenic seeds of Chinese cabbage-type rapeseed, the specific screening process is as follows:

[0018] (1) Preparation of kanamycin solution Dissolve 200mg of kanamycin solid in 1L of water to obtain a 0.2% kanamycin solution;

[0019] (2) Seed soaking 10,000 T. 1 Substitute seeds (from the Rapeseed Research Center of Sichuan Agricultural University) were put into kanamycin solution and soaked for 16 hours, and were taken out when they turned white;

[0020] (3) Screening Seeds are sown on soil that does not contain kanamycin, soak the soil before sowing, and directly sow on the soil surface, observe the results after 2-3 days, select normal green seedlings, and get 12 transgenic plants .

Embodiment 2

[0022] The screening of Brassica napus transgenic plants, the specific screening process is as follows:

[0023] (1) prepare kanamycin solution, 280mg kanamycin solid is dissolved in 1L water, get concentration and be the kanamycin solution of 0.28%;

[0024] (2) Seed soaking: 20,000 grains of transgenic T seeds marked by the kanamycin resistance gene obtained by the floral-dip method (translated as inflorescence soaking method) 1 Substitute seeds (from the Rapeseed Research Center of Sichuan Agricultural University) were soaked in a kanamycin solution for 16 hours, the solution just submerged the seeds, and were taken out when they turned white;

[0025] (2) Screening Soak the soil before sowing, and sow the seeds on the soil without kanamycin; observe the results after 2-3 days, select normal green seedlings, and obtain 20 transgenic plants.

Embodiment 3

[0027] Screening of Transgenic Plants of Brassica napus Using Traditional Medium Containing Kanamycin

[0028] Proceed as follows:

[0029] (1) The screened materials are transgenic seeds of Brassica napus (from the Rapeseed Research Center of Sichuan Agricultural University).

[0030] (2) Preparation of culture medium Prepare MS medium containing 270 mg / L kanamycin according to the usual method, and mark 1500 grains obtained by the floral-dip method (translated as inflorescence soaking method) with the kanamycin resistance gene transgenic T 1 The generation seeds were sterilized and inoculated into the medium for screening, and the results were observed one week later.

[0031] (3) Screening Normal green seedlings were selected to obtain 18 transgenic plants.

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Abstract

The invention discloses a method of screening transgene cole through dipping seeds into kanamycin solution. The cole seeds having undergone transgene processing are dipped into the 0.18-0.30 percent kanamycin solution for processing for 12-24 hours and then are sowed in the soil which does not contain kanamycin, and the green seedlings growing normally are screened as the transgene plants after 2-3 days. The method of screening transgene cole is applicable to screening of a large amount of transgene cole seeds with simplicity, quickness, high efficiency, and reliability.

Description

technical field [0001] The invention belongs to a transgenic plant screening method, in particular to a method for screening transgenic rapeseed by using a kanamycin seed soaking method. Background technique [0002] In plant genetic engineering, selectable marker gene is a kind of exogenous gene that helps to screen and identify transgenic plants. Under appropriate selection pressure, the plants without the marker gene die, while the transformed plants containing the selectable marker gene can continue to survive due to their resistance, which is beneficial to screening transformed plants from a large number of non-transformed plants. Kanamycin is currently one of the most widely used selection marker genes in plant genetic engineering (Gai Shupeng et al. Journal of Shandong Agricultural University, 2000, 31(1): 95-100). The selection of transgenic plants by using kanamycin works essentially through the kanamycin resistance gene. Kanamycin resistance (Kan r ) gene is the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/48
Inventor 牛应泽韦献雅郭世星付绍红余青青
Owner SICHUAN AGRI UNIV
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