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Transformation method for introducing Basta resistance of agrobacterium-mediated herbicide into tobacco

An Agrobacterium-mediated, herbicide-based technology, applied in the field of plant genetic engineering, can solve the problems of low positive rate of herbicide screening resistance and easy pollution, and achieve the effect of reducing Agrobacterium pollution and increasing the positive rate of screening resistance

Inactive Publication Date: 2019-05-31
上海迈其生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a transformation method of Agrobacterium-mediated herbicide Basta resistance into tobacco, to solve the problems of low positive rate of herbicide screening resistance and easy pollution in the prior art

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The leaves of tobacco variety "Nicotiana tabacum NN" were used as explants, transformed with Agrobacterium EHA105 containing herbicide Basta resistance, and the positive plants were detected by genomic PCR.

[0038] Step 1: Tobacco explant pre-cultivation

[0039] To prepare the pre-culture medium:

[0040] MS 4.43g / L+sucrose 30g / L+6-BA 1mg / L+NAA 0.1mg / L+7.8g / L Agar, pH5.8; sterilized at 121°C for 20min.

[0041] Tobacco plants can be used to make leaf discs when they grow to a plant height of 6-8 cm. At this time, each plant has about 5-8 leaves, and the middle-sized and healthy leaves are used to make leaf discs.

[0042] Cut tobacco leaves into 1cm 2 Leaf discs were placed on the pre-medium with the leaf side down, and cultured in the dark for 2 days, with 100 leaf discs per batch.

[0043] Step 2: Infect tobacco explants with Agrobacterium transfected with basta resistance plasmid

[0044] The transformation method is as follows: the pre-cultivated tobacco expla...

Embodiment 2

[0076] The leaves of tobacco variety "Nicotiana tabacum NN" were used as explants, transformed with Agrobacterium EHA105 containing herbicide Basta resistance, and the positive plants were detected by genomic PCR.

[0077] Step 1: Tobacco explant pre-cultivation

[0078] To prepare the pre-culture medium:

[0079] MS 4.43g / L+sucrose 30g / L+6-BA 1mg / L+NAA 0.1mg / L+7.8g / L Agar, pH5.8; sterilized at 121°C for 20min.

[0080] Tobacco plants can be used to make leaf discs when they grow to a plant height of 6-8 cm. At this time, each plant has about 5-8 leaves, and the middle-sized and healthy leaves are used to make leaf discs.

[0081] Cut tobacco leaves into 1cm 2 Leaf discs were placed on the pre-medium with the leaf side down, and cultured in the dark for 2 days, with 100 leaf discs per batch.

[0082] Step 2: Infect tobacco explants with Agrobacterium transfected with basta resistance plasmid

[0083] The transformation method is as follows: the pre-cultivated tobacco expla...

Embodiment 3

[0115] The leaves of tobacco variety "Nicotiana tabacum NN" were used as explants, transformed with Agrobacterium EHA105 containing herbicide Basta resistance, and the positive plants were detected by genomic PCR.

[0116] Step 1: Tobacco explant pre-cultivation

[0117] To prepare the pre-culture medium:

[0118] MS 4.43g / L+sucrose 30g / L+6-BA 1mg / L+NAA 0.1mg / L+7.8g / L Agar, pH5.8; sterilized at 121°C for 20min.

[0119]Tobacco plants can be used to make leaf discs when they grow to a plant height of 6-8 cm. At this time, each plant has about 5-8 leaves, and the middle-sized and healthy leaves are used to make leaf discs.

[0120] Cut tobacco leaves into 1cm 2 The leaf discs were placed on the pre-medium with the leaves facing down, and cultured in the dark for 2 days, with 100 leaf discs per batch.

[0121] Step 2: Infect tobacco explants with Agrobacterium transfected with basta resistance plasmid

[0122] The transformation method is as follows: the pre-cultivated tobacc...

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Abstract

The invention discloses a transformation method for introducing the Basta resistance of an agrobacterium-mediated herbicide into a tobacco. The transformation method comprises the following steps: pre-culturing tobacco explants; impregnating tobacco explants with agrobacterium transferred into basta resistant plasmids; executing co-culture; executing differentiation culture, wherein the concentration of Basta in a differentiation medium is 3-5 mg / mL; executing rooting induction and positive plant detection. Leaf disks are cleaned before co-culture by selecting ticarcillin with appropriate concentration, so that the pollution of agrobacterium is effectively reduced; and the optimal antibiotic concentration aiming at the growth of resistant seedlings is screened out, so that the positive rate of herbicide screening resistance is improved.

Description

【Technical field】 [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a transformation method for introducing herbicide Basta resistance into tobacco mediated by Agrobacterium. 【Background technique】 [0002] Agrobacterium (Agrobacteriμm) is a kind of Gram-negative bacteria that lives on the surface of plant roots, relies on the nutrients infiltrated from the root tissue, and is ubiquitous in the soil. Agrobacterium has the ability to naturally infect plant cells and can induce plant crown gall tumors. A huge plasmid exists in Agrobacterium, called Ti plasmid. Ti plasmid can be used as a carrier of foreign genes to mediate the transformation of foreign genes. At present, Agrobacterium-mediated genetic transformation has been widely used in dicotyledonous and monocotyledonous plants, and the applied Agrobacterium are mainly Agrobacterium tumefaciens and Agrobacterium rhizogenes. [0003] For Agrobacterium transform...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/82
Inventor 贺其其陈晓庆武秀明
Owner 上海迈其生物科技有限公司
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