CN-122012594-A - Method for obtaining transgenic or gene editing plant

CN122012594ACN 122012594 ACN122012594 ACN 122012594ACN-122012594-A

Abstract

The invention discloses a method for obtaining a transgenic or gene editing plant, which comprises the steps of firstly culturing plant seedlings, and obtaining a plant to be infected when the stem length is larger than 1 cm. Transforming a target vector into agrobacterium, selecting a monoclonal colony with the target vector, preparing agrobacterium liquid, finally cutting a wound on the stem of a plant to be infected, carrying out infection by using the activated agrobacterium liquid to apply the wound externally, carrying out illumination culture, removing growing points after a period of time, continuing to culture until a callus grows out from the wound, and obtaining a new bud containing the target gene, thus obtaining the transgenic or gene editing plant. The method comprises the steps of generating a wound on the stem of a plant, removing growing points after true leaves grow out, and enabling wound callus to develop into buds under the stimulation of plant self-hormones. The method is not limited to tissue culture and aseptic operation, does not need to resort to root regeneration buds, does not need to externally apply hormone, does not need to induce root regeneration, has simple operation steps, and can obtain transgenic or gene editing plants in a short time.

Inventors

LI JUN
HU YANGYANG
ZHANG MING
LI WENJING

Assignees

河北农业大学

Dates

Publication Date: 20260512
Application Date: 20260306

Claims (8)

1. A method of obtaining a transgenic or genetically edited plant comprising the steps of: Step 1, sowing plant seeds, and obtaining plants to be infected after the seeds germinate and grow stems; Step 2, transforming the target vector of the transgene or gene editing into agrobacterium, selecting a monoclonal colony with the target vector, and preparing agrobacterium liquid; Step 3, cutting a wound on the stem part of a plant to be infected, and infecting the wound by using activated agrobacterium tumefaciens bacteria liquid; Step 4, after infection is finished, carrying out illumination culture, and carrying out operation of removing growing points after true leaves grow out; And 5, continuing culturing and maintaining the operation of removing the growing points until the wound grows new buds containing genes corresponding to the target vector, and obtaining the transgenic or gene editing plant.
2. The method of obtaining a transgenic or genetically edited plant according to claim 1, wherein said plant to be infected is a plant capable of producing callus at a stem wound.
3. The method for obtaining a transgenic or genetically edited plant as claimed in claim 1, wherein the plant to be infected is a brassica plant or a Solanum plant.
4. A method of obtaining a transgenic or genetically edited plant according to claim 3, wherein: the brassica plant is cabbage, broccoli or rape, and the eggplant plant is eggplant.
5. The method of claim 1,2, 3 or 4, wherein in the step 4, the operation of removing the growing point is performed after the true leaf is grown, the cotyledon is retained if the growing point is completely removed while the cotyledon is retained, and the cotyledon is removed together with the growing point if the cotyledon is not retained after the growing point is completely removed.
6. The method for obtaining transgenic or genetically modified plants according to claim 1, wherein in step 3, a plurality of wounds are cut on the stem of the plant by using a blade, the depth of the wounds is not more than half the diameter of the stem, cotton immersed with the activated agrobacterium liquid is wrapped on the wounds for infection for a certain period of time at room temperature and humidity of 65-80%, in step 4, the cotton is removed for light culture at room temperature, and in step 5, the light continues to be cultured at room temperature.
7. The method for obtaining a transgenic or genetically edited plant as claimed in claim 1, wherein the Agrobacterium is Agrobacterium rhizogenes or Agrobacterium tumefaciens.
8. The method for obtaining a transgenic or genetically edited plant according to claim 7, wherein said Agrobacterium tumefaciens is K599, AR.1193, AR.A4, AR.Qual, C58C1 or MSU440 and said Agrobacterium tumefaciens is GV3101, GV2260, EHA105, AGL1, LBA4404, EHA101.

Description

Method for obtaining transgenic or gene editing plant Technical Field The invention relates to the field of genetic transformation, in particular to a method for obtaining a transgenic or gene editing plant. Background Genetic transformation of plants is a core technology of genetic engineering of plants, and stable genetic transformation systems have been established for many species, and agrobacterium transformation is widely used because of low cost and stable characteristics. For example, in the tissue culture process, callus, hypocotyl, stem segments, leaves, etc. are infected with agrobacterium tumefaciens, and transgenic buds and roots are induced by using different plant hormone ratios, so that regenerated transgenic plants are obtained, and related reports are made in plants such as rice, wheat, corn, cotton, etc. The CDB (cut-dip-budding) delivery system provides a brand-new soil culture genetic transformation method without aseptic operation, and the method uses agrobacterium rhizogenes to transform plants such as sweet potato, dandelion and the like, obtains transgenic roots, and cultures the roots to obtain transgenic plants. The method is simple to operate, requires no sterile manipulation, but requires the plant itself to have the ability to regenerate shoots from roots. The invention patent application CN 113930441A discloses a method for obtaining a transgenic or gene editing plant body, which utilizes a non-tissue culture mode to obtain a transgenic plant, uses agrobacterium rhizogenes carrying a carrier to infect any part of sweet potato, cultures the infected sweet potato tissue until hairy roots grow, screens the transgenic or gene editing root for propagation to obtain the transgenic sweet potato or gene editing sweet potato. Sweet potatoes, however, are obtained by direct root development, whereas most plants do not possess this capability. For most plants, infection with agrobacterium rhizogenes to obtain transgenic roots and then induction of shoot regeneration by transgenic roots of the plant is very challenging. The invention patent application CN 118272428A discloses a method for efficient genetic transformation and gene editing of brassica crops mediated by agrobacterium rhizogenes, genetic transformation and establishment of a gene editing system of the brassica crops are realized through three growth regulatory factors of agrobacterium rhizogenes mediated ZmWUS, attP and AtPLT, and the three growth regulatory factors of agrobacterium rhizogenes mediated ZmWUS, attP and AtPLT induce explants to form callus to directly form buds, so that species with poor root regeneration capability realize effective transformation, but the method is still finished based on a tissue culture mode, a sterile environment is needed, and the operation is relatively complex and tedious. Disclosure of Invention The invention aims to provide a method for obtaining a transgenic or gene-edited plant, which is not limited to tissue culture and aseptic operation, does not need to resort to root regeneration buds, has simple operation steps and can obtain the transgenic or gene-edited plant in a short time. In order to achieve the above purpose, the present invention adopts the following technical scheme: a method of obtaining a transgenic or genetically edited plant comprising the steps of: Step 1, sowing plant seeds, and obtaining plants to be infected after the seeds germinate and grow stems; Step 2, transforming the target vector of the transgene or gene editing into agrobacterium, selecting a monoclonal colony with the target vector, and preparing agrobacterium liquid; Step 3, cutting a wound on the stem part of a plant to be infected, and infecting the wound by using activated agrobacterium tumefaciens bacteria liquid; step 4, after infection is finished, carrying out illumination culture, and carrying out growth point removing operation after true leaves grow out; And 5, continuing culturing and maintaining the operation of removing the growing points until the wound grows new buds containing genes corresponding to the target vector, and obtaining the transgenic or gene editing plant. Preferably, the plant to be infected is a plant which can produce callus and can produce new buds at the wound of the stem. Preferably, the plant to be infected is a brassica plant or a solanum plant. Preferably, the brassica plant is cabbage, broccoli or rape, and the eggplant plant is eggplant. Preferably, in the step 4, the brassica plants are subjected to a growing point removing operation after the true leaves grow, and the cotyledons are reserved at the same time, and the cotyledons are removed together when the growing points of the eggplant plants and the like are removed. Preferably, in the step 3, a plurality of wounds are cut on the neck of the plant by using a blade, the depth of the wounds is not more than half of the diameter of the stem, cotton soaked with the activated agrobacterium liquid is wrapped on