Method for creating transgenic receptor system of corn and application of same

Abstract

The present invention discloses a method for creating transgenic receptor system of corn and its application. The stem tip of corn is inoculated to the inducing culture medium added with combined hormones to induce the cultured stem tip to directly grow tussock uds or tissue block of tussock bud. Its next generation is cultured and its tussock buds or the tissue block of tussock bud are used as receptor. After the exogenous genes are transferred into cultured cells, such steps as recovery and screening are used to obtain transgenic plants. Its advantages are use of the most of gene-type cultured cells to regenerate transgenic plant, high frequency and less variation of characteristics.

Description

(1) Technical field

[0001] The invention relates to a method for establishing a corn transgenic receptor system and the application of the receptor system, which belong to the field of agricultural biotechnology or the field of plant genetic engineering. (2) Background technology

[0002] Maize is an important food and feed crop, and it is of great significance to improve maize through genetic engineering. The use of transgenic technology to breed high-quality maize with specific traits has made great progress, and transgenic insect-resistant maize has also been planted on a large scale in North America and Latin America, but the efficiency of maize genetic engineering breeding is still very low, and transgenic technology and receptor systems are still To be improved.

[0003] Scholars at home and abroad have used a variety of genetic transformation techniques to obtain maize transgenic plants, but the main work has focused on genetic transformation using protoplasts as rec...

Examples

example 1

[0066] The present invention will be further described below in conjunction with embodiment: Example 1: the genetic transformation of the corn backbone inbred line mediated by Agrobacterium and the plant regeneration of the anti-piercing-sucking insect

[0067] 1. Materials

[0068] 1) Inbred seeds of corn backbone inbred line 515.

[0069] 2), Agrobacterium A815 (RSs-1-GNA in AGLO). The strain contains a binary vector system, and the T-DNA region of the Mini-Ti plasmid contains a chimeric galanthus agglutinin (GNA) gene and a hygromycin (Hygromycin) resistance gene. The former can be highly expressed in monocotyledonous plants and can kill piercing-sucking insects such as aphids and brown planthoppers. The latter confers a selectable marker on the transformed cells of the plant.

[0070] 2. Establishment of receptor system

[0071] 1) The self-bred selected seeds of the inbred line 515 were soaked in 70% ethanol for 10 minutes, then soaked in 0.1% mercuric chloride for 12...

example 2

[0080] 2) After the rooted seedlings are washed to remove the culture medium, they are transplanted into flowerpots using vermiculite as the cultivation medium. The plants are grown under natural light, the daily temperature is 22-28°C, the night temperature is 15-21°C, and the inorganic salt composition of the seed germination medium is irrigated with 1 / 2 concentration every other day. After about 2 weeks of growth, the root system of transplanted seedlings becomes well developed, and then they are planted in the field. Transgenic plants were screened by PCR detection method and verified by Southern blotting. The transgenic plants are self-fertilized, and the plants resistant to piercing-sucking insects are selected from their offspring. Example 2: Transformation of Maize Backbone Inbred Lines by Biolistic Bombardment

[0081] 1. Materials

[0082] 1) Inbred seeds of corn backbone inbred lines 8112 and Luyuan 92.

[0083] 2) Plasmid p35SCaMV. The plasmid is 7.7kb in leng...

(1) Technical field

[0001] The invention relates to a method for establishing a corn transgenic receptor system and the application of the receptor system, which belong to the field of agricultural biotechnology or the field of plant genetic engineering. (2) Background technology

[0002] Maize is an important food and feed crop, and it is of great significance to improve maize through genetic engineering. The use of transgenic technology to breed high-quality maize with specific traits has made great progress, and transgenic insect-resistant maize has also been planted on a large scale in North America and Latin America, but the efficiency of maize genetic engineering breeding is still very low, and transgenic technology and receptor systems are still To be improved.

[0003] Scholars at home and abroad have used a variety of genetic transformation techniques to obtain maize transgenic plants, but the main work has focused on genetic transformation using protoplasts as rec...

Examples

example 1

[0066] The present invention will be further described below in conjunction with embodiment: Example 1: the genetic transformation of the corn backbone inbred line mediated by Agrobacterium and the plant regeneration of the anti-piercing-sucking insect

[0067] 1. Materials

[0068] 1) Inbred seeds of corn backbone inbred line 515.

[0069] 2), Agrobacterium A815 (RSs-1-GNA in AGLO). The strain contains a binary vector system, and the T-DNA region of the Mini-Ti plasmid contains a chimeric galanthus agglutinin (GNA) gene and a hygromycin (Hygromycin) resistance gene. The former can be highly expressed in monocotyledonous plants and can kill piercing-sucking insects such as aphids and brown planthoppers. The latter confers a selectable marker on the transformed cells of the plant.

[0070] 2. Establishment of receptor system

[0071] 1) The self-bred selected seeds of the inbred line 515 were soaked in 70% ethanol for 10 minutes, then soaked in 0.1% mercuric chloride for 12...

example 2

[0080] 2) After the rooted seedlings are washed to remove the culture medium, they are transplanted into flowerpots using vermiculite as the cultivation medium. The plants are grown under natural light, the daily temperature is 22-28°C, the night temperature is 15-21°C, and the inorganic salt composition of the seed germination medium is irrigated with 1 / 2 concentration every other day. After about 2 weeks of growth, the root system of transplanted seedlings becomes well developed, and then they are planted in the field. Transgenic plants were screened by PCR detection method and verified by Southern blotting. The transgenic plants are self-fertilized, and the plants resistant to piercing-sucking insects are selected from their offspring. Example 2: Transformation of Maize Backbone Inbred Lines by Biolistic Bombardment

[0081] 1. Materials

[0082] 1) Inbred seeds of corn backbone inbred lines 8112 and Luyuan 92.

[0083] 2) Plasmid p35SCaMV. The plasmid is 7.7kb in leng...