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Method for directly growing seedlings through corn inbred line mature seed seedling stem node genetic transformation

A technology of genetic transformation and inbred lines, which is applied to the field of direct seedling through genetic transformation of mature seedlings of maize inbred lines, can solve the problems of time-consuming, difficult differentiation of Agrobacterium, and low transformation efficiency, so as to improve the transformation effect. , the effect of speeding up the reaction rate

Pending Publication Date: 2022-07-01
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The existing Agrobacterium-mediated method has low transformation efficiency when transforming the stem nodes of seedlings. Due to the relatively closed structure of the stem nodes, it is difficult for Agrobacterium to penetrate into the stem nodes of seedlings for differentiation, which takes a lot of time in the cultivation process , need to improve

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] A method for direct seedling by genetic transformation of mature seed seedlings of corn inbred lines, comprising the following steps;

[0020] Step 1: Select explant materials, stem nodes of mature corn seedlings, put them in 70% concentration ether, wash 3 times, and then in a sterile environment, use instruments to mark the stem nodes on the stem nodes of the seedlings with the texture of the stem nodes. For vertical wounds, the size of the wound on the stem node of the seedling is controlled at 3mm;

[0021] Step 2: Place the treated seedling stem node in a petri dish, keep the ambient temperature at 25°C, and let it stand for 2 days in a dark environment, and callus appears at the wound of the seedling stem node;

[0022] Step 3: Select the stem nodes of the seedlings, observe whether the surface is damaged, and whether the stem node culture is normal, place it in the medium, put a layer of test paper on the bottom of the medium, and put the Agrobacterium carrying t...

Embodiment 2

[0026] A method for direct seedling by genetic transformation of mature seed seedlings of corn inbred lines, comprising the following steps;

[0027] Step 1: Select explant materials, stem nodes of mature corn seedlings, put them in 70% concentration ether, wash 3 times, and then in a sterile environment, use instruments to mark the stem nodes on the stem nodes of the seedlings with the texture of the stem nodes. For vertical wounds, the size of the wound on the stem node of the seedling is controlled at 3mm;

[0028] Step 2: Place the treated seedling stem node in a petri dish, keep the ambient temperature at 28°C, and let it stand for 3 days in a dark environment, and callus appears at the wound of the seedling stem node;

[0029] Step 3: Select the stem nodes of the seedlings, observe whether the surface is damaged, and whether the stem node culture is normal, place it in the medium, put a layer of test paper on the bottom of the medium, and put the Agrobacterium carrying t...

Embodiment 3

[0033] Step 1: Select explant materials, stem nodes of mature corn seedlings, put them in 70% concentration ether, wash 3 times, and then in a sterile environment, use instruments to mark the stem nodes on the stem nodes of the seedlings with the texture of the stem nodes. For vertical wounds, the size of the wound on the stem node of the seedling is controlled at 1mm;

[0034] Step 2: Place the treated seedling stem node in a petri dish, keep the ambient temperature at 20°C, and let it stand for 2 days in a dark environment, and callus appears at the wound of the seedling stem node;

[0035] Step 3: Select the stem nodes of the seedlings, observe whether the surface is damaged, and whether the stem node culture is normal, place it in the medium, put a layer of test paper on the bottom of the medium, and put the Agrobacterium carrying the target gene carrier into it. , co-cultivated with seedling stem and node for 3 days, the medium composition includes MS basic medium + 15g / L...

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Abstract

The invention discloses a corn inbred line mature seed seedling stem node genetic transformation direct seedling formation method. The method comprises the following steps: 1, selecting an explant material, and cutting a wound on a stem node of a seedling, 2, placing the processed stem node of the seedling in a culture dish for standing until a callus appears at the wound of the stem node of the seedling, 3, placing the stem node of the seedling in a culture medium, laying a layer of test paper on the bottom layer of the culture medium, placing agrobacterium carrying a target gene vector into the test paper, and carrying out culture at the temperature of 20-30 DEG C; 4, screening, and 5, taking out the stem nodes of the seedlings from the screening culture medium, culturing the stem nodes in the culture medium, and differentiating and budding; corn embryos are subjected to genetic transformation in an agrobacterium-mediated mode, wounds are marked on stem nodes of seedlings and are connected with test paper with agrobacterium components, so that the defect that the wounds are difficult to permeate into stem and leaf cells in a traditional agrobacterium-mediated mode is overcome, the reaction rate is increased, and the transformation effect of the stem nodes of the mature corn seed seedlings is improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for direct seedling formation by genetic transformation of stem nodes of mature seed seedlings of maize inbred lines. Background technique [0002] Corn is not only an important feed and food crop, but also an extremely important industrial raw material crop. At present, the global corn production is growing steadily, but with the rapid economic development and the rapid improvement of people's living standards, the total consumption of corn continues to increase, and the contradiction between supply and demand intensifies. Genetic improvement of maize through conventional breeding is one of the main ways to increase maize yield, but factors such as limited genetic resources and relatively long breeding cycles often limit its application potential. In the genetic transformation of corn seeds, Agrobacterium-mediated transformation is often used. The steps are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/46
CPCC12N15/8205
Inventor 邢少辰王云鹏韦正乙蔡玉红范杰英
Owner JILIN ACAD OF AGRI SCI