Agrobacterium tumefaciens-mediated sesame cotyledon transgenic method

An Agrobacterium-mediated, transgenic technology, applied in the field of genetic engineering operations, can solve the problems of low genetic transformation efficiency of sesame, low survival rate of transgenic regenerated plants, etc., achieve good application and promotion value, improve transgenic efficiency, and shorten the transformation cycle.

Pending Publication Date: 2022-07-29
HENAN SESAME RES CENT HENAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of this application is to provide a relatively efficient Agrobacterium-mediated transgenic method for sesame cotyledons, so as to overcome the technical problems in the prior art such as low efficiency of sesame genetic transformation and low survival rate of transgenic regenerated plants, and further contribute to the development of sesame transgenic breeding and other breeding. Technical development lays a certain technical foundation

Method used

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  • Agrobacterium tumefaciens-mediated sesame cotyledon transgenic method
  • Agrobacterium tumefaciens-mediated sesame cotyledon transgenic method
  • Agrobacterium tumefaciens-mediated sesame cotyledon transgenic method

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Embodiment 1

[0050] In this example, the pBI121 empty expression vector (the vector itself contains kanamycin resistance and the selection marker GUS reporter gene, that is, in the genetic transformation, the GUS gene is equivalent to the target gene) as an example, takes the main sesame variety Yuzhi 11 in my country as an example. As the transgenic raw material, the construction method of Agrobacterium-mediated sesame cotyledon transgenic genetic transformation system is introduced as follows.

[0051] (1) Obtaining explants

[0052] Select plump and clean Yuzhi No. 11 mature grains, and rinse them twice with sterile water after sterilizing with 75% ethanol for 90 s on a sterile operating table; then, after sterilizing with 3% sodium hypochlorite for 15 minutes, wash with sterile water for 5 minutes. Second-rate;

[0053] Put the sterile seeds in sterile water, under the conditions of room temperature (about 25 ℃) and dark conditions, shake and cultivate at 100 r / min for 12 hours to ensu...

Embodiment 2

[0120] In order to illustrate the versatility of the genetic transformation system provided in this application, the inventors further take the pFGC5941 expression vector (containing the glufosinate-ammonium resistance gene Bar) as an example, and briefly introduce the construction process and experimental results of the related genetic transformation system as follows.

[0121] The relevant experimental operations refer to Example 1, and only some of the different operations are briefly described as follows.

[0122] In step (3), since the pFGC5941 expression vector carries the glufosinate-ammonium resistance gene, the formula of the resistance screening medium M3 is adjusted as follows:

[0123] MS + 6-BA 2.5mg / L + IAA 1.0 mg / L + ABA 1.0 mg / L + AgNO 3 5.0 mg / L + cephalexin 300 mg / L + resistance screening substance glufosinate ammonium 1 mg / L + plant supplement 5.0 g / L + agar powder 7.5 g / L, pH=5.8.

[0124] When further PCR detection and judgment are performed on transgeni...

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Abstract

The invention belongs to the technical field of genetic engineering operation, and particularly relates to an agrobacterium tumefaciens-mediated sesame cotyledon transgenic method. The method comprises the following steps: obtaining an explant, preparing an infection solution, performing infection co-culture, performing tissue culture, performing resistance screening, inducing rooting, transplanting and the like. On the basis of a common method for transgenosis by infecting the cotyledons with agrobacterium tumefaciens, the invention provides a sesame cotyledon-based transgenosis genetic transformation method which is short in transformation period and high in transformation rate in combination with the construction of a sesame tissue culture regeneration system. Preliminary experiment results prove that related plant additives are necessary conditions for realizing healthy growth and rooting of sesame regenerated plants, and a large number of sesame transgenic plants can be successfully obtained through optimal combination of related culture medium components and culture conditions. Based on the genetic transformation system, a good technical foundation can be laid for subsequent researches such as sesame important gene function verification, transgenic mutant library construction, excellent germplasm creation and the like.

Description

technical field [0001] The application belongs to the technical field of genetic engineering operations, and in particular relates to a method for transgenic sesame cotyledons mediated by Agrobacterium. Background technique [0002] Sesame seeds of the Flax family Flax ( Sesamum indicum L., 2n=26), is one of the oldest oil crops. Because the (sesame) grains are rich in oil, protein and antioxidant lignans, it is also known as the "Queen of Oilseeds". Sesame is widely distributed in tropical and subtropical regions of the world and is one of the important oil crops. There are more than 80 countries in the world planting sesame, with an annual planting area of ​​about 8 million hectares and an annual output of 4.5 million tons. India, Sudan, Myanmar and China are the major producers of sesame in the world. The annual planting of sesame in my country is about 530,000 hectares, ranking fourth in the world; the average yield is 1,182 kg / ha, ranking first in the world; the av...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H6/66A01H5/00A01H4/00
CPCC12N15/8205A01H4/002A01H4/005
Inventor 张海洋琚铭苗红梅黄盈盈段迎辉韩秀花马琴穆聪
Owner HENAN SESAME RES CENT HENAN ACADEMY OF AGRI SCI
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