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Method for quickly obtaining transgenic peanuts through peanut cotylcdon and application of method

A peanut cotyledon, transgenic technology, applied in application, botanical equipment and methods, angiosperm/flowering plants, etc., can solve the problems of restricting the production of transgenic receptors, low transformation efficiency, low work efficiency, etc., and shorten the development cycle. , shortened time, time-saving effect

Pending Publication Date: 2017-05-31
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the "Non-tissue Culture Gene Transformation Method Using Peanut Halves of Mature Seeds as Receptors" (201110074925.7), it is also necessary to manually remove the seed coat, and also need to separate the cotyledons separately. The work efficiency is very low, which seriously limits the mass production of transgenic receptors , resulting in low conversion efficiency

Method used

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  • Method for quickly obtaining transgenic peanuts through peanut cotylcdon and application of method
  • Method for quickly obtaining transgenic peanuts through peanut cotylcdon and application of method
  • Method for quickly obtaining transgenic peanuts through peanut cotylcdon and application of method

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Experimental program
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Effect test

Embodiment 1

[0033] The method for obtaining transgenic peanuts rapidly by utilizing peanut cotyledons comprises the following steps:

[0034] ①Preparation of Agrobacterium LBA4404 containing Bar gene: Introduce the Pcambia3301 plasmid into LBA4404 cells according to the method of "Molecular Cloning".

[0035] ②Cultivation of Agrobacterium: Pick a single colony of Agrobacterium LBA4404 containing the Bar gene, inoculate it in 5ml of YEB liquid medium containing 50mg / L rifampicin, shake it overnight at 28°C, then inoculate 0.5ml of the bacterial solution in 50mL YEB liquid medium containing 50mg / L rifampicin was cultured until the OD600 was about 0.5. The bacterial liquid was centrifuged at 4°C and 5000 rpm for 5 min to collect the bacterial cells. Then re-suspend the pellet with 5 mL of MS medium containing 100 μM AS, and then transfer the bacterial solution into 50 mL of MS medium containing 100 μM AS, and culture on a shaker at 27°C until the OD600 is 0.5 to obtain the Agrobacterium tra...

Embodiment 2

[0049] The method for obtaining transgenic peanuts rapidly by utilizing peanut cotyledons comprises the following steps:

[0050] ①Preparation of Agrobacterium LBA4404 containing Bar gene: Introduce the Pcambia3301 plasmid into LBA4404 cells according to the method of "Molecular Cloning".

[0051] ②Cultivation of Agrobacterium: Pick a single colony of Agrobacterium LBA4404 containing the Bar gene, inoculate it in 5ml of YEB liquid medium containing 50mg / L rifampicin, shake it overnight at 28°C, then inoculate 0.5ml of the bacterial solution into 50mL Cultivate in YEB liquid medium containing 50mg / L rifampicin until the OD600 is about 0.5. The bacterial liquid was centrifuged at 4°C and 5000 rpm for 5 min to collect the bacterial cells. Then re-suspend the pellet with 5 mL of MS medium containing 100 μM AS, and then transfer the bacterial solution into 50 mL of MS medium containing 100 μM AS, and culture on a shaker at 27°C until the OD600 is 0.5 to obtain the Agrobacterium tr...

Embodiment 3

[0065] The method for obtaining transgenic peanuts rapidly by utilizing peanut cotyledons comprises the following steps:

[0066] ①Preparation of Agrobacterium LBA4404 containing Bar gene: Introduce the Pcambia3301 plasmid into LBA4404 cells according to the method of "Molecular Cloning".

[0067] ②Cultivation of Agrobacterium: Pick a single colony of Agrobacterium LBA4404 containing the Bar gene, inoculate it in 5ml of YEB liquid medium containing 50mg / L rifampicin, shake it overnight at 28°C, then inoculate 0.5ml of the bacterial solution into 50mL Cultivate in YEB liquid medium containing 50mg / L rifampicin until the OD600 is about 0.5. The bacterial liquid was centrifuged at 4°C and 5000 rpm for 5 min to collect the bacterial cells. Then re-suspend the pellet with 5 mL of MS medium containing 100 μM AS, and then transfer the bacterial solution into 50 mL of MS medium containing 100 μM AS, and culture on a shaker at 27°C until the OD600 is 0.5 to obtain the Agrobacterium tr...

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Abstract

The invention discloses a method for quickly obtaining transgenic peanuts through peanut cotylcdon. The method comprises the following steps: 1) taking agrobacterium tumefaciens in a logarithmic phase, and re-suspending, thereby obtaining transformation bacteria liquor; 2) taking mature peanut seeds to bud for 0-5 days, longitudinally cutting cotyledon into two parts, and cutting off germs at the top ends of plumular axes to obtain transformation receptors; 3) soaking the transformation receptors into transformation bacteria liquor to infect for 20-50 minutes, then, taking out the transformation receptors, putting the transformation receptors onto water-absorbing paper, and cultivating for 3-4 days under a dark condition; 4) thoroughly flushing the transformation receptors, and performing selective cultivation, thereby obtaining peanut seedlings; and 5) continuously cultivating the peanut seedlings to obtain peanut plants, identifying the peanut plants, thereby obtaining transgenic plants. The invention further discloses application of the third in preparing transgenic peanuts. According to the method disclosed by the invention, materials are easily available, operations are simple, time and cost are reduced, and transformation efficiency is high, and therefore, the method is suitable for large-scale transgenic peanut development.

Description

technical field [0001] The invention relates to the field of plant transgene technology, in particular to a peanut transgene method mediated by Agrobacterium using peanut cotyledon as a receptor and its application. Background technique [0002] It is most direct and effective to use transgenic technology to carry out crop genetic improvement by using a large number of transgenic populations to select and breed superior strains. A large-scale transgenic population is one of the guarantees for commercial transgenic breeding. Therefore, a quick and easy transformation method is an important part in the process of crop transgenic breeding. [0003] Peanut (Arachis hypogaea L.) is an important oil-bearing and food crop. It is also the main oil-bearing economic crop in my country and an important source of protein. The transgenic breeding of peanut is relatively lagging behind, mainly because it is relatively difficult in genetic transformation. In recent years, through the co...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00
Inventor 王继华梁炫强温世杰洪彦彬刘南松
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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