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Agrobacterium-mediated genetic transformation method with peanut seed domant bud hypocotyl as explant

A genetic transformation method, Agrobacterium-mediated technology, applied in the transformation field of dormant hypocotyl explants, can solve the problems of long regeneration cycle, low transformation and regeneration rate, unstable transformation effect, etc., and achieve high regeneration rate, The effect of shortening the conversion cycle and stabilizing the effect

Inactive Publication Date: 2009-06-03
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the problems of long regeneration period, low transformation and regeneration rate, and unstable transformation effect in the existing peanut genetic transformation method, the object of the present invention is to provide a kind of agricultural transformation method using dormant embryonic axis of mature peanut seeds as transformed explants. Stable and efficient genetic transformation method mediated by Bacillus

Method used

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  • Agrobacterium-mediated genetic transformation method with peanut seed domant bud hypocotyl as explant
  • Agrobacterium-mediated genetic transformation method with peanut seed domant bud hypocotyl as explant
  • Agrobacterium-mediated genetic transformation method with peanut seed domant bud hypocotyl as explant

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Embodiment 1

[0036] Example 1: The Agrobacterium-mediated genetic transformation method using dormant hypocotyls of mature peanut seeds as explants is used for the transformation of AhPR10 gene

[0037](1) Select mature and plump peanut seeds, and in the aseptic environment of the ultra-clean workbench, sterilize the surface of the peanut seeds with 0.1% (W / V) mercuric chloride solution for 10 minutes, and then wash them with sterile water for 3 times. After soaking in warm water at 50°C for 3 hours, remove the seed coat, cut the seeds along the gap between the cotyledons with a sterile scalpel, peel off the complete embryo from the cotyledons with a scalpel, cut off the radicle and bud primordium, and keep the remaining The dormant hypocotyl part of mature peanut seeds was used as the dormant hypocotyl explant to be transformed, and was pre-cultured on a pre-medium (solid MS+3 mg / L 6-benzylaminopurine) for 0.5 days. Such as figure 1 Shown is the preparation diagram of dormant hypocotyl e...

Embodiment 2

[0045] Example 2: The method of Agrobacterium-mediated genetic transformation of dormant embryonic axes of mature peanut seeds as explants is used for the transformation of LjCYC gene in Lotus japonicus

[0046] (1) Select mature and plump peanut seeds, and in the aseptic environment of the superfloating workbench, sterilize the surface of the peanut seeds with 0.1% (W / V) mercuric chloride solution for 10 minutes, and then wash them with sterile water for 3 times. After soaking in warm water at 50°C for 3 hours, remove the seed coat, cut the seeds along the gap between the cotyledons with a sterile scalpel, peel off the complete embryo from the cotyledons with a scalpel, cut off the radicle and bud primordium, and keep the remaining The dormant hypocotyl part of mature peanut seeds was used as the dormant hypocotyl explant to be transformed, and was pre-cultured on a pre-medium (solid MS+3 mg / L 6-benzylaminopurine) for 1 day.

[0047] (2) Using the LjCYC gene of Lotus japonicu...

Embodiment 3

[0054] Example 3: The Agrobacterium-mediated genetic transformation method using dormant hypocotyls of mature peanut seeds as explants is used for the transformation of soybean GmFAD3C gene

[0055] (1) Select mature and plump peanut seeds, and in the aseptic environment of the superfloating workbench, sterilize the surface of the peanut seeds with 0.1% (W / V) mercuric chloride solution for 10 minutes, and then wash them with sterile water for 3 times. After soaking in warm water at 50°C for 3 hours, remove the seed coat, cut the seeds along the gap between the cotyledons with a sterile scalpel, peel off the complete embryo from the cotyledons with a scalpel, cut off the radicle and bud primordium, and keep the remaining The dormant hypocotyl part of mature peanut seeds is used as the dormant hypocotyl explant to be transformed, and is pre-cultured on a pre-medium (solid MS+3 mg / L 6-benzylaminopurine) for 0.5-1 day.

[0056] (2) Using the soybean GmFAD3C gene as the transgene, ...

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Abstract

The invention discloses an agrobacterium-mediated genetic transformation method with a peanut seed domant bud hypocotyl as an explant. The method uses the peanut seed domant bud hypocotyl as the explant and adopts the agrobacterium-mediated method for the genetic transformation after the pre-culture dedifferentiation. By optimized bud induction culturing and rooting culturing processes, the transformation regeneration rate and the rooting speed are improved effectively. The genetic transformation method does not depend on expensive apparatuses of a genetic gun, and the like and requires no expensive experimental vector materials of gold powder, etc. Only a super-floating worktable and relative agrobacterium tunefaciens strains are required for realizing the highly efficient transformation of the domant bud hypocotyl explant of a peanut mature seed embryo under the simplest experimental environment, and the period is short and the transformation effect among different genetic species is stable.

Description

technical field [0001] The invention relates to a peanut genetic transformation method, in particular to a simple and efficient transformation method mediated by Agrobacterium tumefaciens into dormant embryonic axis explants of mature peanut seeds. Background technique [0002] Peanut is an important oil crop and economic crop with rich nutritional value. Although it is one of the natural hosts of Agrobacterium, it has the same weakness as most leguminous crops, that is, it is difficult to introduce foreign genes, and the work of genetic transformation on peanut breeding is limited and progress is slow. [0003] Foreign countries began to study peanut tissue culture in the 1940s, and my country has established a variety of peanut nutrition and reproductive organ regeneration systems since the 1970s. Agrobacterium-mediated genetic transformation of exogenous genes is one of the important means of genetic transformation of peanut. Since Eapen and George first reported obtain...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H1/00A01H4/00
Inventor 刘海燕温世杰梁炫强
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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