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A method for genetically transforming Brachypodium distachyon by inflorescence dipping

A technology of Brachypodium distachyon and genetic transformation, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, botanical equipment and methods, etc., capable of solving problems such as somatic cell mutation

Inactive Publication Date: 2021-05-21
SHANGHAI CHENSHAN BOTANICAL GARDEN
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  • Application Information

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Problems solved by technology

At present, the genetic transformation of Brachypodium distachyon is to induce callus tissue from immature or mature embryos, infiltrate the callus tissue with Agrobacterium, and then induce the callus to differentiate into shoots and roots. Such complex procedures are used to obtain transgenic plants. The fastest process will take 3-4 months, and there are certain somatic mutations

Method used

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  • A method for genetically transforming Brachypodium distachyon by inflorescence dipping
  • A method for genetically transforming Brachypodium distachyon by inflorescence dipping
  • A method for genetically transforming Brachypodium distachyon by inflorescence dipping

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Embodiment 1

[0050] 1. Transformation of Agrobacterium and identification of positive clones

[0051] Add the hygromycin resistance gene (HYG R ), the nucleotide sequence is shown in SEQ ID NO.3) plasmid DNA (pCAMBIA1300, the map is as shown in figure 1 Shown) 0.1-1μg, then ice-bath for 30min; put in liquid nitrogen for 5min, then immediately put in 37℃ water bath for 5min; take out the centrifuge tube, ice-bath for 2-5min, add 0.5ml YEB liquid medium, 28℃, 220rpm Shake the culture for 3-5 hours; take out the bacterial solution and apply it on the YEB solid medium containing the corresponding antibiotics kanamycin and rifampicin (both at a concentration of 50mg / L), and incubate it upside down at 28°C for 2-3 days in an incubator; Take a single colony and do colony PCR identification. The PCR system is: in a 20μL reaction system, Taq DNA polymerase 1.0U, dNTP 0.25μmol / L, primer 0.5μmol / L, 50ng template DNA; PCR reaction program: 94℃ Pre-denatured for 5 minutes, denatured at 94°C for 30s, ...

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Abstract

The invention discloses a method for genetically transforming Brachypodium distachyon by inflorescence dipping, comprising: A, transformation of Agrobacterium EHA105 and identification of positive clones; B, dipping of Agrobacterium inflorescences of Brachypodium distachyon; C, transgenic two Positive plants of T0 generation of Brachypodium paniculata were obtained. The invention avoids the culture process of the callus through the genetic transformation method of the inflorescence mediated by the agrobacterium, saves a lot of time, and provides an effective and fast method for obtaining transgenic plants for the genetic improvement of crops.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for genetically transforming Brachypodium distachyon by inflorescence dipping. Background technique [0002] Poaceae plants include more than 600 genera and more than 10,000 species, and Poaaceae such as wheat, barley, rye and oats are the most economically valuable groups. Poaaceae crops mostly have large and complex genomes, such as the barley genome is 5096Mb, x=7; while the genome of hexaploid common wheat is 17000Mb, with three completely independent genomes A, B and D, 3x= 21, which makes genome and functional genome research of wheat crops face great challenges. [0003] Rice is the first grass whose genome has been sequenced. However, rice is a model plant for studying temperate cereal crops, but there are many insurmountable difficulties. First of all, rice has a distant relationship with wheat plants. It was on a different evolutionary branch from tem...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/00A01H6/46
CPCC12N15/8205
Inventor 李鹏储昭庆
Owner SHANGHAI CHENSHAN BOTANICAL GARDEN
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