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High-efficiency genetic transformation and plantlet regeneration method of hot peppers

A genetic transformation and plant technology, applied in the field of plant cell engineering, can solve the problems of seedling rooting difficulty, bud elongation difficulty, root death, etc., achieve the effect of reducing bacterial infection death, overcoming long regeneration cycle, and improving survival rate

Inactive Publication Date: 2016-11-16
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after repeated experiments, it was found that there are still many shortcomings in the regeneration of pepper plants mediated by Flamingo bill explants: (1) it is also limited by the difficulty of bud elongation; (2) the seedlings cut from flamingo bill explants Difficulty in rooting; (3) Transfer of Flamingo bill explants in solid MS medium tends to cause root death; (3) Agrobacterium-mediated genetic transformation tends to cause the death of Flamingo bill parent plants during the selection process; (4 ) The root system is prone to bacterial contamination and death

Method used

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  • High-efficiency genetic transformation and plantlet regeneration method of hot peppers
  • High-efficiency genetic transformation and plantlet regeneration method of hot peppers
  • High-efficiency genetic transformation and plantlet regeneration method of hot peppers

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Embodiment 1

[0052] The method disclosed in this embodiment is a preferred embodiment of the present invention.

[0053] A method for efficient genetic transformation and plant regeneration of capsicum, comprising the following steps:

[0054] (1) Vector construction: Design target gene primers through NCBI, compare and screen through the pepper public database, synthesize upstream and downstream primers of the target gene, KODFX (high-fidelity PCR enzyme) amplification template, recover gel, connect T vector, and sequence, if If the sequencing is incorrect, re-amplify and connect the sequence until the amplified sequence is completely correct. After the sequence is correct, perform the expression vector connection;

[0055] (2) After the ligation reaction is completed, take 5ul of the PCR product and transform it into E. coli, smear the plate with LB (bacterial basal medium) plate containing Kan antibiotic (kanamycin), pick out the single clone shaker for detection, and select the correct...

Embodiment 2

[0063] A method for efficient genetic transformation and plant regeneration of capsicum, comprising the following steps:

[0064] (1) Vector construction: Design target gene primers through NCBI, compare and screen through the pepper public database, synthesize upstream and downstream primers of the target gene, KODFX amplification template, gel recovery, connect T vector, sequence, if the sequence is incorrect, re-amplify Add connection sequencing until the amplified sequence is completely correct, after the sequence is correct, perform expression vector connection;

[0065] (2) After the ligation reaction is completed, take 5ul of the PCR product to transform Escherichia coli, smear it with an LB plate containing Kan antibiotics, pick a single clone for detection, select the correct one to extract the plasmid, and digest it with a plasmid or use a gene primer. Plasmid PCR detection, the correct detection is sent for sequencing, and the correct final expression vector is stor...

Embodiment 3

[0073] A method for efficient genetic transformation and plant regeneration of capsicum, comprising the following steps:

[0074] (1) Vector construction: Design target gene primers through NCBI, compare and screen through the pepper public database, synthesize upstream and downstream primers of the target gene, KODFX amplification template, gel recovery, connect T vector, sequence, if the sequence is incorrect, re-amplify Add connection sequencing until the amplified sequence is completely correct, after the sequence is correct, perform expression vector connection;

[0075] (2) After the ligation reaction is completed, take 5ul of the PCR product to transform Escherichia coli, smear it with an LB plate containing Kan antibiotics, pick a single clone for detection, select the correct one to extract the plasmid, and digest it with a plasmid or use a gene primer. Plasmid PCR detection, the correct detection is sent for sequencing, and the correct final expression vector is stor...

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Abstract

The invention discloses a method for high-efficiency genetic transformation and plant regeneration of pepper, which belongs to the technical field of plant cell engineering, and specifically relates to a method for genetic transformation and plant regeneration mediated by explants of pepper Flamingo bill. The invention improves the culture medium , infection mode, explant cutting mode, delayed selection and improved rooting mode, under the mediation of Agrobacterium, transform Flamingo bill explant, induce, differentiate and plant the callus at the wound of Flamingo bill explant Regeneration and other ways to obtain sustainable growth of genetically transformed regenerated plants, and effectively improve the genetic transformation rate of pepper.

Description

technical field [0001] The invention belongs to the technical field of plant cell engineering and relates to genetic transformation and plant regeneration of plants, in particular to a method for efficient genetic transformation and plant regeneration of peppers. Background technique [0002] Capsicum is an important vegetable and industrial raw material crop. The establishment of a genetic transformation system is an important basis for its genetic improvement and functional genomics research. To carry out genetic transformation of capsicum, the establishment of an efficient in vitro regeneration system is one of the important conditions. one. At present, the genetic transformation system and in vitro regeneration system of pepper are not perfect, which limits the effective application of genetic engineering in pepper genetic improvement and the progress of pepper functional genomics research. At present, the plant regeneration of plants is mainly through three ways: seedl...

Claims

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Application Information

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IPC IPC(8): A01H4/00C12N15/82A01H5/00
CPCA01H4/001A01H4/005A01H4/008C12N15/8205
Inventor 聂术君覃成黄丽潘超李辉龚敏史鉴何伟强黄洪张志明
Owner SICHUAN AGRI UNIV
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