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Construction method of screening condition in rice chloroplast genetic transformation process

A technology of genetic transformation and screening conditions, applied in the field of construction of screening conditions, can solve problems such as failure to achieve success

Inactive Publication Date: 2018-05-11
JILIN ACAD OF AGRI SCI
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  • Application Information

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Problems solved by technology

[0004] However, the above-mentioned plants that achieve chloroplast transformation are all dicotyledonous plants. The chloroplast transformation of monocotyledonous plants has not been successful, and many important crops, such as rice, corn, wheat and sorghum, are all monocotyledonous plants.

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  • Construction method of screening condition in rice chloroplast genetic transformation process
  • Construction method of screening condition in rice chloroplast genetic transformation process
  • Construction method of screening condition in rice chloroplast genetic transformation process

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Experimental program
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Effect test

Embodiment 1

[0044] 1 Materials and methods

[0045] 1.1 Test material

[0046] 1.1.1 Plant material

[0047] The test materials for testing are 20 early-maturing rice lines (code-named: 12, 14-17, 15, 17, 19, 35, 37-14, 47, 58, 111, 117, 198, 201, 239, 450, 501 , 808, 848, 1471, Z291), selected and bred by the Rice Breeding Laboratory of Jilin Agricultural University.

[0048] 1.1.2 Plasmids and strains

[0049] Design of the Prrn-T7g10-MSI99-smGFP-HPT-Trps16 expression cassette required for rice chloroplast expression. The Prrn promoter and Trps16 terminator are the 16S rRNA promoter and the 3' end mature sequence respectively (GenBank accession number: BA000010.8); the three genes MSI99, smGFP, and HPT included are based on the rice chloroplast Codon optimization for codon bias. The attP site and attB site recognized by homologous recombinase were inserted in the upstream of the smGFP gene and downstream of the HPT gene to excise the selection marker gene. The fragment was synthes...

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Abstract

The invention discloses a construction method of a screening condition in the rice chloroplast genetic transformation process, which comprises the following steps of step (1) detecting influence of ascreening reagent on growth of rice callus; step (2) screening a resistance gene-screening reagent combination. According to the invention, by screening, a rice material suitable for chloroplast transformation is obtained; the improvement has the following characteristics that the rice material has high-frequency regeneration capacity; the II callus generated by induction still has excellent differentiative capacity after long-time (more than six months) secondary culture. In addition, by comparison, a case that hygromycin is more suitable to be used as the screening reagent for rice chloroplast genetic transformation than common spectinomycin and other screen reagents (antibiotics or herbicides) is proved.

Description

technical field [0001] The invention relates to a method for constructing screening conditions in the process of rice chloroplast genetic transformation. Background technique [0002] Plant chloroplasts are semi-autonomous organelles with their own genomes and are therefore transformable. Chloroplast transformation is usually achieved through direct transformation by biolistic method or PEG method. Although there have been reports of transformation through Agrobacterium-mediated transformation, they have not been repeated. Chloroplast transformation is the direct delivery of exogenous gene DNA to chloroplasts, and then site-specific integration of exogenous genes into the chloroplast genome by means of homologous recombination, which was gradually established with the invention of the gene gun method. [0003] The research on chloroplast transformation started later than that of nuclear transformation. Boynton et al. transformed atpB gene mutant cells with wild-type Chlamyd...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
CPCG01N33/5097
Inventor 邢少辰王云鹏韦正乙
Owner JILIN ACAD OF AGRI SCI
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