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Method for culturing transgenic dianthus through agrobacterium-mediated embryogenic callus transformation

A technology of embryogenic callus and Agrobacterium, applied in the field of ornamental plant tissue culture and genetic transformation, to achieve high transformation efficiency, easy seedling growth, and abundant planting resources

Inactive Publication Date: 2013-11-20
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many research reports on the transformation of Dianthus chinensis, but the research on transgenic plants obtained by genetic transformation using embryogenic callus as a receptor has not been reported yet.

Method used

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  • Method for culturing transgenic dianthus through agrobacterium-mediated embryogenic callus transformation
  • Method for culturing transgenic dianthus through agrobacterium-mediated embryogenic callus transformation
  • Method for culturing transgenic dianthus through agrobacterium-mediated embryogenic callus transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Induction and subculture of embodiment 1 dianthus embryogenic callus

[0047] 1) Material collection and low-temperature treatment. At around 9:00 a.m. on a sunny and dry day, collect dianthus flower buds growing in the field with a length of 7.0-8.5 cm, put them in a petri dish lined with moist filter paper, and place them in a 4°C refrigerator Refrigerate for 4-6d.

[0048] 2) Disinfection and inoculation of materials. The flower buds in step 1) are first washed with washing powder water, then rinsed under tap water for about 1 hour, and then disinfected on an ultra-clean workbench. The process is as follows: First, soak the flower buds in a 70% ethanol triangular flask for 1 minute, wash them with sterile water twice; then sterilize them with 0.1% mercuric chloride for 10-12 minutes. To make the flower buds fully contact with the mercuric chloride solution; then rinse with sterile water for 3-5 times, take out the sterilized flower buds and place them on sterile fil...

Embodiment 2

[0051] Example 2 Transformation of GUS reporter gene into embryogenic callus of Dianthus chinensis by Agrobacterium-mediated method

[0052] The dianthus embryogenic callus obtained in Example 1 was used as a marker of the GUS reporter gene for transformation experiments. Specific steps are as follows:

[0053] 1) Under the aseptic environment of the ultra-clean workbench, take the dianthus embryogenic callus of embodiment 1 on the sterilized filter paper, crush it with tweezers, and disperse it; pick which is bright yellow, with compact tissue, Granular embryogenic callus was used as material for transformation.

[0054] 2) Inoculate the embryogenic callus obtained in step 1) in the pre-culture medium on the ultra-clean workbench, and perform pre-culture before infection. The culture conditions are: temperature 25 ± 1 ° C, relative humidity 50% ~ 60 %, the light intensity is 2000-3000lux, and the light / darkness is 16h / 8h. The embryogenic callus of precultivation treatment ...

Embodiment 3

[0082] Example 3 Transformation of antisense ACO gene into embryogenic callus of Dianthus using Agrobacterium-mediated method

[0083] The embryogenic callus obtained in Example 1 was subjected to the transformation experiment of the antisense ACO gene (the accession number is gi|520801|, see SEQ ID NO: 3 in the sequence listing). The steps are as follows:

[0084] 1) Under the aseptic environment of the ultra-clean workbench, take the embryogenic callus of Example 1 on sterilized filter paper, crush it with tweezers, and disperse it; Strong embryogenic callus was used as the material for transformation.

[0085] 2) Hygromycin (Hyg) sensitivity screening for embryogenic callus: the embryogenic calli from step 1) were inoculated in 6 pre-designed MS media respectively. These 6 kinds of media all use MS as the basic medium (ie MS), add 6-BA1.0mg / L; NAA0.1mg / L; sucrose 30g / L; agar 7.0g / L, pH5.8. When carrying out the concentration gradient screening test of antibiotics, add Hy...

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Abstract

The invention belongs to the field of ornamental plant transgenic technology, particularly relates to a method for culturing transgenic dianthus and more particularly discloses a method for culturing transgenic dianthus by taking agrobacterium-mediated embryogenic callus of the dianthus as a material. The method comprises the following steps of embryogenic callus induction, genetic transformation, screening and regeneration, GUS (glucuronidase) coloration detection and PCR (polymerase chain reaction) detection. The method is characterized in that the GUS report gene and the ACO target gene are introduced into the receptor which is the embryogenic callus of the ianthus through agrobacterium mediation, different factors affecting the embryogenic callus transformation are screened, kanamycin or hygromycin is used as a screening agent to screen the transformed embryogenic callus, and the screening is carried out through GUS coloration, PCR detection and other auxiliary methods to obtain the transgenic dianthus. The invention provides a novel technology platform for the innovation of the germplasm resources for the genetic transformation of the dianthus.

Description

technical field [0001] The invention belongs to the technical field of ornamental plant tissue culture and genetic transformation, and in particular relates to a method for cultivating caryophyllum transgenic plants through embryogenic callus transformation mediated by Agrobacterium. Background technique [0002] There are 17 species, 1 subspecies and 9 varieties of Dianthus plants, most of which are important materials in the flower industry, landscaping and urban beautification. Among them, Dianthus is a perennial plant, which is often cultivated as one or two years. The genetic transformation of dianthus at home and abroad is based on the leaves and young plants as receptors, and the young plants are used as receptors. The top of the plant is cut off to form a wound on the terminal bud, and the suspension of Agrobacterium tumefaciens is injected with a micro-injector. On the wound, the material obtained by this method is tender and easy to die during the transformation i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 包满珠傅小鹏张静静
Owner HUAZHONG AGRI UNIV
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