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Method for genetic transformation of soybean chloroplast

A technology of genetic transformation and chloroplast, applied in the field of agricultural planting, can solve the problems of small number of chloroplasts, difficulty in transformation, etc., and achieve the effect of normal growth and development

Inactive Publication Date: 2013-09-11
JILIN ACAD OF AGRI SCI
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Problems solved by technology

Different from nuclear genome transformation, the chloroplast transformation technology requires explants to be able to proliferate or regenerate continuously on the selection medium (the homogenization process of transformed cells). Therefore, there are certain difficulties in using cotyledon nodes and embryo tips as chloroplast genetic transformation.
In addition, although the somatic cell mass induced by soybean immature cotyledons can continue to proliferate on the proliferation medium, it is different from the genetic transformation of chloroplasts using green leaves as explants, due to the number of chloroplasts in the somatic cell mass (or embryogenic callus). Smaller and mostly undifferentiated protoplasts, making transformation more difficult

Method used

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Experimental program
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Embodiment Construction

[0031] Obtaining of homogenized chloroplast transgenic soybean plants

[0032] 1. Test materials:

[0033] The tested plant material was soybean model variety JACK, the transformation vector was pGm-gfp-aadA(-), and the vector carried the target gene gfp and the selection marker gene aadA.

[0034] 2. Conversion method:

[0035] 1) Take the young pods of soybean (variety JACK) about 20 days after flowering, sterilize with 0.5% sodium hypochlorite for 10 minutes, rinse with sterile water for 5 times, then take out the immature soybean seeds under aseptic conditions and place them in a sterile petri dish Treat for 2 days at 4℃;

[0036] 2) Remove the seed coat, divide the cotyledon into two petals and inoculate it on the induction medium according to the dorsal axis face down, connect 20 explants to each dish, under low light intensity of less than 1000 Lux, 16 / 8h light / dark cycle, Cultivate for 3 to 4 weeks at 26°C. At this time, most of the cotyledons grow around the spherical embryo...

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Abstract

The invention discloses a method for genetic transformation of soybean chloroplasts, belonging to the technical field of agriculture planting. On the basis that multiple factors such as target material treatment, gene gun bombardment parameters, screening pressure and screening period are optimized, homogeneous chloroplast transgenic soybean plants are ultimately obtained by sufficiently utilizing the characteristic that soybean embryonic somatic cell clusters can be continuously multiplied, by carrying out appropriate natural dehydration treatment on a target material and by carrying out continuous, multiple and high-pressure screening. The soybean chloroplast genetic transformation method disclosed by the invention not only has important practical application values in soybean transgenic improvement and biologic reactor study, but also has an important reference significance in genetic transformation of other crop chloroplasts generated in a somatic embryogenesis mode.

Description

Technical field [0001] The invention belongs to the field of agricultural planting technology and relates to a genetic transformation technology. Background technique [0002] Since the birth of the world’s first transgenic plant in 1983, nuclear genome transformation has always been the main research direction in the field of plant genetic engineering. However, with the deepening of research, the limitations of nuclear genome transformation technology have also attracted increasing attention. Due to the large nuclear genome of plant cells and the complicated genetic background, it is difficult to control the integration site and copy number of foreign genes in the nuclear genome. As a result, the expression efficiency of foreign genes is low and the expression is unstable, and "gene silencing" and " At the same time, exogenous transgenes easily spread with pollen to nearby relative species, leading to gene escape and endangering species diversity and ecological safety in nature...

Claims

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Application Information

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IPC IPC(8): C12N15/63A01H4/00A01H5/00
Inventor 杨向东赵桂兰郭东全钱雪艳杨静李海云张原宇齐广勋邢国杰李闯姚瑶
Owner JILIN ACAD OF AGRI SCI
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