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Method of establishing early-maturing ripe hereditary transform system and application

A genetic transformation system and technology of genetic transformation, applied in the fields of application, horticultural methods, botanical equipment and methods, etc., can solve the problem of difficult subculture of embryogenic callus, affecting the development of transgenic work of bluegrass, low differentiation frequency, etc. question

Inactive Publication Date: 2007-06-20
SHANDONG UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Although the induction rate can reach 100% (Zhu Genfa et al., 1994) with young panicles as explants in the work of Zhu Genfa et al., many people still use seed embryos as explants to induce callus because the materials are limited by seasons.
Due to the low callus differentiation frequency of mature embryos of Poa annua and the difficulty of subculture of embryogenic callus, the development of transgenic work in Poa annua

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Induction and subculture of clustered buds: get the seeds of the bluegrass variety prize and sterilize them with 70% alcohol for 1-2 minutes, 0.2% mercury liter (mercuric chloride) for 10 minutes, rinse with sterile water 4 times, and sow in Germinate on moist sterile filter paper, cut out the shoot tip and put it on the induction medium (adding 2,4-D 0.2mg L -1 , 6-BA 2mg L -1 ) to induce base expansion, and the expansion rate was 100%. After culturing on the induction medium for 20 days, cut off the expanded tissue (diameter 1-3mm) at the base of the shoot tip and transfer it to the subculture and clustering medium (take 2,4-D 0.1mg L -1 , 6-BA 2mg L -1 ) to induce clustered buds to occur, and after 15-20 days, most of the expanded bases differentiated clustered buds, the clustering rate was about 75%, and the number of clustered buds was mostly 15-20. After dividing the clumping bud block, transfer it to the subculture and clumping medium, the cells of the clumpin...

Embodiment 2

[0066] Cluster bud induction and subculture: the procedure is the same as in Example 1, but the bluegrass variety is Xingeriad. The selected subculture and clump-forming hormone combination is 2,4-D 0.07mg L -1 , 6-BA 3.0mg L -1 .

[0067] Determination of the concentration of the screening agent: the screening agent is hygromycin. After hygromycin aqueous solution was filtered and sterilized, it was added to the induction medium at concentrations of 0, 2, 4, 6, 8, 10, 12, and 14 mg L -1 , that is, 8 gradient concentrations. Screening procedures and methods are the same as in Example 1. According to the survival rate after screening, determine the hygromycin selection concentration of Xingelaide seedlings as 10mgL -1 . At this concentration, the seedling survival rate is about 3%.

[0068] Gene gun bombardment transforms clustered buds:

[0069] The plasmid is pCAMBIA1300-hpt-antiport. Standard procedures and methods were used for extraction and purification (see "Mol...

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Abstract

A process for configuring a transgenic line of early-mature grass includes such steps as sampling the stem tip of early-mature seedling, in vitro culturing to induce its gibbosity, taking its tissue, culturing to induce its bunch sprouts, reproducing the bunch sprouts rooting culture to obtain complete plant, and using gene gun to bombard the sprout tuber to obtain its transgenic plant.

Description

technical field [0001] The invention relates to a method and application of a bluegrass genetic transformation system, and belongs to the field of agricultural biotechnology or the field of plant genetic engineering. Background technique [0002] Bluegrass (Poa pratensis L.) is a cool-season turfgrass and one of the important grass species in temperate regions. Because of its beautiful color, strong cold resistance, shade tolerance, and pruning resistance, it is often used as one of the lawn grass species in northern my country. Bluegrass also has its own disadvantages, such as being vulnerable to pests and diseases, not tolerant to heat, poor in high and low temperature tolerance, and weak in drought resistance. These traits can be improved to some extent by means of genetic transformation. Successful gene transformation firstly depends on the establishment of a good plant receptor system. In 1985, Vasil first proposed the feasibility of using genetic transformation tech...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80A01H4/00
Inventor 张举仁杨爱芳张可炜尹小燕谷晓峰
Owner SHANDONG UNIV
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