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Agrobacterium tumefaciens-mediated barley mature embryo callus transformation method

An Agrobacterium-mediated, callus technology, applied in the field of plant genetic engineering, can solve the problems of difficulty in controlling the flowering period of barley, time-consuming, labor-intensive, and high cost, and achieve the effects of simple and feasible separation, easy guarantee of quantity, and low pollution rate.

Active Publication Date: 2013-05-01
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of immature barley embryos requires continuous cultivation of donor plants throughout the year, which is time-consuming and costly. At the same time, the transformation of immature embryos has specific requirements for the developmental period of explants, and the flowering period of barley is often difficult to control.

Method used

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  • Agrobacterium tumefaciens-mediated barley mature embryo callus transformation method
  • Agrobacterium tumefaciens-mediated barley mature embryo callus transformation method
  • Agrobacterium tumefaciens-mediated barley mature embryo callus transformation method

Examples

Experimental program
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Effect test

Embodiment 1

[0059] 1), induction of mature barley embryo callus:

[0060] Select mature seeds of healthy and full-bodied barley variety Golden Promise harvested in the same year, wrap them in gauze and wash them in running water for 1 hour in an Erlenmeyer flask, cut off part of the endosperm of the seeds with a scalpel on absorbent paper, and keep only the isolated embryos (length 4-5mm), and then peel off the shell of the isolated embryos by hand and put them into 10mL centrifuge tubes (pre-sterilized). Generally, 15-20 shelled isolated embryos are placed in each 10mL centrifuge tube. On the ultra-clean bench, first pour 75% (v / v) ethanol into the centrifuge tube and shake it for 30 seconds; after pouring off the ethanol, use freshly prepared 15% sodium hypochlorite (v / v) to stand and sterilize for 15 minutes to obtain Sterilize the isolated embryos; wash the above-mentioned sterilized isolated embryos with sterile water for 2 times, each time for 3 minutes. The detached embryos were b...

Embodiment 2

[0087] The healthy and mature seeds of two barley varieties Golden Promise and Zaoshu No. 3 were selected for callus induction, isolation and subculture respectively. The steps were the same as in Example 1, but the vectors carried by Agrobacterium were different during transformation. In Example 2, the Agrobacterium strain LBA4404 was used, carrying the transformed pCambia1300 plasmid, carrying the glyphosate resistance gene driven by the maize ubiquitin promoter.

[0088] Use pcr technology to verify whether there is the target gene transferred in the transformed seedlings, as follows:

[0089] Plant regeneration is carried out after the callus is transformed, bacteriostasis and recovery culture; the steps are as follows:

[0090] Plant differentiation and regeneration:

[0091] The callus after recovery culture was transferred to the differentiation medium for plant differentiation and regeneration, and the culture conditions were high light intensity (60-80 μE m -2 the s...

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Abstract

The invention relates to the field of plant genetic engineering, and discloses an agrobacterium tumefaciens-mediated barley mature embryo callus genetic transformation method and a corresponding tissue culture system. In the method, mature embryo callus is used as a receptor, and agrobacterium tumefaciens LBA4404 is used for mediating, so that allogenetic green fluorescent protein (GFP) genes andglyphosate-resistant genes are successfully introduced into different barley varieties. The method specifically comprises the following steps of: inducing the barley mature embryo callus; separating and subculturing first-generation callus; culturing and activating the agrobacterium tumefaciens; infecting and co-culturing the mature embryo callus; and performing bacteriosasis and recovery cultureof the callus. Detection of reporter gene (GFP) and polymerase chain reaction (PCR) shows that transformation of DNA (Deoxyribonucleic Acid) is realized in a transgenic plant. The method provided by the invention develops a new approach for cloning, functional analysis, character improvement and the like of barley genes.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. Specifically, the invention relates to a genetic transformation method and corresponding tissue culture for obtaining transgenic plants by using Agrobacterium tumefaciens to infect embryogenic callus induced by mature barley embryos system. Background technique [0002] Since Wan and Lemaux took the lead in transforming the barley variety Golden Promise by bombardment with DNA microparticle fragments in the early 1990s, there have been many international transformations of barley using particle bombardment or Agrobacterium-mediated methods and successfully integrating some target traits into Reports from barley (Wan and Lemaux, 1994, Plant Physiol. 104: 37-48; Tingay et al., 1997, Plant J. 11: 1369-1376; Lemaux, 1999, Great Britain: Kluwer Academic Publishers: 255-316) . At present, the vast majority of barley transformation systems use Golden Hope as the model variety, but it...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 韩勇王静巫小建金晓丽张国平
Owner ZHEJIANG UNIV
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