Method for transforming rice

A rice and rice seed technology, applied in biochemical equipment and methods, horticultural methods, botanical equipment and methods, etc., can solve the problems of large demand for explants, strong genotype dependence, limited receptor materials, etc. Achieve the effect of reducing time and manpower consumption, reducing dependence and high conversion efficiency

Inactive Publication Date: 2014-04-23
BEIJING DABEINONG TECH GRP CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to provide a method for transforming rice, which can effectively overcome the technical defects of the prior art such as strong genotype dependence, limited receptor materials, large demand for explants, low transformation efficiency, long transformation cycle, and high precision.

Method used

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  • Method for transforming rice
  • Method for transforming rice

Examples

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Effect test

no. 1 example

[0051] The first embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

[0052] 1. Construct a recombinant cloning vector containing the target gene

[0053]The HPT nucleotide sequence was connected to the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector DBN01-T, and its construction process Such as figure 1 Shown (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ start codon; SP6 is the promoter of SP6 RNA polymerase; T7 is the promoter of T7 RNA polymerase; HPT is the promoter of hygromycin Phosphotransferase gene nucleotide sequence (SEQ ID NO: 1); MCS is a multiple cloning site).

[0054] Then, the recombinant cloning vector DBN01-T was transformed into Esch...

no. 2 example

[0061] The second embodiment, the acquisition of transgenic rice plants

[0062] Sterilization of mature seeds: Take a number of mature rice seeds (Minghui 63), select brown rice with plump grains and intact embryos after shelling, put them in a triangular flask, and sterilize them with 75% (v / v) alcohol for 1 minute to remove Impurities such as rice bran on the surface of brown rice should be filtered off, then add sodium hypochlorite (containing Tween 20 (1 drop / 30mL)) with a mass fraction of 50%, shake and shake continuously for 20-40 minutes, and rinse with sterile water for 4- 5 times to remove residual sodium hypochlorite.

[0063] Callus induction: the brown rice after the above treatment was slightly dried and then inoculated in callus induction medium (a large amount of salt in N6 medium, a small amount of salt in B5 medium, organic matter in B5 medium, 30 g / L sucrose, 500 mg / L casein, grain Aminoamide 500mg / L, proline 500mg / L, 2-morpholineethanesulfonic acid (MES) 5...

no. 3 example

[0073] The third embodiment, using TaqMan to verify transgenic rice plants

[0074] About 100mg of the leaves of transgenic rice plants were taken as a sample, and the genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the copy number of the HPT gene was detected by the Taqman probe fluorescent quantitative PCR method. At the same time, wild-type rice plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.

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Abstract

The invention relates to a method for transforming rice. The method comprises the following steps: obtaining callus by using a method for inducing rice callus or a method for greatly amplifying the rice callus, and infecting the callus by using agrobacterium. By adopting the method for transforming the rice, nonglutinous rice varieties which are not easy to have genetic transformation can obtain embryogenic callus, primary callus is firstly induced by using a solid culture medium and subsequently suspension culture is performed by using a liquid culture medium, so that a great deal of rice embryogenic callus with good quality and rapid growth can be amplified so as to be subjected to infection transformation by the agrobacterium; the transformation frequency can be greater than 10%, the pressure in nonglutinous rice explant supply is remarkably alleviated, and the time and the labor consumption in nonglutinous rice tissue cultivation are greatly reduced.

Description

technical field [0001] The present invention relates to a method for plant transformation, in particular to a method for transforming rice tissue. Background technique [0002] Rice (Oryza sativa) is one of the main food crops in the world, and it is also the largest food crop in my country. However, with the increase in population and social development, the arable land area is getting smaller and smaller, and the annual growth rate of rice production has dropped to less than 1%, and the output of crop varieties has almost reached the production limit. It is difficult to make major breakthroughs in technology, and the progress of crop genetic engineering technology is the hope of breeding high-yielding crop varieties. [0003] The genetic transformation methods of rice mainly include Agrobacterium-mediated method, particle gun bombardment method, electric shock method, PEG method and pollen tube passage method, among which Agrobacterium-mediated method is currently the com...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N15/84A01H5/00A01H4/00
Inventor 金许成
Owner BEIJING DABEINONG TECH GRP CO LTD
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