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Wheat genetic transformation method

A genetic transformation method and wheat technology, which are applied in the field of increasing the genetic transformation efficiency of wheat through antibiotic gradient screening, can solve the problems of strong dependence on tissue culture technology, single starting explants, and low transformation frequency, and achieve the prospect of large market promotion. , Simple operation, high conversion efficiency

Inactive Publication Date: 2014-03-26
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Agrobacterium method has the advantages of low transgene copy, genetic stability, ability to transform relatively large fragments of DNA, and low cost, but it is limited by genotype, highly dependent on tissue culture techniques, low transformation frequency, and a single initial explant And other problems, so the application is subject to certain restrictions

Method used

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  • Wheat genetic transformation method
  • Wheat genetic transformation method
  • Wheat genetic transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, the preparation of culture medium

[0056] The pH value of each culture medium in this example is 5.8, and it is autoclaved at 120° C. for 20 minutes after the preparation is completed.

[0057] 1. Preparation of MS Medium

[0058] See Table 1 for the components and their amounts added per liter of MS medium.

[0059] The contents of each solute in each liter of MS medium in table 1

[0060]

Amount added (g)

Potassium nitrate (KNO 3 )

1.90

Ammonium nitrate (NH 4 NO 3 )

1.65

Potassium dihydrogen phosphate (KH 2 PO 4 )

0.17

Magnesium Sulfate Heptahydrate (MgSO 4 ·7H 2 O)

0.37

Calcium Chloride Dihydrate (CaCl 2 2H 2 O)

0.44

Potassium iodide (KI)

0.00083

Boric acid (H 3 BO 3 )

0.0062

Manganese sulfate tetrahydrate (MnSO 4 4H 2 O)

0.0223

Zinc sulfate heptahydrate (ZnSO 4 ·7H 2 O)

0.0086

Sodium mo...

Embodiment 2

[0070] Embodiment 2, construction of recombinant plasmid

[0071] 1. Construction of pUN1301 vector

[0072] Take the pCAMBIA1301 vector (as shown in sequence 4 of the sequence table; see the structure schematic diagram Figure 12 ; purchased from Shanghai Dior Biotechnology Co., Ltd., catalog number BDRH209) as the backbone vector, the small fragment between the BamHI and HindIII restriction sites was replaced with the UbiPro (promoter) shown in Sequence 1 of the sequence table, and the SacI The small fragment between the EcoRI restriction site and the EcoRI restriction site was replaced by the Noster (terminator) shown in sequence 2 of the sequence listing.

[0073] 2. Construction of TaCYP-pUN1301 vector

[0074] The small fragment between the KpnI and SacI restriction sites of the pUN1301 vector was replaced with the TaCYP gene shown in sequence 3 of the sequence table (TaCYP gene is a gene related to wheat plant type, overexpression will make the plant dwarf).

Embodiment 3

[0075] Embodiment 3, genetic transformation of wheat

[0076] Example 3 was carried out using the culture medium prepared in Example 1.

[0077] 1. Preparation of wheat callus

[0078]1. Take the immature seeds of the wheat variety "Jingdong No. 1" 2 weeks after flowering, peel off the paddles, soak in 70% (volume ratio) alcohol aqueous solution for 30 seconds, rinse twice with sterile distilled water, and then place Soak in 0.1g / 100mL mercuric chloride aqueous solution for 10 minutes (disinfection), rinse 4-5 times with sterile distilled water, then pick out the embryo with surgical tweezers, put the shield side up on the embryogenic callus induction medium , cultured in the dark at 25°C for 4-5 months (subculture once every 2 weeks, and embryogenic callus proliferation medium was used for subculture), and the callus with small particles, compact and fragile, light yellow and bright (see photo figure 1 ).

[0079] 2. From the callus obtained in step 1, take a callus with a...

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Abstract

The invention discloses a wheat genetic transformation method, and particularly relates to a method for increasing wheat genetic transformation efficiency through antibiotic gradient screening. The method comprises the following steps of: (1) carrying out pre-culturing on wheat embryonic calluses; (2) introducing expression vectors comprising target genes and hygromycin resistant genes into the embryonic calluses through a gene gun method; (3) recovering culture; (4) culturing on a screen culture medium for 35-55 days, wherein 35-45 mg.L<-1> of hygromycin is included in the screening culture medium; (5) culturing on a differentiation culture medium including 35-45 mg.L<-1> of hygromycin for 10-20 days to obtain the embryonic calluses for producing bud points; (6) culturing on a differentiation culture medium including 15-25 mg.L<-1> of hygromycin for 30-60 days to obtain plantlets; and (7) culturing on a rooting culture medium until roots are grown. The wheat genetic transformation method has the advantages of simplicity and easiness in operation, high transformation efficiency, good growth state of wheat plantlets obtained after transformation and extremely great actual application value and market promotion prospect.

Description

technical field [0001] The invention relates to a method for genetic transformation of wheat, in particular to a method for increasing the efficiency of genetic transformation of wheat through antibiotic gradient screening. Background technique [0002] Wheat is one of the most important food crops in the world, second only to rice, and is widely grown all over the world. Wheat is a crop that our people rely on for their livelihood. With the increase of our population and the improvement of people's living standards, higher and higher requirements have been put forward for the yield and quality of wheat. Traditional conventional breeding can no longer meet the requirements for wheat variety and quality. The development of transgenic technology has broken the limitation of conventional breeding, and has successfully bred high-yield new varieties in cotton, corn, soybean, rapeseed and other crops, creating a good economic and social benefits. Wheat is relatively backward in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 种康牛遇达李春华张景昱
Owner INST OF BOTANY CHINESE ACAD OF SCI