Culture medium and culture method for breeding transgenic maize

A technology of transgenic corn and culture medium, which is applied in horticultural methods, genetic engineering, chemical instruments and methods, etc., can solve the problems of long cycle of breeding technology, and achieve the effects of short cycle, high positive rate and simple operation.

Active Publication Date: 2017-08-08
CHINA NAT SEED GRP
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AI Technical Summary

Problems solved by technology

Traditional breeding technology has a long cycle and requires continuous backcrossing to obtain homozygous offspring

Method used

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  • Culture medium and culture method for breeding transgenic maize
  • Culture medium and culture method for breeding transgenic maize
  • Culture medium and culture method for breeding transgenic maize

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The preparation method of embodiment 1 transgenic corn

[0041] The corn inbred line used in this example is AY63, provided by China Seed Group Co., Ltd.

[0042] 1. Corn ear processing and separation of immature embryos

[0043] (1) 6-15 days after the pollination of the corn plant, when the immature embryo grows to 0.5-2.0mm, harvest the young ears of corn, remove the bract leaves, and prepare for sterilization;

[0044] (2) Dilute the sodium hypochlorite mother liquor with a concentration of 6.15% to 15%-20% by volume with sterilized water, add 5-10 μ L Tween-20 to each liter of solution and mix to make a sterilized solution;

[0045] (3) Soak the young ears of corn in the sterilizing solution for 15 minutes, rinse with sterile water for 3-5 times, and set aside;

[0046] (4) Peel off the top of the seed with a sterile surgical blade in an ultra-clean workbench, dig out the endosperm with a sterile spatula to expose the immature embryo from the seed, peel off the i...

Embodiment 2

[0072] Example 2 Optimization of AY63 young embryo Agrobacterium transformation system

[0073] 1. Add NAA to co-cultivation medium and callus induction medium

[0074] In the stage of co-cultivation and callus induction, in addition to adding an appropriate amount of plant growth regulator 2,4-D to the medium, the addition of different concentrations of NAA had certain effects on the transformation efficiency. The effect of its addition is shown in Table 1. Adding 0.5 mg / L NAA or 1.0 mg / L NAA to the co-cultivation and callus induction medium greatly improved the transformation rate, with the average value increased from 1.63% to 3.66% and 3.42%.

[0075] Table 1 The conversion rate (%) comparison of adding different concentrations of NAA in the co-cultivation medium and the callus induction medium

[0076]

[0077] 2. Add KT to co-cultivation medium and callus induction medium

[0078] In the stage of co-cultivation medium and callus induction culture, in addition to ad...

Embodiment 3

[0089] The detection of embodiment 3 transgenic corn

[0090] 1. Observation on the expression of exogenous gene GFP in transgenic maize tissue

[0091]Take the AY63 transformed callus of Example 1 to observe the expression of the exogenous gene GFP in the tissue, and see green fluorescence under the fluorescence, indicating that the exogenous gene is successfully expressed, otherwise it is a negative material, and the negative material is compared with the positive material. There is no typical green fluorescence, and the callus is yellow ( figure 1 ).

[0092] 2. Real-time PCR detection

[0093] ABI 7900 fluorescent quantitative PCR instrument was used.

[0094] The internal reference gene was IVR, the forward primer was 5-ACTAGGCATCCAAGGCGAACG-3; the reverse primer was 5-AGTGCGAGAAGAACGAGTGTCC-3'. The target detection gene is Bar, the forward primer is 5-GACCTCCACCGTGAACTTCC-3; the reverse primer is 5-GTCCAGTCGTAGGCGTTGC-3'.

[0095] PCR reaction system (10 μL): 5 μL o...

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Abstract

The invention provides a culture medium for breeding transgenic maize. The culture medium includes a co-culture medium, a callus induction medium, a selection medium, a differentiation medium and a rooting medium, wherein the co-culture medium and the callus induction medium are added with NAA which is 0.5-1.0mg / L in final concentration, TDZ which is 0.005-0.05mg / L in final concentration and / or KT which is 0-0.1mg / L in final concentration. The invention also provides a culture method of the transgenic maize; with a maize immature embryo as an explant, a transgenic maize plant is obtained by virtue of an agrobacterium-mediated transformation method, so that a novel germplasm resource is provided for genetic breeding of maize. The method has the characteristics of being short in cycle, high in positive rate and simple to operate; therefore, a novel method is provided for large-scale commercial production of the transgenic maize.

Description

technical field [0001] The invention relates to the field of plant transgenic technology and the field of crop genetic breeding, in particular to a culture medium and a cultivation method for cultivating transgenic corn. Background technique [0002] Corn is an important food crop and feed crop in my country, and occupies an important position in China's grain production and national economy. However, since 2010, my country has transformed from a net exporter of corn to a net importer. In 2012, my country imported 5.21 million tons of corn, worth 1.7 billion US dollars. Therefore, improving corn yield through the application of breeding technology and mastering the initiative of corn industry is the key to maintaining the balance between domestic corn supply and demand, and will also have a profound impact on my country's food security and response to possible food crises. [0003] With the continuous development of biotechnology, more and more new breeding techniques that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
CPCA01H4/001A01H4/008C07K14/415C07K14/43595C12N15/8205C12N15/8274
Inventor 许洁婷周倩杨桥魏楚楚胡燕琳黄磊旷乐左丹汤益杨晓凤何实涂年影王萍成雄鹰章旺根
Owner CHINA NAT SEED GRP
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