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A high-efficiency plant receptor parthenogenic haploid screening method

A technology of parthenogenesis and haploid induction system, which is applied in the field of high-efficiency induction of plant receptor parthenogenesis induction and haploid screening, and can solve the problems of unseen applications, many procedures, and inability to distinguish haploids, etc.

Active Publication Date: 2021-01-05
LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the constitutive expression of its fluorescent markers, all parts of the endosperm aleurone layer and starch endosperm have strong fluorescent expression. Under the fluorescence microscope, the whole grain emits strong eGFP green fluorescence, so it cannot be detected at the grain level To distinguish the presence or absence of embryonic tissue fluorescence, that is, haploids cannot be distinguished, and must rely on identification at the seedling stage. The same process involves many procedures and increases the cost of manpower and material resources.
Due to the above limitations, no application has been seen

Method used

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  • A high-efficiency plant receptor parthenogenic haploid screening method
  • A high-efficiency plant receptor parthenogenic haploid screening method
  • A high-efficiency plant receptor parthenogenic haploid screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1, the construction of carrier

[0083] 1. Construction of embryo-endosperm-specific double fluorescent marker expression cassette

[0084] The embryo-endosperm-specific dual fluorescent marker dual expression cassette provided by the present invention includes the following elements sequentially from upstream to downstream: embryo-specific promoter ZmESP, fluorescent protein eGFP gene, NOS terminator, endosperm aleurone-specific promoter HvASP , fluorescent protein DsRED gene and NOS terminator; the expression of fluorescent protein eGFP gene is started by the embryo-specific promoter ZmESP, and the expression of fluorescent protein DsRED gene is started by the endosperm aleurone-specific promoter HvASP.

[0085] The embryo-specific promoter ZmESP is shown in the sequence 1 of the sequence listing from the 5' end 340-2275 nucleotides; the fluorescent protein eGFP gene is shown in the sequence listing 1 from the 5' end 2276-2995 Nucleotides are shown; the en...

Embodiment 2

[0092] Example 2. In vitro activity identification of tissue-specific promoters

[0093] Experimental materials: corn inbred line B73, rice Nipponbare, wheat KN199 and barley Liushizhun.

[0094] 1. Disinfect the 25-day-old ears of the experimental materials with 70% alcohol for 5 minutes, sterilize with 5% sodium hypochlorite solution for 30 minutes, and then wash several times with sterile water. Use a scalpel and dissecting needle to peel off the corn kernels, and remove the embryo and endosperm. Stripping, the endosperm stripped off the embryo is cut longitudinally into two.

[0095] 2. Place the embryos and endosperms obtained in step 1 into pre-prepared MS solid hypertonic medium (MS salt, MS organic, agar 10g / l, sorbitol 0.25mol / l) culture dish plate (embryo and endosperm, place 36 flat plates respectively, and place the tissue within 2cm diameter of the center of the flat plate), put about 20 embryos in each flat, put about 10 half endosperms in each flat, and put the...

Embodiment 3

[0108] Embodiment 3, maize stable genetic transformation

[0109] 1. Obtaining of transgenic plants

[0110] 1. Construct a plant expression vector containing a dual fluorescent expression cassette, named pCAMBIA3301-ZmESP-Egfp-HvASP-dsRED vector, as shown in sequence 4 of the sequence listing, wherein positions 16-4764 are a dual fluorescent marker expression cassette. The pCAMBIA3301-ZmESP-Egfp-HvASP-dsRED vector was introduced into BMEHA105 Agrobacterium competent cells (Beijing Biomed Gene Technology Co., Ltd., catalog number: BC303-01) to obtain recombinant bacteria.

[0111] 2. Cultivate the recombinant Agrobacterium obtained in step 1 using N6 liquid medium to obtain the OD of the bacterial solution 600nm 0.8 recombinant Agrobacterium bacterium liquid, centrifuge at 5000r / min for 10min, collect the bacteria, and use the prepared infection buffer (1L infection buffer is 4g of N6 salt containing N6vitamin, 2mg of 2,4-D, 100mg of inositol , 0.7gL-proline, 68.4g sucrose, ...

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Abstract

The invention discloses a high-efficiency inducing screening method for parthenogernesis haploid of a plant receptor. The high-efficiency screening method is characterized in that an expression box with double markers of embryo-specific promoter ZmESP-driven fluorescent protein eGFP and embryo milk aleurone layer specific promoter HvASP-promoted fluorescent protein DsRED forms an embryo-embryo milk double-fluorescent marker carrier; based on the corn, rice, wheat and barley parthenogernesis haploid inducing series with embryo-embryo milk tissue specific double fluorescent markers, the haploidcan be accurately and efficiently verified and screened in a large-scale way, and the particular advantage is realized in the breeding of haploid; the limitation to the expression of seed anthocyaninor content of oil by the embryo-embryo milk color or oil content screening markers due to genetic background can be overcome, and the haploid seeds can be stably and efficiently screened; the high-efficiency screening method can be applied to the materials with any color and oil content under any genetic background, the limitation to the receptor in the prior art is overcome, and the important meaning is realized on the breeding of double haploids.

Description

technical field [0001] The invention relates to a method for efficiently inducing parthenogenetic induction and haploid screening of plant receptors. Background technique [0002] Maize is an important food crop, and it is also the most widely used crop for the utilization of heterosis. Maize double haploid (DH) breeding technology is to use the haploid produced naturally or artificially, and then obtain diploid homozygous inbred lines through natural or chemical doubling. This method realizes the homozygous selection of one generation of inbred lines. Compared with the 6-7 generations required for traditional self-bred breeding, it has obvious technical advantages and is becoming one of the main strategies for line selection. DH breeding technology mainly includes three links, namely haploid induction, haploid screening, and haploid doubling. Efficient and accurate screening of haploids is a key technical link. Currently, haplotype identification methods mainly include m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/10A01H1/02A01H1/04A01H6/46
CPCA01H1/02A01H1/04A01H5/10C07K14/415C12N15/8212C12N15/8218C12N15/8234
Inventor 谢传晓董乐刘昌林李新海黄长玲
Owner LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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