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Corn seed bud growth point transgene method

A growth point, transgenic technology, applied in the field of transgenic, can solve problems such as stability of unfavorable transformation rate, accuracy of unfavorable transformation rate, unfavorable transformation, etc., to reduce the proportion of plants that cannot grow into plants, to easily regenerate plants, and to increase the proportion of transformation. Effect

Inactive Publication Date: 2017-08-08
HEBEI ACADEMY OF AGRI & FORESTRY SCI INST OF GENETICS & PHYSIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conversion rate of the method is low, and it is greatly affected by the genotype of maize
[0006] The Chinese patents "A Method for In Situ Transgenesis of Mature Maize Embryos" (Application No.: 201110051783.2) and "Transgenic Breeding Method Using Haploid Corn Stem Tips as Recipients" (Application No.: 201210448146.3) are both invention patents The growth point of corn buds is used as the transformation object, but there are some problems that affect the transformation efficiency. The germs during transformation are relatively large. The bud sizes used in the two patents are 1-10 cm and 2-4 cm respectively.
In the patent, the co-cultivation of corn mature embryo growth point cells in the Agrobacterium-infected solution and Agrobacterium is carried out in a dark place at a room temperature of 25-28 degrees Celsius, and the second patent is placed at 21-25 degrees Celsius for 4-6 days in the dark. The temperature used is too high or the temperature range is too large, which is not conducive to the conversion or the stability of the conversion rate; for the wound of the growth point, one patent does not clearly state the specific method, and the other patent has a serious wound (pointed out in the patent: from Cut the growth point of the shoot tip longitudinally in the middle, and the depth of the wound is about 1 mm)
[0007] In short, the previous patents or reports that used the growth point of corn buds as the transformation object are generally not conducive to the improvement of the transformation rate and the accuracy of detection.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Transgenic Experiment of Maize Bud Growth Point

[0049] Experimental materials: seeds of the maize inbred line "Zheng 58".

[0050] Gene carrier: Transform the vector PEGAD containing the target gene EGFP, replace the 35S promoter with the ubiqution promoter containing intron, the ubiqution promoter comes from the intermediate vector pAHC25, and transform the transformed ubi-PEGAD plasmid into EHA105 Agrobacterium tumefaciens.

[0051] Seed disinfection and germination: Rinse the seeds with sterile water for 5 min and wash with 0.1% HgCl 2 Disinfect for 12 minutes, rinse with sterile water 5 times, 2 minutes each time, put the seeds in a high-temperature sterilized petri dish with 2 layers of filter paper (specification: diameter 9 cm), put 20 corn seeds in each petri dish, Add 56 µL of acetosyringone (AS) at a concentration of 2 mg / ml, add 8 ml of sterile water, and incubate in the dark at 25°C.

[0052] Receptor preparation: when the corn sprouts are 0.3...

Embodiment 2

[0060] Example 2 Transgenic experiment of corn bud growth point - AS seed soaking concentration experiment

[0061] Experimental materials: seeds of the maize inbred line "Zheng 58".

[0062] Gene carrier: Transform the carrier PEGAD containing the target gene EGFP, replace the 35 S promoter with the ubiqution promoter containing intron, the ubiqution promoter comes from the intermediate vector pAHC25, and transform the transformed ubi-PEGAD plasmid into EHA105 Agrobacterium tumefaciens.

[0063] Preparation of Agrobacterium-mediated transformation solution: Pick a single colony and inoculate it into 50 ml of LB liquid medium containing 50 mg / L kanamycin + 40 mg / L rifampicin, and culture it at 28°C and 210 rpm until the bacteria liquid OD 600 = 0.5, 4000rpm, 5 min, 5 ℃ centrifugation to collect bacteria, discard the supernatant and suspend in 0.5 ml hypertonic infection base solution (1 / 10 MS medium salt + 68.5 g / L sucrose + 30g / L Glucose + 400 mg / L MES + 100 μmol / L acetosy...

Embodiment 3

[0073] Example 3 Maize seed bud growth point transgenic experiment

[0074] Transformation object: seeds of maize inbred line "Zheng 58".

[0075] Gene carrier: Transform the vector PEGAD containing the target gene EGFP, replace the 35S promoter with the ubiqution promoter containing intron, the ubiqution promoter comes from the intermediate vector pAHC25, and transform the transformed ubi-PEGAD plasmid into EHA105 Agrobacterium tumefaciens.

[0076] Seed germination: with 0.1% HgCl 2Disinfect for 15 minutes, rinse with sterile water for 5 times, each time for 2 minutes, put the seeds in a high-temperature sterilized glass petri dish with 2 layers of filter paper (the diameter of the petri dish is 9 cm), put 20 corn seeds in each petri dish, Add 8 ml of 210 μM acetosyringone (AS) aqueous solution to each Petri dish, culture in dark at 25°C for 3 days to germinate, and germinate 4 Petri dishes in total.

[0077] Infection solution preparation: activate Agrobacterium in 50 ml...

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Abstract

The invention relates to a corn seed bud growth point transgene method, which comprises the following steps of (1) receptor preparation; (2) infection solution preparation; (3) conversion treatment; (4) coculture; (5) germination culture; (6) young seedling transplanting; (7) plant management; (8) T0 generation molecular detection and identification; (9) T1 generation molecular detection and identification. The method has the advantages that the tissue culture is not performed; the limitation of the genotype is small; the conversion efficiency is high; the conversion effect is stable; the operation is simple and convenient; the practicability is high; the large-scale application can be easily performed; the consumed cost is low.

Description

technical field [0001] The invention relates to a genetically modified method, in particular to a genetically modified method for corn seed bud growth points. Background technique [0002] In the practice of plant transgenics, the Agrobacterium-mediated method is widely used. This method has the advantages of high fertility of the obtained transgenic plants, most of the foreign genes are integrated in the recipient in single copy or low copy, and large fragments of DNA can be transferred. Etc. However, at present, most of the plant transgenic technologies based on Agrobacterium-mediated transformation need to go through the tissue culture process, and there are many problems such as restriction by the genotype of the transformed recipient, complex operation, long cycle, low transformation efficiency, and unstable transformation results. , has seriously affected the application of Agrobacterium-mediated transformation in maize transgenesis. [0003] The corn seed bud growth...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01G1/00
CPCC12N15/8205
Inventor 吕孟雨王海波董福双赵和柴建芳徐显张俊敏周硕刘永伟杨帆孙果忠王金萍
Owner HEBEI ACADEMY OF AGRI & FORESTRY SCI INST OF GENETICS & PHYSIOLOGY