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Plant seed transformation technology

A technology of plant seeds and technology, applied in the biological field, can solve problems such as limitations, difficult regeneration systems, differences in genetic background, etc., and achieve great application value and improve the effect of transformation efficiency.

Inactive Publication Date: 2007-07-11
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to factors such as difficult regeneration systems and low conversion rates due to differences in genetic backgrounds, they are still in the process of continuous improvement.
The use of physical means (such as ultrasonic wave, negative pressure method, pulse electrophoresis, heavy ion irradiation and gamma ray irradiation, etc.) also has major problems such as limited use of materials and low conversion rate.
Among them, the γ-ray irradiation similar to the present invention, because the irradiation material is a callus, the regeneration system, the irradiation period and pollution and other issues should be considered, and the actual operation is greatly restricted.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0007] Agrobacterium-mediated transient expression of the gus gene from irradiated seeds

[0008] 1.1 Materials and methods

[0009] 1.1.1 Materials

[0010] Species: take plump and intact seeds of cabbage, wheat, pumpkin, celery, coriander, corn, japonica rice, indica rice, rye, spinach, amaranth and cowpea; about 500 seeds of each are used for experiments.

[0011] Agrobacterium and plasmid: LBA4404; the plasmid carried is pKUC (Professor Shu Qingyao's laboratory of Zhejiang University); the antibiotic selection gene is kanamycin resistance gene and hygromycin resistance gene; the reporter gene is GUS gene (including containing subsequences); their promoters are all CaMV 35S promoters.

[0012] 1.1.2 Method

[0013] Seed irradiation: plant seeds were irradiated with gamma rays at a dose of 0.01-10Gy (dose rate 0.1-2Gy / h).

[0014] Shaking bacteria: Agrobacterium was cultured on a LB liquid medium (containing 50 mg / l kanamycin) at 25° C. on a shaker at 120 rpm. In the mi...

example 2

[0026] Transformation Performance of Exogenous Plasmid (gus Gene) in Irradiated Seeds

[0027] 2.1 Materials and methods

[0028] 2.1.1 Materials

[0029] Species: Take plump and intact seeds of wheat, tomato, cabbage and Chinese toon; about 500 seeds of each kind are used for experiments.

[0030] Agrobacterium and plasmid: LBA4404; the plasmid carried is pKUC (from the laboratory of Professor Shu Qingyao of Zhejiang University); the antibiotic selection gene is kanamycin resistance gene and hygromycin resistance gene; the reporter gene is GUS gene (including intron sequence); their promoters are all CaMV 35S promoters.

[0031] 2.1.2 Method

[0032] Seed irradiation: plant seeds were irradiated with gamma rays at a dose of 0.01-10Gy (dose rate 0.1-2Gy / h).

[0033] Shaking bacteria: Agrobacterium was cultured on a LB liquid medium (containing 50 mg / l kanamycin) at 25° C. on a shaker at 120 rpm. In the middle and late stages of exponential growth, the OD of the bacterial ...

example 3

[0046] Transient expression of potted seedlings-Kanamycin selection to obtain the performance of T0 and T1 generation plants

[0047] 3.1 Materials and methods

[0048] 3.1.1 Materials

[0049] Source of material: Take the seedlings of wheat, tomato, Chinese cabbage and Chinese toon in Example 2, stain with GUS, and select blue positive plants for transplanting.

[0050] 3.1.2 Method

[0051]Transplanting in pots: Positive plants were transplanted in pots containing fertile sandy soil. Every 3-5 days, drop 50-200 μl of kanamycin (concentration: 50-250 mg / L) on all shoot tips until the formation of flower buds.

[0052] Identification of plants by GUS staining: GUS-positive plants at the seedling stage were cut from the top young stems at the flowering stage, a part was used for GUS staining, and the other part was used for DNA extraction for PCR identification; the seeds harvested from the plants were germinated in sterile water (T1 generation) , A root tip of a plant was ...

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PUM

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Abstract

The invention discloses a switching technique of multiple-typed plant, which is characterized by the following: switching plant seed irradiated by gamma-ray (dose: 0.01-10Gy; dose rate: 0.1-2Gy / h); germinating in the selective gene; testing; sieving instantaneous expressive seed of GUS gene; adopting positive seed as explant; obtaining regenerative plant on the culture medium; planting positive seed in the flowerpot or field directly for 3-5d; dripping 50-200 mul relative antibiotics with density at 50-250mg / l on the stem top until the flower bud forms; collecting seed; identifying the gene; planting positive material.

Description

technical field [0001] The invention patent belongs to the field of biotechnology. Background technique [0002] Exogenous DNA enters the plant genome, and currently there are mainly technologies such as Agrobacterium-mediated callus and gene gun method. However, due to factors such as difficult regeneration systems and low conversion rates due to differences in genetic backgrounds, they are still in the process of continuous improvement. The use of physical means (such as ultrasonic wave, negative pressure method, pulse electrophoresis, heavy ion irradiation and γ-ray irradiation, etc.) also has major problems such as limited materials and low conversion rate. Wherein the gamma ray irradiation similar to the present invention, because the irradiation material is callus, problems such as regeneration system, irradiation period and pollution have to be taken into consideration, and the actual operation is greatly restricted. Therefore, in the face of the transformation tech...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00
Inventor 李兴林舒庆尧路福平夏英武吴殿星高年发张健崔海瑞傅俊杰
Owner TIANJIN UNIV OF SCI & TECH
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