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Efficient genetic transformation and rapid identification method for cabbage type rapes

A genetic transformation method and technology of Brassica napus, applied in the field of high-efficiency genetic transformation and rapid identification of Brassica napus, can solve the problems of high-efficiency and rapid acquisition of transgenic seedlings, avoid adverse effects, ensure probability, and increase universality Effect

Active Publication Date: 2018-08-28
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

None of these methods can solve the problem of obtaining transgenic seedlings efficiently and quickly.

Method used

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  • Efficient genetic transformation and rapid identification method for cabbage type rapes
  • Efficient genetic transformation and rapid identification method for cabbage type rapes
  • Efficient genetic transformation and rapid identification method for cabbage type rapes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Tissue culture rapid seedling raising method of semi-winter line J2016 of Brassica napus:

[0039] (1) Vector construction: the DsRed gene was inserted into the vector pLH-1390-RNAi containing the target gene LPAT2, and the obtained target vector with DsRed was transformed into Agrobacterium strain GV3101, and stored at -80°C.

[0040] (2) Cultivation of sterile explants: On the first day, select about 20 plump Brassica napus seeds, put them in a 2mL centrifuge tube, add 1mL sterile water to clean the seeds on the ultra-clean workbench for 1-2 Then, discard the suspension; then add 1 mL of 75% alcohol to sterilize for 1 min, discard the suspension; then quickly add 1 mL of 50% 84 disinfectant to sterilize for 3 min, shake the seeds continuously during this period, and discard the suspension; Rinse with bacterial water for 3-5 times, transfer the seeds to filter paper and wait for the surface moisture to dry, then sow in the seed germination medium, and culture in dark f...

Embodiment 2

[0057] Tissue culture rapid seedling raising method of semi-winter line J2016 of Brassica napus:

[0058] (1) Vector construction: the DsRed gene was inserted into the vector pCRISPR-Cas9 containing the target gene LPAT5, and the obtained target vector with DsRed was transformed into Agrobacterium strain GV3101, and stored at -80°C.

[0059] (2) Cultivation of sterile explants: On the first day, select about 20 plump Brassica napus seeds, put them in a 2mL centrifuge tube, add 1mL sterile water to clean the seeds on the ultra-clean workbench for 1-2 Then, discard the suspension; then add 1 mL of 75% alcohol to sterilize for 1 min, discard the suspension; then quickly add 1 mL of 50% 84 disinfectant to sterilize for 3 min, shake the seeds continuously during this period, and discard the suspension; Rinse with bacterial water for 3-5 times, transfer the seeds to filter paper and wait for the surface moisture to dry, then sow in the seed germination medium, and culture in dark fo...

Embodiment 3

[0076] Tissue culture rapid seedling raising method of Brassica napus spring line J9707 (Yang et al, Plant Biotechnology Journal (2018), pp.1–14):

[0077] (1) Vector construction: the DsRed gene was inserted into the vector pcambia1303 containing the target gene SOD7, and the obtained target vector carrying DsRed was transformed into Agrobacterium strain GV3101, and stored at -80°C.

[0078] (2) Cultivation of sterile explants: On the first day, select about 20 plump Brassica napus seeds, put them in a 2mL centrifuge tube, add 1mL sterile water to clean the seeds on the ultra-clean workbench for 1-2 Then, discard the suspension; then add 1 mL of 75% alcohol to sterilize for 1 min, discard the suspension; then quickly add 1 mL of 50% 84 disinfectant to sterilize for 3 min, shake the seeds continuously during this period, and discard the suspension; Rinse with bacterial water for 3-5 times, transfer the seeds to filter paper and wait for the surface moisture to dry, then sow in...

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Abstract

The invention discloses an efficient genetic transformation and rapid identification method for cabbage type rapes. The genetic transformation method for cabbage type rapes comprises the following steps: (1) cultivation of sterile explant; (2) preparation of a target carrier; (3) preparation of agrobacterium conversion liquid; (4) co-cultivation of agrobacterium and hypocotyledonary axis; (5) induction of explant dedifferentiation to form callus; (6) induction of callus dedifferentiation to form buds; (7) transgenic positive selecting; (8) rooting culture; and (9) plantlet hardening-off and transplanting. Integral process settings of the genetic transformation method, parameter conditions of key steps, and the like are improved and are further preferably selected, so that cabbage type rapetransgenic seedlings can be efficiently and quickly obtained through effective selecting means in comparison with the prior art. A DsRed gene is inserted into a carrier containing a target gene, so that the target carrier with DsRed is transferred into agrobacterium strain, and therefore, transgenic positive screening can be quickly and efficiently performed by observing color of the hypocotyledonary axis.

Description

technical field [0001] The invention belongs to the technical field of plant tissue culture, and more specifically relates to a high-efficiency genetic transformation and rapid identification method of Brassica napus. The method can rapidly propagate and efficiently obtain transgenic rapeseed through plant tissue culture technology. Background technique [0002] Brassica napus (AACC, 2n=38) is one of the oil crops with strong adaptability, wide application, high economic value and great development potential. my country is a major producer of rapeseed in the world, accounting for about 26% of the world's rapeseed area, and its total rapeseed output accounts for about 28% of the world's total rapeseed output. The winter rapeseed area in the Yangtze River Basin is the main production area of ​​rapeseed in my country, accounting for about 85% of the national rapeseed area. In recent years, the yield and quality of rape has reached the international leading level. Nevertheless...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H4/00A01H6/20
CPCA01H4/00C12N15/8205
Inventor 栗茂腾张凯付春华尹永泰李怀鑫聂丽逻程琦琪亓福玉邹大山
Owner HUAZHONG UNIV OF SCI & TECH
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