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Efficient broccoli genetic transformation method taking pedicel as explant

A genetic transformation method and explant technology are applied in the field of high-efficiency genetic transformation of cauliflower with pedicels as explants, and can solve the problems of shortening the time required for bud regeneration, cauliflower genetic transformation, etc. The effect of stable results and short regeneration and budding time

Active Publication Date: 2022-08-02
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a kind of cauliflower high-efficiency genetic transformation method using pedicel as explant, which solves the problem of genetic transformation of cauliflower, and using pedicel as explant, does not need callus induction, can directly regenerate and sprout, greatly The time required for bud regeneration is shortened, and the positive rate of genetically transformed plants can reach 10-15%.

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  • Efficient broccoli genetic transformation method taking pedicel as explant
  • Efficient broccoli genetic transformation method taking pedicel as explant
  • Efficient broccoli genetic transformation method taking pedicel as explant

Examples

Experimental program
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Effect test

Embodiment 1

[0029] 1. Preparation of culture medium

[0030] Including the medium of each stage of genetic transformation, their components and contents are as follows:

[0031](1) Explant pre-medium: MS medium+sucrose 30g / L+agar 9g / L+6-BA 2 mg / L+NAA 0.02mg / L+Trans-ZT 0.5mg / L (trans-zeatin, CAS No.: 1637-39-4, purchased from McLean), pH=5.8, autoclaved at 121°C for 20min;

[0032] (2) Explant co-culture medium: MS medium+sucrose 30g / L+agar 8g / L+6-BA 2 mg / L+NAA 0.02mg / L+Trans-ZT 0.5mg / L, pH=5.8,121 ℃ Autoclave for 20min;

[0033] (3) Explant delay medium: MS medium+sucrose 30g / L+agar 9g / L+6-BA 2 mg / L+NAA0.02mg / L+Trans-ZT 0.5mg / L+300mg / L Temei Ting, pH=5.8, autoclaved at 121°C for 20min (wherein, the antibiotic Timentin was added when the medium was cooled to 60°C after high-temperature sterilization);

[0034] (4) Explant screening medium: MS medium+sucrose 30g / L+agar 9g / L+6-BA 2 mg / L+NAA 0.02mg / L+Trans-ZT 0.5mg / L+300mg / L Temei Ting+5mg / L hygromycin, pH=5.8, autoclaved at 121°C for 20...

Embodiment 2

[0128] 1. Preparation of culture medium

[0129] Including the medium of each stage of genetic transformation, their components and contents are as follows:

[0130] (1) Explant pre-medium: MS medium+sucrose 30g / L+agar 9g / L+6-BA 2 mg / L+NAA 0.02mg / L+Trans-ZT 0.4mg / L, pH=5.9, 121 ℃ Autoclave for 20min;

[0131] (2) Explant co-culture medium: MS medium+sucrose 30g / L+agar 8g / L+6-BA 2 mg / L+NAA 0.02mg / L+Trans-ZT 0.4mg / L, pH=5.9,121 ℃ Autoclave for 20min;

[0132] (3) Explant delay medium: MS medium+sucrose 30g / L+agar 9g / L+6-BA 1.5 mg / L+NAA0.02mg / L+Trans-ZT 0.4mg / L+300mg / L Temei Ting, pH=5.9, autoclaved at 121°C for 20min (wherein, the antibiotic Timentin was added after the medium was sterilized at high temperature and cooled to 60°C);

[0133] (4) Explant screening medium: MS medium+sucrose 30g / L+agar 9g / L+6-BA 2 mg / L+NAA 0.02mg / L+Trans-ZT 0.4mg / L+300mg / L Temei Ting+5mg / L hygromycin, pH=5.9, autoclaved at 121°C for 20min (among them, antibiotics Timentin and hygromycin were ad...

Embodiment 3

[0141] 1. Preparation of culture medium

[0142] Including the medium of each stage of genetic transformation, their components and contents are as follows:

[0143] (1) Explant pre-medium: MS medium+sucrose 30g / L+agar 9g / L+6-BA 2 mg / L+NAA 0.02mg / L+Trans-ZT 0.4mg / L, pH=5.9,121 ℃ Autoclave for 20min;

[0144] (2) Explant co-culture medium: MS medium+sucrose 30g / L+agar 8g / L+6-BA 2 mg / L+NAA 0.02mg / L+Trans-ZT 0.4mg / L, pH=5.9,121 ℃ Autoclave for 20min;

[0145] (3) Explant delay medium: MS medium+sucrose 30g / L+agar 9g / L+6-BA 2 mg / L+NAA0.02mg / L+Trans-ZT 0.4mg / L+300mg / L Temei Ting, pH=5.9, autoclaved at 121°C for 20min (the antibiotic Timentin was added after the medium was sterilized at high temperature and cooled to 60°C);

[0146](4) Explant screening medium: MS medium+sucrose 30g / L+agar 9g / L+6-BA 2 mg / L+NAA0.02mg / L+Trans-ZT 0.4mg / L+300mg / L Temei Ting+5mg / L hygromycin, pH=5.9, autoclaved at 121°C for 20min (antibiotics Timentin and hygromycin were added when the medium was st...

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Abstract

The invention discloses an efficient broccoli genetic transformation method taking pedicels as explants, which comprises the following steps: (1) selection of pedicel explants: taking loose broccoli as donor plants, and selecting pedicels with the transverse diameter of 0.3-0.8 cm as explants; (2) sterilizing the explant; (3) pre-culturing the explant; (4) dip dyeing of explants; (5) co-culturing the explant and the bacterial liquid; (6) carrying out delayed culture on the explant; (7) screening and culturing the explants; (8) rooting culture of resistant buds; and (9) performing positive detection on the regenerated plant. According to the method, efficient genetic transformation with cauliflower pedicels as explants is established for the first time, the positive rate of genetic transformation plants reaches 10-15% in combination with PCR detection and GUS dyeing results, in the pedicel bud regeneration process, callus induction is not needed, direct regeneration and budding can be achieved, and the time needed by bud regeneration is greatly shortened.

Description

technical field [0001] The invention relates to a high-efficiency genetic transformation method for cauliflower, in particular to a high-efficiency genetic transformation method for cauliflower using pedicels as explants. Background technique [0002] Cauliflower (Brassica oleracea L. var. botrytis, 2n=2x=18) is an important characteristic vegetable in Brassica genus Brassica, which is produced by curd, and is widely grown and eaten around the world. However, cauliflower originated from the Mediterranean coast, and the collection and identification of cauliflower germplasm resources are very limited, resulting in the defects of cauliflower varieties independently bred in China in terms of the commerciality of curds, resistance to diseases and insect pests, and tolerance to high temperature or cold climates. Therefore, how to use genetic engineering to improve the existing germplasm rapidly and directionally has become one of the most important topics in the field of breeding...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00C12N15/84C12Q1/6895
CPCA01H4/008C12N15/8205C12Q1/6895C12Q1/686
Inventor 盛小光顾宏辉虞慧芳王建升沈钰森赵振卿
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES