Method for establishing high-efficiency regeneration system by using broccoli microspore embryo as explant

A high-efficiency regeneration and microspore technology, applied in the field of plant tissue culture, can solve the problems of low genetic transformation rate, small number of regenerated buds, and low proliferation ability, and achieve the goal of increasing genetic transformation rate, reducing browning, and promoting bud induction rate Effect

Active Publication Date: 2013-04-24
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a kind of explant regeneration for traditional cauliflower regeneration bud induction with its hypocotyl and cotyledons as explants, the existing defects of few regeneration buds, low proliferation ability, low genetic transformation rate, etc. A method for high-frequency plant regeneration of cauliflower with a large number of buds, strong multiplication ability, good genetic background consistency, and high-efficiency genetic transformation potential

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: (cultivation method 1 for broccoli microspore embryo high-frequency bud regeneration)

[0026] The method proceeds as follows:

[0027] (1) Preparation of culture medium: including the culture medium of each stage of explant bud regeneration, their components and the weight of each component in each liter of medium are:

[0028] 1) Callus induction medium: MS basal medium + 2,4-D 1.0 mg / L + ZT 0.8 mg / L + AgNO 3 4.0 mg / L + 30g / L sucrose + 9g / L agar, pH 5.8, high temperature sterilization;

[0029] 2) Callus differentiation medium: MS basal medium + ZT 0.5 mg / L + BAP 2.0 mg / L + NAA 0.05mg / L + AgNO 3 4.0 mg / L + 30 g / L sucrose + 9 g / L agar, pH 5.8, autoclaved;

[0030] 3) Callus redifferentiation medium: MS basal medium + AgNO 3 4.0 mg / L + 30 g / L sucrose + 10 g / L agar, pH 5.8, autoclaved;

[0031] 4) Rooting medium: 1 / 2 MS basal medium + NAA 1 mg / L + 30 g / L sucrose + 9 g / L agar, pH 5.8, high temperature sterilization;

[0032] (2) Cultivation of...

Embodiment 2

[0039] Example 2: (cultivation method 2 for broccoli microspore embryo high-frequency bud regeneration)

[0040] In this example, step (1) medium preparation: wherein, 1) 2,4-D in the callus induction medium is 1.5 mg / L, ZT is 1.0 mg / L, AgNO 3 4.5 mg / L; 2) ZT in callus differentiation medium was 0.8 mg / L, BAP was 1.5 mg / L, AgNO 3 4.5 mg / L; 3) The agar in the callus redifferentiation medium was 10.5 g / L; Step (2) Cultivation of broccoli microspore embryo regenerated plants: 1) The selected microspores were cultured in dark at 25°C After 35 days, 180 well-grown cotyledon embryos were taken; 2) Cotyledon embryos were cultured for 1 week at 27°C with 16 hours of light per day; 3) Calli were cultured for 2-3 weeks at 28°C with 16 hours of light per day until they appeared Visible bud point; All the other step techniques are the same as in Example 1.

Embodiment 3

[0041] Example 3: (cultivation method 3 for broccoli microspore embryo high-frequency bud regeneration)

[0042] In this example, step (1) medium preparation: 1) 2,4-D in the callus induction medium is 2.0 mg / L, ZT is 0.5 mg / L, AgNO 3 5.0 mg / L; 2) ZT in callus differentiation medium was 1.0 mg / L, BAP 1.0 mg / L, AgNO 3 5.0 mg / L; 3) The agar in the callus redifferentiation medium was 11 g / L; Step (2) Cultivation of broccoli microspore embryo regenerated plants: 1) The selected microspores were cultured in the dark at 28°C for 30 Take 180 well-grown cotyledon embryos after 1 day; 2) Culture the cotyledon embryos at 28°C with 16 hours of light per day for 1 week; 3) Culture the callus at 27°C with 16 hours of light per day for 2-3 weeks until visible Bud point; All the other step techniques are the same as in Example 1.

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Abstract

The invention discloses a method for establishing a high-efficiency regeneration system by using broccoli microspore embryo as explant, and belongs to the technical field of plant tissue culture. The method comprises two main steps of preparing a culture medium and culturing a broccoli microspore embryo plantlet. According to the method, the microspore embryo is used as the explant, the bud inductivity is as high as 100%, the bud number of each explant is 30-50, after the culture medium is added with 0.5-1.0mg/L of zeatin, the bud inductivity and the induction number are respectively increased by 1.1 times and 3.2 times; and after 4.0-5.0mg/L of LAgNO3 is added, the browning rate is reduced by about 10%. By utilizing the method, the efficiency in broccoli tissue culture is greatly improved, and a new technical way for improving the heredity conversion rate is provided. The method can be popularized and applied in broccoli tissue culture and gene engineering heredity improvement.

Description

technical field [0001] The invention relates to the technical field of plant tissue culture, in particular to a method for establishing a high-efficiency regeneration system of cauliflower using microspore embryos as explants. Background technique [0002] brocoli( Brassica oleracea var. botrytis ) belongs to the Brassica genus of Brassicaceae. Its curds are rich in nutrients. In addition to protein, cellulose and various minerals, it also contains a variety of indole derivatives, which have significant anti-cancer and anti-cancer effects. More and more The more it is favored by consumers, the cultivated area has expanded rapidly in recent years, and it has become one of the main vegetable crops in my country. However, during the growth period of cauliflower, serious diseases and insect pests, intolerance to storage, poor quality and other problems have always plagued the majority of vegetable farmers and merchants. Therefore, it is imperative to improve cauliflower va...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
Inventor 盛小光顾宏辉赵振卿虞慧芳王建升许映君
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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