A method for establishing an Agrobacterium-mediated short-branch casuarina callus transgenic system

An Agrobacterium-mediated technology for establishing methods, which is applied in the field of establishment of short-branched casuarina callus transgenic systems, can solve problems such as inapplicability of short-branched casuarinas, no display of transgenic plant data, etc., so as to improve the transformation rate and shorten the the effect of time

Active Publication Date: 2022-06-21
ZHEJIANG FORESTRY UNIVERSITY
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Problems solved by technology

In 2012, Lin Yongsheng et al. (Lin Yongsheng, Jiang Jing, Qiao Guirong, et al. Casuarina twigs tissue culture and transgenic research (English) [J]. Agricultural Science&Technology, 2012,40(1):1313-1314.) on twigs Callus culture and transgenic systems of casuarina have been studied, but no data have been shown to obtain complete transgenic plants
However, the existing transgenic systems are limited to whorl casuarina, casuarina twigs, and casuarina twigs, and the methods are not applicable to casuarina twigs

Method used

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  • A method for establishing an Agrobacterium-mediated short-branch casuarina callus transgenic system

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Embodiment 1

[0037] A method for establishing an Agrobacterium-mediated callus transgenic system of Casuarina chinensis, the specific steps are as follows

[0038] (1) get short branch casuarina branch (the young branch more than 1cm of casuarina seedling root that grows in artificial greenhouse for 30-90 days is explant material) as explant, after cleaning and sterilization, will cut off Casuarina japonica branches are diagonally cut into 0.8-1cm long segments (the cut surface is as large as possible), placed on a callus induction medium to induce callus, and callus is obtained, and the callus induction rate can reach about 95%;

[0039] The callus induction medium is: 0.1mg / L NAA+0.1mg / L TDZ+1 / 2MS+30g / L sucrose+3.5g / L phytogel, pH 5.7±0.1;

[0040] (2) Select the callus with good growth status (light yellow and translucent) and put it into the Agrobacterium GV3101 infection solution with OD600=0.6-0.8 for infection culture; the conditions for infection culture are: 28°C shaking table inf...

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Abstract

The invention discloses a method for establishing an Agrobacterium-mediated short-branch casuarina callus transgenic system. The method comprises: taking short-branch casuarina shoots as explants, inducing callus to obtain callus tissue; selecting callus tissue Put it into the Agrobacterium infection solution for infection culture; co-cultivate the infected callus, and then carry out screening culture; use the light yellow, plump and obviously enlarged resistant calli to screen for adventitious buds Cultivation and rooting culture; finally, judge whether the regenerated seedlings of the casuarina short-branches are transgenic positive plants by GUS staining method, and obtain the transgenic plants of the casuarina short-branches. The present invention adopts specific callus induction medium, co-cultivation medium, resistant callus selection medium and adventitious bud selection medium to construct the Agrobacterium-mediated short-branched Casuarina callus transgenic system, which can not only obtain short The transgenic plants of casuarina twigs can greatly shorten the time of the transformation system and increase the transformation rate.

Description

technical field [0001] The invention relates to the technical field of transgenic trees, in particular to a method for establishing an Agrobacterium-mediated callus transgenic system of Casuarina chinensis. Background technique [0002] Casuarina (Casuarina) is the collective name of the genus Casuarina. It is native to some tropical islands in Australia and the Pacific Ocean. Casuarina can grow in extremely salty soil and is known as the "pioneer of the world's plants". [0003] Casuarina japonica is tall and straight, with soft and drooping twigs, rapid growth, strong wind resistance, salt and alkali resistance, drought resistance and barrenness, not afraid of sea tide impregnation, and less pests and diseases. Since the 1950s, my country's coastal areas such as Taiwan, Guangdong, Hainan, Guangxi, and southern Fujian have been introduced and cultivated, mainly three species of Casuarina chinensis, Casuarina vulgaris, Casuarina vulgaris and some hybrids. At present, casuar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84A01H4/00A01H5/00A01H6/00
CPCC12N15/8205A01H4/008A01H4/001
Inventor 任慧敏许妍赵晓红崔富强柳参奎亓果宁
Owner ZHEJIANG FORESTRY UNIVERSITY
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