Method for establishing agrobacterium tumefaciens-mediated casuarina equisetifolia callus transgenic system

A technique of Agrobacterium-mediated and establishment method, which is applied in the field of establishment of short-branched casuarina callus transgenic system, can solve problems such as inapplicability of short-branched casuarinas, no display of transgenic plant data, etc., to improve transformation rate, shorten the effect of time

Active Publication Date: 2021-08-17
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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  • Method for establishing agrobacterium tumefaciens-mediated casuarina equisetifolia callus transgenic system

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Embodiment 1

[0037] A method for establishing an Agrobacterium-mediated short-branch casuarina callus transgenic system, the specific steps are as follows

[0038] (1) Get short casuarina shoots (the young shoots more than 1 cm in the root of casuarina seedlings grown in an artificial greenhouse for 30-90 days are explant materials) as explants, after cleaning and disinfection, cut the Casuarina twigs are cut into 0.8-1cm long subsections (cutting surface is as large as possible), placed on the callus induction medium to induce callus, to obtain callus, 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 state (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 shaker infection 2h; the preparation method ...

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Abstract

The invention discloses a method for establishing an agrobacterium tumefaciens-mediated casuarina equisetifolia callus transgenic system, which comprises the following steps: taking a casuarina equisetifolia branch as an explant, and inducing a callus to obtain a callus; selecting and putting the callus into agrobacterium infection liquid for infection culture; carrying out co-culture on the infected calluses, and then carrying out screening culture; performing adventitious bud screening culture and rooting culture on the faint yellow, plump and obviously expanded resistant calluses; and finally, judging whether the Casuarina equisetifolia regenerated seedling is a transgenic positive plant or not through a GUS staining method to obtain a Casuarina equisetifolia transgenic plant. The specific callus induction culture medium, the co-culture culture medium, the resistant callus screening culture medium and the adventitious bud screening culture medium are adopted to construct the agrobacterium tumefaciens-mediated casuarina equisetifolia callus transgenic system, so that not only can the transgenic plant of the casuarina equisetifolia be obtained, but also the time of transforming the system can be greatly shortened; and the conversion rate is improved.

Description

technical field [0001] The invention relates to the technical field of tree transgenics, in particular to a method for establishing an Agrobacterium-mediated short-branch casuarina callus transgenic system. Background technique [0002] Casuarina (Casuarina) is the collective name for species of Casuarina genus. It is native to Australia and some tropical islands in the Pacific Ocean. Casuarina can grow in extremely salinized soil, and is known as "the pioneer of plants in the world". [0003] Casuarina has a tall and straight stem shape, soft and drooping branchlets, rapid growth, strong wind resistance, salt and alkali resistance, drought and barren resistance, not afraid of sea tide soaking, and few pests and diseases. Since the 1950s, the coastal areas of Taiwan, Guangdong, Hainan, Guangxi and southern Fujian have begun to introduce and cultivate, mainly three species of Casuarina twigs, Casuarina common, Casuarina twigs and some hybrids. At present, casuarina has becom...

Claims

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Application Information

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Patent Type & Authority Applications(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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