Construction and optimization of simple and efficient carya illinoinensis agrobacterium transformation system

A thin-shell pecan and Agrobacterium rhizogenes technology, applied in agriculture, gardening tools/equipment, horticulture, etc., can solve the problems of limited application space of injection method, long cycle of transgenic plants, long cycle of transformation system, etc., and achieve easy production The promotion of practice, broad development and application prospects, and the effect of short cycle

Active Publication Date: 2021-07-23
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the different Agrobacterium infection operations, the existing Agrobacterium-mediated plant transgenic methods can be divided into bacterial liquid infection method and syringe needle insertion method, and the transformation rate is generally low. For woody plants, the stem Hardness and lignification further limit the application space of the injection method
[0004] Conventional Agrobacterium-mediated transgenes mainly

Method used

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  • Construction and optimization of simple and efficient carya illinoinensis agrobacterium transformation system
  • Construction and optimization of simple and efficient carya illinoinensis agrobacterium transformation system
  • Construction and optimization of simple and efficient carya illinoinensis agrobacterium transformation system

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0039] Example 1: Thin Shell Mountain Walnut Seedlings in Differences

[0040] (1) Collection of seeds, harvest the mature seeds of thin shell mountain walnuts;

[0041] (2) Germination of seeds

[0042] Seed seed species in the matrix (70-80% grass carbonilo, 5-10% perlite, 2-5% vermiculite, 5-10% 沙 土, add appropriate amount of water and mix, and use organic compulsory microbial agents for 10 days In the basin of the decomposing process, the germination process of two months in the gensented chamber (26 ± 2 ° C) was carried out, and the shell mountain walnut seedlings were obtained in different periods.

Example Embodiment

[0043] Example 2: Preparation of bacteria

[0044] (1) Firstly, the expression vector with 35s :: GFP will be transferred to the K599 root apex strain, which is used at -80 ° C with 50% glycerol storage.

[0045] (2) The above-mentioned Agrobacterium with 35s :: GFP is overlined in TY solid medium, and inverted in 28 ° C for 2-3 days.

[0046] (3) When OD 600 When = 0.5, the cultured bacteria was centrifuged at room temperature, and the centrifugal speed was 6500 rpm, 10 min enriched Agrobacterium.

[0047] The rotation speed of the centrifugation is preferably from 4,000 to 8000 rpm, more preferably 6500 rpm. In the present invention, the centrifugation is preferably 5-15 min, more preferably 10 min.

[0048] (4) Abandon the supernatant, recover the enriching bacteria (10mm Mes-KOH, PH = 5.2, 10mm MgCl 2 100μm AS).

[0049] (5) Stressing bacteria measuring OD 600 = 0.5.

Example Embodiment

[0050] Example 3: Induction of Human Roots

[0051] like figure 1 (a) Single-sided blade with a petrococcus solution is sloppy 45 ° in the thickness of the seeds of the shell mountain walnuts, 1-5 cm, is sloped at 1-5 cm, and the horizontal is looped, and each knife is pulled. Soak the single-sided knife in the bacterial liquid to ensure that the wound is infected by the bacterial liquid.

[0052] figure 1 (b) Injection of the hazelnut bacterium liquid in the thin shells or stem of the stem or stem of the stem of the stem of the seedling pecker, and 3 parts per seed seedlings were injected.

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Abstract

The invention discloses construction and optimization of a simple and efficient carya illinoinensis agrobacterium tumefaciens transformation system. The method comprises the following steps: carrying out streak culture on agrobacterium rhizogenes on a TY solid culture medium to obtain single colonies, selecting the single colonies, and culturing the single colonies in a TY liquid culture medium to obtain an activated agrobacterium rhizogenes solution; infecting hypocotyl or stems, close to seed parts, of carya illinoensis seedlingswith agrobacterium rhizogenes, wherein the positions of hypocotyls or stems, close to the seed parts, of the carya illinoensis seedlings are 1-5 cm away from the hypocotyls or stems of the roots of the seedlings; and carrying out co-culture and rooting culture until transgenic hairy roots of the carya illinoensis grow at the positions where hypocotyls or stems are infected. The seedlings are used as propagation materials for infection, and a series of tissue culture technologies such as culture of aseptic seedlings in an aseptic environment for subculture operation are not needed.

Description

technical field [0001] The invention belongs to the technical field of pecan carya transgenic system and relates to the construction and optimization of a simple and efficient transformation system of pecan agrobacterium. Background technique [0002] At present, the transformation methods of transgenic plants mainly include Agrobacterium transformation method, pollen tube passage method, nuclear microinjection method, gene gun method, etc. The Agrobacterium-mediated genetic transformation system is one of the relatively complete and mature methods in plant genetic engineering. one. Agrobacterium rhizogenes is a highly infective Gram-negative bacterium of the genus Agrobacterium in the Rhizobium family. After infecting plants, it can induce plants to produce a large number of highly branched adventitious roots, commonly known as hairy roots. It has a wide range of applications in plant genetic engineering. The Ri plasmid it carries can effectively infect many plants. The T-...

Claims

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Application Information

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IPC IPC(8): A01G17/00A01G7/06
CPCA01G17/005A01G7/06
Inventor 王晓飞谢晓婷黄巧宇温广超郑炳松黄坚钦袁虎威何漪闫道良
Owner ZHEJIANG FORESTRY UNIVERSITY
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