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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
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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 use Agrobacterium tumefaciens, but this method of constructing transgenic plants still requires a lot of time for callus induction, differentiation, and subculture, and the cycle for obtaining transgenic plants is long and cumbersome. low efficiency
[0005] At present, there are few reports on the transgenic system of pecan walnut, and the transformation system mediated by Agrobacterium tumefaciens has a long cycle and low efficiency.

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

Embodiment 1

[0039] Embodiment 1: the thin shell hickory seedling of different periods

[0040] (1) the collection of seed, harvests the mature seed of pecan;

[0041] (2) Germination of seeds

[0042] Seeds are planted in substrate (70-80% peat soil, 5-10% perlite, 2-5% vermiculite, 5-10% sandy loam, add appropriate amount of water and mix, and select organic decomposed microbial bacteria agent for 10 days) decomposing treatment) pots and in the germination chamber (26 ± 2 ℃), the germination process was carried out for two months, to obtain different periods of pecan seedlings.

Embodiment 2

[0043] Embodiment 2: Bacterial liquid preparation

[0044] (1) First, the expression vector carrying 35S::GFP was transformed into the K599 Agrobacterium rhizogenes strain, and the bacterial solution was preserved at -80°C with 50% glycerol.

[0045] (2) The above-mentioned Agrobacterium rhizogenes carrying 35S::GFP were streaked on TY solid medium, and cultured upside down at 28° C. for 2-3 days.

[0046] (3) When OD 600 When = 0.5, the cultured bacterial solution was centrifuged at room temperature at 6500 rpm for 10 min to enrich Agrobacterium rhizogenes.

[0047] The rotational speed of the centrifuge is preferably 4000-8000rpm, more preferably 6500rpm. In the present invention, the centrifugation time is preferably 5-15 minutes, more preferably 10 minutes.

[0048] (4) Discard the supernatant, resuspend the enriched bacteria in MES-KOH (10mM MES-KOH, PH=5.2, 10mM MgCl 2 , 100 μM As).

[0049] (5) Determination of OD of the bacterial solution after resuspension 600 =...

Embodiment 3

[0050] Example 3: Induction of Hairy Roots

[0051] Such as figure 1 (a) Gently tilt the single-sided blade dipped in the Agrobacterium rhizogenes solution at 1-5 cm from the hypocotyl or stem of the pecan seed at a horizontal angle of 45°, and cut three cutting edges horizontally, one cut at a time Soak the single-sided knife in the bacterial solution to ensure that the wound is infected by the bacterial solution.

[0052] figure 1 (b) Inject the Agrobacterium rhizogenes bacterium liquid into the hypocotyl or stem portion 1-5cm of the pecan seed near the seed with a 1ml syringe, and inject 3 positions laterally for each plant seedling.

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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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