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Method for breeding root-knot nematodes by using transgenic adventitious roots

A root-knot nematode and transgenic technology, applied in the directions of botanical equipment and methods, application, and introduction of foreign genetic material using vectors, to overcome the influence of biological factors in the soil environment, facilitate long-term preservation, and be easy to identify.

Inactive Publication Date: 2012-05-30
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the use of transgenic adventitious roots to propagate root-knot nematodes

Method used

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  • Method for breeding root-knot nematodes by using transgenic adventitious roots
  • Method for breeding root-knot nematodes by using transgenic adventitious roots
  • Method for breeding root-knot nematodes by using transgenic adventitious roots

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the acquisition of recombinant Agrobacterium rhizogenes K599

[0029] 1. Materials

[0030] The wild-type Agrobacterium rhizogenes K599, the plasmids pBIN19 and pGFP were all preserved by the Key Laboratory of Botany, Hangzhou Normal University; the plasmid pCHS was donated by Dr. Tao Jianying, Institute of Biological and Agricultural Sciences, Academia Sinica, Taiwan. Plasmid mini-extraction kit and DNA gel recovery kit were purchased from Shanghai Sangon Company. Taq DNA polymerase, dNTP, restriction endonuclease and T4 DNA ligase were purchased from Promega, USA. Antibiotics and acetosyringone were purchased from Beijing Biotech Co., Ltd.

[0031] 2. Method

[0032] (1) Construction of plant transgene expression vector containing gfp gene

[0033]Plasmid DNA mini-extraction, enzyme digestion, and fragment recovery were all carried out according to the product instructions; the screening and identification of ligated and transformed clones were carri...

Embodiment 2

[0039] Embodiment 2: the acquisition of transgenic adventitious roots and the propagation of root-knot nematodes

[0040] 1 Materials and methods

[0041] 1.1 Experimental materials

[0042] The seeds of cucumber (Cucumis sativus) Zhongnong No. 6 and the recombinant Agrobacterium rhizogenes K599 carrying the plasmid pBIN-35S-GFP (obtained according to the method in Example 1) were all preserved by the Key Laboratory of Botany, Hangzhou Normal University.

[0043] 1.2 Induction, identification and propagation of transgenic adventitious roots

[0044] Cucumber seeds were washed with sterile water for 3 times, soaked in 75% alcohol for 30s, rinsed with sterile water for 3 times, and then treated with 20% commercially available antiformin (sodium hypochlorite) solution for 20 minutes, during which every 5 minutes Stir once, and finally rinse with sterile water for 3 times, inoculate on 1 / 2 MS medium (MS medium with halved macroelement concentration), temperature 24±2°C, and cult...

Embodiment 3

[0059] Embodiment 3: the acquisition of transgenic adventitious roots and the propagation of root-knot nematodes

[0060]The wild-type Agrobacterium rhizogenes K599 was preserved by the Key Laboratory of Botany, Hangzhou Normal University. After wild-type Agrobacterium rhizogenes K599 was activated by streaking on LB+Str 50mg / L plate at 28°C, single clones were picked and cultured in LB+Str 50mg / L liquid medium at 28°C and 200r / min for 24h. Take 1 mL of the bacterial liquid and transfer it into 50 mL of LB+Str 50 mg / L liquid medium at 28°C and 200 r / min to incubate until the A600 is 0.5, collect the bacterial liquid in a centrifuge tube and suspend and wash it with MS liquid medium for 3 times. Then infect the cucumber cotyledons with wounds for 10 minutes, absorb the bacterial solution on the cotyledons with sterile filter paper, and then transfer to MS+As (acetosyringone) 25 mg / L solid medium for co-cultivation for 2-3 days, and then pass MS +Cef (cephalosporin) 500mg / L liq...

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Abstract

The invention provides a method for breeding root-knot nematodes by using transgenic adventitious roots. The method comprises the following steps of: firstly, infecting cucumber cotyledon with agrobacterium rhizogene K599; and inducing the adventitious roots by high frequency, wherein rooting rate of the excised cotyledon (transgenic frequency) reaches 90 percent, and the adventitious roots induced can emit strong green fluorescence under a fluorescence microscope. The method breeds the southern root-knot nematodes by using the cucumber adventitious roots induced, successfully forms root knots, fulfills the aims of breeding the root-knot nematodes, can provide a great amount of uniform root-knot nematode experimental materials for researching protection of the root-knot nematodes, and overcomes the influences of season limit and biotic factors of soil environment of the seedling-bred root-knot nematodes. Moreover, the method is favorable for preserving the root-knot nematodes for a long time, and allows the root-knot nematodes to be used whenever needed. The method has the advantages of easy identification of various kinds of root-knot nematodes, and difficult confusion during operation and provides a new way for fast and efficiently breeding and separating the root-knot nematodes.

Description

(1) Technical field [0001] The invention relates to a method for propagating root-knot nematodes by using transgenic adventitious roots. (2) Background technology [0002] Root-knot nematodes mainly include southern root-knot nematode (Meloidogyne incognite), Javan root-knot nematode (M.javanica), peanut root-knot nematode (M.arenaria) and northern root-knot nematode (M.hapla). A devastating pest of Cucurbitaceae, Solanaceae, and Leguminous crops. The harm of root-knot nematodes is mainly manifested in the formation of root knots, causing root development to be blocked and rotted, and the aboveground parts of plants are weak and dead; at the same time, the harm of root-knot nematodes has aggravated soil-borne fungal diseases such as wilt and root rot and some The occurrence of bacterial diseases. According to the estimates of the Food and Agriculture Organization of the United Nations, nematodes cause losses of more than 20% of the output of vegetable crops every year. In...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/06A01K67/033
Inventor 向太和孟莎莎王琳
Owner HANGZHOU NORMAL UNIVERSITY