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Inoculation method for infecting hibiscus cannabinus with multan cotton leaf curl virus

A technology of cotton leaf curl virus and Multan cotton, which is applied in the field of inoculation of kenaf infected by Multan cotton leaf curl virus, can solve problems such as impact, difficulty in application, and high price

Inactive Publication Date: 2016-01-06
PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that it needs to separate, purify, raise and maintain the Bemisia tabaci population, which is highly professional and time-consuming. The success of the inoculation is also significantly affected by environmental factors such as seasons. At the same time, Bemisia tabaci may spread the poison source Microorganisms such as various viruses including CLCuMuV are all transmitted to the inoculated plants indiscriminately
The disadvantage of this method is that it needs to purchase an expensive professional instrument gene gun, and it needs professional personnel to operate, the operation cost is expensive, and it is difficult to apply to the operation of a large number of samples

Method used

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  • Inoculation method for infecting hibiscus cannabinus with multan cotton leaf curl virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Construction of Invasive Clones of CLCuMuV and β Satellite Molecules

[0035] Infectious clones pBinPLUS-1.85A and pBinPLUS-2.0β of CLCuMuV (molecular characteristics of geminiviruses infecting okra in Guangdong and construction of infectious clones, master thesis, Gansu Agricultural University, 2010) were supported by Plant Protection Research of Guangdong Academy of Agricultural Sciences Institute of Vegetable Diseases Research Laboratory was established.

[0036]Genomic DNA of Multan cotton leaf curl virus Guangdong isolate (GenBank: FJ770370) was used as a template, and primers OPAL: 5'-GGTACCTCCCAGATATATGCGCCAT-3' (introducing the KpnⅠ site) and OPAR: 5'-GGTACCAATCCCCTTTATTTCAAG-3' (including KpnⅠ site) for PCR amplification to obtain 1 times the full-length DNA-A. After the sequence verification is correct, select the SalⅠ enzyme digestion site contained in the sequence itself and introduce KpnⅠ enzyme digestion to obtain 0.85A, insert the same enzyme digestion DN...

Embodiment 2

[0040] CLCuMuV and β-satellite molecular infective clones inoculated kenaf

[0041] (1) Experimental materials

[0042] The invasive clones pBinPLUS-1.85A and pBinPLUS-2.0β of CLCuMuV were constructed by the Vegetable Disease Research Laboratory of the Institute of Plant Protection, Guangdong Academy of Agricultural Sciences;

[0043] Infectious clones of CLCuMuV, pGreenⅡ049-1.6A and pGreenⅡ049-2.0β, were constructed by the Vegetable Disease Laboratory of the Institute of Plant Protection, Guangdong Academy of Agricultural Sciences;

[0044] (2) Experimental method

[0045] Activation on LB medium plate with 50 mg / L kanamycin and 50 mg / L rifampicin Take out the Agrobacterium strain EHA105 containing pBinPLUS-1.85A and pBinPLUS-2.0β and the strain containing pGreenⅡ049-1.6A from the -80°C refrigerator , pGreenⅡ049-2.0β Agrobacterium GV3101 strain, after culturing at 28°C for 48 hours, pick a single colony and inoculate it in 5mL LB liquid medium containing 50mg / L kanamycin an...

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Abstract

The invention belongs to the field of plant protection, and particularly relates to an inoculation method for infecting hibiscus cannabinus with multan cotton leaf curl virus. The method comprises the steps that a manual injection method is adopted for conducting infectivity clone inoculation on mesophyll tissue and stem vascular bundle tissue of hibiscus cannabinus plants in the 4-5 leaf stage through multan cotton leaf curl virus mediated by agrobacterium tumefaciens, and then hibiscus cannabinus is infected by the multan cotton leaf curl virus. Compared with a gene gun blasting method frequently adopted by the pathogenicity research of the multan cotton leaf curl virus at present, the method has the advantages of being free of special instruments, easy to implement, accurate in result, good in repeatability, low in cost and the like, and is simple, convenient to implement, economical and effective.

Description

technical field [0001] The invention belongs to the field of plant protection, in particular to an inoculation method for infecting kenaf with Multan cotton leaf curl virus. Background technique [0002] Cotton leaf curl Multan virus (CLCuMuV) was first discovered as a plant virus on cotton in Multan, Pakistan, and it is one of the main pathogenic viruses that cause the epidemic of cotton leaf curl disease in Pakistan. The virus belongs to the genus Begomovirus of the family Geminiviridae; it is transmitted by Bemisia tabaci in a persistent manner, but cannot be transmitted by mechanical friction inoculation and seed infection. The viral genome only contains component A (DNA-A), which is single-stranded and circular, and is a single-component geminivirus. The isolates of CLCuMuV that have been reported so far are generally accompanied by β-satellite molecules (previously called DNAβ). CLCuMuV is mainly distributed in Pakistan, India, the Philippines, Laos, Vietnam, and my ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84
Inventor 何自福汤亚飞佘小漫蓝国兵
Owner PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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