Method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and application thereof

Abstract

The invention discloses a method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and an application of the method. The method comprises the steps of coating geminivirus infectious clone-containing Agrobacterium tumefaciens EHA105 on YEP solid culture medium containing 100 mg / L kanamycin and 50 mg / L rifampicin; culturing into colony at 28 DEG C; picking colony with toothpicks; piercing 3-4 points on phloem of plant after three-leaf stage, at position 1-2 cm from root; culturing inoculated plant in a greenhouse; and observing virus symptom. The inoculation method provided by the invention has the advantages of no need for raising Bemisia tabaci, high inoculation efficiency and repeatability, simple and rapid operation, and suitability for large-scale popularization. The method provided by the invention is applicable to 16 kinds of geminivirus with severe breakout in our country. The inoculated plants are in families of Solanaceae, Cucurbitaceae and Convolvulaceae. The method can be used for pathogenicity identification of geminivirus and virus gene function research, anti-geminivirus plant variety determination and its breeding.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for pricking and inoculating plants with a solid bacterium colony containing geminivirus infective clones and an application thereof. Background technique [0002] Geminiviruses are a class of plant viruses that occur widely. With global warming, changes in agricultural farming systems, the rapid strengthening of international trade activities and the unprecedented expansion of insect vectors around the world, geminiviruses are widely occurring around the world. At present, geminivirus has been widely distributed and prevalent in Asia, central America, central and northern South Africa, and the Mediterranean, and is further expanding to surrounding uninfected areas. According to preliminary statistics, cotton, cassava, tomato and other crops in at least 40 countries have suffered devastating damage from such viruses. [0003] Since the 1990s, with the global warming and the...

AI Technical Summary

Problems solved by technology

For a long time, the research on geminivirus has mostly used the method of bemisia tabaci transmission and inoculation, but this method requires bemisia tabaci to complete the virus transmission, so there are many problems that are difficult to overcome: 1. Bemisia tabaci are difficult to raise; 2. Whether the whitefly is carrying the virus, whether the virus is pure and single, and the detection of the virus-carrying rate of the whitefly is difficult, etc., resulting in that the source of the virus in each test cannot be unified; 4. The transmission efficiency of Bemisia tabaci is even more difficult to control, which affects the accuracy of the experimental results; 5. Since Bemisia tabaci can transmit a variety of geminiviruses and other diseases at the same time, the inoculation results are often very disturbing

The recently developed Agrobacterium liquid injection method needs to use the inoculation buffer for post-processing the bacterial liquid, the steps are cumbersome, the dependence on the instrument is strong, the operation is inconvenient, and the experimental efficiency is reduced.

In addition, the syringes and excess bacterial fluid consumed during the experiment caused environmental pollution, which is not conducive to environmental protection, and the injection inoculation method is poorly operable for plants with thick stems, which affects the efficiency of geminivirus inoculation

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