A method for cultivating and propagating sterile banana nematodes by using carrot pure callus
A technology for banana perforating nematodes and callus, which is applied in the field of plant pathogen culture, can solve the problems that the banana perforating nematodes cannot be truly sterile, the concentration of use is difficult to control, and is unstable, and the biological activity and fecundity of the nematodes are small, The effect of mild and controllable processing conditions and safe use and operation
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Embodiment 1
[0067] Example 1 Using Carrot Pure Callus to Culture and Propagate Aseptic Banana Perforata nematode
[0068] 1. Aseptic treatment of banana perforator nematode
[0069] (1) Collection of sources of banana borer nematodes
[0070] 1) Obtain the insect source (non-sterilized) from the banana perforator nematode cultured from carrot slice callus with carrot rhizome traditionally. The specific method is: after culturing the carrot slice callus inoculated with the banana perforator nematode in the dark for 40 days, take it out from the incubator, wash the culture dish and the nematode on the surface of the carrot slice callus with clean water into another clean petri dish, Draw the nematodes under the dissecting microscope (to avoid culture impurities) into a 1.5ml centrifuge tube, centrifuge at 5000rpm for 45s to absorb the supernatant, and sink the nematodes into the bottom of the tube for aseptic treatment. The best collection amount per tube is 2000 Nematodes around.
[007...
Embodiment 2
[0099] The optimization of embodiment 2 disinfectant hydrogen peroxide solution concentration and disinfection time
[0100] 1. In the process of aseptic treatment of banana perforator nematodes, hydrogen peroxide solution is used to disinfect the surface of nematodes. In this embodiment, the concentration of hydrogen peroxide disinfection and the time of disinfection are optimized. The specific method is:
[0101] (1) Wash the collected banana perforator nematodes (refer to Example 1 for the collection method) twice with sterile water, centrifuge at 5000rpm for 45s, suck off the supernatant in the centrifuge tube, and use concentration gradients of 1% and 3% respectively , 5%, 8% and 10% (V / V) hydrogen peroxide solution to soak the nematodes (slowly shake the centrifuge tube horizontally during the soaking process), and take samples for each concentration treatment at 1 min, 3 min, 5 min, 8 min and 10 min after soaking .
[0102] (2) Wash the taken nematode suspension three...
Embodiment 3
[0108] Example 3 Screening of Antibiotics and Optimization of Using Concentration and Treatment Time in Aseptic Treatment of Banana Forerunner Nematode
[0109] 1. Screening of the antibiotic ofloxacin
[0110] (1) In Example 2, after the banana perforator nematode was sterilized by the hydrogen oxide solution, there was still bacteria contamination under the premise of maintaining the viability of the nematode, and further aseptic treatment of the nematode was required. In this example, different antibiotics were screened to further inhibit the growth of miscellaneous bacteria of the banana borer nematode, so as to achieve the purpose of nematode sterility. The specific method is:
[0111] In Example 2, banana threadworms were sterilized by 5% (V / V) hydrogen peroxide solution for 5 minutes, washed twice with sterile water, and the supernatant was sucked off; 1ml of 0.1wt% of the following antibiotic solutions were added respectively: Streptomyces sulfate Ampicillin, amoxyci...
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