Method for evaluating resistance of pine tree calli to bursaphelenchus xylophilus
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A technology of pine wood nematode and callus, which is applied in the biological field and can solve the problems that there is no method for evaluating the characteristics of pine callus resistance to pine wood nematode
Inactive Publication Date: 2019-06-25
NANJING FORESTRY UNIV
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Problems solved by technology
Jang et al. (1990) compared however, there is no method for evaluating the resistance of pine callus to B. xylophilus
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Embodiment 1
[0034] A method for evaluating pine callus resistance to pine xylophilus, comprising the following steps:
[0035] (1) Prepare PDA medium, inoculate Botrytis cinerea on the medium, and cultivate it at 25°C for 5-7 days. After the Botrytis cinerea hyphae cover the plate, inoculate the pine wood nematode AMA3C1 strain into it, and cultivate it at 25°C 5-7 days, after the pine wood nematodes have multiplied in large numbers, separate the pine wood nematodes using the Bellman funnel method, collect them in sterile centrifuge tubes, centrifuge, remove the supernatant, then wash with sterile water, centrifuge, and repeat 3-5 times , finally keep 2-3m1 pine xylophilus suspension, adjust the concentration to 20000 / mL, and set aside;
[0036] (2) Cultivate and obtain the pine wood nematode AMA3C1 nematode suspension according to step (1), prepare bacteria-free pine wood nematode with reference to the method of Zhu Lihua et al. (ZL201010550230.7), adjust the concentration to 20000 / mL, a...
Embodiment 2
[0041] A method for evaluating pine callus resistance to pine xylophilus, comprising the following steps:
[0042] (1) Prepare PDA medium, inoculate Botrytis cinerea on the medium, and cultivate it at 25°C for 5-7d. After covering the plate with Botrytis cinerea mycelia, inoculate Pine Xylophilus into it, and cultivate it at 25°C for 5-7d , after the large number of pine wood nematodes multiplied, the Baerman funnel method was used to separate the pine wood nematodes, collected in sterile centrifuge tubes, centrifuged, removed the supernatant, washed with sterile water, centrifuged, repeated 3-5 times, and finally retained 2-3ml pine wood nematode suspension, prepare bacteria-free pine wood nematode according to the method of Zhu Lihua et al. (ZL201010550230.7), adjust the concentration to 20000 / mL, and set aside;
[0043] (2) Adopt existing conventional methods or refer to Chen Tingting et al. [Optimization of somatic embryogenesis in Pinus massoniana for resistance to pine w...
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Abstract
The invention discloses a method for evaluating the resistance of pine tree calli to bursaphelenchus xylophilus. The method is characterized in that the sterile bursaphelenchus xylophilus is utilizedfor being inoculated to induced pine tree embryogenic cell lines, the responses of different cell lines to the bursaphelenchus xylophilus are compared through the appearance shape change of the calli,a TTC cytochemical staining result and a bursaphelenchus xylophilus population increase speed, thus the resistant cell lines are sifted out, the sensitive cell lines are eliminated, and early-stage evaluation on the resistance of the pine tree embryogenic cells to the bursaphelenchus xylophilus can be conducted in the callus stage. According to the method, the sterile bursaphelenchus xylophilus is utilized for being inoculated to the induced pine tree embryogenic cell lines, the responses of different cell lines to the bursaphelenchus xylophilus are compared through the appearance shape change of the calli, the TTC cytochemical staining result and the bursaphelenchus xylophilus population increase speed, thus the resistant cell lines are sifted out, the sensitive cell lines are eliminated, and early-stage evaluation on the resistance of the pine tree embryogenic cells to the bursaphelenchus xylophilus can be conducted in the callus stage. By means of the method, the resistance of thepine tree calli to the bursaphelenchus xylophilus is simply and quickly evaluated.
Description
technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a method for evaluating the resistance of pine callus to pine xylophilus. Background technique [0002] Pine wood nematode disease is a devastating major forest disease caused by pine wood nematode (Bursaphelenchus xylophilus). At present, it is mainly distributed in the United States, Canada, Japan, China, South Korea, Portugal and Spain (Mota et al., 1999; Abelleira et al., 2011). Pine wood nematode disease was first discovered in my country in 1982. Over the past 30 years, the disease has spread rapidly to many southern provinces (regions), and gradually spread to northern provinces. In 2018, a total of 588 county-level administrative regions in 18 provinces across the country had pine wood nematode epidemics. The disease not only harms the species of Pinus, but also the species of Larix (Yu Haiying, 2018). It poses a serious threat to the 900 million mu of p...
Claims
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