Screening method of cucumber agrobacterium tumefaciens sensitive genotype
A technology of Agrobacterium tumefaciens and a screening method, which is applied in the field of screening of sensitive genotypes of Agrobacterium tumefaciens in cucumber, can solve the problem of not being able to distinguish the sensitivities of different varieties of Agrobacterium tumefaciens, and achieves the effect of simple operation.
Active Publication Date: 2018-03-30
QINGDAO ACAD OF AGRI SCI
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[0004] In view of this, the purpose of the present invention is to provide a screening method for sensitive genotypes of Agrobacterium tumefaciens in cucumber, which can accurately evaluate the sensitivity of cucumber materials of different genotypes to Agrobacterium infection, and solve the problem of traditional GUS transient expression rate differentiation. The problem of susceptibility of different species of Agrobacterium
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Embodiment 1
[0069] In this example, 8 South China type cucumber varieties / lines 'Yulong', 'Lu Cucumber No. 3', 'Cuilong', 'C369', 'C661', 'C677', 'C699' and 'C215×118' were used as Materials, using the method of Agrobacterium solid-state inoculation to infect the cotyledonary node explants, after the end of the co-cultivation, perform GUS histochemical staining and grading evaluation, the specific implementation methods are as follows:
[0070] 1. Preparation of cucumber sterile seedlings:
[0071] Pick healthy and plump cucumber seeds, soak them in warm water at 55°C for 20 minutes, and make the seeds settle completely; then in an ultra-clean workbench, sterilize with 70% ethanol for 1 minute, and quickly wash them with sterile water for 3 times; add 10% NaClO (add 1 drop of Tween-20 ) disinfection for 15 minutes; the sterile water was firstly washed 4 times quickly, and continued to wash for about 20 minutes. After the seeds were soaked for 4 hours, they were inoculated on the germinati...
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The invention provides a screening method of a cucumber agrobacterium tumefaciens sensitive genotype, and belongs to the technical field of biotechnological breeding of vegetables. The screening method comprises the following steps: carrying out solid inoculation on grobacterium tumefaciens containing a GUS gene plant expression vector into explants infected with cucumber cotyledonary nodes; putting the infected explants on a co-culture medium for dark culture; after the co-culture is finished, excising two thirds cotyledons of the explants and only retaining cotyledonary node zones of the explants; immersing the cotyledonary node zones into a GUS staining solution for staining and decolorizing; carrying out staining classification according to chemical staining sites and staining areas ofGUS tissues and determining the number of the explants belonging to each Gus transient expression level, calculating according to a formula I and classifying conditions to obtain an instantaneous expression index of the GUS gene; when a GUS instantaneous expression index value is in the interval [55.56 to 77.77], judging the GUS gene as a sensitive genotype. The screening method provided by the invention has the advantages that the sensitivity of cucumbers of different genotypes to agrobacterium tumefaciens infection can be accurately evaluated, and the problem that the sensitivity of the agrobacterium tumefaciens of different genotypes is difficultly distinguished by traditional GUS instantaneous expression rate is solved.
Description
technical field [0001] The invention relates to the technical field of vegetable biotechnology breeding, in particular to a method for screening sensitive genotypes of Agrobacterium tumefaciens in cucumber. Background technique [0002] Cucumber is an important vegetable crop in the world, and it is also one of the important vegetable crops in my country's facility production. In 2013, my country's facility cucumber planting area was 10.51 million mu, second only to tomato's 12.15 million mu, ranking second (Li Tianlai. Current situation and trend of China's protected vegetable technology and industry development [J]. China Rural Science and Technology, 2016, 05:75-77.). Due to the narrow genetic background of cultivated cucumbers, the lack of resistant germplasm, and the incompatibility of distant hybridization with wild resistant germplasm resources, it is difficult to introduce excellent genes for disease resistance and insect resistance into cultivation by traditional hyb...
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IPC IPC(8): G01N21/84C12N15/82
CPCC12N15/8212G01N21/84G01N2021/8466
Inventor 张功臣张守才李磊秦玉红王波江志训崔健孙吉禄王珍青刘玲
Owner QINGDAO ACAD OF AGRI SCI