A method for establishing the genetic transformation system of Agrobacterium-mediated hair fern gametophytes
A technology of Pteridopsis fragariae and Agrobacterium, which is applied in the directions of biochemical equipment and methods, botanical equipment and methods, ferns, etc., can solve the problem of failing to find a method for establishing a genetic transformation system of Pteridopsis fragrantii, and failing to establish genetic transformation. system issues
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Embodiment 1
[0035] 1. Preparation of test materials
[0036] The whole plant of Trichofern is collected in Wudalianchi area, Heilongjiang Province. It was identified by researcher Zhang Xianchun, Institute of Botany, Chinese Academy of Sciences as Trichofernaceae, a plant of the genus Trichofern. The collected spores of D. elegans were stored in a refrigerator at 4°C for later use.
[0037] The Agrobacterium tumefaciens strain used was LB4404, and the plant binary expression vector was PBI 121, which carried a GUS reporter gene, a CaMV promoter, and a selection marker gene NptⅡ. Preserved by the Department of Plants, Northeast Agricultural University.
[0038] Antibiotic reagents such as kanamycin (Km) and rifampicin (Rif) were purchased from Sinopharm Group. Antibiotic stock solution: the water used for preparation is sterile water. Cephalosporin was produced by Shanghai New Pioneer Pharmaceutical Co., Ltd.; acetosyringone was imported analytically pure; sucrose was purchased from Tia...
Embodiment 2
[0060] Embodiment 2: each influence factor experiment
[0061] 1. Selection of the Optimum Kanamycin Resistance Concentration of D.
[0062] Since kanamycin is used as a screening agent, it is first necessary to master the kanamycin resistance index of D. chrysalis itself. The specific experimental process and results are as follows:
[0063] The selection marker gene of the vector pBI121 used in the experiment is NptII, and the product encoded by it is resistant to kanamycin. Therefore, it can be set to add 0, 50mg / L, 100mg / L, 150mg / L, 200mg / L, and 5 kana concentration gradients are placed on the basic medium and placed in the cultivation room for light cultivation. After 30 days, the statistics of different treatments on the medium The comparative analysis of the survival situation determined that the lowest concentration that could make the explants die was the appropriate kana concentration. 3 dishes were treated for each treatment, and 10 explants were inoculated in eac...
Embodiment 3
[0086] This embodiment provides the optimal method for establishing the genetic transformation system of Agrobacterium-mediated Trichophylla gametophyte obtained after the above-mentioned optimization, which specifically includes the following steps:
[0087] 1) Pre-cultivation: place the gametophytes of Trichophyton chrysalis on the culture medium for pre-cultivation for 2 days;
[0088] 2) Preparation of Agrobacterium bacterium liquid: transform Agrobacterium LB4404 with carrier PBI 121, then expand and culture Agrobacterium LB4404 containing PBI 121 carrier, collect the cells, resuspend and make OD 600 is 0.5, obtain the Agrobacterium bacterium liquid;
[0089] 3) Soaking of the Agrobacterium bacterium liquid; immersing the pre-cultivated Trichophyllum gametophytes in step 1) in the Agrobacterium bacterium liquid obtained in step 2) to infect for 10 minutes;
[0090]4) Wash the gametocytes after the Agrobacterium infiltration with 200mg / L-500mg / L cephalosporin sterile wate...
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