A Directed Genetic Transformation Method for Improving the Transient Expression Rate of Wucai Explants
A genetic transformation method and transient expression technology, applied in the field of directional genetic transformation of Wucai explants, can solve the problems of explant damage, low transient expression rate of Wucai explants, etc., to promote cell division and improve transient expression and transformation rate, improving the effect of infection efficiency and effect
Active Publication Date: 2021-01-29
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology
In this method, the explants are placed in the liquid Agrobacterium, which will cause the explants to be hypoxic in a short period of time, causing damage to the explants, and the Agrobacterium will infect the parts that do not need to be infected on the explants, and the same cause explant damage; in addition, when this method is applied to Wucai, the transient expression rate of Wucai explants is very low
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Embodiment 1
[0029] (1) Get the aseptic seedling of 5 days seedling age, cut cotyledon-cotyledon petiole and hypocotyl explant;
[0030] (2) Place the explants on the pre-medium, and culture them under light at 25°C for 2 days;
[0031] (3) Spread the Agrobacterium containing the plant expression vector (containing the GUS reporter gene) evenly on the activation medium with an inoculation needle, and culture at a constant temperature of 28°C until the cells grow out;
[0032] (4) Moisten the sterile cotton swab with a liquid co-culture medium;
[0033] (5) Dip the activated Agrobacterium with a moistened cotton swab, and gently smear it on the incision of the pre-cultured explant;
[0034] (6) Place a piece of sterile filter paper on the surface of the solid co-culture medium;
[0035] (7) Spread the smeared explants on the solid co-culture medium, and culture them in the dark at 25°C for 2 days;
[0036] (8) After dark culture, the explants were chemically stained with GUS.
[0037] T...
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The invention discloses a method for directional genetic transformation of Wucai explants with simple operation and high transient expression rate. Its features include the following steps: (1) aseptic seedlings of Wucai for 5 days, cutting cotyledons-cotyledon petioles and hypocotyl explants. (2) The explants of Wucai were cultured on the pre-medium for 2 days. (3) Agrobacterium (containing the plant expression vector) is activated on the activation medium. (4) The activated Agrobacterium is dipped in a sterile cotton swab moistened with the liquid co-culture medium, and smeared on the incision of the explant in a targeted manner. (5) The explants after smearing were placed on a solid co-culture medium, cultured in the dark at 25°C for 2 days to complete the co-culture, and the transient expression rate could be detected. The directional genetic transformation method adopted in the invention has the advantages of no need for liquid cultivation of Agrobacterium, simple operation, high transient expression rate of explants, etc., and lays the foundation for efficiently obtaining transgenic plants of Wucai.
Description
technical field [0001] The invention belongs to the technical field of plant genetic transformation, and particularly provides a method for directional genetic transformation of Wucai explants with simple operation and high transient expression rate. Background technique [0002] Black cabbage, native to my country, is a variety of Brassica cabbage subspecies, and is one of the vegetables with the largest planting area in the Jianghuai region. For a long time, Wucai varieties have been improved mainly through variety hybridization and distant hybridization. There are many disadvantages such as long breeding period, high cost, easy degeneration of parental self-crossing for multiple generations, and difficulty in male sterility transfer. system. At present, in cabbage crops, Agrobacterium-mediated genetic transformation is mostly used. After the explants are co-cultured with a certain concentration of liquid Agrobacterium for 15-20 minutes, the Agrobacterium is removed, and ...
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IPC IPC(8): C12N15/82
CPCC12N15/8205
Inventor 陈国户汪承刚汪健朱世东袁凌云侯金锋
Owner ANHUI AGRICULTURAL UNIVERSITY