A Rapid and Efficient Agrobacterium-mediated Genetic Transformation Method for Tobacco Seeds
A genetic transformation method, Agrobacterium-mediated technology, applied in the field of tobacco seed transformation mediated by Agrobacterium, can solve the problems of killing plant cells, affecting the effect of tissue culture, and short disinfection time, so as to achieve easy repeatability and reduce the cost of genetic transformation , Reduce the effect of manpower and material resources input
Active Publication Date: 2019-07-12
GUIZHOU UNIV
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Problems solved by technology
Traditional tobacco Agrobacterium genetic transformation often requires the use of plant tissue culture technology, and the disinfection of explants is one of the key factors. If the disinfection time is too short, the tissue culture process is prone to bacterial contamination; if the time is too long, the disinfection substances will kill plant cells. Affect the effect of tissue culture
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[0038] Example: This method was used to obtain tobacco K326 transgenic plants.
[0039] 1. Construction of plant expression vector and activation of engineered Agrobacterium
[0040] The plant expression vector pSH737 ( figure 1 Middle A) is the transformation of the original vector. The pSH737 vector contains a multiple cloning site (MCS), including restriction sites such as Xba I, BamH I, Xma I, Sma I, Asp718, Kpn I and EcoR I. The plant expression vector pSH737 and the artificially synthesized QPT gene fragment with BamH I and Kpn I restriction sites were double-digested with BamH I and Kpn I respectively, and then in T 4 Linked under the action of DNA ligase, and constructed into plant expression vector pQPT( figure 1In B, the carrier can be released to the public). The QPT gene encodes quinolinic acid phosphoribosyltransferase, which catalyzes quinolinic acid to form nicotinic acid mononucleotide (NAMN), thereby entering the pyridine nucleotide cycle, and then forming...
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The invention discloses a fast and efficient agrobacterium-mediated tobacco seed genetic transformation method, which comprises the main steps that after the proper grinding, mature tobacco seeds are used as genetic transformation materials; the concentration of an agrobacterium solution is regulated to a proper value, so that the agrobacterium solution can be used for infecting receptor materials; after the sequential performing of co-culture, seeding, GUS tissue chemical dyeing and PCR amplification, transgenic tobacco plants are identified. The method has the advantages that the steps of sterilization, disinfection, tissue culture and the like are not needed; most operation processes are performed under the sterile condition; the defects of complexity, time consumption and the like due to sterile and tissue culture operation are avoided; the method belongs to a simple fast low-cost genetic transformation method with good repeatability; important significance and values are realized for fast creating the transgenic tobacco new germplasm.
Description
technical field [0001] The invention belongs to the field of agricultural biotechnology and the field of plant genetic engineering, in particular to an agrobacterium-mediated tobacco seed transformation method. Background technique [0002] Tobacco is a crop with high economic value and occupies a huge economic market in our country. In addition to being mainly used for smoking, protein food and medicine, tobacco also has a wide range of uses. With the development of plant science, tobacco, as a model plant in basic research and application, has played an important role in the study of plant genetics, development, physiology, biochemistry and secondary metabolism. Although my country's tobacco planting area and output rank first in the world, tobacco is an economic crop that pays equal attention to both output and quality. Factors such as different climatic conditions, diseases and insect pests will seriously affect the growth of tobacco plants and the quality of tobacco le...
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Login to View More IPC IPC(8): C12N15/84A01H5/10A01H6/82
CPCC12N15/8205
Inventor 赵德刚张祎秦利军赵丹杜致辉祖庆学余婧
Owner GUIZHOU UNIV