Method for genetic transformation of zoysia matrella mediated by agrobacterium tumefaciens
A method of genetic transformation, the technology of Zoysia ditchleaf, applied in the fields of biochemical equipment and methods, horticultural methods, botanical equipment and methods, etc., can solve the problems of incomplete genetic transformation system and limit the development of molecular breeding, etc., and achieve simplification Experimental process, effect of reducing workload
A method of genetic transformation, the technology of Zoysia ditchleaf, applied in the fields of biochemical equipment and methods, horticultural methods, botanical equipment and methods, etc., can solve the problems of incomplete genetic transformation system and limit the development of molecular breeding, etc., and achieve simplification Experimental process, effect of reducing workload
CN110577966AInactive Publication Date: 2019-12-17INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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[0022] The present invention will be further described below in conjunction with the examples, but not as a limitation of the present invention.
[0023] For the test methods that do not indicate the specific experimental conditions in the following examples, usually follow the routine experimental conditions or the experimental conditions suggested by the manufacturer.
[0024] 1.1 Method
[0025] 1.1.1 Induction and subculture of callus from Zoysia spp.
[0026] Embryogenic callus was induced from the stolons of Zoysia rugosa, and the callus induction and subculture were carried out according to the methods of Chai Mingliang et al.
[0027] Table 1 Basic medium for plant regeneration and Agrobacterium culture
[0028]
[0029] 1.1.2 Antibiotic susceptibility test
[0030] Determination of the inhibitory concentration of Timentin: the calli soaked in the Agrobacterium solution for 30 min were inoculated in the callus containing 0, 100, 150, 200, 250 and 300 mg / L Timenti...
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The invention discloses a method for genetic transformation of zoysia matrella mediated by agrobacterium tumefaciens. The method includes the steps of the concentration of a bacterial solution, the infection time, the co-culture time, the antibiotic concentration, acclimatization and transplantation, PCR detection of transgenic plants and the like. The method includes the following steps that an optimal genetic transformation system is that an OD600 value of the bacterial solution is 0.4, infection is conducted for 30 min, and selective culture is conducted after co-culture is conducted for 3d; the optimal bacteriostatic concentration of Timentin in a selective culture phase is 250 mg / L, the optimum selection pressure for hygromycin callus screening is 40 mg / L, and the optimum concentration of root taking screening is 15 mg / L; and histochemical analysis and PCR identification of GUS activity show that a target gene is successfully transferred into a genome of the zoysia matrella. Theabove research provides important technical support for gene function research and molecular breeding of the zoysia matrella. The method has the advantages that a stable and high-efficiency zoysia matrella genetic transformation system is obtained, the convenience is provided for genetic improvement of the zoysia matrella, and meanwhile, a key preliminary foundation is laid for verification of functional genes of the zoysia matrella.
Description
technical field [0001] The invention relates to a genetic transformation method in the biological field, in particular to a method for the genetic transformation of Zoysia sativa mediated by Agrobacterium. Background technique [0002] Zoysia ditchleaf ( Zoysia matrela ) is Poaceae ( Gramineae ) Perennial warm-season turfgrass of the genus Zoysia in the subfamily Choridoideae. It has the characteristics of slender leaves, dense grass layer, well-developed root system and long green period. It is also one of the most salt-tolerant halophytes. It is mostly used in urban greening, sports field lawns and water and soil conservation, and is widely used in tropical and subtropical regions of the world. [0003] With the rapid development of biotechnology, transgenic technology has gradually matured, and specific target genes can be genetically transformed to shorten the breeding cycle. Among many plant transgenic methods, the Agrobacterium-mediated method has become the pref...
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Patent Timeline
17 Dec 2019
Publication
CN110577966A
- IPC
- C12N15/84; A01H4/00; A01H5/00; A01H6/46
- CPC
- A01H4/00; C12N15/8205
- Inventors
- 王凯; 曲爱爱