A kind of gene transformation method of Chinese chestnut
A technology of gene transformation and chestnut, applied in the fields of biochemical equipment and methods, genetic engineering, plant gene improvement, etc., can solve the problem of undetermined reliability and repeatability, and achieve stable transfection effect, stable expression and high transfection efficiency. Effect
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Embodiment 1
[0050](1) Plant materials
[0051]The material used in the experiment was'Yanshanhongli' (Castanea mollissima cv. Yanshanhongli), which was collected from the Chinese chestnut experimental station in Huairou District, Beijing. Times), one month after flowering, it is an ovule, and with time, the ovule will grow into an immature embryo. Store in an ice box and bring it back to the laboratory. Store at 4°C for later use.
[0052]The sterilized immature embryo tip is inoculated into the induction medium for induction culture, and cultured in the dark at 23-25°C. The fresh medium was replaced every 3 weeks and cultured until somatic embryos were formed.
[0053]It was found that 45 to 55 days after the full blooming stage has a higher probability of developing adult embryos. The Chinese chestnut fruits picked at this time were cultured as described above, and somatic embryos formed after about three months. Continue to culture the somatic embryos (such asfigure 1 ) As an infestation material.
[00...
Embodiment 2
[0094](1) Plant materials
[0095]The material used in the experiment,'Castanea mollissima cv. Yanshanhongli', was collected from the chestnut test station in Huairou District, Beijing. The embryo tips of the immature embryos 45 days after the full blooming period were collected, stored in an ice box and brought back to the laboratory, and stored at 4°C for later use .
[0096]The sterilized immature embryo tip is inoculated into the induction medium for induction culture, and cultured in dark at 23-25°C. The fresh medium was replaced every 18 days. After about three months, somatic embryos formed. The somatic embryos were cultured on the induction medium for 1 week as the infection material.
[0097](2) Transfection bacterial solution
[0098]2.1 Preparation of the vector
[0099]The pBI121 plant expression vector can be purchased commercially, and the target gene fragment MYB10 is inserted into the pBI121 plant expression vector by conventional methods.
[0100]2.2 Preparation of bacteria
[0101]The ...
Embodiment 3
[0124](1) Plant materials
[0125]The material used in the experiment,'Castanea mollissima cv. Yanshanhongli', was collected from the Chinese Chestnut Experimental Station in Huairou District, Beijing. The embryo tips of immature embryos 55 days after the full bloom period were collected, stored in an ice box and brought back to the laboratory at 4°C spare.
[0126]The sterilized immature embryo tip is inoculated into the induction medium for induction culture, and cultured in the dark at 23-25℃. The fresh medium was changed every 25 days. After about three months, somatic embryos formed.
[0127]The somatic embryos were cultured on the induction medium for 2 weeks as the infection material.
[0128](2) Transfection bacteria solution
[0129]2.1 Preparation of the vector
[0130]The pBI121 plant expression vector can be purchased commercially, and the target gene fragment MYB10 is inserted into the pBI121 plant expression vector by conventional methods.
[0131]2.2 Preparation of bacteria
[0132]The prepa...
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