Agrobacterium-mediated jatropha curcas gene transformation method
An Agrobacterium-mediated and gene transformation technology is applied in the field of obtaining A. oleifera transgenic plants through the Agrobacterium-mediated gene transformation technology, which can solve the problems of chimera generation and gene silencing, and achieve the effect of simplifying the procedure and shortening the transformation period.
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Embodiment 1
[0035]Take mature seeds of tung tree, peel off the outer testa, soak in sterile water for 10 hours, then sterilize with 75% alcohol for 30 seconds, rinse with sterile water for 2 to 3 times, and then treat with 1.0% sodium hypochlorite solution for 10 minutes , rinsed with sterile water 3-4 times, placed on sterile filter paper to remove excess water; peeled off the endosperm with sterile tweezers and a scalpel, took out the embryo, and cut off 1 / 3-1 / 4 of the cotyledons at the end of the hypocotyl , and the remaining cotyledons are used as transformation recipients ( figure 1 A).
[0036] Pick the LB4404 Agrobacterium single clone containing the pCAMBIA 2301 plasmid and activate it in the YEP liquid medium containing 50 mg / L kanamycin sulfate and 25 mg / L streptomycin, shake it at 28°C at 200 rpm for 12 hours, transfer and merge Concentration of secondary activated Agrobacterium to OD 600 =0.4, collect the cells by centrifugation at 4000rpm at 4°C for 5 minutes, discard the s...
Embodiment 2
[0043] Repeat Example 1, with the following differences: the time for callus induction culture is 3 weeks, and then subcultured to the selection medium containing kanamycin sulfate for culture.
Embodiment 3
[0045] Repeat Example 1, with the following differences: the callus induction culture time is 2 weeks, and then subcultured to culture on the selection medium containing kanamycin sulfate.
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