Shenzhen No.5 flameray gerbera transgene technology
A transgenic, gerbera technology, applied in the direction of recombinant DNA technology, cells modified by introducing foreign genetic material, and introduction of foreign genetic material using vectors, etc., can solve the limitation, low frequency of plant regeneration, low heat resistance, cold resistance, etc. question
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Embodiment 1
[0023] Step 1, the plasmid is introduced into Agrobacterium tumefaciens: according to the method of direct introduction of the intermediate vector plasmid DNA into Agrobacterium tumefaciens, pCAMBIA1301 is introduced into Agrobacterium tumefaciens LBA4404; plate culture, the medium adopts the formula of YEB+50mg / L kana Solid medium A of mycin + 50mg / L streptomycin, cultured at 28±1°C for 46 hours;
[0024] Step 2, preparation and preservation of LBA4404 seed liquid: pick the single colony of LBA4404 obtained from the above cultivation containing the pCAMBIA1301 plasmid, and inoculate it into medium B: the liquid of YEB+50mg / L kanamycin+50mg / L streptomycin In the culture medium, shake culture at 28±1°C and 180r / min for 24 hours; mix the prepared glycerol with a volume percentage concentration of 80% with the cultured bacterial liquid to make the final glycerol concentration in the glycerol bacterial liquid mixture up to 20%. Dispense the glycerin bacteria liquid mixture into 1...
Embodiment 2
[0032] Embodiment 2: other is with embodiment 1, and difference is:
[0033] Step 1: medium A is a solid medium of YEB+100mg / L kanamycin+100mg / L streptomycin, and the culture time is 50h;
[0034] Step 2: Medium B is a liquid medium of YEB+100mg / L kanamycin+100mg / L streptomycin, the culture conditions are 28±1°C, 220r / min, and the culture time is 36h; LBA4404 seed liquid is divided into 1.5 ml eppendof tubes and stored at -80°C for future use.
[0035] Step three:
[0036] (1) Activation of Agrobacterium: Take 8 μl of the LBA4404 seed liquid obtained in step 2 and inoculate it into 15 ml of medium B in step 2, and cultivate it for 25 hours according to the temperature and shaking culture conditions of step 2; take 1 ml of the bacterial liquid and transfer it to 50 ml of fresh medium In B, continue to cultivate for 14 hours according to the temperature and shaking culture conditions of step 2; transfer the cultured bacterial liquid into a 10ml eppendof tube, centrifuge at 500...
Embodiment 3
[0042] Embodiment 3: other is with embodiment 1, and difference is:
[0043] Step 1: medium A is a solid medium of YEB+100mg / L kanamycin+100mg / L streptomycin;
[0044] Step 2: medium B is a liquid medium of YEB+100mg / L kanamycin+100mg / L streptomycin, and the culture time is 36h;
[0045] Step 3: (1) After completion, OD 600 The value is 0.5; (2) the standby bacterial solution shaking culture time is 2h; (3) the explants are sterilized and cultured for 3 days under the condition of the sterilized medium pH is 5.8; (4) the medium pH is 5.8; (5) The pH value of the adventitious bud rejuvenation and proliferation medium in ) is 5.6; the time of culturing on the rooting medium is 30 days.
[0046] Test results: The transient expression rate of GUS in single-bud explants was 20.3%, the occurrence frequency of resistant adventitious buds was 20.3%, and the frequency of transgenic adventitious buds finally obtained was 7.35%.
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