Method for converting lucerne by vacuum penetrating auxiliary agrobacterium-mediated
An Agrobacterium-mediated, vacuum infiltration technology, applied in the fields of biotechnology and modern agriculture, can solve the problems of only 50% high, 12-month transformation period, and low transformation efficiency.
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Embodiment 1
[0047] The plant material is the No. 1 seed of Medicago sativa (purchased from the Institute of Animal Husbandry, Chinese Academy of Sciences), which is packed into a 1.5ml centrifuge tube, washed with tap water for 2 minutes, sterilized with 70% ethanol for 60 seconds, and then sterilized with 0.1% mercuric chloride. Sterilize for 6 minutes, then wash 6 times with sterile water, inoculate the seeds on MSB + 2.0% sucrose + 0.45% agar, pH 5.8 medium after sterilization, cut the seedlings after 7 days and continue to transfer to the above medium for differentiation After culturing for 18 days, the young true leaves were cut off.
[0048] The seed germination and differentiation culture conditions are: day and night light cycle culture, light intensity 40μmol·m -2 ·s -1 , light time 14h·d -1 , day temperature 25 ℃, night temperature 18 ℃.
[0049]Select a single colony inoculation of Agrobacterium tumefaciens LBA4404 strain carrying the binary vector pBin438 (the vector contai...
Embodiment 2
[0070] Inoculate the sterilized alfalfa seeds on the germination medium (MSB+2.0% sucrose+0.5% agar, pH value 5.8), cut the seedlings after 10 days and continue to transfer them to the above-mentioned medium for 15 days, then cut off the young true leaves , culture condition with embodiment 1.
[0071] Pick a single colony of Agrobacterium tumefaciens GV3101 carrying the binary vector pCAMBIA3301 (the vector contains the CaMV35s promoter, the rd29A gene of Arabidopsis thaliana, the selectable marker gene bar and the reporter gene 35sGUS) and inoculate in 5ml containing Kan 50mg L -1 In the YEB liquid medium, cultivate for 24 hours (28°C, 180rpm), take 1ml and transfer it to 50ml of antibiotic-free YEB medium and culture it for 16 hours under the same conditions, then take 15ml of bacterial liquid and add it to 50ml of medium I , cultured for 4 hours to OD at a shaking speed of 200rpm 600 0.7 spare.
[0072] Put the young true leaves of alfalfa tissue-cultured seedlings in th...
Embodiment 3
[0082] Inoculate the sterilized alfalfa seeds on the germination medium (MSB+0.1mg / L6-BA+3.0% sucrose+0.55% agar, pH 5.8), cut the seedlings after 8 days and continue to transfer to the above medium for differentiation and culture for 20 days , cut off the young true leaves.
[0083] Select the single colony of Agrobacterium tumefaciens EHA105 strain (its carrier contains CaMV35s promoter, SOS2+3 gene of Arabidopsis thaliana, selectable marker gene bar and reporter gene 35sGUS) carrying the binary vector pCAMBIA3301 to inoculate in 5ml containing Kan 50mg. L -1 In the YEB liquid medium, cultivate for 28 hours (28°C, 190rpm), take 1ml and transfer it to 50ml of YEB medium without antibiotics and culture it for 10 hours under the same conditions, then take 20ml of bacterial liquid and add it to 50ml of medium I , cultivated for 2 hours to OD at a shaking speed of 220rpm 600 0.4 spare.
[0084]Put the young true leaves of alfalfa tissue-cultured seedlings in the prepared Petr...
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