Method for efficiently obtaining transgenic plants of Euphorbia and its Agrobacterium-mediated transformation system
A transgenic and euphorbia technology, which is applied in botany equipment and methods, genetic engineering, plant gene improvement, etc., can solve the problems of low absolute content and no transgenic plants of euphorbia have been found, and achieve the effect of wide application value
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Embodiment 1
[0033] Embodiment 1: induction of embryogenic callus of Euphorbia euphorbia;
[0034] Take the soon-to-open or newly-opened flower buds of Euphorbia and disinfect them. The procedure is as follows: first rinse with tap water for 2 hours, rinse with sterile water twice, disinfect with 75% ethanol for 45 seconds, rinse with sterile water for 3 times, and disinfect with 0.1% mercury chloride for 6 minutes. , then scratched on it, inoculated in callus induction medium (MS+6-BA 2.0mg / L+NAA 0.3mg / L+sucrose 30g / L+phytogel 0.4g / L), first 26±1 Cultivate in the dark at ℃ for 7 days, and then culture in the light, the wound edge of the inoculated flower buds begins to expand after 7 days, and the callus tissue is induced after 14 days. The callus was subcultured on the embryogenic callus induction medium, and the embryogenic callus induction medium consisted of: MS+6-BA 2.0mg / L+NAA 0.1mg / L+sucrose 30g / L+0.4g / L vegetable gel. The embryogenic calli with good and fast growth, no browning...
Embodiment 2
[0035] Embodiment 2: the acquisition of the plant expression vector pCAMBIA1301 Agrobacterium tumefaciens engineering bacterium containing hygromycin gene;
[0036]The plant expression vector pCAMBIA1301 was transformed into Agrobacterium tumefaciens (such as EHA105, which is a publicly available biological material in the market), and PCR verification was performed. Specifically, first prepare competent Agrobacterium tumefaciens (Agl-1): culture Agrobacterium tumefaciens to OD600=0.5 of the bacterial solution, after cooling the bacterial solution on ice for 30 minutes, transfer the bacterial solution to a 1.5mL centrifuge tube, at 4°C, Centrifuge at 5000rpm for 5min, remove the supernatant; suspend the cells with pre-cooled 0.1M CaCl2 sterilized by suction, place on ice for 30min, then centrifuge at 5000rpm for 5min, remove the supernatant, and suspend the cells with 100μL pre-cooled 0.1M CaCl2 Afterwards, it was stored at 4°C for future use, and this was the prepared compete...
Embodiment 3
[0038] 1. Agrobacterium tumefaciens-mediated transformation
[0039] 1.1 The cultivation of Agrobacterium tumefaciens;
[0040] Pick a single colony of Agrobacterium tumefaciens engineering bacteria containing the plant expression vector in the YEB liquid medium containing 100mg / L kanamycin and 40mg / L rifampicin, cultivate overnight on a shaker at 28°C, and when the bacteria liquid When the concentration OD600 reaches 0.6-0.8, centrifuge at 4000rpm for 10min, remove the supernatant, resuspend the Agrobacterium precipitated at the bottom with MS containing 100μM acetosyringone, culture and activate it on a shaker at 28°C for 2h at 180rpm, and then use it to transform Euphorbia embryogenicity heal.
[0041] 1.2 Co-cultivation of Agrobacterium tumefaciens and explants;
[0042] Inoculate the embryogenic callus mass of Euphorbia euphorbia in the Agrobacterium tumefaciens bacterial liquid, culture it on a shaker at 28°C and 120rpm for 30min, take out the callus mass, blot the exc...
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