High-efficiency induction of production of hairy roots of gromwell by seedling stem node needle puncturing method and application
A technology of hairy roots and comfrey, applied in the field of genetic engineering, can solve the problems of unstable cell lines, slow cell growth, and production of metabolites that are not resistant to shearing
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Embodiment 1
[0040] The cultivation of embodiment 1, comfrey aseptic seedling
[0041] Clean the surface of comfrey seeds, soak them in tap water at low temperature for two days, change the water 2-3 times during this time, then mix them with wet sand and seeds, put them in a refrigerator at 4°C for stratification treatment for about 1 month, after the seeds germinate ( figure 1 A), rinse off the sand with tap water, sterilize the germinated seeds with 0.1% mercury liter for 5 minutes, then rinse with sterile water for 5-7 times, blot the water to remove part of the hypocotyl, and inoculate in 1 / 2 MS hormone-free On solid medium, cultured at 25°C, 16 hours light and 8 hours dark for 2 weeks ( figure 1 B). When the seedlings grow to a height of 2-4 cm and stretch out the 2-3 pairs of true leaves, they are used as infested materials ( figure 1 C).
Embodiment 2
[0042] Example 2 Culture of Agrobacterium rhizogenes
[0043] Streak inoculation of Agrobacterium rhizogenes ATCC15834 (the strain contains Ri plasmid without resistance marker) on YEB solid medium, activate at 28°C, pick a single colony on the plate, and inoculate in 25 mL of YEB liquid medium , 28°C 120 rpm shaker culture in the dark, in the OD of Agrobacterium rhizogenes 600 nm When the value reaches 0.8-1.0 (12 hours before adding acetosyringone to the bacterial solution to a final concentration of 100 μM to improve the infection efficiency of the bacterial solution), it is used to infect comfrey.
Embodiment 3
[0044] Example 3 "Acupuncture Seedling Stem Node Method" Induces the Production of Comfrey Hairy Roots
[0045] Dip the above-mentioned activated bacterial solution with infection characteristics with a sterile needle, and use the needle tip to puncture the stem node of the first to second pair of true leaves of the comfrey seedlings. Puncture 2-3 times ( figure 1 C). Then the infected seedlings were transferred to MS solid medium and cultured in weak scattered light at 25°C. In about a week, white protrusions can be found on the infection site ( figure 1 D), followed by white protrusions at the site of infection that first grow a villous root system ( figure 1 E), followed by one or more white hairy roots ( figure 1 F, G). Cut the induced hairy roots from the infection site, culture them on MS medium containing 250 mg / L cephalosporin, and transfer them once a week until the bacteria are eliminated, then move to MS medium without antibiotics upper culture ( f...
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