Composition used for plants direct gene conversion
A technology of gene transformation and composition, applied in the field of bioengineering, can solve the problems of low protoplast regeneration frequency, popularization restrictions, safety issues, etc., and achieve the effect of avoiding the establishment of bottlenecks, expanding the scope, and having biological safety
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Embodiment 1
[0026] Example 1: Rapid and efficient transient expression of GUS gene in onion epidermal cells
[0027] Take the lower epidermis of the onion and use a blade to divide it into tissue pieces of appropriate size, sterilize in 70% ethanol for 1 minute, rinse with MS medium once, sterilize with mercury chloride for 5 minutes, rinse with MS several times, and then culture in hypertonic buffer MS liquid Base + 47g / L mannitol + 47g / L sorbitol for 10-20 minutes.
[0028] The Agrobacterium containing the Ti plasmid was inoculated in the YEB medium containing antibiotics, and cultured with shaking at 28°C and 250rpm until the logarithmic phase of growth (the absorbance of the bacterial solution OD 550 1.8-2.0); ③Centrifuge the cultured bacterial solution at 5000rpm for 10min, and then resuspend the bacterial cell in 1 / 2MS liquid medium (with 20mg / L acetosyringone) + transformation element + 0.01% Dow corning Q2-5211+10mmol / L CaCl 2 +0.1mmol / L arginine, the concentration of the dilute...
Embodiment 2
[0029] Example 2: Efficient and Stable Transformation of a Linear Fragment Containing the GUS Gene Through the Pollen Tube Passage Method
[0030] Preparation of the buffer solution with the Agrobacterium toxin protein virD2 / E2: Agrobacterium LBA4404 containing the Ti plasmid was inoculated in the YEB medium containing 50 mg / L rifampicin and 25 mg / L streptomycin, at 28 ° C and Under the condition of 250rpm, shake culture to the logarithmic phase of growth (absorbance of bacterial solution OD 550 1.8-2.0); Centrifuge the cultured bacterial liquid at 5000rpm for 10min, resuspend the bacterial cells in 1 / 2MS liquid medium to make the bacterial liquid, the concentration of the diluted bacterial liquid is about 2×10 8 cfu / ml, then add acetosyringone to the bacterial solution to a final concentration of 20mg / L, continue shaking culture at 28°C and 250rpm for 3-4 hours, centrifuge at 3000rpm for 10min to remove the bacteria, supernatant is the buffer with virD2 and virE2. Then, the...
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