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Agrobacterium mediated transformation

LZ Liu Zhang
YZ Yang Zhao
HL Haiyan Liang
XL Xugang Li
KG Kimberly L. Gallagher
SW Shuang Wu
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All of the H2B destination vectors were transformed into Agrobacterium strain, GV3101-pSoup-pMP. 5 µl of plasmid was mixed with 50 µl chemically competent Agrobacterium (prepared by what method-citation) and incubated on ice for 30 min. The mixture was then heat-shocked at 37 °C for 5 min followed by incubation on ice for 10 min. After incubation on ice, 500 µl of LB medium was added and the bacterial cells were incubated for 2 h at 30 °C on a shaking incubator. The bacterial cultures were spread onto solid LB medium with antibiotics (50 μg/ml rifampicillin, 100 μg/ml spectinomycin, 30 μg/ml gentamycin and 5 μg/ml tetracycline) for selection. The transformed cells were grown at 28 °C for 2–3 days until colonies appeared. The Agrobacterium strains were used to transform Arabidopsis (Col-0) following the floral dip method [21]. Transgenic plants were screened by using resistance to glufosinate-ammonium (Basta) in soil.

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