pPZP-RCS2-ocs-nptII

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	Agrobacterium tumefaciens strain LBA4404 harboring the control binary vector pPZP-RCS2-ocs-nptII and the vectors containing expression cassette of each starch-hydrolyzing enzymes (α-amylase, amylopullulanase, and glucoamylase genes) with or without glgC were used for transformation. Single colonies of LBA4404 carrying each construct were cultured in 2 mL LB medium containing appropriate antibiotics for about 10-h while shaking at 220 rpm at 28°C. About 1–1.5 mL of the suspension was used to initiate 25 mL of YM medium containing appropriate antibiotics and 100 μM acetosyringone. The culture was allowed to grow overnight to reach an OD600 of 0.5–0.75.

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Agrobacterium tumefaciens strain LBA4404 harboring the control binary vector pPZP-RCS2-ocs-nptII and the vectors containing expression cassette of each starch-hydrolyzing enzymes (α-amylase, amylopullulanase, and glucoamylase genes) with or without glgC were used for transformation. Single colonies of LBA4404 carrying each construct were cultured in 2 mL LB medium containing appropriate antibiotics for about 10-h while shaking at 220 rpm at 28°C. About 1–1.5 mL of the suspension was used to initiate 25 mL of YM medium containing appropriate antibiotics and 100 μM acetosyringone. The culture was allowed to grow overnight to reach an OD600 of 0.5–0.75.

Publication protocol

Agrobacterium tumefaciens strain LBA4404 harboring the control binary vector pPZP-RCS2-ocs-nptII and the vectors containing expression cassette of each starch-hydrolyzing enzymes (α-amylase, amylopullulanase, and glucoamylase genes) with or without glgC were used for transformation. Single colonies of LBA4404 carrying each construct were cultured in 2 mL LB medium containing appropriate antibiotics for about 10-h while shaking at 220 rpm at 28°C. About 1–1.5 mL of the suspension was used to initiate 25 mL of YM medium containing appropriate antibiotics and 100 μM acetosyringone. The culture was allowed to grow overnight to reach an OD600 of 0.5–0.75. The bacteria suspension was prepared according to Taylor et al. (2012). The non-ionic surfactant Pluronic F-68 was added to the bacterial suspension at a concentration of 0.03%. Embryogenic tissues obtained from GD2-50P agar media (Supplementary Table S1) or suspension culture was inoculated with the bacterial suspension in 6-well plates (Figure ​Figure22). At least two independent transformations were done for each construct. The tissues were vacuum-infiltrated for a total of 15 min with two pauses every 5 min, and incubated at room temperature for 30 min. Removal of excess Agrobacterium and coculturing was performed according to Taylor et al. (2012). After 5 days of-coculture, the tissues were transferred to Stage I selection medium GD2-50P containing 25 μM paromomycin and 500 mg/L carbenicillin. Putative transgenic tissues were further selected on GD2-50P containing 30 μM (Stage II) and 40 μM (Stage III) paromomycin each for 3 weeks. Transgene insertion was validated by PCR from genomic DNA samples isolated from proliferating putative transgenic calli.

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