Transformation assays of hypocotyl-derived callus with A. tumefaciens

Assays of transformation of ice plant callus with infection by A. tumefaciens strains were as described (Ishimaru 1999) with minor modifications. Bacteria strains were first grown in 523 media with appropriate antibiotics at 28 °C to OD₆₀₀ 0.8–1.0. The bacteria cells were then washed and resuspended in AB-MES media (2% glucose, 0.3% K₂HPO₄, 0.03% MgSO₄·7H₂O, 0.1% NaH₂PO₄, 0.1% NH₄Cl, 0.015% KCl, 0.001% CaCl₂, 0.00025% FeSO₄·7H₂O, and 0.97% MES, pH 5.5) (Hwang et al. 2010) without antibiotics at 28 °C to mid-log phase for 6 h. After adding 200 μM acetosyringone (AS; Sigma-Aldrich, St. Louis, MO, USA), bacteria cultures were further cultivated at 28 °C for 16–18 h and collected, resuspended in liquid CIM at the desired bacteria concentrations for infection.

The hypocotyl-derived callus was transferred from solid to liquid medium for subsequent Agrobacterium infection and GUS staining. During repeatedly shaking and washing of callus, a large quantity of “single cells” was released into the liquid medium and named as “callus-derived cells”. The callus cultures were maintained in 25 mL of liquid CIM medium in 125 mL Erlenmeyer flasks and subcultured every 14 days.

The A. tumefaciens EHA105 strain containing the T-DNA binary pBISN1 plasmid (Additional file 1: Table S1) was used to co-incubate with the 5-, 7-, 10-, or 14-day-old callus for 2 days. Two days after co-incubation with bacteria strains, ice plant callus was washed and sonicated for 10 min in liquid CIM medium containing timentin (100 μg mL⁻¹) five times to remove bacteria, then transferred into the CIM medium containing timentin (100 μg mL⁻¹) and kanamycin (100 μg mL⁻¹). More than 2 × 10⁵ ice plant callus-derived cells were infected with A. tumefaciens strain for each independent transformation assay. After 1- or 2-day recovery, ice plant callus was stained with X-gluc staining solution [50 mM sodium phosphate buffer, 0.1% Tween 20, 3% sucrose, and 1–2 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc), pH 7.0] for 1 day at 37 °C. An Olympus IX71 fluorescence microscope (Olympus Optical Co. Ltd., Tokyo, Japan) with the DP Controller program was used to observe GUS expression in ice plant callus-derived cells and determine transient transformation efficiencies. We treated GUS-stained samples with 0.5% pectinase (macerase) for 1 day to disrupt the aggregates before counting cell number by hemacytometer. More than 5 × 10³ callus-derived cells were examined and counted for each transformation experiment to determine transient transformation rates. Transformation rates were mean ± SD (standard deviation) from at least 3 independent experiments. The transformed callus was then continuously cultured in CIM medium containing 100 μg mL⁻¹ kanamycin.