The Agrobacterium tumefaciens strain LBA4404 harboring the plasmids of synthetic reporters -TCS::GUS/pKGWFS7.0 which have been used to report cytokinin output was used in our study (Yang et al., 2018). Approximately 0.5g fresh Wolffia fronds and 1 g sterilized glass beads (1 mm) were placed in 2 ml sterilized eppendorf microcentrifuge tube adding 500 uL Agrobacterium suspension, 1uL silwet L-77 and 100 μM Acetosyringone (AS). The tubes were first subjected to ultrasound at a frequency of 40 kHz for 1 min at 28 °C. After sonication treatment, a vacuum of approximately 0.7 kg/cm2 was applied for 10 min. Then the tubes were shaken around 150 rpm for 15 min at 28℃. Finally, plants were transferred onto filter papers wetted with liquid 1/2 SH medium adding 1% sucrose (w/v) and 100 μM AS at pH 5.2 in the dark for 5 d at 25 °C.
After five days co-cultivation, the infected fronds were transferred to selection medium: solid 1/2 SH medium supplemented with 300 mg/L cefotaxime, 20mg/L G418 (Geneticin), and 1% (w/v) sucrose under a 16/8 h photoperiod of approximately 85 μmolm-2s-1of white light at least for 4 weeks. Then the survived fronds were transferred to frond induction medium: solid 1/2 SH medium adding 150mg/L cefotaxime, 20mg/L G418 (Geneticin) and 1% (w/v) sucrose. The resistant fronds were identified by the β-Glucuronidase (GUS) assay (Figure 2). The transformation efficiency was calculated as percentage of GUS positive fronds in the total number of explants. The highest transformation efficiency in our study was 46.49%.
Reference: P.P.M. Heenatigala et al. Development of Efficient Protocols for Stable and Transient Gene Transformation for Wolffia Globosa Using Agrobacterium. Front. Chem., 2018,