Cefotaxime

Gamborg B-5 basal medium, Murashige and Skoog (MS) medium (Murashige and Skoog, 1962 (link)), yeast extract beef broth (YEB), Agargellan™, and cefotaxime were obtained from PhytoTechnology Laboratories (Lenexa, KS, USA). YE powder was obtained from HiMedia (Maharashtra, India). MeJA (1 g/mL in water), CHI (from shrimp shells), vanillin, and acetosyringone were purchased from Sigma-Aldrich (St. Louis, MO, USA). SA was procured from Ajax Fine Chem (Sydney, Australia). The friedelin analytical standard was obtained from Extrasynthese (Genay, France), and the epifriedelanol analytical standard was acquired from Biopurify (Sichuan, China). Glacial acetic acid, sulfuric acid, toluene, dichloromethane, and ethyl acetate were obtained from Merck (Darmstadt, Germany). Chloroform was procured from Labscan (Bangkok, Thailand). For DNA isolation and amplification, a DNAsecure Plant Kit (Tiangen, Beijing, China), Taq Phusion polymerase, and GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA) were used, and the primers were synthesized by Macrogen (Seoul, Korea).

Firstly, 100 seeds were used to produce explants as described above and these were cultured for 3 days on RM73 medium. Then, explants were subcultured onto Petri dishes containing 30 mL of RM73 medium plus various concentrations (0, 25, 50, 75, 100 mg L⁻¹) of kanamycin (PhytoTechnology Laboratories^®, Germany). Explants were maintained at 22 °C with a photoperiod of 16 h light/8 h dark and subcultures were made every two weeks as described previously. After 15, 30 and 45 days, the number of surviving explants was scored according to Figure 3. The experiment was repeated twice with kanamycin. The same experiment was performed for hygromycin, testing 0, 10, 25, and 50 mg L⁻¹ of this antibiotic and repeated twice.
During transformation with Agrobacterium tumefaciens, another antibiotic is often used to eliminate the presence of this bacteria. In this case, the antibiotic was cefotaxime (PhytoTechnology Laboratories^®, Germany). Because hygromycin or kanamycin are used concurrently with cefotaxime, the combined effect of these antibiotics was analysed and compared with a control (0 mg L⁻¹ kanamycin, 0 mg L⁻¹ hygromycin and 0 mg L⁻¹ cefotaxime). In this case, the number of surviving explants was also counted, starting with 100 seeds and following the protocols previously described.

Seeds of subterranean clover cv. Daliak (sourced from Dr Phillip Nichols, (DPIRD) former Department of Agriculture and Food WA [DAFWA], 3 Baron-Hay Ct, South Perth, WA 6151, Australia) were washed for 3 min in 50 mL of sterile deionised (DI) water + 2 drops Tween−20 solution (Amresco^®, Solon, OH, USA). Then, seeds were sterilised in 70% ethanol for 5 min. At this stage, 0.3% chlorine solution + 2 drops Tween 20 were added for 15 min. Subsequently, seeds were washed twice with DI water, and then moved into a 0.8 mg mL⁻¹ cefotaxime (PhytoTechnology Laboratories^®, Lenexa, KS, USA) solution for 10 min, modifying the protocol reported for Ding et al. [10 (link)]. Finally, seeds were washed 3 times with DI water (5 min each time) and left to imbibe overnight in a Petri dish with sterile water (Figure 2).