Timentin

The tetraploid potato cultivar Desiree (ZPC, Joure, The Netherlands) was in vitro propagated in a controlled environmental chamber at 19 °C under a 16 h light/8 h dark photoperiod and transformed as previously described [25 (link)]. Plant regeneration was performed using a selection medium containing 250 µg mL⁻¹ cefotaxime (Duchefa, Haarlem, The Netherlands), 100 µg mL⁻¹ timentin® (Duchefa, Haarlem, The Netherlands) and 50 µg mL⁻¹ kanamycin (Duchefa, Haarlem, The Netherlands).
Plants from the WVA106 tomato cultivar were cultured in sterile conditions in a growth chamber with controlled temperatures of 22 °C/18 °C under a 16 h/8 h (day/night) photoperiod. Agrobacterium-mediated transformation using the C58 pGV2260 strain was performed on cotyledon segments from 8-12 day-old seedlings, as previously described [29 (link)]. Plant regeneration was performed using a selection medium containing 225 µg mL⁻¹ timentin® (Duchefa, Haarlem, The Netherlands) and 100 µg mL⁻¹ kanamycin (Duchefa, Haarlem, The Netherlands).

R. rhizogenes ATCC 15,834 strain was used for the transformation by heat-shock method^{124 (link)}. Individual cotyledons were excised from 15- to 20-day-old tomato seedlings grown as described above and immersed in a 2-day-old R. rhizogenes suspension for incubation at 28 °C for 2 h, with agitation at 100 rpm. The excised cotyledons then were placed on standard-strength Gamborg’s B5 salt media for 3 days for co-cultivation, and then transferred to B5 agar media supplemented with the antibiotics kanamycin (50 mg/mL) (DUCHEFA, Haarlem, the Netherlands) and timentin (15:1) at 300 mg/mL (DUCHEFA, Haarlem, the Netherlands). After 7–10 days of incubation in the dark at 25 °C, roots emerged from the wounded surface of the cotyledons. Hairy roots were transferred to Gamborg’s B5 medium containing 0.8% Gelrite and kanamycin (50 mg/mL). For nematode-infection experiments, transformed roots were subcultured in antibiotic-free media for 2 weeks, and 300 freshly hatched sterile M. javanica juveniles were used to inoculate the transgenic root lines, and root samples were taken at the designated time points for GUS assessment.

The binary vector pCAM-LOXD, pCAM-CWP and the empty-vector control pCAMBIA2300 were electrotransformed into A. rhizogenes ATCC 15834 [85 (link)]. Individual cotyledons were excised from 8–10-day-old tomato seedlings and immersed in an A. rhizogenes suspension (OD₆₀₀ 1.0) for 15 min. The excised cotyledons were blot-dried on sterile filter paper, then co-cultivated on standard-strength Gamborg’s B5 salt medium for 3 days. Explants were then washed with liquid B5 medium supplemented with the antibiotics kanamycin (50 μg ml⁻¹) (Duchefa Biochemie) and Timentin (300 μg ml⁻¹; ticarcillin disodium:potassium clavulanate, 15:1) (Duchefa Biochemie) and incubated at room temperature for 1 h with mild shaking. The explants were blot-dried on sterile filter paper and placed on B5 agar medium supplemented with the same antibiotics. Within 7 to 10 days of incubation at 25°C in the light, roots emerged on the surface of the cotyledons. Hairy roots were transferred to Gamborg’s B5 medium supplemented with 0.8% (w/v) Gelrite and kanamycin (50 μg ml⁻¹).