Figure 4. Antimicrobial peptide expression had no negative fitness consequences for field-grown plants.

N. attenuata ICE-lines were compared with empty vector controls (EV) in a field experiment at the Lytle ranch preserve Utah (USA). (A) Elongated flowers and flower buds were removed before opening in a three-day interval and normalized against days post planting to compensate the planting delay of 12 days for the ICE 8 plants. For the cumulative flower production all removed flowers per genotype were summed from 25 to 40 dpp (±SEM, n = 20 plants; Mann-Whitney U Test following Kruskal-Wallis Test: **p≤0.01, n.s. = not significant). (B) Larval performance of the tobacco hornworm (Manduca sexta) did not differ in WT and transgenic ICE lines in the glasshouse (±SEM, n = 18–22 larvae). (C) Herbivore damage from the native herbivore community did not differ among EV and ICE 6 plants. The graphs show the estimated % canopy damage averaged from the assessments of two independent researchers (±SEM, n = 20 plants). Except for the sap-sucking herbivore Tupiocoris spp. (Heteroptera: Miridae) most herbivores showed damage with low abundance (<1%). The median is shown as the centered line, limited by the 25^th and 75^th percentiles and 1.5 times extended whiskers after Tukey. Only line ICE 8 (planted with delay) showed significantly less herbivore damage compared to EV (Mann-Whitney U test following Kruskal-Wallis Test; **p≤0.01; ***p≤0.001). (D) At the time of the herbivore screening ICE 8 and EV plants showed an average height difference of 46.5 cm (±2.7 cm) due to the delay in planting, EV and ICE 6 plants did not differ.

Figure 4—figure supplement 1. Off-target effects of the plant transformation process dominate growth performance of line ICE 1.

Growth comparisons of transgenic N. attenuata ICE 1 and ICE 6 in the field and the glasshouse. (A) During the first field season, ICE 1 plants showed a severe reduction in rosette diameters and stalk heights compared to the controls (EV) (±SEM, n = 14–16 plants). Asterisks indicate statistically significant differences. (Mann-Whitney U Test, following Kruskal-Wallis Test; ***p≤0.001). (B) Elongated flowers and flower buds were removed and counted every 3 d before opening. For total flower numbers, the cumulative flower productions per genotype was summed from 29 to 41 d post planting (dpp). ICE 1 showed significant reductions in flower numbers. Asterisks indicate statistically significant differences to the controls (Mann-Whitney U Test; ***p≤0.001) (C) Glasshouse experiments comparing diameter and stalk height of transgenic plants to WT controls. Both ICE 1 lines (from a single regeneration event) showed a minor reduction in growth (red and green arrow) (±SEM, n = 8 plants, WT and ICE 6 n = 16). Asterisks indicate statistically significant differences compared to the controls (Mann-Whitney U Test, following Kruskal-Wallis Test; *p≤0.05; **p≤0.01; ***p≤0.001).

Figure 4—figure supplement 2. Growth performance of transgenic N. attenuata ICE 6 and ICE 8 lines did not differ from the controls in the field. .

Growth performances of N. attenuata ICE-lines were compared with empty vector controls (EV) in a field experiment at the Lytle ranch preserve Utah (USA). (A) Due to a mishap in germination the ICE 8 plants had been planted with a delay of 12 d compared to the controls and all growth parameters were normalized against days post planting (dpp), as illustrated with the rosette diameter. (B) Normalized rosette diameter and stalk heights of the transgenic plants showed no difference to the controls (±SEM, n = 20 plants). (C) The plant shoot and root fresh biomass was determined 43 dpp and did not differ in the ICE 6 plants compared to the controls (±SEM, n = 20 plants) (Mann-Whitney U test p>0.3, n.s. = not significant).