Water Stress Induction: Determination of PEG Selection Dose

Water stress induction: determination of PEG selection dose

PEG 6000 was added in different amounts to the growth medium in order to establish the toxic selection dose. To this purpose 20 mL of PEG solution at different concentrations, 5, 10 and 20% in LS liquid medium, were poured on the top of an equal volume of LS solid medium in a petri dish and after 24 h, the solution was poured off and the dish used for the experiments. Percentage of survival and shoot differentiation capability of leaf discs collected from N. langsdorffii wild type plants and grown for 90 d on a regeneration medium conditioned or not with PEG solution were used as the index of PEG toxicity effect. Results showed that 20% PEG decreased N. langsdorffii wild type leaf discs survival from 100% of unstressed ones (0%PEG) to 26%, while shoot regeneration capacity was strongly inhibited in the presence of 5% PEG and fully inhibited in the presence of higher PEG concentrations (Supplementary Fig. S1).

Determination of the temperature selection range for heat stress induction

In order to choose the temperature conditions to be used for the induction of heat stress, a series of preliminary experiments were carried out. Electrolyte leakage (EL) of leaf discs of N. langsdorffii wild type plants, was used as an index of stress injury. In vitro whole plants of Nicotiana langsdorffii were subjected to heat stress, after four week of incubation on LS medium. The plants were maintained in a SANYO incubator, mod. MIR-153 (Richmond Scientific Ltd) under different heat stress intensities (25, 35, 44, 45, 46, 48 and 50°C) for 2 h. 10 leaf discs per test tube were placed in 5 mL 1 M sucrose and incubated at each temperature for 2 h. Samples were then allowed to equilibrate at room temperature for 30 min before measurement of EL. After measurement the discs were stored overnight in a -80°C freezer and EL was measured again to evaluate the total EL. Results obtained showed that temperatures up to 48°C did not cause any injury to leaf tissue (Supplementary Fig. S2).

Figure Legends

Figure S1. Dose response curve of leaf discs from N. langsdorffii wild type plants grown on RM media conditioned with different PEG concentrations. Standard deviations bars refer to three replicates; each replicate consists of 30 leaf discs.

Figure S2. Response of N. langsdorffii wild type plants (NLWT) to heat stress induction: electrolyte leakage of leaf discs from NLWT measured at different temperatures.