Plant Material, Root Induction and RNA Extraction

Pine (P. radiata D. Don) seeds were germinated, and seedlings were grown as previously described (Sánchez et al., 2007). The seedlings were watered daily with water, and, after 21 days, weekly with 2 g/l of a commercial soluble fertilizer [NPK 20-7-19 (w/w/w)]. Cuttings for adventitious root induction were prepared in accordance with Sánchez et al. (2007). Briefly, rooting-competent hypocotyl cuttings, including the intact epicotyl, from 21-day-old seedlings (cH21) and non-competent hypocotyl (ncH91) or epicotyl (ncE91) cuttings from 91-day-old pine seedlings were prepared by severing the hypocotyl or epicotyl at its base, trimming it to 2.5 cm from the cotyledons (hypocotyls) or from the apical bud (epicotyls). All but one apical tuft of needles were removed from the epicotyls to obtain a foliar surface similar to that of the hypocotyls. Root induction was conducted by exposing the cuttings to 10 μM indole-3-butyric-acid (IBA) continuously. The IBA was obtained from Sigma (St. Louis, MO, United States) as IBA-K and dissolved in distilled water. For the analysis of gene expression during adventitious rooting, 50 basal segments, 1 cm in length, of the hypocotyl or epicotyl cuttings were pooled for each treatment at time 0 (time of excision) and after 1, 2, and 6 days of IBA treatment. Basal segments from 50 hypocotyl or epicotyl cuttings maintained in distilled water were also collected at the same time points and used as controls (mock). Basal segments from treated and non-treated cuttings were immediately frozen in liquid nitrogen and stored at −70°C until used for RNA isolation. Protocols for total RNA isolation and quantification from cuttings have been previously described (Sánchez et al., 2007). Total RNA was extracted using an RNeasy^® Plant mini kit (Qiagen GmbH), following the manufacturer’s instructions. The RNA concentration and quality were determined using a ND-1000 Spectrophotometer (NanoDrop Technologies Inc., United States). RNA was prepared from three independent biological replicates.