Chromeleon v 7

In order to study the phylloxera effect on carbohydrates content and partitioning, soluble NSC (glucose, fructose, sucrose) and starch were measured at the end of the treatment in different plant tissues (leaf apex, noninfested leaf, leaf galls, infested and noninfested root tips, see Section 2.3). After being ground to fine powder in liquid nitrogen and freeze-dried for 48 h, about 15 mg of sample were suspended in 1.5 mL of 80% ethanol. The soluble sugars were extracted through three cycles of heating (at 90 • C), centrifugation and supernatant collection [20, (link)21] (link). The starch contained in the remaining pellet was digested with α-amylase (70 u, 1 mL per sample) followed by amyloglucosidase (6 u dissolved in 0.5 mL, per 0.1 mL of subsample) solutions. In total, 0.6 mL of chloroform were added to each sample to precipitate the enzymes [21] (link); all the supernatants were then filtered through a syringe nylon filter with pore size of 0.45 µm and, after appropriate dilution, analyzed with ion exchange chromatography (Dionex, ICS-5000, Thermo Fischer Scientific, MA, USA). Glucose, fructose, sucrose, and starch content (digested to glucose hydrolysate) were quantified using their reference standards with the software Chromeleon (v 7.2, Thermo Scientific, MA, USA). For additional details see Savi et al. [5] (link).