The evidence we provide here for long-length transportation from AsA when you look at the potato flowers, corroborates prior to results into the A

Bush material and you can growth criteria

Potato plants cv. ceny sugar faddy for me Desiree was in fact sex in the cuatro0 cm containers within the unheated glasshouses significantly less than sun light in compost. So you’re able to topic plant life so you’re able to artificial light/black schedules, plants have been directed immediately following 35 months so you can Sanyo Fitotron 1700 controlled ecosystem cabinets and you will maintained getting a deeper 14 days on the fourteen h–ten h light-dark schedules that have day and night temperature of twenty two°C and you can fifteen°C respectively. Light is actually provided with sixty W incandescent lamps to include an excellent photon flux out of 900 ?mol meters dos s -step 1 on top of the new cover. Relative humidity are handled during the a stable 70% and you can vegetation had been watered every single day. In all circumstances tests was basically did to your tuberising vegetation just after 40–two months out of growing. Regarding text stolons is identified as low-swelling (uniform diameter together terminal fifteen mm) or tuberising (lump 2–5 mm diameter). Swellings between 5–ten mm diameter try identified as developing tubers.

Measurement out-of AsA in plant structures

Tissue was extracted in a mortar and pestle with ice-cold 5% metaphosphoric acid (MPA) containing 5 mM tris(2-carboxyethyl)phosphine hydrochloride TCEP (9:1 v/w). Samples were then held on ice for 60 min to allow reduction of dehydroascorbic acid to AsA therefore, all data are reported as total AsA pool (AsA_t) i.e. reduced L -ascorbic acid + dehydroascorbic acid. Samples were then centrifuged at 16000 g for 5 min at 1°C and AsA_t in the supernatant quantified by HPLC according to the method of Hancock et al . Briefly, 20 ?l of sample supernatant were injected onto a 300 ? 7.8 mm ID Coregel 64H ion exclusion column (Interaction Chromatography, San Jose, CA, USA) with a 4 ? 3 mm ID carbo-H + guard cartridge (Phenomenex, Macclesfield, UK) maintained at 50°C. Mobile phase was 8 mM H₂SO₄ at 0.6 ml min -1 and AsA_t was detected at 245 nm using a Gynkotech UVD 340S diode array detector (Dionex, Camberley, UK).

Detection out of AsA in the phloem

Phloem exudates were collected from the petiole of source leaves or tuberising stolons using an adaptation of the method developed by King and Zeevart . Following excision of the organs, a portion of the petiole (5 mm) or stolon (10 mm) was removed under water, the sample was rinsed and the cut end transferred to a 0.6 ml reaction tube containing 200 ?l 15 mM EDTA pH 7.5. In the case of petioles, samples were transferred to a pre-humidified atmosphere at 20°C and exudate collected for 90 min in the dark. In the case of stolons, exudates were collected from the cut end which remained attached to the plant and moist paper was wrapped around the top of the reaction tube to minimise evaporation. Control samples were run in parallel in which petioles or stolons were incubated in 5 mM CaCl₂ pH 7.5 to induce callose gellation and reduce exudation . At the end of the incubation, MPA and TCEP were added to the samples to a final concentration of 5% and 5 mM respectively. Following centrifugation (16000 g, 1°C, 5 min), AsA_t concentration was determined by HPLC as described above. Histochemical localization of AsA in tubers using the AgNO₃ method was carried out as previously described . Briefly, tubers were hand sliced to form approximately 2 mm sections, washed in distilled water and fixed and stained in 5% (w/v) AgNO₃ dissolved in 66% (v/v) aqueous ethanol containing 5% (v/v) glacial acetic acid at 3°C in the dark for up to 24 h. The reaction was stopped by washing the tissue twice for 15 min in ethanolic ammonium hydroxide (95% (v/v) 70% ethanol, 5% (v/v) NH₄OH ACS reagent, Sigma-Aldrich, Dorset, UK) . Finally the tissue was transferred to 70% (v/v) ethanol and stored at 3°C prior to photography.