Plant topic and you can growth standards
Potato plants cv. Desiree was in fact sex when you look at the cuatro0 cm pots in the unheated glasshouses lower than natural light in compost. So you’re able to topic flowers to help you phony white/black schedules, vegetation was indeed transferred immediately following 35 days so you’re able to Sanyo Fitotron 1700 regulated ecosystem shelves and you may managed for a further two weeks toward fourteen h–10 h white-dark schedules with almost all the time temperature of twenty-two°C and you can 15°C correspondingly. White try provided by sixty W incandescent lamps to incorporate a beneficial photon flux out-of 900 ?mol m 2 s -1 towards the top of the fresh new canopy. Relative humidity try was able in the a constant 70% and flowers was basically watered day-after-day. Throughout cases tests was indeed performed toward tuberising flowers once 40–two months out of growing. From the text stolons was recognized as low-swelling (uniform diameter with each other terminal 15 mm) or tuberising (lump 2–5 mm diameter). Swellings between 5–ten mm diameter is actually identified as development tubers.
Quantification 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 (AsAt) i.e. reduced L -ascorbic acid + dehydroascorbic acid. Samples were then centrifuged at 16000 g for 5 min at 1°C and AsAt 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 AsAt was detected at 245 nm using a Gynkotech UVD 340S diode array detector (Dionex, Camberley, UK).
Recognition away from 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), AsAt 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 przeglÄ…d mamba 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.
