A11 basic analytical mill

The lower (bases), middle (stipe), and upper (pileus) parts of dried young G. sp. 2a (9) basidiocarps were separated, homogenized sequentially using an IKA A11 Basic analytical mill (IKA Werke GmbH & Co. KG, Staufen im Breisgau, Germany) and a MM 301 oscillating mill (RETSCH GmbH, Haan, Germany), and used for the analysis of the elemental and chemical composition.

For metabolomic analysis, apple and pear fruits were cut into halves and the true fruit parts (core and seeds) were removed using a small spoon (Schnitzmesser-set Professional; Triangle, Solingen, Germany). From each half, we prepared four equal pieces including the exocarp (skin) and mesocarp (pulp) using a professional tool for fruit sculpture (Schnitzmesser-set Professional). Two pieces were frozen in liquid nitrogen and stored at −80 °C as technical replicates. The other two pieces were frozen in liquid nitrogen and powdered using an A11 basic analytical mill (IKA-Werke, Staufen, Germany) and stored at −80 °C. For quality analysis, the °Brix, pH and hardness were determined for 30 fruits as previously described [14 (link)]. Briefly, the hardness was measured with a PCE-PTR 200N sclerometer (PCE Instruments, Lucca, Italy) at four fruit positions, then six pools of five fruits were created and the juices were extracted with a mixer. A micropH 2001pH meter (Crison, Carpi, Italy) and a DBR 35 digital refractometer (Kingstic, Ningbo, China) were used to assess the pH and °Brix, respectively.

After sampling, whole plants were immersed in liquid nitrogen and lyophilized, then ground to powder using a mill (A11 basic analytical mill, IKA^®-Werke GmbH &Co., KG, Staufen, Germany), and stored until further analysis.
The extraction was performed according to the protocol described by Medic et al. [5 (link)]. Briefly, 0.2 g of the previously lyophilized sample was extracted at a tissue-to-solution ratio of 1:30 (w/v) using an extraction medium of 80% methanol and 3% formic acid in water.

Tomato plants at the flowering stage grown in the above-mentioned conditions were used for sampling roots, hypocotyl, cotyledons, first leaves following cotyledons, true leaves from six different nodes, the stem (separate internodes), flower buds and flowers at anthesis. Roots were further separated into the primary root, primary-proximal secondary root and primary-distal secondary root. Another group of plants was grown to complete the life cycle and, following fruit set, fruits were collected at various developmental and ripening phases based on phenological observation: immature-green (berry still growing), mature-green (fruit at maximum size), early breaker (green-white/yellow fruit), late breaker (red-orange turning), ripe and over-ripe. Immature-green and mature-green fruits were pooled for the analysis of exocarp (peel), mesocarp (flesh) and seeds, and the same strategy was applied to ripe and over-ripe fruits. All samples consisted of three biological replicates, each represented by a pool of material collected from five different plants. The samples were immediately frozen in liquid nitrogen and ground to a fine powder using an A11 basic analytical mill (IKA-Werke, Staufen, Germany). The powder was stored at −80°C.

The AED panel was analyzed for seed, carbon and sulfur contents. Seed sample preparation was performed by drying the seeds, pooling approximately six seeds from two plant replicates and milling into fine powder using an electric milling machine (IKA A11 basic analytical mill, IKA-Werke GmbH & Co. KG, Staufen, Germany) at maximum speed for about 1 min to obtain a homogenous powder. Analysis was carried out using the Elementar Analyzer Vario EL Cube (Elementar Americas Inc., Ronkonkoma, NY, USA) according to the company’s user’s manual. The analysis was carried out in triplicates. For each sample, seed protein content was estimated by multiplying seed nitrogen content with a conversion factor of 6.25 (AOAC, 1990 ).

Determination of the chlorophyll (chl) level in P. dulcis and P. arabica stems and leaves was done as described in Porra (2002 (link)) with the following modifications. Leaves were sampled, scanned, and immediately frozen in liquid nitrogen. Approximately, 3 cm (in length) stem-cuts were collected from the 1^st-year (current year growth), and the 2^nd year stems (stems from last year growth). Each sample was weighed, measured for its diameter using a caliber, and immediately frozen in liquid nitrogen to arrest any metabolic activity. The surface area of both leaves and stems was calculated as described above (in surface area evaluation). Leaves and stems were then grounded in liquid nitrogen, using a mortar and pestle (leaves) or Ika A11 basic Analytical mill (IKA®-Werke GmbH & Co. KG, Germany; stems). Chl was extracted in 80% acetone in phosphate buffer pH 7.8, at 4°C in the dark. The supernatant from two consecutive overnight extractions was combined for each sample. Absorbance (A663.6/A646.6) was measured (CARY 50 Bio UV-visible Spectrophotometer, Agilent Technologies Inc., Santa Clara, CA, USA), and chl concentration was calculated (Porra, 2002 (link)).