*2.4. Grafting Success, Biometric Parameters, Yield, and Apparent Fruit Quality Evaluation*

Grafting success was recorded after two weeks from grafting and was calculated on 100 grafted seedlings for each rootstock used. Plant height and root collar diameter (via a digital caliper) at 50, 80, and 110 days after transplanting (DAT), number of leaves at 50 DAT, and aboveground biomass produced at the end of fruit harvest (including total yield and vegetative part produced (weight of the plant at the end of harvests plus vegetative part removed by pruning after plant branching)) were determined. First flower formation (expressed as DAT) were also recorded.

Immediately after harvesting fruits were weighed. Total yield (kg plant-1), marketable yield (kg plant<sup>−</sup>1), and number of marketable fruits per plant were collected. Average marketable fruit weight (g) was calculated.

Color (a\* and b\* parameters -CIELab) was measured on four replications of five fruits per rootstock–scion combination. The records were taken on two opposite point of eggplant fruit skin (equatorial zone) by a colorimeter (Chroma-meter CR-400, Minolta Corporation, Ltd., Osaka, Japan). Hue angle (H◦) was calculated as follows: H◦ = arctan(b\*/a\*).

Fruit firmness was determined by measuring its resistance to the plunger of a digital penetrometer (Trsnc, Italy). Each fruit was subjected to a compression in two opposite point in the equatorial part using a 6 mm diameter stainless steel cylinder probe. The mean peak force was calculated in Newton (N).

Apparent fruit quality traits of Scarlatti eggplant fruit were measured in four replications of ten representative commercially mature fruits from non-grafted and self-grafted plants, and from plants derived from Scarlatti scions grafted onto *S. torvum* and accession 1 and 2 of *S. aethiopicum* gr. *gilo* hybrid rootstocks. Fruit length/width ratios were calculated. Several traits were measured in an arbitrary scale according to the European Eggplant Genetic Resources Network (EGGNET) descriptors [23]. These traits included fruit curvature (1 = none; 9 = U-shaped), fruit cross section (1 = circular; 9 = very irregular), fruit calyx length (1 = very short (>10%); 9 = very long (>75%)), and fruit calyx prickles (0 = none; 9 = very many (>30)). In addition to these EGGNET descriptors, seed index (0 = none; 5 = very many (>80) seeds visible in a longitudinal fruit section) was measured.

### *2.5. Pulp Browning, Soluble Solid Content, and Chemicals*

The colorimeter was also used to determine the lightness of fruit pulp by measuring L\* value (0 = black and 100 = white). Fruits were sectioned in the equatorial part and the color of the pulp was measured immediately after cutting (L0) and after 30 min (L30) in two areas (central and lateral) of the section. The oxidation potential was estimated using Larrigaudiere et al. [24] method with little modifications as in part suggested by Concellòn et al. [25]. The oxidation potential was expressed as ΔL30 = (L30 − L0).

Sampling for the quality analysis of the fruits was carried out as described by Sabatino et al. [10] and Sabatino et al. [26]. Thus, 3–5 commercially mature fruits for each replication from the second and third harvest were used; only healthy fruits were chosen. Care was taken to ensure that each sample contained the same percentage weight of apical, middle, and distal parts of the fruits. Qualitative fruit characteristic analyses were conducted on fruits harvested from labeled fruits (the flowers were labeled at the fruit set stage) and all fruits were harvested after 35 days from labeling (fruit commercial maturity stage).

Sample of the fruit pulp were squeezed by hand with a garlic squeezer. The juice was filtered and soluble solids content (SSC) was measured using a digital refractometer (MTD-045nD, Three-In-One Enterprises Co. Ltd. Taiwan).

Fruit dry matter percentage was determined in samples dried at 105 ◦C until constant weight as 100% × (dry weight/fresh weight).

Proteins, metals, total anthocyanins, chlorogenic acid, and glycoalkaloids were determined only in the second trial (2015). Protein concentration was determined from N content obtained from the Kjeldahl method. In particular, a sample rate was subjected to acid-catalyzed mineralization to turn the organic nitrogen into ammoniacal nitrogen. The ammoniacal nitrogen was then distilled in an alkaline pH. The ammonia formed during this distillation was collected in a boric acid solution and determined through titrimetric dosage. The protein concentration was reported as N × 6.25. Phosphorus content were assessed using colorimetry [27]. Ca, Mg, and K were determined using atomic absorption spectroscopy following wet mineralization [28].

Polyphenols were extracted and analyzed according to Stommel and Whitaker [29] with minor modifications. The analyses were performed through a Waters E-Alliance HPLC system constituted by a 2695 separations module with quaternary pump, auto sampler, and a 2996 photodiode array detector; data were acquired and analyzed with Waters Empower software on a personal computer. A binary mobile phase gradient of methanol in 0.01% aqueous phosphoric acid was used according to this procedure: 0–15 min, linear increase from 5 to 25% methanol; 15–28 min, linear increase from 25 to 50% methanol; 28–30 min, linear increase from 50 to 100% methanol; 30–32 min, 100% methanol; 32–36 min, linear decrease from 100 to 5% methanol; 36–43 min, 5% methanol. The flow rate was 0.8 mL/min. Quantification of chlorogenic acid (CA), carried out after a RP-HPLC separation, was based on absorbance at 325 nm relative to the sesamol internal standard and an external standard of authentic CA (Sigma-Aldrich, St. Louis, MO). The results were expressed as mg·100 g−<sup>1</sup> of fw.

The extraction and the analysis of anthocyanins were carried out on 200 mg of lyophilized and powdered peel as reported in Mennella et al. [30]. Briefly, the chromatographic separations were performed at a flow rate of 0.8 mL/min and at 0.1 absorbance units full scale (AUFS). Purified delphinidin-3-rutinoside (D3R, Polyphenols Laboratories AS, Sandnes, Norway) was used as external standard in RP-HPLC analyses, with a different retention time (23.9 min) compared to delphinidin-3-(p-coumaroylrutinoside)-5-glucoside (nasunin), that was eluted at a longer retention time (25.8 min for cis-nasunin and 26.1 min for trans-nasunin, respectively). As for nasunin quantification, a partially purified standard was used according to Lo Scalzo et al. [31]. The results were expressed as mg·100 g−<sup>1</sup> of peel fw; the limit of detection was 2.00 mg·100 g−<sup>1</sup> of peel dw.

Glycoalkaloids were extracted as described by Birner [32] with some modifications. Glycoalkaloid extraction was detected from 0.5 g lyophilized samples and powdered flesh tissue by 95% ethanol. The analyses were carried out by means of RP-HPLC according to Kuronen et al. [33] using partially purified solasonine and solamargine as the external standard. The data were expressed as mg·100 g−<sup>1</sup> fw; the limit of detection was 0.03 mg·100 g−<sup>1</sup> of dw.
