Effect of Rootstock on the Volatile Profile of Mandarins

Mandarin production has increased in recent years, especially for fresh consumption, due to its ease of peeling, its aroma, and its content of bioactive compounds. In this sense, aromas play a fundamental role in the sensory quality of this fruit. The selection of the appropriate rootstock is crucial for the success of the crop and its quality. Therefore, the objective of this study was to identify the influence of 9 rootstocks (“Carrizo citrange”, “Swingle citrumelo CPB 4475”, “Macrophylla”, “Volkameriana”, “Forner-Alcaide 5”, “Forner-Alcaide V17”, “C-35”, “Forner-Alcaide 418”, and “Forner-Alcaide 517”) on the volatile composition of “Clemenules” mandarin. For this, the volatile compounds of mandarin juice were measured using headspace solid-phase micro-extraction in a gas chromatograph coupled to a mass spectrometer (GC-MS). Seventy-one volatile compounds were identified in the analyzed samples, with limonene being the main compound. The results obtained showed that the rootstock used in the cultivation of mandarins affects the volatile content of the juice, with “Carrizo citrange”, “Forner-Alcaide 5”, “Forner-Alcaide 418”, and “Forner-Alcaide 517” being those that presented the highest concentration.


Introduction
Citrus is one of the main cultivated fruits worldwide [1]. Among the different citrus fruits (oranges, lemons, limes, grapefruit, and mandarins), the mandarin (Citrus reticulata) is gaining popularity due to its economic and nutritional value [2]. Mandarin production has reached 38 million tons in 2020 [3]. Currently, China is the largest producer of mandarins (23.12 mln. tons), followed by Spain (2.17 mln. tons), Turkey (1.58 mln. tons), and Brazil (1.02 mln. tons) [3]. Throughout the world, citrus fruits are one of the most important fruits, especially in juice production [4,5]. However, mandarins are mainly consumed fresh, although they have a shorter shelf life than other citrus fruits [6]. In this sense, Spain has had notable success with its seedless clementine varieties in Europe and the United States [7]. The main reasons for the fresh consumption of mandarins are that they are easy to peel; have a desirable flavor; and their content of vitamin C (≈25.8 mg/100 mL), flavonoids (≈38.97 mg rutin equivalent g −1 DW), and total phenolics (≈59.3 mg GAE/100 mL) [6,8]. The presence of citrus phenolic compounds contributes to the sensory quality of the fruit, in addition to being associated with the reduction of cardiovascular diseases and some types of cancer [9,10]. Moreover, aromas and volatile compounds play a fundamental role since they are responsible for the flavor of the fruit, so aromas are an important contributor to the sensory quality of these fruits and their derivatives [11][12][13].
On the other hand, farmers depend not only on the yield but also on the quality of the fruit [14]. In this sense, rootstocks play an important role since they help crops adapt to climate and soil conditions, as well as being a method of defense against climate change [9]. The selection of the appropriate rootstock is crucial for the success of the crop [15]. The identification of markers linked to citrus flavor and aroma can facilitate genetic improvement and the release of new superior varieties [8]. Some authors have shown that rootstocks affect the quality of citrus fruits, for example soluble solids content, acidity, ripening index, composition sugars and organic acids, antioxidant activity, and total phenolics, among others [14,16,17]. Currently, consumers demand higher-quality fruit that is produced sustainably [18]. Therefore, obtaining higher-quality citrus (internal and external) is essential. Then, the new studies carried out no longer focus exclusively on the yield and optimization of crops but instead choose to evaluate the effect of rootstocks on the quality of fruits [9]. Furthermore, there is little information on the effect of rootstock on volatile compounds in citrus.

Plant Material
"Carrizo citrange", "Swingle citrumelo CPB 4475", "Macrophylla", "Volkameriana", "C-35", and four new hybrid selections, obtained in the rootstock breeding program carried out at IVIA (Instituto Valenciano de Investigaciones Agrarias) since 1974 (Table 1), were tested as rootstocks for "Clemenules" (selection virus-free INIASEL 22). Seeds of "Carrizo" citrange and "Cleopatra" mandarin were obtained from the germplasm collection of rootstocks at IVIA, and the seeds of the hybrids were obtained from the plants obtained in the citrus rootstock breeding program. Table 1. Pedigree of the nine rootstocks tested for "Clemenules" mandarin.

Preparation of Juice
The mandarin "Clemenules" (Citrus clementina Hort. ex Tan.) fruits were harvested at optimum maturity (>12 • Brix). The juice preparation was carried out according to the methodology proposed by Legua et al. [17].

Volatile Composition
The determination of volatile compounds in the mandarin juice was carried out following the method described by Cano-Lamadrid et al. [19], using the headspace solidphase micro-extraction (HS-SPME) method with slight modifications. A SPME 50/30 mm DVB/CAR/PDMS (Divinylbenzene/Carboxen/Polydimethylsiloxane) fiber (Supelco) was used for the extraction. The exposure time was 50 min at a temperature of 40 • C and with constant agitation (600 rpm). Then, desorption of the volatile compounds from the fiber was carried out in the injection port of the gas chromatograph for 3 min at 230 • C. Volatile compounds were analyzed and identified using a Shimadzu GC-17A gas chromatograph coupled to a Shimadzu QP-5050A mass spectrometer (Shimadzu Corporation, Kyoto, Japan). The analysis was carried out from 45 to 400 m/z with an electronic impact (EI) of 70 eV in 1 scan/s mode. The GC-MS system consisted of a TRACSIL Meta X5 column containing 95% dimethylpolysiloxane and 5% diphenylpolysiloxane (Teknokroma S. Co., Ltd., Barcelona, Spain; 30 m × 0.25 mm i.d., 0.25 µm film thickness). The oven program started at 80 • C with an increase of 3 • C/min from 80 • C to 210 • C and hold for 1 min. After this, an increase of 25 • C/min from 210 • C to 300 • C was maintained for 3 min. The injector and detector temperatures were 230 and 300 • C, respectively. Helium was used as the carrier gas (column flow rate of 0.6 mL/min).
Three methods were used to identify volatile compounds: (i) retention rates and their comparison with the literature; (ii) retention times of pure chemical compounds; (iii) mass spectra of authentic chemical compounds and the spectral library of the National Institute of Standards and Technology (NIST) database. Only fully identified compounds have been described. The analysis of the volatile composition was run in triplicate.

Statistical Analysis
To carry out the statistical analysis, the software XLSTAT (Addinsoft 2016.02.270444 version, Paris, France) was used. Two-way analysis of variance (ANOVA) and Tukey's multiple range test were used to compare experimental data and determine significant differences between rootstocks (p < 0.05). Principal component analysis (PCA) using Pearson correlation was also run.
These results demonstrate that rootstocks significantly affect the volatile composition of citrus. Similar results were found by Aguilar-Hernández et al. [16] in lemon fruits. In the same way, Castle [14] showed that rootstocks have effects on the quality factors of citrus fruits. The rootstocks under study were also studied by Legua et al. [17], showing their influence on the composition of bioactive constituents in mandarins. Furthermore, Saini et al. [30] found that "Kinnow" mandarin juice grafted on "Pectinifera" had the highest levels of limonene and therefore the highest values of total volatile compounds, while the same mandarin grafted on "Shekwasha" had the highest levels of β-pinene, dodecylaldehyde, octanal, α-terpineol, terpinen-4-ol, peraldehyde, nonanal, isoleucine, linalool, and hexanal. Furthermore, Raddatz-Mota et al. [31] discovered that rootstocks not only affect the volatile profile but also have an effect on the presence or absence of certain volatile compounds in the fruit. This was the case for "Persian" lime, in which β-myrcene was only found in two of the five rootstocks studied, while the compounds β-thujene and dodecane were only found in the rootstocks "Volkamer" lemon and "C-35".

Conclusions
The results obtained show that the rootstock used in the cultivation of mandarins affects the volatile content of its juice. In this case, the rootstocks that showed the highest volatile concentration were "Carrizo citrange", "Forner-Alcaide 5", "Forner-Alcaide 418" and "Forner-Alcaide 517", while "Macrophylla", "Volkameriana", and "C-35" were the least. However, more research is needed to assess the effects of the environment and other factors on rootstocks and their effect on citrus juice properties. Funding: This research was funded by the Ministerio de Ciencia e Innovación with the project: "Obtención, selección y evaluación de cítricos para conseguir plantaciones más sostenibles frente a las nuevas amenazas debidas al cambio global" (PID2021-124145OR-C21). Paola Sánchez-Bravo was funded by the grant for the recall of the Spanish university system for the training of young doctors (Margarita Salas, 04912/2021), funded by the European Union-Next Generation EU, Ministry of Universities of Spain.