Introduction

Trans-resveratrol (t-RSV; 3,5,4′-trihydroxy-trans-stilbene) is a phytoalexin of stilbenes group. It is found at considerable amounts in grape berries and products. t-RSV has been detected in different parts of the grapevines such as seed and pulp (Pezet and Cuneat 1996), cluster stems (Bavaresco et al. 1997a), cane (Karacabey and Mazza 2008), and especially berry skin (Creasy and Coffee 1988; Jeandet et al. 1991; Roggero and Garcia-Parrilla 1995; Romero-Pérez et al. 2001). The genotypic potential is one of the most important factors affecting t-RSV production in grape berries (Okuda and Yokotsuka 1996). Preliminary data clearly revealed that some grape cultivars describe to have low t-RSV production capacity (‘Kalecik Karası’, ‘Aglianico’ ‘Schiava Grossa’ and ‘Nebbiolo’) while some (‘Erciş’ ‘Öküzgözü’, ‘Cabernet Sauvignon’, ‘Pinot noir’, ‘Lemberger’, ‘Marsanne’) have higher contents (Keskin and Kunter 2007, 2008, 2009, 2010; Castellarin et al. 2012). Environmental factors and cultural practices notably affects t-RSV production potential not only among the grape cultivars but also in a cultivar depending on the vintage year (Negri et al. 2008; Gökçen et al. 2017; Keskin 2017; Keskin and Kunter 2017). So, t-RSV accumulation in the berries of a cultivar can increase as a reply of biotic and abiotic stress conditions (Revilla and Ryan 2000; Adrian et al. 2000).

The purpose of clonal selection is to select superior individuals of a grape variety in its original populations considering the yield and quality characteristics. Therefore, variations among the clones of a grape cultivar may contribute to a better understanding of their genetic potential for t-RSV accumulation (Bavaresco and Fregoni 2001). ‘Kalecik Karası’ is one of the leading red wine grape variety of Turkey, grown along the valley of Kızılırmak river near Kalecik located at 70 km east of Ankara. This cultivar with its violet-ruby colored and aromatic berries gives soft, lively, easy-to-drink and medium-sized bodied wines with elegant and delicate structure (Çelik et al. 2019). T-RSV content in ripened grapes and wines of ‘Kalecik Karası’ has been studied in some previous studies (Anlı and Vural 2009; Çelik et al. 2019). Although agronomic traits of ‘Kalecik Karası’ clones are well known, our knowledge on their t-RSV capacity is still limited. A unique and detailed clone selection study was conducted by Çelik et al. (2019) on 23 clones of ‘Kalecik Karası’ including the t-RSV contents of their berry skins and seeds in its original ecology.

The objectives of the present study were to determine the t-RSV production performance of 23 ‘Kalecik Karası’ clones by use of total berry extraction method and to compare the performances with statistical comparison technique (Analysis of mean ANOM).

Material and Method

Material

The study was carried on the 23 Clones of ‘Kalecik Karası’ (Vitis vinifera L. cv.) which were selected as the result of clone selection project (Fidan et al. 1975, 1988, 1991; Çelik et al. 2019) and were cultivated in the clonal selection vineyard which was established in 1999 at the Research Station for Viticulture in Kalecik, Ankara, Turkey.

Method

Collecting the Grape Berry Samples

Berries were collected randomly when total soluble solids contents of the berries reached at 22–24 °Brix, according to Amerine and Cruess (1960) during the vintage of 2016–2017, and then were stored in a deep freezer at −25 oC within plastic bags, up to extraction.

Determination of t-RSV Content of the Berries

Grape berries were thoroughly ground with a mortar and pestle, then extracted in darkness at room temperature with an orbital shaker vigorously for 20 min in 30 ml of 95% methanol. After the mixture was filtered through GF/A Whatman filter paper, the filtrate was dried in vacuum at 40 °C using a rotary evaporator. During the drying process, the water phase was extracted twice with ethyl acetate and 5 ml of 5% NaHCO3. The organic phase was evaporated till to dryness and t-RSV was recovered twice with 1 ml of methanol (100%). The extract was stored in brown glass bottles at 4 °C until analysis (Bavaresco et al. 1997b).

T-resveratrol was analyzed with HPLC (High Pressure Liquid Chromatography) and standard for this analysis was supplied by Sigma (Germany).

HPLC device was used with Agilent 1100 (Agilent, USA), Phenomenex/Luna guard column (5 µm, 12.5 × 4.6 mm, ID), Phenomenex/Luna C18 column (5 µm, 250 × 4.6 mm, ID) and UV-VIS detector system in accordance with Jeandet et al. (1997). T-RSV values were obtained at 330 nm wavelength.

Statistical Analysis

Descriptive statistics for t-RSV content of the clones were expressed the mean and standard error. One-way analysis of variance (ANOVA) was performed to compare means of clones. Following the ANOVA, Duncan multiple comparison test was used to determine the comparative performances of the clones. Additionally, the ANOM test was also performed to determine the differences of the clones from the overall mean and to present visually the results. For all statistical comparisons, the significance level was considered as 5% and MINITAB statistical package program was used for all statistical computations.

Results and Discussion

The t-RSV contents of 23 clones of ‘Kalecik Karası’ red wine grape cultivar are shown in Table 1. The highest t-RSV content was measured in Clone 7 (1.67 mg kg−1) and the lowest in Clone 22 (0.31 mg kg−1).

Table 1 Descriptive statistics and comparative performances of the clones of ‘Kalecik Karası’ in terms of t-RSV contents of the berries
Full size table

Fig. 1 has also presented the similarities of the clones in terms of t-RSV. Upper and lower decision lines were 0.807 mg kg−1 and 0.616 mg kg−1, respectively. Clones numbered as 1, 2, 3, 5, 6, 7, 8, 10, 16 and 21 were significantly different from the 0.712 mg kg−1 overall mean with the outside of the upper decision line. Similarly, clones numbered as 4, 9, 11, 12, 13, 14, 15, 17, 18, 20, 22 and 23 were found to be significantly different from the overall mean by out of the lower decision line. In addition, the differences among the clones that below and above of the decision lines were also statistically significant. Thus, it can be stated that there is a considerable difference among the clones in terms of t-RSV content.

Fig. 1
figure 1

ANOM diagram for t-RSV content of ‘Kalecik Karası’ clones

Full size image

Clone selection studies have focused more on yield and quality. T-RSV content of the grape berries that can be considered as a quality factor in grape cultivars has been affected by cultivars (Bavaresco et al. 2007; Gatto et al. 2008), clone (Gatti et al. 2014), meteorological conditions (Bavaresco et al. 2007), soil (Bavaresco et al. 2005) and cultural practices (Gebbia et al. 2003; Bavaresco et al. 2007, 2008; Gatti et al. 2011). There are almost no studies to compare the clones in terms of t-RSV content.

Iacopini et al. (2008) found quite low t-RSV content in the clones of ‘Italian Sangiovese’ cultivar (AP SG 1, F9, ISV 2, ISV, RC1). There was no difference in t-RSV content of ‘Cabernet Sauvignon’ clones (169, 191, 338, 341, 685, ISV105, ISV117, ISV2, R5, VCR8) although there were significant differences between the clones in terms of other stilbene compounds such as piceid, piceatannol and ε‑viniferin (Gatti et al. 2014). Çelik et al. (2019), compared 23 clone of ‘Kalecik Karası’ grape cultivar for t-RSV content in the berry skin and seed separately. According to their data, the berry skin t-RSV content showed significant difference between years. Clone 1 had the highest value with 1.54 µg g skin FW−1 in 2008 while Clone 23 with 1.67 µg g skin FW−1 in 2009. Similarly, t-RSV content in the seed ranged from 1.10 µg g skin FW−1 (Clone 1) to 0.40 µg g skin FW−1 (Clone 17) in 2008.

Conclusion

Genotype, rootstock, climate, soil and cultural practices as well as biotic and abiotic stress factors affect directly or indirectly the production of t-RSV in grapes. The investigation of t-RSV content whether in lower and upper limits can be a criterion for improving clonal selection. Therefore, this study was carried out to contribute better understanding of the t-RSV production whether depends on the genetic performance of the clones of ‘Kalecik Karası’ that is one of the leading red wine cultivars of Turkey. t-RSV content in 23 clones of ‘Kalecik Karası’ varied between 0.31 mg kg−1 and 1.67 mg kg−1. It was concluded that clonal variation is considerable in 23 clones and Clone 7 has the highest capacity with 1.67 mg kg−1 for t-RSV content. Also statistical similarity analysis showed that selected clones of ‘Kalecik Karası’ can be grouped for their t-RSV accumulative capacities.