An Alternative to Increase Accumulation of Phenolic Compounds in Grapevine Callus Cultures: Chemical Mutagen Applications

This research was carried out to investigate the effects of chemical mutagens on producing phenolic compounds in calli obtained from Royal grape variety. Petioles were cultured in B5 medium (0.5 mg/L benzyl amino purine and 0.5 mg/L indole acetic acid) to obtain callus. Calli were subcultured in the same nutrient medium three times at one month intervals. In the fourth subculture, the calli were transferred to media with the same composition containing ethyl methanesulfonate, sodium azide, azacitidine and acridine orange in three different doses (0.5 mM, 1.0 mM, and 2.0 mM) and cultured in these mediums for 60, 120 and 180 minutes and then they were cultured for four weeks in mutagen-free nutrient mediums with the same content as their previous mediums. After that total phenolic compound, total flavanol, total flavonol and anthocyanin analyzes were performed. As a result, all mutagens applied are effective in increasing the production of phenolic compounds depending on the dose and time.

Anthocyanins in the flavonoid class of phenolic compounds contain natural red, blue, and purple color pigments. Although 22 different anthocyanin types are known, the most important are delphinidin, malvidin, pelargonidin, peonidin, petunidin, and cyanidin. Anthocyanins can be used as natural food additives to increase the attractiveness of food and beverages by providing natural coloring on the one hand (Jackman et al., 1993), and in the food industry to increase the shelf life of foods on the other hand. Anthocyanins, like other phenolic compounds, are anticarcinogenic (Rossi et al., 2003) and antioxidant (Kahkönen et al., 2001;Kahkönen et al., 2003;Viljanen et al., 2004) and are used in the pharmaceutical industry (Zhang and Furusaki, 1999).
Various difficulties are encountered during the production of these compounds, which have a wide range of uses from plants under natural conditions. The main problems encountered are that the collection of these plants is sometimes difficult and expensive, the danger of extinction of some species as a result of the collection of rare plants from nature in large quantities, the quantity, and quality of the compounds are affected by climatic conditions or the need for large agricultural areas and intensive labor for the production of effective substances in economic amounts due to their synthesis at certain stages of development and in very small amounts. In recent years, studies have been carried out to ensure that these compounds can be obtained in high amounts and purity by in vitro techniques. Producing secondary metabolites with biotechnological methods has many advantages. With these methods, environmental factors (climate, geographical difficulties, seasonal restrictions) are eliminated, less land use is provided, the danger of extinction due to the collection of the plant from nature is prevented, the ability to produce sufficient amounts of economically valuable metabolites found in plants in low quantities, homogeneity, standard quality and efficiency in production, and effectiveness in understanding the biosynthesis mechanisms of metabolites are provided. The production of secondary metabolites with biotechnological methods can be done faster and more reliably than classical methods. Among in vitro methods, especially callus and cell culture techniques, enable the rapid and reliable production of secondary metabolites on a large scale compared to other methods. This advantage provided by in vitro techniques has led researchers to work on these issues. In one of these studies, Hovhannisyan et al. (2011) found that oleandrin, oleandrigenin, and odoroside compounds were stimulated in the callus culture of oleander; Çölgeçen et al. (2012) stated that in the iridescent flower (Centaurea tchihatcheffii), callus cultures and the production of flavonoids and terpenoids from secondary metabolite groups increased. Estrada Zuniga et al. (2012) stated that the content of fatty acids (lauric, myristic, pentadecanoic, palmitic, and stearic acids) and phenolic compounds increased in callus culture in Ibervillea sonorae, which is a succulent. Besher et al. (2014) recorded an increase in the production of tropane alkaloids, hyoscyamine, and scopolamine content in callus culture in henbane (Hyoscyamus aureus). Bibi et al. (2018) showed that the amount and antioxidant capacity of phenolic compounds and flavonoids among secondary metabolites increased in black cumin in callus culture; Arijanti and Suryaningsih (2019) found that the biosynthesis of gingerol, shogaol, and zingerone, which are secondary metabolites, increased in callus cultures of the ginger plant.
Studies have also shown that biotic or abiotic elicitors (drought, temperature, salinity, heavy metal, etc.) applied to plants in in vitro conditions cause significant increases in the number of secondary metabolites as a response of the stress mechanism in plants ( Verporte et al., 2002;Commun et al., 2003;Vanisree ve Tsay, 2004;Grzegorczyk-Karolak et al., 2015;Sharma et al., 2015;Cardoso et al., 2019). It has been determined that various stress-inducing elicitors such as polysaccharides, jasmonic acid/methyl jasmonate, heavy metal, light radiation (Çetin, 2010), UV rays (Çetin, 2014;Çelik et al., 2020;Oğuz et al., 2020) can be successfully applied in the synthesis of secondary metabolites. In these studies, Bulgakov et al. (2002) recorded an increase in anthraquinone production with salicylic acid, methyl jasmonate, ethephon, and cantharidin applications in callus cultures of madder; Blando et al. (2005) obtained an increase in the number of cyanidin glucosides using jasmonic acid elicitor in cherry callus culture. Katerova et al. (2013) reported that UV-B and UV-C radiation stimulated plant secondary metabolite production in callus cultures; Kochan and Chmiel (2013) have managed to increase the production of ginsenocytes by using different photoperiods in ginseng callus culture. Benítez García et al.  Purwianingsih et al. (2019) reported that chitosan extract obtained from shrimp shell as an elicitor in callus cultures of Noni (Morinda citrifolia L.) plant increased the anthraquinone content. Apart from these stress-inducing elicitors, another factor that causes stress in plants is mutagens that cause mutation.
The application doses of mutagens depend on the type of mutagen and the material to be used. Some mutagens are lethal when used in high doses. Others cannot produce the desired mutations at low doses. The main reason for this is that chromosomes can repair weak mutations in their bodies over time. With the ability of chromosomes to repair at low doses, the desired mutations can be prevented. In this research, the effects of mutagen applications that cause stress in the plant on the production of phenolic compounds were investigated. Studies have shown that vine, grape, and products derived from grapes are rich in phenolic compounds (Revilla and Ryan, 2000;Murthy et al., 2002) and known to contain benzoic acids, hydroxycinnamic acids, stilbene derivatives (resveratrol), flavanols (catechin, epicatechin), flavonols (kaempferol, quercetin) and anthocyanins (Vinson and Hontz, 1995;Ghiselli et al., 1998). In this study, the grapevine was used as plant material. It is known that mutagen applications are mainly used in viticulture to create a genetic variation to improve mutation breeding, cluster density, berry color, aroma, seed properties, and ripening time, increase resistance to diseases and pests, and strengthen tolerance to environmental stress. This study aimed to increase the level of phenolic compounds by mutagen application in calli obtained from petiole of the Royal grape variety.
Within the scope of the research, chemical mutagens of acridine orange, azacitidine, ethyl methanesulfonate (EMS), and sodium azide were applied to calli at different concentrations and durations, and it was tried to reveal the effects of these mutagens on the total phenolic substance, total flavanol, total flavonol and anthocyanin content.

Material
In the research, Royal grape variety was used as plant material. Royal is a variety with very large and round berries it has purplish-black color, matures in the mid-late season, has a variety-specific aroma, and is for table use.
This study, which was carried out in the tissue culture laboratories, culture preparation, and culture development rooms within the Yozgat Bozok University Faculty of Agriculture, Department of Horticulture.

Methods
The leaves were taken from the collection vineyard of our university and brought to the laboratory. The petioles were separated and subjected to surface disinfection in a 15% commercial sodium hypochlorite solution for 10 minutes and then rinsed three times with sterile distilled water. They were then cut in lengths of approximately 0.5-1 cm, inoculation in B5 (Gamborg et al., 1968) nutrient mediums containing 0.5 mg/L benzyl amino purin (BAP) and 0.5 mg/ indole acetic acid (IAA), and cultured at 25 ± 1ºC under dark conditions. After the callus was obtained in these culture conditions, the calli were subcultured three more times at one-month intervals in the same environment and under the same culture conditions. After providing a sufficient amount of callus production for mutagen applications, calli were transferred to nutrient mediums containing acridine orange, azacitidine, EMS, and sodium azide at three different concentrations of 0.5 mM, 1.0 mM, and 2.0 mM and cultured in these mediums for 60, 120 and 180 minutes. Mediums without mutagen were used as a control. Then, they were cultured in mutagen-free nutrient mediums with the same content for four weeks at 25 ± 1 °C under 16/8 light/dark conditions. At the end of this period, the calli were taken from the nutrient medium they were in, and kept in a deep freezer at -20 o C until the analysis was done. Total phenolic compound, total flavanol, total flavonol, and anthocyanin analyzes were carried out to determine the effects of mutagen applications on phenolic substance production in calli.

Phenolic compound extractions
The extraction processes were made based on the method of Kiselev et al. (2007) to determine the total phenolic substance, total flavanol, and total flavonol amounts.

Determination of total phenolic compounds
Total phenolic compound analyzes were performed using Folin-Ciocalteu colorimetric method according to Singleton and Rossi (1965), and the readings were done at 765 nm wavelength in a spectrophotometer. The total phenolic compound amounts were determined as mg/g fresh weight (FW) as gallic acid equivalent by using the curve prepared from the standard gallic acid solution.

2.2.3.Determination of total flavanols
Total flavanols were produced by using DMAC (dimethylaminocinnamaldehyde) method, according to Arnous et al. (2001). The results are given in the form of mg/g (FW) as the equivalent of catechin by using the curve prepared from the catechin standard.

2.2.4.Determination of total flavonols
Total flavonols were made using Neu solution according to Dai et al. (1995). The number of total flavonols was determined in mg/g (FW) as a rutin equivalent by using the curve prepared from the rutin standard.

2.2.5.Determination of anthocyanin
Anthocyanin analyzes were performed using Mcllvaine's buffer (pH =3) according to the method used by Qu et al. (2006). Spectrophotometer readings were done at a wavelength of 535 nm, and anthocyanin amounts were calculated as color values (CV) according to the formula below. CV = 0.1 x absorbance x dilution factor 2.2.6. Statistical analysis All applications and analyzes in the research were carried out in three replications. SPSS (20.0) statistical analysis program was used to evaluate numerical data, and Duncan's multiple range test was used to determine the differences between applications.

Results and Discussion
The study determined that chemical mutagens applied to calli positively affect the number of phenolic compounds, depending on the dose and time.

Application of acridine orange
The first of the chemical mutagens applied in the study was acridine orange, and it was determined that this application was effective on the total phenolic compound, total flavanol, total flavonol, and anthocyanins in different ways (Table 1). There are differences in metabolite accumulation according to the dose and duration of the mutagen in terms of total phenolic compound amount; It is seen that 60 minutes of 0.5 mM dose and 60 and 180 minutes of 2.0 mM dose are the most suitable combinations in terms of total phenolic compound. It is also noteworthy that this application increased the total phenolic compound content 16 times compared to the control of the same periods. In terms of total flavanols, a 0.5 mM/60 minutes application was effective on total flavanol amounts and in total phenolic compounds (0.022 mg/g), and in the same period, 2.75 times more flavanol production was realized compared to the calli in the control group. In terms of total flavonols, 60 minutes application of 2.0 mM dose, which is the highest dose, was determined as the combination, which was the only effective one among the applications, and 25 times more flavonols compared to the control group were obtained. In the anthocyanins determined in terms of color value, the highest increase in acridine orange application with a value of 25.015 CV/g was obtained from calli in the 1.0 mM/120 min group.

Application of azacitidine
The changes in phenolic compound contents of calli to which azacitidine mutagen was applied are presented in Table 2. As can be understood from the examination of the table, the application of 0.5 mM azacitidine for 180 minutes provided the highest values in total phenolic, total flavanol, and total flavonol contents. In terms of total flavonol, it is seen that doses of 2.0 mM are also effective for all periods. When an examination is made in terms of anthocyanin, it is seen that the highest anthocyanin values were obtained in 60 and 120 minutes of a 1.0 mM dose and 60 and 180 minutes of a 2.0 mM dose, and the values obtained here were approximately 20 times higher than the lowest content.

Application of EMS
When the effect of EMS application on phenolic compound contents in calli was examined, the highest total phenolic substance contents were obtained from the applications of 0.5 and 1.0 mM doses for 180 minutes with 0.708 and 0.757 mg/g (Table 3). 1.0 mM EMS application for 180 minutes was also the combination in which the highest flavanol content was obtained. Combinations of 0.5 mM/120 min and 2.0 mM/180 min were the most effective groups in total flavonols. Anthocyanin contents were highest in combinations of 0.5 mM/60 min and 1.0 mM/120 min.

Application of Sodium azide
Another mutagen whose effect was examined in the study is sodium azide. The administration of sodium azide at a dose of 1.0 mM for 60 minutes increased the amount of total phenolic substances approximately 12 times (0.733 mg/g) compared to the control at the same time (0.057 mg/g). The application of 1.0 mM sodium azide for 120 minutes resulted in high values in total flavanols and total flavonols. The highest value in anthocyanin (19.433 CV/g) was obtained when the highest dose of 2.0 mM was applied for 60 minutes. Like our study, Journal of Biology, Agriculture and Healthcare www.iiste.org ISSN 2224-3208 (Paper) ISSN 2225-093X (Online) Vol.11, No.22, 2021 Chandran and Pillai (2018) reported that the application of sodium azide in callus cultures of green chiretta increased secondary metabolite production. When the study's results are evaluated collectively, the mutagen application of different durations and doses causes significant statistical differences in the amount of anthocyanin, total phenolic compound, total flavanol, and total flavonols in calli. It was found that; -The mutagen in which the best result was observed in terms of the total phenolic compound amount was Acridine orange with 0.912 mg/g; -In terms of total flavanols, the mutagen that provides the highest accumulation is Azacitidine with 0.023 mg/g, followed by Acridine orange with 0.022 mg/g; -The highest result in terms of total flavonol amount was obtained from Acridine orange mutagen with 0.100 mg/g; -The highest mutagen in terms of anthocyanin amount is EMS with 32,578 and 32,291 CV/g. Therefore, among the mutagens used, it can be said that sodium azide is insufficient in terms of promoting secondary metabolite accumulation compared to other mutagens in the applied doses and durations. However, it should be considered that it may show a greater effect in different dose and time trials.
Studies are using different mutagens to create chemical mutations in plants. Pathirana et al. (2014) applied 1% EMS for 1 hour to calli obtained from petiole explants in kiwi; Krupa-Malkiewicz et al. (2017) investigated the effects of 2 different concentrations of EMS application of 0.5 and 5.0 mM on calli in petunia for 60, 120 and 180 minutes; While Kannan et al. (2015) used sodium azide on calli in bahiagrass, Toyada et al. (2013) used EMS, 6-azacitidine and acridine orange mutagens to rose buds at concentrations of 50, 100 and 200 µg/ml. Shah et al. (2019) also applied EMS to calli in the Hyoscyamus niger plant. The aim of these studies is not to directly increase the production of secondary metabolites but to create mutations for different purposes. In a study conducted to improve callus properties in vitro without creating mutations, Mohammed and İbrahim (2016) applied UV-B and EMS to callus cultures of rice plants (Oryza sativa L.). The effects of the applications on callus formation and development were examined, and as a result of the research, it was stated that mutagen applications were effective on callus properties. There is an extremely limited number of studies investigating secondary metabolite accumulation by applying mutagen. In one of these studies, Chandran and Pillai (2018) applied sodium azide at a concentration of 0.01% for 3 hours in callus cultures of green chiretta (Andrographis paniculata). As a result of their research, they stated that sodium azide applications improved some callus properties compared to control and increased the production of andrographolide, neo andrographolide, 14-deoxy-11,12-dihydro andrographolide, and andrographonin positively. Singh and Sharma (2020) stated that creating mutations may be effective in induction of secondary metabolite synthesis mechanism, the production of phenolic compound and cinnamoyl putrescine was increased in mutant cells of Nicotiana tabacum and Nicotiana glauca to which p-fluorophenylalanine was applied, and Xray radiation was applied to cell cultures of Anisodus acutangulus to increase scopolamine production. Similarly, it has been stated that the biotin accumulation in Lavendula vera cell cultures is increased by irradiation of γ rays; the somaclonals created by a mutation in Catharanthus roseus were used to increase the production of ajmalicin and serpentine. In a study in which p-fluorophenylalanine was used as a mutagen to obtain cell lines with high efficiency in terms of phenolic production, it was reported that capsaicin production increased in Capsicum annuum cell cultures where p-fluorophenylalanine was applied (Singh and Sharma, 2020).

Conclusion
The results obtained from our research show that chemical mutagens, which are used to create more genetic variation, can act as an elicitor in the callus cultures of the leaf of grapevine causing significant increases in the concentration of secondary metabolites.
It is thought that this research, which was carried out using different chemical mutagens in callus cultures, will constitute a reference to the researches on the biosynthesis of secondary metabolites with the use of chemical mutagens. However, it is thought that higher amounts of secondary metabolite production can be achieved by evaluating various doses and durations of different mutagens. mouse skin two stage initiation promotion protocol and identification of procyanidin B5-3-gallate as the most effective antioxidant constituent. Carcinogenesis. 20: 1737-1745.