Plants produce "primary metabolites" (carbohydrate, protein, fat, etc.), which are necessary for their growth and development, as well as "secondary metabolites" from the intermediate products of these metabolism pathways. These compounds produced by special metabolic pathways and generally classified according to biosynthesis methods are compounds that do not have direct effects on the vital functions of plants but determine quality criteria such as color, taste, and odor. Secondary metabolites are defined as compounds that play an active role in developing the defense mechanism of plants against microorganisms and herbivores and protect against various biotic and abiotic stresses (Wink, 1999; Theis and Lerdau, 2003). Although some of these compounds, which are extremely important in the adaptation of plants to their environment, are produced in relatively higher quantities, it is also known that they are generally produced in low quantities and that the production of some is limited to certain species and certain organs (Verpoorte et al., 1999; Sökmen and Gürel, 2001). It is known that compounds, which have important functions in the plant, also have a great role in human health (Kahkönen et al., 2001; Kahkönen et al., 2003; Rossi et al., 2003; Viljanen et al., 2004).
One of the most important members of secondary metabolites representing a rich group is phenolic compounds. These compounds are aromatic ring compounds containing at least one hydroxyl group and functional groups thereof with antioxidant properties. Phenolic compounds are known to inhibit the formation of low-density lipoproteins (Frankel et al., 1995), have protective effects against cardiovascular diseases (Renaud et al., 1999; Gronbaek et al., 2000), and are among the important secondary metabolites with their antimicrobial (Nychas et al., 2003, Göktürk Baydar et al., 2004) and anticarcinogenic (Zhao et al., 1999; Waffo-Teguo et al., 2001) properties. They also bind free radicals to themselves and prevent them from attacking nucleic acids, somatic cells, and the immune system (Han, 1997; Khalil et al., 2007). Also, these compounds are used in the cosmetic industry with their anti-aging effects.
Flavonoids, an important group of phenolic compounds, are responsible for many plant life-related functions as secondary metabolites in plants. They are the most common phenolic compounds in the human diet, and more than 5000 flavonoids have been identified (Bronze et al., 2012). Flavonoids are divided into six different classes. These are flavones (apigenin), flavonoids (eriodictyol, hesperetin, and naringenin), flavonols (quercetin, kaempferol, myricetin, and isorhamnetin), isoflavonoids (genistein, daidzein), anthocyanins (cyanidin, delphinidin, malvidin, pelargonidin, petunidin, peonidin) and flavanols (epicatechin, proanthocyanidins) (Peterson and Dwyer, 1998).
The most common flavonoid group in foods are flavanols, and they are colorless compounds found in most fruits. They take place as intermediates in flavonoid biosynthesis. They are systematically called flavan-3-ol because they contain an -OH group on the C3 atom (Aron et al., 2008). They are mostly found in glucoside form in plants. Approximately 450 flavonol glucosides have been identified (Corradini et al., 2011; Crozier et al., 2009). The main sources of flavonols are onions, garlic, cabbage, leeks, cauliflower, broccoli, blueberries, cherries, tomatoes, apples, and red grapes (Perez Vizcaino and Duarte, 2010).
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. (2014) increased carotenoid production in callus cultures of marigold; Awad et al. (2014) applied methyl jasmonate and bacterial and fungal elicitors to the root cultures of Indian licorice and stated that the amount of glycyrrhizic acid increased. Alhasnawi et al. (2017) found that β-glucan and salt stress applications stimulated antioxidant accumulation in callus cultures of rice plants (Oryza sativa L.). In another study in this topic, Ullah et al. (2019) stated that UV-C radiation and melatonin applications in callus cultures of cress increase the biosynthesis of antioxidant and antidiabetic metabolites; 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.
Mutations can be obtained in three different applications as physical, chemical, and transposable. Physical mutagens are X-rays, Gamma rays (Co60, Sz137), Neutrons, Beta cathode rays, Alpha particles, and protons. Chemical mutagens are Diethyl sulfate (DES), ethyl methanesulfonate (EMS), methyl methane sulfate (MMS), ethyleneimine (EI), N-nitrous N-ethylurea (NEU), and azides. Chemical mutagens are generally suitable for generating micro mutations (Sağel, 1994). Transposable elements exist in different forms: transposons, retrotransposons, T-DNA, and retroviruses (Pakyürek, 2019). Chemical mutagens used in the mutation are divided into seven groups according to their activity patterns. These are basic compounds (5-bromine uracil, 5-bromodeoxyuridine, 2 amino purines); antibiotics (azeserin, mitomycin C, streptonigrin, actinomycin D), alkali compounds (such as EMS, ethyl-2-chloroethyl sulfide, ethylene oxide), azides (sodium azide), hydroxylamine, nitrite acid and acridines (acridine orange).
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.