Carbohydrate-induced biomass accumulation and elicitation of secondary metabolites in callus cultures of Fagonia indica
Graphical abstract
Introduction
The medicinal plant, Fagonia indica is well known for its anticancer potential (Waheed et al., 2012). It is rich in medicinally important secondary metabolites, such as phenolic compounds, flavonoids, and terpenoids. F. indica products have been employed as tea or decoction for the treatment of various disorders (El-hadidi et al., 1988; Hope, 2012; Shaker et al., 2000; Shehab et al., 2011). The importance of F. indica has been highlighted in numerous studies for being active as the antimicrobial, antihemorrhagic, hepatoprotective, antidiabetic, anthelminthic, thrombolytic plant (Bagban et al., 2012; Rasool et al., 2014; Shehab et al., 2011; Soomro and Jafarey, 2003). The most prominent activity of F. indica has been its anticancer potential. Lam et al. (2012) have demonstrated the potent role of F. indica extract against breast cancer cell lines, MCF-7 and MDA-MB-231 via FOXO3a and p53 expression. Owing to its medicinal importance, a drastic increase in market demand of F. indica has been observed in recent years. Over-exploitation for the preparation of pharmaceuticals can endanger the plant. Research programs must be initiated and implemented for sustainable utilization and conservation of this important medicinal plant (Khan et al., 2015). The plant is being investigated in vitro through different culture systems for increased production of medicinally important secondary metabolites (Ebrahimi and Payan, 2013; Khan et al., 2017, 2016). Cell cultures, especially callus cultures, can produce a handful of secondary metabolites (Nikolaeva et al., 2009). Callus cultures can be exploited further to provide inoculum to establish and develop cell suspension (Mustafa et al., 2011), as a source of explant for plantlet regeneration (Ikeuchi et al., 2013), for induction of somatic embryos (Abbasi et al., 2016), and adventitious root cultures (Sivakumar et al., 2005). Callus cultures are usually affected by various in vitro conditions including explant type, plant growth regulators, nutrient supply, carbohydrate source and other environmental conditions (Khan et al., 2016; Kumar et al., 2015; Lee et al., 2011; Yan et al., 2009). In vitro cultures depend on the growth-room light for their photosynthesis (Tariq et al., 2014). However, the growth-room light is not enough for photosynthetic activity and thus the plant cells become heterotrophic and start relying upon the exogenously supplied carbohydrate source. Monosaccharides and disaccharides have been employed in vitro in various concentrations for their stimulating effects on biomass accumulation and production of useful compounds in various commercially important plant species (Kretzschmar et al., 2007; Kumar et al., 2015).
Amorim et al. (1977) evaluated the effects of exogenous glucose on phenol synthesis in Paul's Scarlet Rose cells grown in liquid suspension or solid culture and concluded that higher concentrations of glucose in the culture medium resulted in an increased synthesis of phenols. In another study, Pourtau et al. (2006) showed that increased intake of external sugars promotes symptoms of senescence such as the leaf yellowing in Arabidopsis thaliana. This has been linked with the production of phenolic compounds and thus subsequent oxidation, yellowing, and deterioration of leaves of the plant. Similarly, Lux-Endrich et al. (2000) concluded that the increased sucrose content resulted in an enhanced amount of phenolic compounds. Conventionally, 3% sucrose is defined as a standard carbohydrate amount and type in plant culture medium for feasible in vitro growth (Murashige and Skoog, 1962). Sucrose is preferred for growth because of its rapid intake, long-distance intercellular transport and long half-life inside cells (Tognetti et al., 2013). Other types of carbohydrates are also used for growth and development in plant in vitro cultures. For instance, Maltose has been shown to produce an optimum biomass accumulation in callus cultures of Gossypium hirsutum (Kumar et al., 2015). Similarly, the exogenous application of fructose and glucose have also been reported to influence the growth and development of plant cells, organs or tissues (Ma et al., 2017).
Apart from being crucial components of the general metabolism, carbohydrates are important signaling molecules in cellular and metabolic processes during biotic and abiotic stress situations (Lastdrager et al., 2014; Rolland et al., 2006). Sugars also act as a signal for regulating the plant defense system (Morkunas and Ratajczak, 2014). Sugars stimulate the biosynthesis of secondary metabolites such as phenolic and flavonoids to confer anti-oxidative properties to the plant system. Sugar optimization was found to affect the production of flavonoid in callus and cell suspension cultures of Coleus blumei (Qian et al., 2009). This suggests that changes in sugar supplementation to the callus culture medium can be used as a strategy to stimulate the production of medicinally important phenolic compounds. The alteration in carbohydrate source and concentration during cell cultures, especially callus cultures, is a relatively novel approach for elicitation of secondary metabolites (Kumar et al., 2015). This study, for the first time, reports the impacts of different carbohydrate types and concentrations on the biomass accumulation, antioxidant activity, and the secretion of phenolic and flavonoid compounds in callus cultures of F. indica.
Section snippets
Effect of varying levels of different carbohydrates on callus growth and biomass accumulation in F. indica
Callus cultures were established by following our previously published report (Khan et al. (2016). Briefly, stem explants (∼1.0 cm) from 50 days old lab-grown plantlet of F. indica were cultured in Murashige and Skoog basal medium (MS0, 1962) containing 0.8% (w/v) agar, 3% sucrose and Thidiazuron (TDZ = 1.0 mg/L). In subsequent experiments, the callus cultures were grown in varying concentrations (1% = 10 g/L, 3% = 30 g/L 5% = 50 g/L) of sucrose, glucose, fructose, and maltose supplied in the
Effects of different carbohydrate types on callus induction and biomass accumulation
Sugars play vital regulatory and physiological roles in the growth and development of plants (Stokes et al., 2013). In the present study, 3% sucrose resulted in the highest callus induction frequency (90%) in explants than all other concentrations and types of sugars used. At this treatment, callus formation was observed on the third day of culture cultivation (Table. 1). Moreover, sucrose at higher concentrations (5%) produced the highest FW (52.05 g/100 mL) and the highest DW (2.72 g/100 mL)
Conclusions
Conclusively, disaccharides especially sucrose at 3% concentrations is the most feasible carbohydrate source for higher biomass accumulation in callus cultures of F. indica. Higher concentrations of sucrose favor the division of cells and thus the growth of callus cultures quickly and for a long time. Optimal concentrations of glucose (3%) produced the highest total phenolic content in callus cultures of F. indica. Furthermore, glucose-treated callus cultures showed the highest antioxidant
Conflict of interest
The authors declare that they have no conflict of interests
Acknowledgments
The authors acknowledge the role of the Higher Education Commission, Pakistan, and the University of Florida, the USA for their support.
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