PGRs are involved in all parts of the plant life cycle and profoundly affect plant cell growth, development, and responses to environmental and physiological factors and secondary metabolite production and accumulation (Richard et al. 2002). The PGRs are one of the most critical factors affecting callus formation and plant cell growth in vitro, and the type and concentration of the PGRs required for each plant or even genotype and explant should be experimentally optimized. The presence of auxins and cytokines is necessary for cell division and morphogenesis, especially during G1/S and G2/M transitions. In various plants, it has been reported that auxins are the most influential factor in the induction of callus, and cytokines support this role. Auxins increase the extensibility of the cell wall by stimulating acidification. Auxins also induce transcription of mRNAs encoding proteins involved in cell growth and development. Cytokines also directly affect the cell cycle by regulating the production of proteins involved in spindle fibers (Silveira et al. 2004). The present study and the results of other studies on yew and hazelnut cell cultures (Frense 2007; Bestoso et al. 2006) showed that auxins, especially 2,4-D, play an essential role in the stimulation of the cell division and proliferation and callus production; and its combination with cytokines such as Kin is more effective in the callus production and growth from hazelnut explants. According to the present study results, a low concentration of Kin (0.2 mg/L) performs better than high concentrations of this hormone (0.5 and 1 mg/L). In this regard, it was reported that in a medium containing 2 mg/L Kin and different auxin concentrations, either no callus produced from yew explants or the amount of produced callus is meager. However, in a medium containing 0.2 mg/L Kin, the percentage of callogenesis was very high. It was also found that in cultures containing low concentrations of Kin and high concentrations of 2,4-D, the callus cultures overgrow, and the callus structure is soft and friable, but as the concentration of Kin increases, the callus tissue also becomes stiffer (Rahmati et al. 2017).
Ascorbic acid (AA), casein hydrolysate (CH), and ultrasonic wave exposure for 1 min also positively affected callus induction and growth from hazelnut explants. It has been reported that media containing casein hydrolysates promote callus growth in various plants (Khaleda and Al-Forkan 2006). Casein hydrolysates contain various organic and inorganic materials such as calcium, phosphate, microelements, vitamins, and amino acids. Casein has been reported to promote growth in cultures where phosphate deficiency inhibits growth; hence casein hydrolysates can be considered a phosphate source (George et al. 2008). The utilization of antioxidants such as ascorbic acid in the medium composition would effectively reduce the explants and cultures’ browning phenomenon (Krishna et al. 2008). Ascorbic acid has also been shown to play some roles in plant growth, including cell division, cell wall expansion, and other growth phenomena (Piganocchi and Foyer 2003). There are several reports on the beneficial effects of ascorbic acid on various physiological and biochemical aspects of different plants (Beltagi 2008; Sheteawi 2007).
In the present study, the effect of ultrasonic waves on callus growth was positive for 1 min, but callus growth was reduced with increasing exposure duration up to 3 min. So, in media with the same PGRs composition, cultures whose explants were exposed to ultrasonic waves for 1 min and media containing ascorbic acid and casein hydrolysate had better callus growth than those that did not have these conditions. However, cultures whose explants were exposed to ultrasonic waves for 3 min had less callus induction and growth than the control. Ultrasonic waves can have different physiological and biological effects on the plant cells depending on their exposure duration and intensity. At low energy levels, US causes beneficial and reversible biological changes in cells and plant tissues. Low energy US can increase the permeability of cell membranes through the sonoporation process, which facilitates the mass transfer and molecular uptake by the cells and have a variety of biological effects on plant cells. Low intensity and energy US is of particular importance in biotechnology and has many non-destructive biological effects on living cells, including increased membrane permeability, gene expression changes, increased activity of enzymes, and levels of certain hormones (Miller et al. 1999). Wound formation in plant tissue and cells by US treatment maybe increases the indigenous hormone levels in the cells by affecting the biosynthesis and transport of plant hormones, which finally improves the callus formation and growth (Srivastava 2002). In the previous studies, it was also reported that the US at high intensities significantly reduces cell viability and survival (Safari et al. 2012; Hazrati et al. 2017). This reduction may occur due to the damages caused by the US in the cell structure, including cell membrane and wall, and organelles, which ultimately results in cell death and reduced proliferation and growth (Wu and Lin 2002).
Since callus quality and growth depend on the type and amount of nutrients (organic and inorganic compounds) in the culture medium, the basal medium composition and various PGRs can affect callus cultures’ growth and biochemical characteristics. Although MS medium containing 2,4-D and BAP has been used in hazelnut cell culture (Bestoso et al. 2006), B5 medium (Bemani et al. 2012) has also been reported. In the present study, we investigated the effect of different basal medium formulations, including MS, B5, WPM, MS salts + Nitsch vitamins, and their full and half-concentration (length) on the callus induction and growth from hazelnut explants. In general, the percentage of callus formation was more than 50% in most cultures except for ½ B5 and 1/2 WPM media, which had a lower percentage of callus formation. The highest fresh callus weight was observed in ½ MS and B5 media. MS, B5, WPM, and MS salt + Nitsch vitamins media differ in the concentration or composition of some macro and micro-elements. High concentrations or combinations of salts in the medium may affect nutrient uptake by plant cells (Mihaljevic et al. 2002). The MS and WPM medium has the highest concentration or composition of salts. Besides the differences in macronutrients, the B5 medium does not contain glycine but contains more thiamine, unlike the WPM and MS media (George et al. 2008). Plant species and even different cultivars of a given species may require different nutrients for proper in vitro culture responses. It seems that ½ MS and B5 media are better for callus growth in hazelnut in vitro cultures, maybe because these formulations have a reduced concentration of salts.
Further callogenesis in the B5 medium could also be due to the lack of glycine and the increased level of thiamine, as it has been shown that the increase in thiamine level effectively increases the callogenesis and regeneration in Aloe vera (Zarinpanjeh et al. 2012). Mihaljevic et al. (2002) reported that the induction and growth of yew callus are more successful in the B5 medium than in the MS medium, which reduces cell division. In in vitro culture of woody plant species, MS medium is a growth inhibitor and can be solved by reducing the ammonium concentration and completely removing nitrogen.
Typically, the optimal conditions for callus growth differ from the optimal conditions for secondary metabolite production and require careful consideration. Among the published reports on hazelnut in vitro culture, there is no report on determining the best basal medium to achieve a higher callus induction and growth rate and a more significant amount of secondary metabolites, and most studies focused on the effect of other compounds, including plant growth regulators and elicitors. In this study, some basal media were compared in terms of taxol and baccatin III production in the callus cultures of Corylus avellana L. The highest production and accumulation of taxol and baccatin III were obtained in the WPM and MS media. The effect of macro and micronutrients on cell growth and development is relatively straightforward and proven. Therefore, it can be concluded that these compounds can indirectly affect the biosynthesis pathways and secondary metabolites production in the cell cultures. On the other hand, many of these elements, including potassium, calcium, magnesium, and most microelements, are involved in the various cellular processes and activity of metabolic pathways. So these factors and their concentration in the culture medium can also influence the production and accumulation of secondary metabolites in the plant cells.
Vitamins in the medium composition not only influence cell growth and development but also serve as catalysts in metabolic processes. A comparison of the nature and concentration of nutrients in the used media shows that the WPM medium contains higher amounts of potassium in the form of K2SO4 than other tested basal media, as well as significant amounts of ammonium nitrate, which may directly or indirectly affect the biosynthesis pathway of taxol and other taxanes.
Furthermore, nitrogen source significantly influences the production of secondary metabolites such as terpenes and alkaloids, anthocyanins, and shikonin in the plant cell suspension cultures (Kim and Chang 1990). Ammonium nitrate is one of the most important organic compounds and can be used as a nitrogen source in many essential processes for producing secondary metabolites and the production of antioxidants (George et al. 2008). Similarly, the MS medium is high in ammonium nitrate. Ammonium readily accumulates in tissues and becomes highly toxic if not rapidly metabolized. When the ammonium concentration in the medium is low, most of the accumulated ammonium is metabolized by the cell. However, when the ammonium concentration is high, only a small amount is metabolized. The apparent difference between the B5 medium with the MS and WPM media is in their vitamin type and concentration, especially thiamine. Thiamine plays a vital role as a cofactor in the metabolic pathways of plants, such as glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle. It has also been shown to play a cofactor role in response to biotic and abiotic stresses in plants (Goyer 2010).
The callus cultures in the MS, WPM, and MS salts + Nitsch vitamins media exhibited the higher H2O2 and MDA content and enhanced secondary metabolites production (phenolic compounds, taxol, and Baccatin III) and antioxidant enzymes activity. In contrast, the B5 medium and the 1/2 MS and 1/2 WPM media showed the lowest H2O2 and MDA content and had an inhibitory effect on the production of these compounds. There is also a positive correlation between total phenolic content and their antioxidant activity. Plants have enzymatic and non-enzymatic antioxidant defense systems to counteract the harmful effects of ROS. Enzymatic antioxidants such as SOD, POD, CAT, glutathione reductase, and ascorbate peroxidase are responsible for protecting against the toxic effects of reactive oxygen species (ROS) (Mittler 2002). The amount of ROS in a cell depends on how fast it is produced, how quickly it reacts with target molecules such as proteins, lipids, or nucleic acids, and how quickly it is broken down or neutralized by antioxidant enzymes (Mittler 2002). ROS such as H2O2 has a significant influence on cell growth and the production of secondary metabolites. MDA is a peroxidation product of unsaturated fatty acids in phospholipids. Lipid peroxidation levels have been used as a marker of free radicals level that damages the cell membranes under stress conditions (Jaleel et al. 2007). Increasing salt causes oxidative stress in cells and disrupts the physiological functions of cells. The increased MDA and H2O2 in the cell cultures on MS, MS salts + Nitsch vitamins, and WPM media may be attributed to the higher salts concentration in these basal media. Plant cell and tissue culture techniques, due to disinfection treatments, sucrose, PGRs, or the high concentration of macro-elements in the medium composition, may induce stressor conditions such as oxidative stress in the plant cells and tissues. Plant cells activate a complex adaptive mechanism for adaptation and confrontation with this stressor condition associated with epigenetic, physiological, and metabolic changes (Gaspar et al. 2002). In Catharanthus roseus, the utilization of high concentrations of calcium chloride reduced callus growth (Siddiqui and Mujib 2012).
The positive correlation between total flavonoids, total phenolics, and antioxidant activity indicates the role of these compounds in the antioxidant capacity of the hazelnut cells. In other words, the increased level of H2O2 in the cells activates the signaling pathway by inducing oxidative stress, which may stimulate the plant cell defense mechanisms and the production and accumulation of secondary metabolites (Wang et al. 2009). For instance, the increased level of H2O2 induces the phenylpropanoid compounds biosynthetic pathway and production of phenolic compounds in stevia under salicylic acid and methyl jasmonate treatment, maybe through increasing the transcription of phenylalanine ammonia-lyase (PAL) (Wang et al. 2009). At higher concentrations of phenolic compounds, due to the increase in the number of hydroxyl groups of the aromatic rings of the phenolic compounds in the reaction, the probability of hydrogen being given to free radicals increases, and consequently, the inhibitory power of the extract increase (Zhang et al. 2009).
Generally, it can be concluded that in addition to the appropriate concentration and combination of PGRs, the concentration and type of basal medium have a crucial effect on callus formation, the physiological status of cultures, and the production of secondary metabolites. In contrast to the callus formation and growth, which grew better in media with lower salt concentrations, the production of secondary compounds increased in the basal media with high salt content. This may be due to the oxidative stress condition induced by higher salt concentration in these basal media, which results in an increase in H2O2 production and accumulation in the cells and membrane lipid peroxidation (MDA content), consequently induction of plant cell defense responses. Furthermore, short-term exposure of the explants to ultrasound (low-energy ultrasonic waves) also positively affects callus formation.