Introduction

Wheat has been a crucial cereal crop in providing people’s nutritional needs throughout history (Aydin et al. 2016a). It is a form of food that people in all geographic regions of the world require. The reason for this is that it is adapted to high altitudes, can be cultivated quickly and readily, has a high nutritional value, is easier to store and keep, and has a greater bread quality than other cereal crops. According to the data of FAO 2021, the wheat cultivation area in the world was 220.76 million ha and the production amount was 770.87 million tons (FAO 2021).

The expansion of wheat production to wide areas raises awareness of production techniques, resulting as output under stressful conditions and in marginal locations. This circumstance increases the amount of yield obtained from the unit area. Plants are exposed to many abiotic stresses such as ultraviolet (UV). This situation leads to serious reductions in the yield of agricultural products in particular (Láposi et al. 2009; Sigmaz et al. 2015).

Solar radiation is the universe’s primary source of energy and is essential for plant growth and development. Plants are exposed to varying levels of UV-B light throughout their life cycle. Furthermore, although UV-B radiation is a minor component of solar radiation, it can have multiple effects on life systems due to its high energy (Mariotti et al. 2021). UV-B influences plant growth by modifying morphological, physiological, and molecular responses (Kanungo et al. 2022). Common climatic characteristics such as precipitation patterns and temperature have a considerable influence on the efficiency of UV-B radiation (Puranik et al. 2022). UV-B effects also vary with species, altitude, latitude, time of the day, day of the year, cloud cover, and other meteorological conditions (Rai et al. 2022). UV-B is received by plants as an environmental signal and a possible abiotic stressor that affects plant development and response (Shi and Liu 2021). UV-B rays have the potential to damage cells due to the high energies, which is one of their most critical properties. Reactive oxygen species (ROSs), which builds in the cell as a result of severe UV-B stress, damages numerous cellular components such as DNA, RNA, proteins, and membrane lipids, as well as causing metabolic problems (Foyer and Noctor 2009). It has been shown that exposure to UV-B radiation induces several reactions in higher plants. Diverse enzymatic and non-enzymatic antioxidants help plants survive oxidative stress by removing ROSs. These antioxidants are substances that slow down the oxidation of fats (Mackerness et al. 2001).

α-Tocopherol, which is found in the cell membrane and has a significant antioxidant impact due to its lipophilic nature, inhibits phospholipid peroxidation and cell membrane damage (Gey et al. 1991; McNeil et al. 2004). α-Tocopherol protects cell membranes by reducing the damaging effects of ROS and polyunsaturated fatty acids in membrane phospholipids. α-Tocopherol scavenges lipid peroxyl radicals produced during stress and stops lipid peroxidation chain events (Asthir 2015).

Plant tissue culture is a widely used technology to facilitate and support plant breeding programs. Plant cells are cultured in vitro to create suitable environments, desired characteristics of tissues and organs are determined, and new plants are created (Aydin et al. 2011). During plant growth, in vitro selection is a technique used to select plant genotypes that are resistant to biotic and abiotic challenges such as UV radiation, drought, excessive salt, and disease. It is a natural method for the production of stress-resistant plants that combines traditional and technological aspects. Plant cells, tissues, or organs can be cultured in vitro on a medium containing selective chemicals, allowing for the selection and regeneration of plants with desirable traits (Rai et al. 2011).

Some UV-B light does reach the Earth’s surface, although the stratospheric ozone layer absorbs most of it (Sharma and Kumar 2022). Plants are injured by UV-B stress, which is caused by exposure to excessive levels of continuous full-wavelength UV-B radiation (Chen et al. 2022). This has necessitated research into the effects of UV-B radiation, particularly on plants. In this study, the negative effects of UV-B stress on the plant were investigated in vitro in mature embryo culture of Kırik wheat cultivar, and the effects of α-tocopherol application were determined in order to eliminate the negative effects, and the role of α-tocopherol in tolerance to UV-B stress was explained.

Materials and methods

Plant material was acquired from mature seeds of the Kırik bread wheat variety that was supported with endosperm. Endosperm-supported embryo culture was conducted based on methods descripted by Aydin et al. (2011). Mature seeds were cleaned under sterile circumstances for 30 min with industrial-strength bleach (5% sodium hypochlorite) and two drops of Tween 80 after being submerged in 70% ethanol for 5 min. Afterward, sterilized seeds were rinsed with sterile distilled water several times and kept on +4 °C for 14–16 h in dark conditions. Mature embryos of surface sterilized seeds were divided into 6 parts without detaching from the seed. Endosperm-supported embryos were grown for 10 days at 25 °C in the dark with 12 mg/l dicamba, 0.5 mg/l IAA, 20 g/l sucrose, 2 g/l phthalate, and 1.95 g/l MES hydrate added to the callus formation media. Under sterile conditions, 10-day-old calli were exposed to UV-B stress (300 μW cm−2) using a Philips Special 20W/Narrowband tube (311 nm), as previously mentioned (Manaf et al. 2016; Zhao et al. 2021), positioned directly above cultures for 2, 4, and 6 h at a distance of 30 cm under photoperiod (62 μmol m−2 s−1) and 25 ± 1 °C (Manaf et al. 2016).

The calli exposed to UV-B stress were incubated in a callus formation medium containing different concentrations of α-tocopherol (0, 50, and 100 mg/l) (Sadiq et al. 2019) in the same conditions for 20 days. The relative growth rate of callus (%) (CRGR) and embryogenic callus formation rate (%) (ECF) were determined before calli were transferred plant regeneration. The calli for plant regeneration were kept on plant regeneration medium I (hormone-free callus formation medium) for 2 weeks at 25 ± 1 °C under 16-h photoperiod (62 μmol m−2 s−1) and then transferred to plant regeneration medium II (plant regeneration medium I contain 0.5 mg/l TDZ). At the end of this period, the rate of responding embryogenic callus (REC) (%) and plant number per explant (PN) (number) were determined. Responding embryogenic callus is a callus that occurs in plantlets with roots and shoots. Regenerated plants were grown in glass jars containing hormone-free callus formation medium until they were 10–12 cm. All media were adjusted to pH 5.7 ± 0.1 before autoclaving for 20 min at 121 °C. MS vitamins and plant growth regulators (dicamba, IAA, TDZ, and α-tocopherol) were added to the autoclaved medium (50–60 °C) after sterilized with cellulose nitrate filters (Milipor®) with 0.22 μm.

Data were analyzed variance analysis (ANOVA) based on a completely randomized factorial design. Every petri dish was approved as a replicate and 10 endosperm-supported embryos were used in each petri dish. Five petri dishes were used for each application. The means were compared using the LSD test at the 0.05 level. Statistical analysis was performed using SAS (v 9.3) computer program.

Results

In this study, the effect of UV-B and α-tocopherol on plant growth activity was looked at by measuring callus relative growth, which is measured by changes in size, fresh/dry biomass, and the rate at which cells divide. In addition, to determine their effect of them on plant morphogenesis, ECF rate (%), REC rate, and PN (number) parameters, which are widely used in wheat somatic embryogenesis, were investigated.

The main effect of UV-B exposure time and α-tocopherol concentration on callus relative growth rate was very significant (p < 0.01) (Table 1). When compared to a 0-h exposure period, the results indicated that all exposure times in this parameter reduced dramatically. The highest CRGR with a % of 7.0 was recorded in an exposure time of 0 h, whereas the lowest value was determined in a 6 h exposure time of 2.4%. Moreover, a decrease in the CRGR was observed depending on the increase in exposure time (Table 2). As for the effect of α-tocopherol concentration on CRGR, there is a very significant difference among the α-tocopherol concentrations. The CRGR increased in tandem with the α-tocopherol concentration; additionally, the highest CRGR was detected in 100 mg/l α-tocopherol. UV-B stress had a different effect on CRGR depending on α-tocopherol application, making the UV-B × α-tocopherol interaction (U×T) very significant (p < 0.01). According to the U×T interaction, α-tocopherol increased RGR during all UV-B exposure times (Table 2) (Fig. 1a).

Table 1 Variance analysis results for parameters
Full size table
Table 2 Effect of UV-B exposure time and α-tocopherol concentration on tissue culture parameters
Full size table
Fig. 1
figure 1

Effect of U×T interaction on tissue culture parameters. a Relative growth rate of callus (%) (CRGR), b embryogenic callus formation rate (%) (ECF), c rate of responding embryogenic callus (REC) (%), d plant number per explant (PN) (number). Data are presented as means ± SE (n = 4)

Full size image

Based on a meaningful comparison of different UV-B exposure times, the ECF rate (%) ranged from 60.7% under 6 h to 87.3% under 0 h, and the main effect of UV-B exposure time was determined very significant (p < 0.01) (Table 1). The highest ECF was observed at 0 h of UV-B exposure, while the lowest was observed at 0 h of UV-B exposure time. Furthermore, the main effect of α-tocopherol on embryogenic callus formation rate was very significant (Table 1), and the ECF rate ranged from 52.0 to 60.7 for 0 and 100 α-tocopherol concentrations, respectively (Table 2). The results of the research indicate that the application of α-tocopherol concentrations affects the ECF rate under UV-B stress (Tables 1 and 2). Although the effect of U×T interaction on ECF rate was not significant (p > 0.05), all α-tocopherol concentrations increased ECF rate in all UV-B exposure times. The highest ECF rate (88.0%) was recorded in calli exposed to both 50 and 100 mg/l concentrations of α-tocopherol and the 0 h UV-B exposure time while the lowest rate (52%) was observed at 0 mg/l concentration of α-tocopherol and the 6 h UV-B exposure time (Table 2) (Fig. 1b).

The main effect of UV-B exposure time and α-tocopherol concentration on REC rate was very significant (p < 0.01) (Table 1). The REC rate increased with 50 mg/l and 100 mg/l α-tocopherol concentrations compared to 0 mg/l α-tocopherol (Fig. 2a-c). In addition to this, with the increase in UV-B application time, an increase in the REC rate was observed (Table 2). UV-B stress had a different effect on REC rate depending on α-tocopherol application, making the U×T interaction not significant (p < 0.05). α-Tocopherol generally increased REC rate in UV-B exposure times. The highest REC rate (74.0%) was recorded in calli exposed to 50 mg/l α-tocopherol and the 0 h UV-B exposure time while the lowest rate (28%) was observed at 0 mg/l concentration of α-tocopherol and the 6 h UV-B exposure time (Table 2) (Fig. 1c).

Fig. 2
figure 2

Effects of α-tocopherol and UV-B exposure on responding embryogenic callus and plant regeneration. a Responding embryogenic callus under 4 h UV-B exposure time and 0 mg/l α-tocopherol treatment, b responding embryogenic callus under 4 h UV-B exposure time and 50 mg/l α-tocopherol treatment, c responding embryogenic callus under 4 h UV-B exposure time and 100 mg/l α-tocopherol treatment, d regenerated plants under 4 h UV-B exposure time and 0 mg/l α-tocopherol treatment, e regenerated plants under 4 h UV-B exposure time and 50 mg/l α-tocopherol treatment, f regenerated plants under 4 h UV-B exposure time and 100 mg/l α-tocopherol treatment

Full size image

UV-B exposure time and α-tocopherol concentration affected PN and their main effects were very significant (p < 0.01), as was the effect of the other parameters (Table 1). When the main effect of UV-B stress was evaluated, a decrease in EBBS was determined in parallel with the increase in UV-B exposure time. However, parallel to the increase in α-tocopherol application concentration, an increase in PN was identified (Table 2) (Fig. 2d–f). UV-B stress had a different effect on PN depending on α-tocopherol exposure time, making the U×T not significant (p < 0.05). The highest PN (3.02) was recorded in calli exposed to 100 mg/l α-tocopherol and the 0 h UV-B exposure time while the lowest (1.44) was observed at 0 mg/l concentration of α-tocopherol and the 6 h UV-B exposure time (Table 2) (Fig. 1d).

Discussion

Numerous biotic and abiotic stress factors that plants are subjected to have an adverse effect on their growth and development. Light provides plants with the energy required for photosynthesis and is an essential environmental cue for regulating plant survival and growth. However, light such as high light and UV-B radiation can also be an abiotic stressor for plants (Shi and Liu 2021). Exposure to elevated levels of UV-B radiation induces UV-B stress, which in turn leads to modifications in plant growth, development, morphology, and photosynthesis, as well as damage to DNA, proteins, and membranes (Chen et al. 2022).

The utilization of in vitro culture techniques enables the examination of morphogenesis and its regulatory mechanisms within a controlled experimental setting. The utilization of these models is founded on the fundamental significance of the cell in all morphogenetic occurrences in plants, both in vivo and in vitro. Additionally, the similarity of plant responses in vivo, calli in vitro, and callus regenerants in vitro and ex vitro is based on the universality of plant morphogenesis pathways (Kruglova et al. 2018). In addition, callus cultures are viable models for investigating the responses of plant cells and tissues to various stressors such as salt stress, drought, and UV radiation (Efferth 2019). UV-B radiation has a wide variety of effects on various metabolic processes in the cell (Correia et al. 1999; An et al. 2000; Láposi et al. 2009; Zu et al. 2010). In this study, the increase in UV-B exposure time resulted in a decrease in the relative callus growth rate. Growth inhibition is one of the most common reactions in plants exposed to UV-B. UV-B radiation on the rate and duration of cell division and elongation can reduce the growth of wheat primary leaves (Hopkins et al. 2002). In our work, the decrease in callus formation rate can be related to the suppression of cell division and slowing of cell expansion in UV-B-exposed calli (Searles et al. 2001; Zavala et al. 2001; Flint et al. 2004). The decrease in growth rate can be attributed to the inhibition of cell division and slowing down of cell expansion in calli under UV-B radiation stress. Hopkins et al. (2002) reported that UV-B stress in bread wheat lowered the proportion of mitotically active cells and lengthened the time required for cell division. Furthermore, both cell growth and development are affected by low doses or continual UV-B radiation, both of which are stress-inducing UV-B conditions (Staxén and Bornman 1994; Allen et al. 1998; Laakso et al. 2000; Hofmann et al. 2003; Kakani et al. 2003; Hectors et al. 2007). After exposure to UV-B, the number of mature epidermal cells reduced in several species (Gonzalez et al. 1998; Hopkins et al. 2002). In lettuce (Lactuca sativa), UV-B reduced leaf size and cell expansion rate (Wargent et al. 2011). Absorption of UV-B by IAA leads to the degradation of IAA and the generation of growth-inhibiting photoproducts and thus be reduced cell elongation in response to UV-B (Tevini and Iwanzik 1986). Li et al. (2010) reported that because UV-B lowered the IAA concentration in wheat cultivars, it had a significant impact on the development, morphology, and biomass of wheat plants. UV-B radiation damages DNA, causes cell cycle arrest, inhibits growth, and causes cell death. Pyrimidine dimers, such as cyclobutane pyrimidine dimers and pyrimidine (6-4) photopyrimidone products, are produced as a result of UV-B exposure. As a result, DNA replication and transcription are inhibited, resulting in a rise in mutations, cell death, and cell cycle arrest (Lo et al. 2005; de Lima-Bessa et al. 2008). Moreover, plant cell size regulation is controlled by cell division and cell expansion (Sugimoto-Shirasu and Roberts 2003). Endoreduplication, on the other hand, is a cell size determinant associated with larger cells and greater ploidy levels. UV-B inhibits cell cycle progression by lowering cell count and replication. All of these elements are detrimental to growth. Another reason for decreased growth could be cell membrane damage caused by ROS produced in response to UV-B exposure. ROS accumulate in the cell as a result of severe UV-B stress, causing damage to diverse cellular components such as membrane lipids and disrupting metabolism (Foyer and Noctor 2009). Chen et al. (2019) found that increased UV-B radiation intensified lipid peroxidation in wheat seedlings and changed the activities of antioxidant enzymes involved in scavenging ROSs. Jansen et al. (1998) found that UV-B stress damages membranes and impairs photosynthesis, growth, and development. Moreover, it was determined that watercress (Steinmetz and Wellmann 1986) and tomato seedlings (Ballaré et al. 1995) showed a comparable drop in leaf growth under UV-B stress. Zlatev et al. (2012) determined that plant growth, development, and morphology changed depending on the increase in UV-B, and in addition, the yield of crops decreased, especially in sensitive agricultural plants. A study conducted with 34 plant species showed that there was a decrease in the growth and development of plants due to UV-B stress and 10–15% reductions depending on the plant type, severely limiting crop yield and quality (Torabinejad et al. 1998; Conner and Neumeier 2002; Koti et al. 2004).

In this study, the effect of UV-B exposure time on the rate of formation of embryogenic callus (%), response rate of embryogenic callus (%), and plant number (number) was statistically significant, and it has been determined that these parameters decrease depending on the increase in UV-B exposure time. The reason for the decrease in ECF rate due to UV-B stress in our study may be because of UV-B stress on plant growth regulators, especially auxins, which are important in the formation of somatic embryos. According to the degree of embryo differentiation, somatic embryogenesis is classified into three stages: promoting embryogenesis, growing and developing somatic embryos, and turning mature embryos into whole plants (Malá et al. 2009). Auxins are one of the most important regulators of plant growth in stimulating embryogenesis and somatic reproduction. It is known that UV-B stress affects auxins indirectly. Normally, flavonoids accumulate in tissues and organelles exposed to UV-B (Agati and Tattini 2010). Auxin homeostasis is impacted by flavonoid aglycones either by modifying auxin catabolism or by adversely influencing polar transport through oxidation transporters (Peer and Murphy 2007). Changes in auxin distribution and plant shape have been seen in certain flavonoid mutants of Arabidopsis (Peer et al. 2004; Ringli et al. 2008). Two Arabidopsis auxin mutants demonstrated flavonoid accumulation and a different morphogenic response to UV-B exposure (Hectors et al. 2012). This indicates that the interaction between auxin homeostasis and flavonoid accumulation can influence the morphological responses to UV exposure in a favorable way. UVR8 signaling is responsible for the crosstalk between auxins and flavonoids because it regulates the expression of numerous flavonoid biosynthesis genes (Brown et al. 2005). Furthermore, one of the negative effects of UV-B stress on tissue culture parameters may be due to its effect on polyamines (PA), which are crucial for cell differentiation during somatic embryogenesis as well (Chi et al. 1994; Bajaj and Rajam 1996; Kevers et al. 2000). Stress can cause a decrease in PA synthesis (Smith et al. 2001; Aydin et al. 2016b; Eliášová et al. 2018); in addition, Smith et al. (2001) observed that severe UV-B stress decrease PA levels. These findings suggest that this may be the cause of our study’s decreased embryogenic callus production and embryogenic callus rates. Aydin et al. (2016b) reported that the endosperm-supported mature embryo in wheat plays a key role in somatic embryogenesis and plant regeneration. The somaclonal variation caused by UV-B stress may be one of the explanations for its detrimental influence on the tissue culture parameters examined in this study. ROS formed under UV-B stress cause somaclonal variation by causing base deletion and addition at the DNA level, changes in chromosome level, chromosome number and structure (Czene and Harms-Ringdahl 1995), and hyper- or hypomethylation of DNA (Wacksman 1997).

Stress-tolerant plants contain more tocopherol (Szarka et al. 2012). Tocopherol is essential for several processes, including the transfer of photoassimilates and plant defense (Falk and Munne-Bosch 2010). It also regulates gene expression in signal transduction pathways. Plants usually contain the antioxidant tocopherol, which eliminates additional ROS and singlet oxygen species (Foyer et al. 2005). While tocopherol has an important role in severe stress conditions, other antioxidants play a role in moderate stress conditions (Havaux et al. 2005). Tocopherol’s capacity to transfer phenolic hydrogen to free radicals underlies its antioxidant action. Among tocopherol isomers, the most powerful antioxidants α-tocopherol and δ-tocopherol have the least antioxidant activity. Other β- and γ-tocopherols have moderate antioxidant capacity (Kamal-Eldin and Appelqvist 1996; Evans et al. 2002). Tocopherol provides the stability of the structure of cell membranes by removing lipid peroxyl radicals from the plant (Liebler 1993). In our research, it was discovered that α-tocopherol lessens the harmful effects of UV-B on the parameters of the tissue culture that were examined. Munné-Bosch et al. (1999) reported that α-tocopherol reduces the negative effects of stress factors. It is thought that this effect of α-tocopherol is due to its ion balance and cell membrane stability, as well as its antioxidant properties against ROS that damage DNA, protein, and membrane lipids. There are many studies showing that α-tocopherol inhibits lipid peroxidation (Kapoor et al. 2015). Abbasi et al. (2009) determined that tocopherol deficiency increases peroxidation of lipids in transgenic tobacco leaves. Carletti et al. (2003) reported that α-tocopherol in maize exposed to UV-B stress reduced the production of ROS resulting from this stress. On the other hand, Sattler et al. (2006) also reported that tocopherol regulates the expression of genes involved in lipid peroxidation.

Conclusion

UV-B radiation plays a crucial role in plant growth and development, but excessive exposure can have negative impacts on plants and ecosystems. While plants need UV-B radiation for essential processes like photosynthesis and pigment synthesis, high levels of UV-B radiation can cause damage to DNA, reduce plant growth and productivity, and even lead to plant death. Development of UV-resistant crop varieties can help to mitigate the negative impacts of UV-B radiation on plants. Therefore, it is vital to understand and manage the ecological and global impacts of UV-B radiation on plants. Plant tissue culture is useful for investigating the multiple products of plants, as well as for monitoring their reaction to stress conditions and selecting tolerant species. The effect of UV-B stress on some tissue culture parameters (CRGR, ECF, REC, and PN) related to mature embryo-induced somatic embryogenesis in wheat was evaluated. These parameters decreased with increasing UV-B exposure time. Furthermore, α-tocopherol concentrations of 50 and 100 mg/l have an adaptive effect on plants growing under UV-B stress, and its use can be advised to prevent the detrimental effects of UV-B. The results of the study have shown that in vitro conditions may also be a guide as an alternative to traditional methods in determining UV-B-tolerant genotypes in wheat.