Effects of mammalian sex hormones on regeneration capacity, retrotransposon polymorphism and genomic instability in wheat (Triticum aestivum L.)

Mammalian steroid hormones (MSH) are also inherently synthesized by several plant species. However, external application of steroid hormones further stimulates cell division, pollen germination, plant growth and development. There is little known about the effects of MSH on polymorphism and DNA damage in Triticum aestivum L under in vitro conditions. In this study, inter-primer binding site (iPBS) retrotransposon and coupled restriction enzyme digestion-iPBS (CRED-iPBS) markers were used for detection of the variation in responded embryogenic callus (REC) that were obtained from endosperm-supported mature embryo of wheat in Murashige and Skoog (MS) medium containing different concentrations [0 (control), 10–4, 10–6 and 10–8 m mol L−1] of MSH (progesterone, 17β-estradiol, estrone and testosterone). Responded embryogenic callus (REC) and regenerable callus (RE) induction from mature embryos varied with MSH type and concentrations. The highest level of genomic template stability (GTS) value (80.52%) was obtained from 10–8 mM progesterone treatments to the lowest value (68.83%) from 10–4 mM 17 β-estradiol treatments. Epigenetic changes were more frequent and variable than the genetic changes. While DNA hypermethylation was observed at higher 17 β-estradiol concentrations, DNA hypomethylation was observed in progesterone treatments. It was concluded based on iPBS and CRED-iPBS findings that high MSH concentrations caused DNA changes and methylation. Variability among regenerates caused by MSH treatments in different concentration were observed. iPBS polymorphisms indicated the presence of genetic variation among the wheat regenerates. Application of MSH in high concentration had a clearly effective on polymorphism and genomic instability in the wheat mature embryo.


Introduction
Plant hormones are inherently synthesized organic molecules with great impacts on several physiological processes. They designate the entire plant growth and development. Even at low concentrations, plant hormones synchronize cellular activities. Following the production, plant hormones are moved to other parts of the plants and they may have immediate effects on tissue differentiation, plant growth and development. Plants produce a wide variety of hormones including auxins, gibberellins, cytokinins, abscisic acid, ethylene, brassinosteroids, salicylates, jasmonates and strigolactones (Su et al. 2017).
Mammalian sex hormones (MSHs) inherently occur in some plant species (Turk 2021). While androsterone and progesterone were identified in 80% of plant species, testosterone was identified in 70% and estrogens in 50% of the plant species. Milanesi et al. (2001) investigated the effects of external application of plant-originated steroids on cell growth and tissue differentiation of vascular plants under in vitro conditions. It was reported that plant-originated steroids stimulated cell division, pollen germination, plant growth and development. MSHs may also improve stress and disease resistance of the plants, thus improve yield levels and quality parameters (Nolan et al. 2020). Several research have been conducted to identify type and quantity of MSHs in plant species and to investigate the effects of these hormones on plant growth and development. However, there is little known about the effects of MSHs on plant physiology, genetics and epigenetics under in vivo and in vitro conditions. Previous studies indicated that external MSH treatments had various positive effects on plant physiology. Those studies mostly focused on potential of MSH in alleviation of negative impacts of biotic stress factors. Present literature reviews revealed that there were not any studies about the effects of MSH on polymorphism and DNA damage in Triticum aestivum L under in vitro conditions. Therefore, current study was designed to elucidate the roles of MSH in polymorphism and genomic instability of wheat under in vitro conditions.
Effects of MSHs on genetic polymorphism and DNA methylation can be used with the use of various methods including comet assay, chromosomal aberrations assay, micronucleus assay and DNA analysis methods. Each method has inherent limitations in terms of sensitivity and detection. However, DNA analysis with molecular marker technology offers greater sensitivity and selectivity. Molecular markers have successfully been used to determine polymorphism and DNA metalations and iPBS was used to elucidate the effect of different treatments on plants . The iPBS offers a universal method for DNA fingerprinting (Kalendar et al. (2010). The iPBS primers are designed on primer binding site (PBS) sequences with conserved parts of tRNAs for reverse transcription during replication cycle of retrotransposons (Guo et al. 2014). Transposable elements (TEs) play a great role in genome instability and evolution and offer improved adaptation to adverse conditions (Kalendar et al. 2021). Insertion or transposition of TEs under normal conditions (without mutations, biotic or abiotic stressors) can have detrimental impacts on plants, therefore under normal conditions, TEs are either silenced or inactivated through epigenetic silencing mechanisms including DNA methylation and suppressive chromatin alterations (Lerat et al. 2019). CRED (Coupled Restriction Enzyme Digestion) technique involves DNA profiling with the use of molecular markers. Also, coupled restriction enzyme digestion and inter-primer binding site (CRED-iPBS) is important technique for studying methylation status in plants. It is used to identify changes in cytosine methylation stress-induced toxicity in plant genomes (Hosseinpour et al. 2019a). Present literature reviews naked that there aren't any reports showing the effect of MSHs on plant tissue culture, genomic instability, and DNA methylation of wheat plants. Precisely, this study was conducted to investigate the effects of MSH on REC induction and regeneration of wheat plants as well as CRED-iPBS method coupled with iPBS markers was used to determine DNA methylation status of wheat plants. The primary objective was set as to elucidate the effects of MSHs on plant tissue culture, genomic instability and DNA methylation of wheat plants.

Plant material
Wheat (Triticum aestivum L. cv Kirik) seeds were obtained from Field Crops Department of Ataturk University (Erzurum, Turkey).

Plant growth and treatment conditions
Mature seeds were surface sterilized first by washing in 70% ethanol for 5 min, rinsed with sterile water and then with 10% solution of sodium hypochlorite with two drops of Tween-20 for 20 min and subsequently seeds were rinsed with sterile water three times and incubated at 4 ºC for 24 h in sterile distilled water. Imbibed seeds were prepared as described by (Aydin et al. 2011;Hosseinpour et al. 2020). Seeds were placed in furrow downwards in petri dishes containing MS (Murashige and Skoog) medium supplemented with 20 mg/L sucrose, 2 g/L phytagel and 12 mg/L dicamba for callus induction (Aydin et al. 2011). The media pH was adjusted to 5.8 with sodium hydroxide (NaOH) before autoclaving for 20 min at 121 ℃ and 105 kPa. Vitamin and auxin solutions were filter-sterilized and subsequently added to the autoclaved media. The Petri dishes were incubated at 25 ± 1 ℃ for 30 days in darkness for callus induction from endosperm-supported mature embryos. After 30 days of culture, 10 prepared calli in four replications were placed into petri dishes containing MS (Murashige and Skoog) medium supplemented with 20 mg/L sucrose, 2 g/L phytagel and different concentrations [0 (control), 10 -4 , 10 -6 and 10 -8 m mol L −1 ] of mammalian sex hormones (estrogen, progesterone, 17 β-estradiol and testosterone). In control treatments, 0.5 mg/L TDZ was used instead of MSH. Media solidification, pH adjustment and sterilization were carried out as described for callus induction media. Regeneration callus cultures were incubated in a growth chamber at 25 ± 1 °C in 16 h light (62 μmol m −2 s −1 ) and 8 h dark photoperiod. Responded embryogenic callus (REC) rate and regeneration efficiency (RE) were determined after four weeks. REC (%) was calculated as number of RECs / number of explants × 100 and RE was calculated as the number of regenerated plants / number of RECs (Kodaz et al. 2021).

Isolation of genomic DNA
Genomic DNA was extracted from the responded embryogenic callus raised from responded embryogenic calli, using cetyl trimethylammoniumbromide buffer as recommended by (Zeinalzadehtabrizi et al. 2015). Concentration and quality of genomic DNA was measured by Nanodrop spectrophotometer (Thermo Fisher Scientific) and run on 1.5% (w/v) agarose gel.

iPBS and CRED-iPBS PCR assays
Twenty five primers were tested for iPBS-PCR amplification (Kalendar et al. 2010). For iPBS analysis, PCR reaction was carried out in a total volume of 20 ml PCR mix containing 10 X PCR buffer, 25 mM MgCl 2 , 10 mM dNTP mix, ddH2O, 10 pmol random primer, 1 U Taq DNA polymerase and 50 ng/ml sample DNA. After vertexing, tubes were placed in a thermocycler (Sensoquest GmbH, Labcycler Gradient, Germany) for amplification. PCR steps were 5 min pre-denature step at 95 ℃, followed by 40 cycles of 1 min for denaturing at 95 ℃, 1 min for annealing at 56-63 ℃, 2 min for extension at 72 ºC, subsequently 10 min final primer extension at 72 ℃, then dropped to 4 ℃. Out of 25, only 10 iPBS oligonucleotide primers resulted in specific and stable DNA profiles in all experimental groups (Table 1). For CRED-iPBS analysis, 1 mg of sample DNAs from each treatment were separately digested with 1 µL (1 FDU) HpaII and 1 µL (1 FDU) MspI (Thermo Scientific) endonucleases at 37 ℃ for 2 h according to manufacturer's guidelines. Digested DNA (for each endonuclease) was added in PCR mix instead of nondigested gDNA. The primers listed in Table 1 were employed for amplification. PCR steps were the same as iPBS analysis described above.

Electrophoresis
The iPBSs and CRED-iPBS PCR products were separated with 1.5% agarose gel containing 0.05 mL/mL ethidium bromide in electrophoresis using in 1X SB buffer in 100 V for 120 min and 100-1000 bp (Sigma Aldrich No: P1473-1VL) DNA ladder was used to estimate the molecular weight of the fragments. The gels were photographed under UV light in a Universal Hood II (Bio-Rad, Hercules, CA, USA).

Genetics analysis
The iPBS and CRED-iPBS banding patterns were analyzed using TotalLab TL120 software (Nonlinear Dynamics LtdR). Polymorphism in the iPBS profiles was expressed as the disappearance of a normal band and the appearance of a new band compared to the control. The average polymorphism was calculated for each experimental group (MSH treatments at different concentrations) and changes in these values were calculated as a percentage of their value in the control (set to 100%) (Hosseinpour et al. 2019). The genomic template stability (GTS %) which is a quantitative measurement was calculated for iPBS with the following formula: GTS = 1 − a n x100 ; where a is the average number of polymorphic bands found in each treated template and n is the

Responding embryogenic callus (REC) induction (%)
REC rate was determined based on the presence of embryogenic callus forming plantlets with healthy roots and shoots (Fig. 1b). ANOVA results (Table 2) revealed significant effects of MSH type, MSH concentration and their two-way interactions on REC rates. In terms of MSH types, the highest REC rate was observed in progesterone-containing MS culture medium (53.13%) and the lowest value (37.50%) was observed in testosterone-containing MS culture medium (Table 2). MSH concentrations lead to significant differences in REC rates. The highest REC rate (72.50%) was observed in the control without MSH (Table 2) and the lowest value (3.13%) was obtained from the MS medium containing 10 -4 mM MSH (Table 2). MSH type x concentration interactions had significant effects on REC rates ( Table 2). The highest REC rate was obtained from the MS media containing 10 -8 mM concentration of progesterone (75.00%) and the lowest from the culture medium supplemented with 10 -4 mM concentration of 17-β estradiol and estrogen (0.00%) ( Table 2).

Regeneration efficiency (RE)
In this study, effects of MSH type and MSH concentration, as well as their two-way interactions on RE were also investigated (Table 2). It was observed that among the MS culture media, progesterone-supplemented media yielded higher RE (1.04 number) than the other MSH types (Table 2). RE was significantly depended on MSH concentration. The highest RE (1.15 number) was obtained from the control without MSH, followed respectively by 10 -6 and 10 -4 mM concentrations (Table 2). In terms of MSH type x concentration interactions, the highest RE (2.38 number) was obtained from 10 -6 mM progesterone-containing MS culture medium and the lowest values (0.00 number) were obtained from 10 -8 mM 17-β estradiol, estrogen, progesterone and testosterone and 10 -6 mM testosterone-containing MS media (Table 2).

Genetics analysis iPBS analysis
Here, we report the effect of different MSH type and concentrations on responding embryogenic callus in wheat in terms of genotoxicity, DNA methylation and LTR polymorphism. In total, out of twenty-five iPBS primers tested, only 10 primer (as shown Table 1) produced clear and obvious as well as polymorphic banding patterns in all treatments. These primers gave specific and stable bands in wheat genome. It was seen important changes in MSH type and concentrations in iPBS profiles. The appearance of new bands as well as disappearances of bands and difference in band frequency is the slightly alters in the iPBS designs produced by MSH treatments in wheat. The number of polymorphic bands appearing for the primers used was determined by comparing control and MSH-exposed plants. In this study, totally 77 bands were appeared in control treatment. The number of polymorphic bands in MSH-treated wheat for all primers applied in the study ranged from 3 (iBPS-2217) to 14 (iBPS-2217). Amplified fragment lengths ranging from 140 140 (iPBS-2080) bands to 1000 (iPBS-2402) bp in treated plants. (Table 2 and Table 3). According to the results, control and MSH-treated samples showed significant differences in iPBS profiles. These differences were arising disappearance (−) and/or appearance ( +) of the bands. After MSH treatment, totally 125 normal iPBS bands disappeared and 104 new bands appeared compared to control.

CRED-iPBS analysis
For CRED-iPBS approach, only ten primers products that were applied for the iPBS analysis produced specific and stable bands (Table 4). The result show DNA hypermethylation/hypomethylation was dependent MSH type and       Table 4 Results   concentration compare to the PCR production obtained from the control DNA. Results of CRED-iPBS analysis are expressed as polymorphism percentage in MspI and HpaII digested CRED-iPBS assays (Table 4). The result clearly shows the number of bands in MspI and HpaII digested control treatment were 65 and 72 respectively. The extent of bands in MspI digested experimental groups (251 number) higher than HpaII digested experimental groups (203 number). MspI polymorphism percentages varied between 23.06% and 40.00%. the result show polymorphism values increased with increasing MSH concentrations. HpaII polymorphism percentages ranged from 19.4% to 32.3%. As in MspI in HpaII polymorphism percentages changes are the same (Table 4). In general, 10 -4 mM 17 β-estradiol treatments yielded higher polymorphism percentages of both MspI and HpaII digested CRED-iPBS assays. On the other hand, 10 -8 mM progesterone treatments decreased both HpaII and MspI polymorphisms percentages. In other words, the highest MSH concentration had an impact on cytosine methylation status, thus can be classified as hyper-methylation when the average polymorphism percentage for MspI digestion was taken into consideration. A clear decrease was seen in average polymorphism percentage and methylation statue; thus, it can be concluded that MSH at low concentration had a protective role in hyper-methylation. The polymorphism percentage gradually decreased at low MSH concentrations (Table 4). This statue could be explained as hypo-methylation. These results have shown that MSH in low concentration have the antagonistic effect against epigenetic and genotoxic effects.

Discussion
The primary objective of the present study was to elucidate the effects of MSHs on plant tissue culture, genomic instability and DNA methylation of wheat plants. It was reported that external MSH treatments had positive impact on plant growth and development under both normal and stress conditions (Erdal 2012;Janeczko 2012). Several research have been conducted to investigate presence, quantity and action mechanisms of MSH presence in different plant species (Tarkowská 2019); Erdal 2012;Erdal and Dumlupinar 2011). However, there is little known about the effects of MSH on polymorphism and DNA damage in Triticum aestivum L under in vitro conditions. Thus, current study primarily focused on effects of MSH treatments on polymorphism and genomic instability of wheat under in vitro conditions.
In this study, REC induction rates and regenerable callus from mature embryos varied the MSH type and concentrations. The highest responded embryogenic callus (REC) rate (90.00%) was obtained 10 -8 mM concentration of progesterone-containing media and the greatest regeneration efficiency (RE) (2.38 number) was observed in 10 -6 mM concentration of progesterone-containing media. The lowest REC rates (0.00%) were seen in 10 -4 mM concentration of 17-β estradiol and estrogen-containing media and the lowest RE values (0.00 number) were seen in 10 -8 mM concentration of 17-β estradiol, estrogen, progesterone and testosterone-containing media and 10 -6 mM concentration of testosterone-containing media. Uysal and Bezirganoglu (2017) indicated that MSH treatments influenced regeneration capacity of Triticale. For number of explant forming shoots, the best outcomes were achieved respectively by estrogen and progesterone treatments. Dadasoglu and Tosun (2018) conducted a study with exogenous MSH treatments in Onobrychis sativa L. plants and reported the greatest number of shoots from 10 -6 mM concentration of progesterone, 10 -4 and 10 -6 mM concentrations of estrone hormones and the greatest callus formation from 10 -4 mM concentration of estrone hormone. Additionally, testosterone treatments yielded the best outcomes for rooting traits. Janeczko and Skoczowski (2005) indicated that MSHs had significant effects on root and shoot growth and development. The reason why the responded embryogenic callus rate and regeneration efficiency was higher in progesterone treatments than the control may be the fact that plant tissue culture generated a stress condition and mammalian sex hormones alleviated the stress in plant tissue culture medium in wheat. Erdal (2012) indicated that MSH treatments decreased salt stressinduced reductions in dry weight, sugar, proline, protein, chlorophyll and glutathione contents and concluded that MSH treatments modulated the negative effects of stress conditions.
In present study, high MSH concentrations altered genetic template instability and cytosine methylation in wheat. In addition, low MSH concentrations significantly improved molecular disorders of the plants. DNA metalation and polymorphism differed among the experimental groups. CRED has been successfully used for determination of cytosine methylation in plant genome or toxicity caused by abiotic stresses (Hosseinpour et al. 2019a). CRED-iPBS technique was employed to investigate how the wheat genome alters its cytosine methylation status in response to NaCl stress and any enhancements in DNA methylation statue against to NaCl by using PGPBs with CuO-NPs treatments by Hosseinpour et al. (2021). Also, effects of chemical mutagens on the polymorphism and genomic instability of the wheat mature embryo were more pronounced in ethyl methanesulfonate (EMS) and Sodium azide (NaN 3 ) treatments by CRED-iPBS technique (Türkoğlu et al. 2022a, b).
In this study inter-primer binding site (iPBS) retrotransposon and CRED-iPBS (Coupled Restriction Enzyme Digestion-iPBS) techniques were used to define the DNA damage levels and changes in the pattern of DNA methylation.
Retrotransposons are ubiquitous, active and abundant components of plant genomes and retrotransposon-originated polymorphisms have generally been detected with marker systems. The iPBS retrotransposon method is commonly used for detection of polymorphisms through amplified DNA segments. The iPBS is a marker system operating through transposable elements. Retrotransposons represent stress-induced molecular changes in plants (Ramakrishnan et al. 2021). They are generally inactive during normal growth stages. Biotic and abiotic stressors reactivate LTR retrotransposons in plants (Mansour 2009). Long terminal repeat (LTR) retrotransposons are mobile elements of the plant genome (Kalendar et al. 2021). DNA methylation is an important enzymatic modification and represents a chemical alteration of DNA structure. It plays a key role in regulation of gene expression and genome defense (Erturk et al. 2015). In present study, high MSH concentrations increased retrotransposon movement, thus reduced GTS ratios, probably because plants developed biochemical and molecular defense mechanisms to eliminate destructive effects of MSH. All MSHs at the greatest concentration decreased GTS values. Such a case indicated that high MSH concentrations had genotoxic impacts on wheat genome. According to Citterio et al. (2002), iPBS technique is sufficiently sensitive for detection of DNA damages. A change in iPBS profile as compared to profile of control sample is reflected as reduction in GTS value (Aly 2012;Doğan et al. 2012;Gupta and Sarin 2009;Tanee et al. 2012). Similar with the present findings, Alzohairy et al. (2012) indicated that environmental stress factors induced LTR retrotransposons in barley plants. Taspinar et al. (2018) also indicated that aluminum stress reduced GTS rates in maize plants through induced retrotransposon movement.
CRED-iPBS technique was used in present study to assess the effects of MSH type and concentrations on cytosine methylation status and DNA methylation of wheat genome. Present findings revealed DNA hypermethylation at higher concentrations of 17 β-estradiol and DNA hypomethylation at lower concentrations of progesterone hormone. Present findings clearly demonstrated that low MSH concentrations resulted in hypo-methylation. While hypermethylation is generally associated with gene silencing, hypo-methylation is generally related to active transcription (Steward et al. 2002). Plant tissue culture is largely influenced by epigenetic changes at histone methylation/ demethylation level (Grafi et al. 2007), DNA methylation level (Han et al. 2018), by change in gene expression (Kabita et al. 2019) and by a wide range of small RNAs (Li et al. 2012). There is less known about the mechanisms revealing somaclonal variations. Stressful environments influence cell membrane and wall. Signals are then transmitted to plant organelles and such signals trigger reactive oxygen species (Wachsman 1997) through altered biochemical pathways (Kumaravel et al. 2017). Signaling (retrograde) pathways also transmit the change into the nucleus (Chi et al. 2015) resulting in (epi) genetic reaction, then cell dedifferentiation and differentiation. Cell differentiation includes histone modification (Grafi 2004), DNA methylation (Lee and Seo 2018) and gene expression (Wibowo et al. 2018) alterations. The signal is transmitted back to chloroplasts and mitochondria (anterograde) when a new balance was established. It can be stated based on present findings that antigenotoxic effect of low MSH concentrations could be attributed to radical-scavenging and high antioxidant activity of these hormones. However, role of MSH in epigenetic modifications is still to be elucidated.

Conclusion
Since the discovery of MSHs in plants, effects of exogenous MSH treatments on plant growth and development have been studied by several researchers. So far, this paper is the first report presenting the effects of MSH type and concentrations on responded embryogenic calli (REC) rates, regeneration efficiency (RE) and DNA methylation of wheat plants. In this study, the highest responded embryogenic callus (90.00%) was obtained 10 -8 mM concentration of progesterone-containing media and the greatest regeneration efficiency (2.38 number) was observed in 10 -6 mM concentration of progesterone-containing media. This study was also successful in detection of MSH-induced retrotransposon polymorphism. Present findings revealed that MSH treatments influenced cytosine methylation status and improved genomic template stability of wheat plants. In present study, effects of MSH type and concentrations on DNA methylation profiles were also investigated with the use of CRED-iPBS technique. Present findings revealed changes in DNA methylation at high MSH concentrations. However, further research is recommended to better elucidate the role of MSHs in plant tissue culture.
Author contributions AT conceived and designed the experiments and worked for writing and editing the English of this paper.

Funding
The authors have not disclosed any funding.

Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.