## 3 Results
### CRED-iPBS Assays  
The ten primers listed in Table 1 were used in CRED-iPBS analysis to detect cytosine gDNA methylation modifications. The results of the CRED-iPBS assay are indicated as the  
Figure 2: DNA methylation changes in the wheat exposed to MSH: (**A**) total band; (**B**) polymorphism; (**C**) GTS value as estimated using different MSH experimental groups. percentage of polymorphism in CRED-iPBS assays that were digested by _Mspl_ and _HpaII_ (Figure 3 and Table 3). Compared to the PCR production obtained from control DNA, the results show that whether DNA was hypermethylated or hypomethyllated depended on the type and amount of MSH. The result clearly shows that the number of bands observed in the _Mspl_- and _HpaII_-digested controls were 68 and 78 bands, respectively. The number of bands in the experimental groups digested with _Mspl_ (266) was higher than in the experimental groups digested with _HpaII_ (263). The experimental groups for _Mspl_ showed a net increase of 190 newly formed bands and a net decrease of 76 pre-existing bands compared to the control group. Compared to the control group for _HpaII_, the experimental groups showed a net increase of 106 newly formed bands and a net decrease of 157 pre-existing bands. The percentages of _Mspl_ polymorphism showed a range of variation from 20% to 40%. The results of the CRED-iPBS analysis conducted on _Mspl_ showed that the experimental group treated with 0.05 uM estrogen presented the highest polymorphism value of 40%, while the experimental group treated with 5 uM progesterone presented the lowest value of 20%. The results of the study indicate that there was a decrease in the polymorphism values with an increased in estrogen concentrations for _Mspl_, leading to a state of hypomethyllation. In contrast, increasing doses of 17-\(\beta\)-estradiol, progesterone, and testosterone did not consistently decrease or increase in polymorphism level (%) in the plants. The percentage of _HpaII_ polymorphism showed a variable range from 19.4% to 33.3%. According to the results of the CRED-iPBS analysis performed on _HpaII_, it was observed that the experimental group treated with 0.5 uM 17-\(\beta\)-estradiol showed a maximum polymorphism value of 33.3%. In contrast, the experimental group treated with 0.05 uM 17-\(\beta\)-estradiol and 0.05 uM progesterone showed a minimum value of 19.4% (Figure 4). According to the average percentage of polymorphisms observed in _HpaII_ and _Mspl_ digestion, it can be inferred that raised levels of MSHs (17-\(\beta\)-estradiol, progesterone, and estrogen) may confer a protective effect against hypermethylation. Figure 3: CRED-iPBS profiles for various MSH experimental groups with iPBS 2217 primers; 1: M, 100–1000 bp DNA ladder; 2: control _Hpa II_; 3: control _Mspl I_; 4: 0.05 uM 17-\(\beta\)-estradiol _Hpa II_; 5: 0.05 uM 17-\(\beta\)-estradiol _Mspl I_; 6: 0.5 uM 17-\(\beta\)-estradiol _Hpa II_; 7: 0.5 uM 17-\(\beta\)-estradiol _Mspl I_; 8: 5 uM 17-\(\beta\)-estradiol _Hpa II_; 9: 5 uM 17-\(\beta\)-estradiol _Mspl I_; 10: 0.05 uM progesterone _Hpa II_; 11: 0.05 uM progesterone _Msp I_; 12: 0.5 uM progesterone _Hpa II_; 13: 0.5 uM progesterone _Msp I_; 14: 5 uM progesterone _Hpa II_; 15: 5 uM progesterone _Mspl I_; 16: 0.05 uM testosterone _Hpa II_;17: 0.05 uM testosterone _Msp I_; 18: 0.5 uM testosterone _Hpa II_; 19: 0.5 uM testosterone _Msp I_; 20: 5 uM testosterone _Hpa II_; 21: 5 uM testosterone _Msp I_; 22: 0.05 uM estrogen _Hpa II_; 23: 0.05 uM estrogen _Msp I_; 24: 0.5 uM estrogen _Hpa II_; 25: 0.5 uM estrogen _Msp I_; 26: 5 uM estrogen _Hpa II_ and 5 uM estrogen _Msp I_.