2.3.1. CHS Gene Expression Studies 10 Days after ODN Incubation

The impact of the unmodified, and modified 1. met and 2. pto ODNs on the gene expression of both studied isoforms is presented in Figure 3. In order to determine whether the effect obtained by modified ODNs lasts longer, the gene expression was investigated in the material harvested after at least 10 days after the incubation with oligodeoxynucleotides. The value of *CHS* expression after ODN1 and ODN11 treatments was at a similar level (RQ ~2), regardless of the sequence modification, in comparison to the control. However, the ODN6 modified by methylation and thiophosphate triggered repression more prominently than unmodified ODN6, respectively by 41% and 63% in comparison to the control. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 7 of 17 triggered repression more prominently than unmodified ODN6, respectively by 41% and 63% in comparison to the control.

**Figure 3.** CHS gene expression in in vitro plants treated with modified ODNs after 10 days of incubation**.** The 4-week-old plants cultured in vitro were incubated with particular sequences of previously analyzed ODN 1, 6 and 11: unmodified ODN, ODN met (sequence with methylated cytosines) and ODN pto (sequence with thiophosphate bonds). The total expression of the two CHS genes *LuCHS6* and *LuCHS7* was determined by the real-time PCR reaction in plants harvested 10 days after incubation. The values are referred to the reference gene expression actin. The relative quantity (RQ) presents the transcript level in comparison to the control (set as 1, black). Data represent the mean value ± SD from at least three independent experiments. The significance of the differences between each mean and control was determined by Student's *t*-test. Asterisk indicates \* *p* < 0.05. **Figure 3.** CHS gene expression in in vitro plants treated with modified ODNs after 10 days of incubation. The 4-week-old plants cultured in vitro were incubated with particular sequences of previously analyzed ODN 1, 6 and 11: unmodified ODN, ODN met (sequence with methylated cytosines) and ODN pto (sequence with thiophosphate bonds). The total expression of the two CHS genes *LuCHS6* and *LuCHS7*was determined by the real-time PCR reaction in plants harvested 10 days after incubation. The values are referred to the reference gene expression actin. The relative quantity (RQ) presents the transcript level in comparison to the control (set as 1, black). Data represent the mean value ± SD from at least three independent experiments. The significance of the differences between each mean and control was determined by Student's *t*-test. Asterisk indicates \* *p* < 0.05.

2.3.2. Pattern of DNA Methylation in Variable CCGG Motifs of the CHS Gene after Treatment by Unmodified and Modified Oligos 2.3.2. Pattern of DNA Methylation in Variable CCGG Motifs of the CHS Gene after Treatment by Unmodified and Modified Oligos

In order to determine a specific pattern of the CHS gene methylation, crucial -CCGG- motifs in the CHS gene sequence were analyzed. As presented in the previous work, among the studied sites, the stable demethylated and highly variable in methylation -CCGG- were observed. Stable lack of cytosine methylation was observed in the following regions: 5'UTR (−232), non-coding (+217) and exon 2 (+1606), which were basically unchanged in comparison to the control. Variable sites were noted only in the coding region (+996, +1219, +1273). The figures show the percentage of lack of methylation, methylation of single inner cytosine and both cytosines. Since the stable sites do not differ in the cytosine methylation profile between studied plants, only results for variable CCGG sites In order to determine a specific pattern of the CHS gene methylation, crucial -CCGG- motifs in the CHS gene sequence were analyzed. As presented in the previous work, among the studied sites, the stable demethylated and highly variable in methylation -CCGG- were observed. Stable lack of cytosine methylation was observed in the following regions: 5'UTR (−232), non-coding (+217) and exon 2 (+1606), which were basically unchanged in comparison to the control. Variable sites were noted only in the coding region (+996, +1219, +1273). The figures show the percentage of lack of methylation, methylation of single inner cytosine and both cytosines. Since the stable sites do not differ in the cytosine methylation profile between studied plants, only results for variable CCGG sites are presented.

are presented. The analysis of the CCGG sites for unmodified and modified sequences of ODN11 is presented in Figure 3. Similarly as in Figure 4, the experiment was performed in the material harvested 10 days after the moment of incubation with oligodeoxynucleotides. After 10 days, the original sequence ODN11 did not lead to maintaining the changes in the methylation profile. The data presented by [9], shown in Figure 4D, indicate the potent increase in CmCGG methylation after 48 h in unmodified The analysis of the CCGG sites for unmodified and modified sequences of ODN11 is presented in Figure 3. Similarly as in Figure 4, the experiment was performed in the material harvested 10 days after the moment of incubation with oligodeoxynucleotides. After 10 days, the original sequence ODN11 did not lead to maintaining the changes in the methylation profile. The data presented by [9], shown in Figure 4D, indicate the potent increase in CmCGG methylation after 48 h in unmodified ODN11,

percentage was noted in the site +1219, by 15.0% of single cytosine methylation and 2.01% of double cytosines methylation. An increase in CmCmGG methylation after ODN11 pto was also observed at

ODN11, in comparison to the control. However, at 10 days after the flax incubation with the ODN11, the methylation profile became similar to that observed in the control. In case of ODN11 met and

not presented).

in comparison to the control. However, at 10 days after the flax incubation with the ODN11, the methylation profile became similar to that observed in the control. In case of ODN11 met and ODN11 pto, the perpetuation of initially induced cytosine methylation was observed (Figure 4). In ODN11 met, a potent increase of CmCGG was observed in all three variable sites +996, +1219, +1273, respectively by 19.3%, 23.0% and 13.8%, whereas for ODN11 pto in comparison to the control a higher percentage was noted in the site +1219, by 15.0% of single cytosine methylation and 2.01% of double cytosines methylation. An increase in CmCmGG methylation after ODN11 pto was also observed at the +996 site (by 8.1 %). *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 8 of 17

**Figure 4.** Methylation profiles for variable CCGG motifs in control plants (**A**). Plants treated with unmodified ODN11 (**B**) and modified ODN11 met (**C**) and ODN11 pto (**D**), 10 days after the treatment. The figure presents the percentage of the cytosine methylation in the variable CCGG sites (+996, +1219, +1273) located in the coding region of the CHS gene sequence. The genomic DNA was digested by restriction enzymes HpaII and MspI. The amount of non-digested DNA was determined by real-time PCR. The percentage of particular modification was presented for studied plants and control: CCGG—lack of methylation (dark grey), CCmGG—methylation of internal cytosine (light grey) and CmCmGG—methylation of both cytosines (medium grey). The site positions were presented according to the *LuCHS6* sequence, due to the presence of all CCGG sites. Data represent the mean value ± SD from at least three independent experiments. The significance of the differences between each mean and control was determined by Student's *t*-test. Asterisk indicates \* *p* < 0.05. **Figure 4.** Methylation profiles for variable CCGG motifs in control plants (**A**). Plants treated with unmodified ODN11 (**B**) and modified ODN11 met (**C**) and ODN11 pto (**D**), 10 days after the treatment. The figure presents the percentage of the cytosine methylation in the variable CCGG sites (+996, +1219, +1273) located in the coding region of the CHS gene sequence. The genomic DNA was digested by restriction enzymes HpaII and MspI. The amount of non-digested DNA was determined by real-time PCR. The percentage of particular modification was presented for studied plants and control: CCGG—lack of methylation (dark grey), CCmGG—methylation of internal cytosine (light grey) and CmCmGG—methylation of both cytosines (medium grey). The site positions were presented according to the *LuCHS6* sequence, due to the presence of all CCGG sites. Data represent the mean value ± SD from at least three independent experiments. The significance of the differences between each mean and control was determined by Student's *t*-test. Asterisk indicates \* *p* < 0.05.

2.3.3. Heredity of CHS Gene Expression in the F1 and F2 Generations of Modified ODN-Treated Flax Cultivated In Vitro 2.3.3. Heredity of CHS Gene Expression in the F1 and F2 Generations of Modified ODN-Treated Flax Cultivated In Vitro

The flax plants treated with modified ODNs were cultivated in the experimental field. A part of the seeds obtained were sterilized and introduced to the in vitro culture. The stabilization of the modulation of CHS gene expression by modified oligodeoxynucleotides in the F1 generation of plants was investigated and the results are presented in Table 3. For ODN1 met, 4 out of 5 (80%) studied plants maintained the overexpression of *CHS* in comparison to the control (set as 1). The flax plants treated with modified ODNs were cultivated in the experimental field. A part of the seeds obtained were sterilized and introduced to the in vitro culture. The stabilization of the modulation of CHS gene expression by modified oligodeoxynucleotides in the F1 generation of plants was investigated and the results are presented in Table 3. For ODN1 met, 4 out of 5 (80%) studied plants maintained the overexpression of *CHS* in comparison to the control (set as 1).

Regarding ODN6, the modification of oligonucleotide sequences via methylation made it possible to select one individual (out of five analyzed plants) with a repressed level of *CHS* expression (other four plants demonstrate similar too control level of *CHS* expression). For ODN11, both met

The F2 generation plants obtained after treatment with modified ODNs were analyzed in order to assess maintenance of the induced changes in chalcone synthase gene expression. In comparison to the F1 generation, the number of analyzed plants was higher (25–30 individual plants for each


**Table 3.** Maintenance of gene expression modulation of CHS gene in F1 and F2 generations of plants treated with modified ODNs (met and pto).

Regarding ODN6, the modification of oligonucleotide sequences via methylation made it possible to select one individual (out of five analyzed plants) with a repressed level of *CHS* expression (other four plants demonstrate similar too control level of *CHS* expression). For ODN11, both met and pto modifications were investigated in the next generation. Out of 7 analyzed plants, 6 presented transmission of the primarily induced modification in the chalcone synthase gene expression (data not presented).

The F2 generation plants obtained after treatment with modified ODNs were analyzed in order to assess maintenance of the induced changes in chalcone synthase gene expression. In comparison to the F1 generation, the number of analyzed plants was higher (25–30 individual plants for each ODN, except ODN6 with 6 plants). Despite the percentage of maintaining induced changes being lower, the results of F2 analysis were more significant.
