**2. Results**

#### *2.1. Heredity of CHS Gene Expression in the F1, F2 and F3 Generations of ODN-Treated Flax.*

The nomenclature of the CHS gene isoforms *CHS1* (Phytozome database, Lus10031622) and *CHS2* (Phytozome database, Lus10033717) published in our previous work [9] will be followed in the present paper by the numbers used by [20], sc. *LuCHS6* (Phytozome database, Lus10033717) and *LuCHS7* (Phytozome database, Lus10031622).

The level of the mRNA derived from both studied isoforms, *LuCHS6* and *LuCHS7*, was analyzed in the F1 and F2 generation of plants treated with ODNs. In our previous study, the series of ODN sequences homologous to *CHS* was investigated. The oligodeoxynucleotides directed to the regulatory regions, 5'UTR-ODN1, intron-ODN6 and 3'UTR-ODN11 have shown significant modulation in the CHS gene expression. For ODN1 and ODN11, overexpression of the endogenous CHS gene was observed. Alternatively, for ODN6, the gene repression was noted for the studied gene. These three ODNs were selected for further analyses. Table 2 presents the maintenance of the modulation of CHS gene expression induced by ODNs in the next generations (F1–F3).

**Table 2.** Maintenance of gene expression modulation of CHS gene in F1, F2 and F3 generations of plants treated with short oligodeoxynucleotides (ODNs).


A part of seeds obtained from the F0 plants treated with ODNs were introduced into the in vitro culture in order to select the lines that maintained the initially induced changes in the *CHS* expression. The percentage proportion of the plants that maintained the tendency of the initial modulation to all analyzed plants was as follows: ODN1-71%, ODN6-5%, ODN11-86%, where the studied groups varied between 7 to 20 individuals in the F1 plant generation. The most plants with the stable modification in the *CHS* gene were noted for the ODN11, whereas the least for ODN6. Seeds of selected plants that maintained the generated characteristics served to obtain the next generation (F1) under field conditions.

Seeds collected from the F1 generation of plants were also partially introduced to the in vitro conditions. The total gene expression of *LuCHS6* and *LuCHS7* (the sum) was determined in the F2 flax. Among the series of plants, individuals that presented a tendency of *CHS* expression similar to the parental generation were selected. The most plants that perpetuated the alteration in the CHS gene expression was observed according to the ODN11 treatment (75%—6 of 8 ODN11 treated plants). A minor percentage (33%) of the ODN1 treated plants maintained in the F2 generation induced primarily changes. The repression noted after ODN6 in the F0 and F1 plants was not observed in the F2 generation and the massive overexpression in those plants was investigated.

The F3 field generation of plants treated with ODNs was also determined in the context of the maintenance of the induced changes in the CHS transcript level. A decreased percentage of modulation maintenance was observed for ODN1 (38%) and ODN11 (6%) in relation to the previous generations. In contrast, the number of plants that perpetuated repression of the CHS gene was the highest among the F3 generation of ODN-treated plants and the maintenance of the induced changes reached 61%.
