*4.6. Gene Expression Analysis*

The expression of investigated genes was analyzed via real-time PCR. The total RNA was isolated from freeze-ground green tissue of the transgenic plants. Isolation was performed using the Trizol method (Invitrogen, Carlsbad, CA, USA), following the protocol of the manufacturer. The isolated RNA was deprived of DNA contamination by DNase I (Invitrogen, Carlsbad, USA). The purified RNA was used as a template for cDNA synthesis using reverse transcriptase—High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA).

Real-time PCR reactions were performed using a DyNAmo SYBR Green qPCR kit (Thermo Scientific, Waltham, MA, USA). Primers used for the reaction are presented in the Supplementary data Table S1. The used system was the Applied Biosystems StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, USA). The reaction was conducted according to the protocol provided by the manufacturer. Each reaction was performed in three repeats. As the reference, the actin gene was used. The results were presented as the relative quantification (RQ) to the reference gene.
