This research used cloned sequences of the desA gene, which encodes the Δ12-acyl-lipid desaturase from Synechocystis sp. PCC 6803, and the thII gene from T. daniellii, which encodes a sweet-tasting thaumatin II protein structurally related to the TLP family. The desaturase genes were translationally fused to the licBM3 gene, which encodes a reporter protein derived from the thermostable lichenase of Clostridium thermocellum. The hybrid genes, desA:licBM3, were cloned into pBISN-based vectors containing the selectable nptII gene, regulated by the cold-induced promoter CBF1 from the A. thaliana. The thaumatin II gene was integrated into a pNMD46732-based vector, with a selectable bar, under the control of the constitutive 35S cauliflower mosaic virus (CaMV) DNA promoter.

Control groups included transgenic plants with vector constructs incorporating the GFP reporter gene instead of the desaturase gene (desA), as well as wild-type tobacco (N. tabacum). All genetic constructs and transgenic plants were sourced from the collection at the Institute of Cell Biology and Genetic Engineering.

Leaf blades measuring 1–1.5 cm² were excised and placed into a bacterial suspension, which was incubated for 1 hour at +25 °C in the dark. The explants were then collected, washed to remove excess bacterial culture, and transferred to MS medium. They were co-cultivated with Agrobacterium for 2 days at +25 °C (to promote Agrobacterium growth). After co-cultivation, the explants were washed in sterile distilled water, dried on sterile filter paper for 10–15 min, and then placed in MS medium supplemented with the phytohormones BAP (1 μg mL^-1) and NAA (0.1 μg mL^-1), along with 700 μg mL^-1 cefotaxime to inhibit Agrobacterium growth. Regeneration of potential transgenic shoots was monitored over a period of 2–3 weeks, with regeneration occurring in vitro at 25 °C ± 3 °C under a 16-h photoperiod with light intensity of 100 μmol quanta (m²s).

Plant DNA was isolated using a standard CTAB method (Mark et al., 2024), and its concentration was determined by measuring optical density at 260 nm with a BioPhotometer spectrophotometer (Eppendorf, Germany). PCR was conducted using a 2720 Thermal Cycler (Applied Biosystems, USA), with primers thII-f (cac​ctt​cga​gat​cgt​caa​ccg​ctg) and thII-r (aag​ctt​agg​cag​tag​ggc​aga​aag​tg). Amplification conditions included 5 min at 94 °C; 30 cycles of 30 s at 94 °C, 45 s at 63 °C, and 45 s at 72 °C; followed by 5 min at 63 °C. Amplification products were separated via 1% agarose gel electrophoresis using TAE buffer and visualized with ethidium bromide. The O’GeneRuler 1 kb DNA Ladder (Fermentas, Lithuania) served as a DNA marker.

The preparation of plant material and the reaction were carried out according to the protocol described earlier (Gerasymenko et al., 2015). A qualitative lichenase assay and part of analysis of the FA was conducted 40 min after exposure to cold stress (0 °C for 30 min, followed by −5 °C for 80 min).

Samples for gas chromatography and mass spectrometry analysis were prepared according to previously published protocols (Gerasymenko et al., 2015). The analysis was conducted using an Agilent 6890N/5973 inert chromatographic-mass spectrometry system equipped with a DBFFAP capillary column (J&W Scientific, United States). Fatty acid methyl esters (FAMEs) were identified by comparing the obtained spectra with entries from the NIST 02 mass spectrum library and standard mixtures of bacterial FAMEs (47080U, Supelco).

For fatty acid analysis, mixed plant material from three lines of N. tabacum CBF1:desA:licBM3+thII, three lines of N. tabacum CBF1:desA:licBM3, two lines of N. tabacum CBF1:GFP:licBM3 obtained from transgenic plants, and N. tabacum were used. Six biological replicates and one analytical replicate were selected (upper unfolded leaves). The statistical significance of differences between average means was estimated using Student’s paired t-test, p-values were calculated using Excel standard functions.

The genetic transformation of transgenic N. tabacum, which demonstrated the insertion and expression of the desA gene, was carried out using a plasmid vector containing the thII gene. The active regeneration phase of the plants was observed within 1–1.2 months (Figure 1A). Two months post-transformation, regenerants were cultured on standard MS medium. After biomass growth, the presence of the thII gene insertion was confirmed via PCR (Figure 1B).

The expression of the desaturase gene was indirectly assessed through the activity of the thermostable lichenase reporter protein (licBM3), since both the desaturase and lichenase genes are located within the same reading frame under the control of the cold-inducible promoter. The assay evaluates the lichenase’s ability to degrade the complex carbohydrate lichenan (Figure 1C). The number of replicates for gene insertion detection and lichenase activity testing was 3, all of which were positive.

Results indicated that the plants maintained lichenase activity, suggesting the continued expression of the desA gene and lichenase activity. Tobacco plants demonstrating confirmed insertion and expression of the 12-acyl-lipid desaturase genes exhibited heightened linoleic acid content. To compare fatty acid spectra, tobacco plants with an insertion both the desA and thII genes, those only the desA gene, those with the GFP:licBM3 gene, and wild-type tobacco plants were analyzed. The upper open leaves were used for this analysis, which was performed under normal physiological conditions and following cold stress exposure. An increase in linoleic acid (C18:2) proportion was observed in plants with the desA gene insertion, differing from the fatty acid spectrum analysis results of the transgenic control and wild-type tobacco plants. No significant distinctions in fatty acid composition between plants with the desaturase gene and those with both desaturase and thaumatin gene insertions were observed (Table 1).