Oilseed rape is considered relatively recalcitrant for genetic modification. This work was performed to establish conditions for effective transformation and regeneration of commercially used cultivars Campino, Haydn, Heros, Hunter and Topas (Brassica napus L.). Cotyledonary petioles and hypocotyls (obtained from the seedlings grown under dark conditions) as a source of explants were used. Our experiments revealed that a lower selection pressure in combination with postponing of selection by 14 days after co-cultivation resulted in recovery of transgenic plants from all cultivars. Transformants were obtained with efficiency from 1 to 5.5%. Histochemically GUS-positive plants were analysed by PCR using primers corresponding to the internal fragments of gus and nptII genes. The transgenic nature was confirmed by Southern blot analyses using a specific nptII probe. This work enriches the list of oilseed rape cultivars available for genetic modification.

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The (1,3)-β-D-glucan also referred to as callose is a main component of cell walls of higher plants. Many physiological processes are associated with the changes in callose deposition. Callose is synthesised by the callose synthase complex while its degradation is regulated by the hydrolytic enzymes β-1,3-glucanases. The latter one specifically degrade (1,3)-β-D-glucans. This work is aimed to study β-1,3-glucanase activities in the leaves of plants at two leaf stage in two diploids (Agilops tauschii, Triticum monococcum L.), four tetraploids (Ae. cylindrica, Ae. triuncialis, T. araraticum, T. dicoccum) and two hexaploids (T. aestivum L, T. spelta L.). The leaves were subjected to qualitative and quantitative β-1,3-glucanase activity assays. Our results showed that the total β-1,3-glucanase activities were variable and genotype dependent. No significant correlation between β-1,3-glucanase activities and ploidy level was observed. The gel activity assays revealed a single fraction of ~52 kDa Glu1 that was found in all genotypes. The Glu1 fraction corresponds to a single or two acidic Glu isoforms in dependence on genotype. However, none of the acidic Glu fractions can be assigned as a specific for di-, tetra- or hexaploid genotypes. A single basic GluF isoform was detected and found as present in all genotypes.

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The basic β-1,3-glucanase of the carnivorous plant Drosera binata was tested as a purified protein, as well as under the control of a double CaMV35S promoter in transgenic tobacco for its capability to inhibit the growth of Trichoderma viride, Rhizoctonia solani, Alternaria solani, and Fusarium poae in an in-vitro assay. The purified protein inhibited tested phytopathogens but not the saprophytic fungus T. viride. Out of the analysed transgenic plants, lines 13, 16, 19, and 22 exhibited high DbGluc1 transcript abundance normalised to the actin transcript. Because of DbGluc1 transgene expression, lines 13 and 16 showed a 1.7-fold increase and lines 19 and 22 showed more than a 2-fold increase in total β-1,3-glucanase activity compared to the non-transgenic control. In accordance with the purified β-1,3-glucanase in-vitro antifungal assay, crude protein extracts of lines 19 and 22 significantly inhibited the growth of phytopathogens (14–34%). Further analyses revealed that the complementary action of transgenic β-1,3-glucanase and 20% higher activity of endogenous chitinase(s) in these lines were crucial for maximising the antifungal efficiency of crude protein extracts.

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Defense components such as chitinases (EC 3.2.1.14) are crucial for plants to cope diseases. Despite of that the pattern and activities of these enzymes in agronomically important Triticale is unexplored. This work is aimed to study chitinase activities in the leaves of plants of early developmental stages in two diploids (Aegilops tauschii Coss., Triticum monococcum L.), four tetraploids (Ae. cylindrical Host, Ae. triuncialis L., T. araraticum Jakubyz, T. dicoccum Schrank) and two hexaploids (T. aestivum L., T. spelta L.). The leaves were subjected to quantitative and qualitative activity assays using synthetic 4-methylumbelliferyl-β-D-N,N´,N´´-triacetylchitotrioside and glycolchitin as substrates, respectively. Our results showed that the activities of chitinases with specificity towards short oligomers were variable and genotype dependent. The enzyme activities in the tetra- and hexaploid genotypes were significantly higher than in diplod counterparts. In the gel detection assays were revealed up to four fractions (~20, 30, 42 and 95 kDa) of proteins with the chitinase activity towards long chain polymers. The isoform of ~30 kDa was identified in all analyzed genotypes. Among the seven acidic and three basic chitinase fractions identified, three acidic (ChiA, ChiB, ChiC) and two (ChiH, ChiI) fractions were present in all genotypes. None of the isoforms can be assigned as specific with respect to ploidy.

Dehydrins, one of the Late Embryogenesis Abundant (LEA) proteins, are specialized proteins related to environmental stress tolerance in plants. They are multifunctional proteins that can bind free metal ions, interact with macromolecules and specific membrane regions or participate in reduction of reactive oxygen species. Here, we report preparation of a plant transformation vector containing Arabidopsis dehydrin gene At1g54410 under the control of the strong constitutive double dCAMV 35S promoter and the selectable marker neomycin phosphotransferase gene driven by the nopaline synthase promoter. The gene At1g54410 was isolated from Arabidopsis genomic DNA by PCR approach. The resulting binary vector pND3 was introduced into the Agrobacterium tumefaciens strain LBA 4404 and verified for its stability in agrobacterial cells.