Activation of the peroxidase gene in potato plants expressing the gene of antimicrobial peptide MsrA1

Antimicrobial peptide MsrA1 is a synthetic hybrid molecule based on cecropin A from giant silk moth larvae and on melittin from melliferous bee venom. Transgenic potato plants of the Belarusian variety Odyssey with the constitutive expressiоn of msrA1 gene are shown to exhibit increased resistance to fungal pathogens Phytophthora infestans and Alternaria solani. Peroxidase genes expression studies using cDNA-PCR and DNA sequencing revealed the activation of the POX peroxidase gene in transgenic plants in the absence of pathogens. This may be indirect evidence of the increased formation of reactive oxygen species, which may explain special resistance to fungal pathogens. The data obtained also confirm a possible role of intracellular antimicrobial peptide in making the plants more resistant to oxidative stress by the way of activation of the host plant defense system.

Development of Efficient, Reproducible and Stable Agrobacterium-Mediated Genetic Transformation of Five Potato Cultivars

The developments in transformation technology have enabled the scientists to incorporate, mutate or substitute gene(s) leading to a particular trait; advancing it to a point where only few technical limitations remain. Genotype dependency and explant types are important factors affecting transformation efficiency in potato. In the present study, a rapid, reproducible and stable Agrobacterium-mediated transformation procedure in potato was developed by a combination of different plant growth regulators. Leaf discs and internodal explants of five cultivars of potato, i.e. Lady Olympia, Granola, Agria, Désirée and Innovator were infected with Agrobacterium tumefaciens strain LBA4404 containing pBIN19 expression vector with β-glucuronidase gusA gene under the control of 35S CaMV promoter. Kanamycin was used as plant selectable marker for screening of primary transformants at concentration of 100 mg/L. Both explants responded positively; internode being more suitable explant for better transformation efficiency. Based on GUS histochemical assay, the transformation efficiency was 22, 20, 18.6, 15 and 10 % using the internodal explant, and 15, 12, 17, 8 and 6 % using leaf discs as explant in Lady Olympia, Granola, Agria, Désirée and Innovator respectively. Furthermore, PCR assays confirmed the presence of gusA and nptII genes in regenerated plants. The molecular analysis in succeeding progeny showed proper integration and expression of both genes. The results suggest Lady Olympia as the best cultivar for future transformation procedures. Overall, the short duration, rapidity and reproducibility make this protocol suitable for wider application of transgenic potato plants.

Expression of Jerusalem artichoke (Helianthus tuberosus L.) fructosyltransferases, and high fructan accumulation in potato tubers

Fructans are polymers of fructose that are present as storage carbohydrates in various plants. Jerusalem artichoke (Helianthus tuberosus L.) contains a high amount of inulin. Two enzymes are involved in inulin biosynthesis. The sucrose:sucrose 1-fructosyltransferase (1-SST) enzyme mainly catalyzes the synthesis of 1-kestose from sucrose. In the next step, fructan:fructan 1-fructosyltransferase (1-FFT) catalyzes the synthesis of inulin from 1-kestose. In this study, the Ht1-SST and Ht1-FFT genes were isolated from Jerusalem artichoke and expressed in potato (Solanum tuberosum L.), either separately or together, via Agrobacterium-mediated transformation. Transgenic potato tubers overexpressing Ht1-SST accumulated 1-kestose to a high level (up to 3.36 mg/g), while tubers overexpressing both Ht1-SST and Ht1-FFT accumulated up to 3.14 mg/g short-chain inulin-type fructans, with the degree of polymerization (DP) ranging from 3 to 5, excluding high DP inulins. Transgenic potato plants accumulated fructo-oligosaccharides to a high level, following the fructan biosynthetic pathway of Jerusalem artichoke, and therefore present a high potential for the mass production of inulin through established potato breeding and cultivation methods.