Using Mathematical Optimization Models to Improve the Efficiency of Duckweeds (Wolffia arrhiza and Lemna minor) Micropropagation.

The method of plant micropropagation is widely used to obtain genetically homogeneous and infection-free plants for the needs of various industries and agriculture. Optimization of plant growth and development conditions plays a key role in economically successful micropropagation. Computer technologies have provided researchers with new approaches for modeling and a better understanding of the role of the factors involved in plant growth in vitro. To develop new models for optimizing growth conditions, we used plants with a high speed of vegetative in vitro reproduction, such as duckweed (Wolffia arrhiza and Lemna minor). Using the development of the optimal modeling of the biological processes, we have obtained the prescriptions for an individually balanced culture medium that enabled us to obtain 1.5-2.0 times more duckweed biomass with a 1.5 times higher protein concentration in the dry mass. Thus, we have demonstrated that the method of optimization modeling of the biological processes based on solving multinomial tasks from the series of quadratic equations can be used for the optimization of trophic needs of plants, specifically for micropropagation of duckweeds in vitro.

Genome-Wide Identification, Characterisation, and Evolution of the Transcription Factor WRKY in Grapevine (Vitis vinifera): New View and Update.

WRKYs are a multigenic family of transcription factors that are plant-specific and involved in the regulation of plant development and various stress response processes. However, the evolution of WRKY genes is not fully understood. This family has also been incompletely studied in grapevine, and WRKY genes have been named with different numbers in different studies, leading to great confusion. In this work, 62 Vitis vinifera WRKY genes were identified based on six genomes of different cultivars. All WRKY genes were numbered according to their chromosomal location, and a complete revision of the numbering was performed. Amino acid variability between different cultivars was assessed for the first time and was greater than 5% for some WRKYs. According to the gene structure, all WRKYs could be divided into two groups: more exons/long length and fewer exons/short length. For the first time, some chimeric WRKY genes were found in grapevine, which may play a specific role in the regulation of different processes: VvWRKY17 (an N-terminal signal peptide region followed by a non-cytoplasmic domain) and VvWRKY61 (Frigida-like domain). Five phylogenetic clades A-E were revealed and correlated with the WRKY groups (I, II, III). The evolution of WRKY was studied, and we proposed a WRKY evolution model where there were two dynamic phases of complexity and simplification in the evolution of WRKY.

Wolffia arrhiza as a promising producer of recombinant hirudin.

The production of recombinant proteins in transgenic plants is becoming an increasingly serious alternative to classical biopharming methods as knowledge about this process grows. Wolffia arrhiza, an aquatic plant unique in its anatomy, is a promising expression system that can grow in submerged culture in bioreactors. In our study 8550 explants were subjected to Agrobacterium-mediated transformation, and 41 independent hygromycin-resistant Wolffia lines were obtained, with the transformation efficiency of 0.48%. 40 of them contained the hirudin-1 gene (codon-optimized for expression in plants) and were independent lines of nuclear-transformed Wolffia, the transgenic insertion has been confirmed by PCR and Southern blot analysis. We have analyzed the accumulation of the target protein and its expression has been proven in three transgenic lines. The maximum accumulation of recombinant hirudin was 0.02% of the total soluble protein, which corresponds to 775.5 ± 111.9 ng g-1 of fresh weight of the plant. The results will be used in research on the development of an expression system based on Wolffia plants for the production of hirudin and other recombinant pharmaceutical proteins.

Development of Wolffia arrhiza as a Producer for Recombinant Human Granulocyte Colony-Stimulating Factor.

To date, the expression of recombinant proteins in transgenic plants is becoming a powerful alternative to classical expression methods. Special efforts are directed to the development of contained cultivation systems based on cell culture or rhyzosecretion, which reliably prevents the heterologous DNA releasing into the environment. A promising object for the development of such systems is the tiny aquatic plant of Wolffia arrhiza, which can be used as a dipped culture in bioreactors. Herein we have expressed the human granulocyte colony-stimulating factor (hG-CSF) in nuclear-transformed Wolffia. The nucleotide sequence of hG-CSF was optimized for expression in Wolffia and cloned into the vector pCamGCSF downstream of double CaMV 35S promoter. Wolffia plants were successfully transformed and 34 independent transgenic lines with hG-CSF gene were obtained, PCR and Southern blot analysis confirmed the transgenic origin of these lines. Western blot analysis revealed accumulation of the target protein in 33 transgenic lines. Quantitative ELISA of protein extracts from these lines showed hG-CSF accumulation up to 35.5 mg/kg of Wolffia fresh weight (0.194% of total soluble protein). This relatively high yield holds promise for the development of Wolffia-based expression system in strictly controlled format to produce various recombinant proteins.