Using the examples of biosynthesis of streptomycin, bialaphos, actinorhodin, oligoketides and autoregulators during the first hours of streptomycete cultivation, it is stressed that the external environment in cooperation with the internal metabolic abilities of the cell determines the metabolic type that would develop during the life cycle of the producing streptomycetes. If we accept that a certain metabolic type (from the point of view of the production of secondary metabolites) was determined already during the first hours of cultivation of the microorganisms, we must also admit that the availability of primary metabolites in the so-called production phase of growth (stationary phase, idiophase, etc.) is to a certain extent determined by the very early stages of strain development.

• MeSH
• anthrachinony metabolismus   MeSH
• antibakteriální látky biosyntéza   MeSH
• časové faktory MeSH
• organofosforové sloučeniny metabolismus   MeSH
• regulace genové exprese u bakterií MeSH
• Streptomyces fyziologie   MeSH
• streptomycin biosyntéza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• actinorhodin MeSH Prohlížeč
• anthrachinony MeSH
• antibakteriální látky MeSH
• bialaphos MeSH Prohlížeč
• organofosforové sloučeniny MeSH
• streptomycin MeSH

We extended the applicability of the BY-2 cell line as a model by introducing two new selection systems. Our protocol provides guidelines for optimising Basta selection in other recalcitrant models. Tobacco BY-2 cell line is the most commonly used cytological model in plant research. It is uniform, can be simply treated by chemicals, synchronised and easily transformed. However, only a few selection systems are available that complicate advanced studies using multiple stacked transgenes and extensive gene editing. In our work, we adopted for BY-2 cell line two other selection systems: sulfadiazine and phosphinothricin (PPT, an active ingredient of Basta herbicide). We show that sulfadiazine can be used in a wide range of concentrations. It is suitable for co-transformation and subsequent double selection with kanamycin or hygromycin, which are standardly used for BY-2 transformation. We also have domesticated the sulfadiazine resistance for the user-friendly GoldenBraid cloning system. Compared to sulfadiazine, establishing selection on phosphinothricin was considerably more challenging. It did not work in any concentration of PPT with standardly cultured cells. Since the selection is based on blocking glutamine synthetase and consequent ammonium toxicity and deficiency of assimilated nitrogen, we tried to manipulate nitrogen availability. We found that the PPT selection reliably works only with nitrogen-starved cells with reduced nitrate reserves that are selected on a medium without ammonium nitrate. Both these adjustments prevent the release of large amounts of ammonium, which can toxify the entire culture in the case of standardly cultured cells. Since high nitrogen reserves can be a common feature of in vitro cultures grown on MS media, nitrogen starvation could be a key step in establishing phosphinothricin resistance in other plant models.

• Klíčová slova
• Bialaphos, Glufosinate ammonium, Selectable markers, Suspension cell lines, Tobacco (Nicotiana tabacum),
• MeSH
• amoniové sloučeniny * MeSH
• dusík MeSH
• geneticky modifikované rostliny genetika   MeSH
• sulfadiazin MeSH
• tabák * genetika   MeSH
• transformace genetická MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• amoniové sloučeniny * MeSH
• dusík MeSH
• phosphinothricin MeSH Prohlížeč
• sulfadiazin MeSH