Resumen de Molecular breeding of synchronized grape cell suspensions for flavonoid overexpression
V. Georgiev, A. Ananga, V. Tsolova
In this study we demonstrated the advantage of plant cell based molecular breeding as a promising alternative for production of grape nutraceuticals and improvement of their biological activity. The data for chemical composition and biological activities of M. rotundifolia in vitro systems are presented. Three stable suspensions of Muscadinia rotundifolia ?Noble? were selected based on growth rate and anthocyanin production. The type and concentration of the carbon source, ammonia/nitrate ratio, growth regulators and the composition of macro- and microelements were used as selective pressures. The red suspension had a total anthocyanin content of 2.50±0.01 mg petunidin-3,5-0-diglucoside equivalents (PDE)/g DW and accumulated dry biomass (ADB)=4.44±0.71 g/L; yellow (no anthocyanins were detected) with significantly improved growth ADB=7.11± 0.27 g/L; and red-brown with significant decreases of both growth (ADB=3.38± 0.34 g/L) and anthocyanin production (total anthocyanin of 0.72±0.05 mg PDE/g DW) were generated. Comparative expression analyses of 18 genes, involved in flavonoid biosynthesis (PAL; STS1; STS2; STS3; STS4; CHS2; CHS3; CHI1; CHI2; F3?H; F3?5?H; F3H1; F3h2; FLS; DFR; LDOX; UFGT and OMT) showed that 9 genes (STS1; STS2; STS3; STS4; CHS2; CHS3; CHI1; F3H1 and DFR) were significantly overexpressed in the red suspension and 17 genes with the exception of FLS were significantly down-regulated in the yellow strain. The genes involved in stilbene synthesis (STS1; STS2; STS3; STS4) were completely shut down, whereas FLS, involved in production of flavonols, was the only gene significantly overexpressed in the yellow suspension. In the red-brown cell line only a few genes (CHI2 and OMT) had expression level close to that of the initial red cell culture. However, several genes (PAL; F3?H; LDOX and UFGT) were found to be significantly overexpressed. To further identify the occurrence of permanent genotypic changes in the cell suspensions, we used 10 random amplified polymorphic DNA (RAPD) and 10 simple sequence repeat (SSR) markers. All of the10 RAPD and 5 of the SSR markers discriminated among the 3 selected grape suspensions (red, yellow and red-brown). The calculated percentages of polymorphism of individual markers varied from 0 to 100% for RAPD and SSRs.
