Growth behavior of Brassica rapa rapa hairy root line
A Murashige and Skoog-based growth medium for Brassica rapa rapahairy roots had been previously established by Samabriva (Brassica Growth Medium (BGM)). In this study, we assayed whether modified Gamborg B5 -based medium (B5mod) could also result in good biomass accumulation with B. rapa rapa hairy roots, since earlier it has been successfully used for cultivation of Solanaceae hairy roots (Häkkinen et al., 2005; Häkkinen et al., 2014). In addition, the growth in basic Gamborg B5 (B5) medium was studied in parallel (Fig. 1A&B ). When the accumulation of fresh biomass weight (FW) was evaluated, it was observed that the biomass accumulation in studied media differed statistically (Fig. 1A ) and the highest biomass was achieved with B5mod media. However, based on dry weight (DW), there were no differences in biomass accumulation in the studied media (Fig. 1B ). Differences in FW and DW results reflected the differences in sucrose concentrations, as BGM medium contained higher sucrose concentration (50 g/l) when compared to the other two (30 g/l). The B. rapa rapa hairy roots grown in BGM medium with high sucrose level did not absorb as much intracellular water as hairy roots grown in B5 and B5mod (Fig. 1A ). Due to the equal growth with lower sucrose level, B5mod was selected as a cultivation medium for biomass production in the rest of the study.
In order to find out the optimal secretion induction phase, the growth behavior of B. rapa rapa hairy root line in B5mod was detrmined (Fig. 1C ). An increase in biomass accumulation was observed until day 14, when stationary stage was reached. Thus, 14 days was selected as the induction point for the test protein, eGFP secretion. As a base line, without induction, amount up to 40 mg/l eGFP was detected in the culture medium already after 21 days when the B5-mod medium was applied (Fig. 1D ).