Optimization of induction of eGFP secretion in B. rapa rapa hairy root lines
B. rapa rapa hairy roots were cultured for 14 days in modified Gamborg B5 medium. Inoculum of 50 mg fresh weight (FW) was taken into 100 ml shake flask with 20 ml of culture medium. Cultivation was performed at + 24 °C, 90 rpm (shake radius of 3.2 cm), in the dark. After growth period, the recombinant protein secretion was induced by changing the culture medium and continuing the cultivation for another 14 days. Induction medium consisted of standard amounts of polyvinyl pyrrolidine (PVP) (1.5 mg/l) and 2,4-D (1.0 mg/l) with varying concentrations of 1- naphthaleneacetic acid acid (NAA) [1-20mg/l], methyl jasmonate (MeJA) [9-250µM], and potassium nitrate (KNO3) [2-35g/l]. Sampling was performed by filtrating the hairy roots under suctions using Miracloth filter, and both hairy roots and culture medium were collected separately, frozen via liquid N2 and stored at -20 °C. Both, fresh and dry weight (FW and DW) were recorded. For the DW measurement, samples were freeze-dried over three days in CHRIST ALPHA1-4 LD plus chamber.