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.