FIGURE LEGENDS
Figure 1. Biomass accumulation, growth behavior and eGFP secretion of Brassica rapa rapa hairy roots carrying eGFP encoding gene. A) Biomass accumulation in fresh weight (FW, g/l) with B5, B5mod and BGM after growth period of 14 days. B)Biomass accumulation in dry weight (DW, g/l) with B5, B5mod and BGM after growth period of 14 days. C) Growth behavior on B5mod medium (Circles: biomass expressed in FW; squares: biomass in DW).D) Extracellular eGFP yield (mg/l) during the cultivation (only one replicate assayed). Error bars represent the standard deviation of four (A, B) or three (C) biological replicates. Letters (a,b,c) indicate the statistically significant differences (p<0.01).
Figure 2. Brassica rapa rapa hairy root eGFP secretion induction experiments. A) A contour-plot based on the model constructed from the empirical secretion data of the 1st secretion induction experiment (Tables 1 & 2) showing secreted eGFP related to MeJA and KNO3.B) A contour-plot based on the model constructed from the empirical secretion data of the 2nd secretion induction experiment (Tables 1 & 2) showing secreted eGFP related to MeJA and KNO3. One outlier (N6) was selected.C) A contour-plot based on the model constructed from the empirical secretion data of the 3rd secretion induction experiment (Tables 1 & 2 ) showing secreted eGFP related to KNO3.
Figure 3. Biomass accumulation and eGFP secretion inBrassica rapa rapa hairy root clone in the secretion induction verification A) Experimental set-up, B) Biomass accumulation (DW, g/l) C) Amount of secreted eGFP to culture medium after induction period (mg/l) [one-way ANOVA, post-hoc Tukey HSD, p < 0.01], D) Secreted eGFP after secretion induction expressed in mg/g DW hairy root biomass. Error bars represent the standard deviation of five biological replicates. Letters (a,b,c,d) indicate statistically significant differences (p<0.01).
Figure 4. Biomass accumulation and α-L-iduronidase (IDUA) secretion in Brassica rapa rapa hairy root clone with optimized parameters. A) Biomass accumulation (DW, g/l). Dry weight biomass after secretion induction (Dunnett T3). Asterisks indicate significant differences in levels p<0.05 (*). Error bars represent the standard deviation of four biological replicatesB) Immunodetection of IDUA protein in equivalent amounts of recovered media from the different treatments. A positive control of a commercial standard protein (+) (ABIN1464142, Antibodies-online) and a negative control (-) of clean non-induction media. The red arrow shows the 70.8 kDa band corresponding to the mature non-glycosylated standard protein. In the contiguous table, the relative densitometries of the different treatments are displayed. [A=PVP; B=2,4-D; C=KNO3;D=PVP+2,4-D; E=PVP+2,4-D; F=PVP+2,4-D+KNO3; G=No induction.] C) IDUA activity assay on the culture medium of the different treatments applied. Error bars indicates standard deviations. * indicates significant difference based on Student test (p<0.05).
Supporting Figure 5. Visual changes in the IDUA-expressingBrassica rapa rapa hairy root clone after the induction of secretion. A) Color and geotrophic changes in representative bottles of each treatment. B) Close-up photographs of representative pieces of hairy roots after the different treatments. Yellow arrows point out swollen tips and hump-like structures in the treatments with 2,4-D. The photographs are augmented four times the actual size. C) Bright field microscopic photography of the hump-like structures resulting from any treatment in which 2,4-D was used. D) Bright field microscopic photographies from ‘No induction’ and ‘PVP+2,4-D+KNO3’. 1 and2 show the non-induced tissue (water and TBO, respectively), there is callus/starch (unstained) in some of the tips which might denote possibility of the tissue to keep growing. Blue in the very tips denotes lignified cell walls and the root apical meristem. 3and 4 show the tissue induced with ‘PVP+2, 4-D+KNO3’ (water and TBO, respectively). 2,4-D induced hump-like structures can be seen as a proliferation of possibly lateral roots primordia from pericyclic cells. Also, the root-tip vascular cylinders seem to abruptly deviate due to the formation of the formed humps. Additionally, root tips are swollen and there is absence of root apical meristems (i.e. not blue parts as those in the non-induced root tips).