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).