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. 2014 Jul 3;9(7):e100144. doi: 10.1371/journal.pone.0100144

Figure 5. Overexpression of GmACSL2 in transgenic hairy roots.

Figure 5

(A) Hairy roots were induced by A. rhizogenes. (a) Soybean seeds were germinated in germination medium; (b) Explants were co-cultivated with A. rhizogenes in MCC medium; (c) Hairy roots were induced in 1/2 MS medium with hygromycin B as selection; (d) Hairy roots appeared at the wounding sites of the explants in 1/2 MS medium; (e) Hairy roots were grown in 1/2 MS solid medium; (f) Hairy roots were propagated in 1/2 MS liquid medium. (B) GUS histochemical staining and GFP fluorescence signal detection. After extended culture, the hairy roots were stained by GUS (upper panel) and signals of fluorescence GFP observation (lower panel). WT represents the wild-type soybean hairy roots hairy roots transformed with the K599 strain only; Trans represents the soybean hairy roots transformed with the control vector pGFPGUS and pGFPGUS-GmACSL2. (C) PCR analysis of hairy roots DNA using the primers to amplify the fragment of the GmACSL2 gene to further identify the transgenic line of GmACSL2. M, Marker; “+”, positive control, GmACSL2 plasmid was used as template; “−”, negative control, soybean hairy roots were transformed with the control vector pGFPGUS as template; 1–8, 1–11, 1–13, 1–17, 2–2, 2–4, 2–9, 2–22, 3–5, 3–14, and 3–33, individual lines transformed with the binary vector pGFPGUS-GmACSL2. (D) Expression analysis of GmACSL2 in different transformed lines by RT-PCR. 1–8, 1–11, 1–13, 1–17, 2–2, 2–4, 2–9, 2–22, 3–5, 3–14, and 3–33 represent different transformed hairy roots overexpression with GmACSL2. Soybean actin gene was used as an internal control. (E) Analysis of lipid contents in the transgenic lines by soxhlet extraction. Control, transgenic hairy roots induced by pGFPGUS control vector; GmACSL2-1-8, GmACSL2-2-4, GmACSL2-3-5, GmACSL2-3-14, and GmACSL2-3-33, the five transgenic hairy root lines, induced by the pGFPGUS-GmACSL2 vector. (F) Fatty acid analysis comparisons between five GmACSL2 transgenic lines and the control. The five major fatty acid species C16∶0, C18∶0, C18∶1, C18∶2, C18∶3 and the total fatty acids content in hairy roots were detected by gas chromatography-mass spectrometry. Data are the mean ± SEM of the three experiments. *p<0.05, **p<0.01.