T zeatin

One-year-old ginseng seedlings were transferred to 1/2 B5 liquid medium with or without plant hormones (auxin, NAA 10 μM, Sigma # n0640 and cytokinin, t-zeatin 1 μM, Sigma # z0876) and incubated for 2 h. After treatment, the samples were immediately frozen in liquid nitrogen. To extract total RNA, the Easy-Spin IIp Plant RNA Extraction Kit (iNtRON Biotecnology, Seongnam, Korea) was used. cDNA was synthesized with a first-strand synthesis KIT (Enzynomics, Daejeon, Korea). One microgram of total RNA was used. The reaction conditions were as follows: 50 °C for 60 min, 95 °C for 5 min, and 1 min of cooling on ice during the synthesis of first strand cDNA. Selected target genes were subjected to quantitative real-time PCR under the same reaction conditions. The primers were designed using Primer Express 3.0.1 software (Table S6, PE Applied Biosystems, Foster City, CA, USA). qRT-PCR analysis was performed using the QuantBase 3 (Applied Biosystem, Foster City, CA, USA) instrument with SYBR Green Real-time PCR Master Mix (Applied Biosystem, Foster City, CA, USA) according to the manufacturer’s recommendations. The primer sequences used in this study are listed in Table S5. The expression levels using the threshold cycle (Ct) value were calculated using the 2^−ΔΔCt method.

Cellulase R10 and Macerozyme R10 for enzymatic digest of leaf tissue were purchased from Melford Biolaboratories Ltd. Buffer W5 was 154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES pH 5.7. MMG solution was 0.4 M mannitol, 15 mM MgCl₂, 4 mM MES pH 5.7. Buffer W1 was 0.5 M mannitol, 20 mM KCl and 4 mM MES pH 5.7. Substances for hormonal treatments were abscisic acid (ABA), 1-naphthylacetic acid (NAA), trans-zeatin (t-zeatin), salicylic acid (SA) and methyl jasmonate (MeJA) and were purchased from Sigma Aldrich. Mock-treated wells received the amount of solvent present in the medium concentration of the three hormonal treatments or water. For analysis of marker specificity the treatment concentrations were 10 μM ABA, 500 nM NAA, 20 μM t-zeatin, 30 μM SA and 50 μM MeJA.
Lysis buffer was prepared as 5-fold stock solution using 125 mM Tris / H₃PO₄ (pH 7.8), 10 mM DTT, 10 mM DACTAA (Sigma D1383), 50% (v/v) glycerol, 5% (v/v) Triton X-100. LUC substrate was prepared using beetle luciferin (Promega E1602) as 1 mM luciferin, 30 mM HEPES (pH 7.8), 3 mM ATP (Sigma 797189) and 15 mM MgSO₄. GUS substrate was prepared using MUG (4-Methylumbelliferyl-β-D-glucuronide, Melford Biolaboratories Ltd. M65900) as 1 mM MUG, 10 mM Tris / HCl (pH 8.0) and 2 mM MgCl₂.