Cannabis sativa callus cultures (1 g) cultivated onB5 medium [25] modified with 50 mg/l myo-inositol, 10 mg/l

thiamine HCl, 1000 mg/l casein hydrolysate and supplemented with 3% sucrose and solidified with 0.4% gelrite, had initiated into adventitious roots on full strength B5 medium supplemented with various concentrations of auxins (1.5, 2.5 and 4 mg/l) of NAA, IBA and IAA (Table 1). The emerging hairy root cultures were isolated from callus cultures and subsequently transferred to solid B₅ medium with 4 mg/l NAA for their further proliferation. Hairy rooting cultures were subcultured every 30 days intervals and maintained in the “Grow Bank” system (Plant Master BB. XXL4 CLF Plant Climatics GmbH, Wertingen, Germany) and cultivation was accomplished in the dark condition at 25˚C. Initiation of adventitious root cultures was evaluated after 30 days of cultivation.

Once established, the adventitious root cultures were cuttransversely and placed on Petri dishes (2 - 3 mm, 4 explants/treatment). Half solid B5 medium with various concentrations of auxins (0.25, 0.5 and 1.5 mg/l) of NAA, IBA and IAA in the dark at 25˚C were applied (Table 2). The morphology of hairy roots grown on the cultured media was evaluated using light microscope (Nikon SMZ800) and images were captured by a microscope digital camera system Nikon type-104 (Nikon, Japan). A liquid culture was obtained by incubating the root tips to B5 medium (100:250 ml Erlenmeyer flask) supplemented with 4 mg/l NAA. Roots were then grown in the dark at 25˚C on a rotary shaker (INFORS. HT, Bottmingen, Switzerland) at about 110 rpm. These roots were subcultured every 30 days and maintained for about 3 years in the liquid medium.

The kinetics of root growth were investigated by transferring (1 g fresh mass of inoculum; 4 - 5 cm of root tips) of two-week-old to liquid B₅ medium (100:250 ml Erlenmeyer flask; pH adjusted to 5.8 prior to autoclave), supplemented with various concentrations of auxins (Table 3). During the culture period of 35 days, hairy growth kinetics was evaluated every 7 days to determine the fresh and dry weights and the content of cannabinoids. The fresh weight was obtained by removal of three independent biological replicates induction flasks and washing with distilled water by suction filtration, then the cells were lyophilized for 24 h in freeze dryer (Alpha I-4 LSc Christ GmbH, Osterode am Harz, Germany) at −55˚C and 0.6 mbar until constant dry weight was obtained.

Dry biomass samples were pulverized and homogenized with methanol (70%, 3 × 10 ml: g roots) at 25˚C for 1 h.

Full solid B5 medium with plant growth regulators (PGR free). ^aFullsolid B5 medium. ^bVarious treatments on solid half strength B5. ^†All data the mean 3 replicates with two calli pieces/treatment; repeated three times; mean ± SE; 0 = no response. Induction of hairy roots from callus cultures was observed after 4 subcultures. NAA, naphthaleneacetic acid; IBA, Indolebutyric acid; IAA, indole-3-acetic acid.

^aWild type on full solid B5 medium (wild type). ^bVarious treatments on solid half strength B5. NAA, naphthaleneacetic acid; IBA, Indolebutyric acid; IAA, indole-3-acetic acid.

^aWild type on full liquid B5 medium (wild type). ^bVarious treatments on liquid halfstrength B5. NAA, naphthaleneacetic acid; IBA, Indolebutyric acid. IAA, indole-3-acetic acid.

The extracted solution for each treatment were combined, filtered and evaporated in vacuo at 35˚C using a Büchi Rotavapor (R-210, Switzerland) and the resulting residue dissolved in chloroform (2 × 10 ml). The chloroform extracts were combined dehydrated over sodium sulfate anhydrous, filtered and evaporated in vacuo at 35˚C to give total cannabinoids mixture. The total mixture was then re-dissolved again in 0.5 ml methanol, centrifuged at 13,000 × g for 4 min, at 4˚C. Then the clear supernatant was removed, filtrated by 0.2 µm filter and kept in a 2 ml clean amber vial, deoxygenated with CO₂, and stored at −20˚C for further qualitative and fingerprinting studies. A calibration curve was established using CBDA, THCA, and THC (THC-Pharm, Frankfurt, Germany) as a standard.

Chromatographic analysis of residue obtained after extraction and evaporation (see above) was carried out in an Agilent HPLC 1260 infinity (Agilent Technologies, Waldbronn, Germany). The HPLC consisted of a binary pump (model no. G1312B), an auto-sampler (model no. G1367D), a degasser (model no. G1379B) and a photodiode array detector (model no. G4212B). The HPLC system was controlled by the ChemStation software (Agilent, v. 04.03). The separation of cannabinoids was achieved on a Gemini C18 column (250 × 4.60 mm, 5 µm particle size; Phenomenex, Aschaffenburg, Germany). The mobile phase consisted of two solvents: 0.1% aqueous trifluoroacetic acid (solvent A) and acetonitrile (solvent B). The compounds were eluted with a step gradient as follows: 0 - 10 min (65% B), 10 - 13 min (95% B), 13 min (65% B); 13 - 15 min (65% B). The flow rate was 1.5 ml/min and the injection volume was 10 μl. Peaks of cannabinoids were detected at 224 nm and quantified on the basis of authentic compounds.

Cannabinoids in the Methanol crude extract of hairy roots were analyzed by LC/(+)ESI-MS LTQ-Orbitrap spectrometer (Thermo Scientific GmbH, Schwerte, Germany). The mass spectrometer was equipped with HPLC system (Agilent 1200) consisting of pump (model no. G1312), and autosampler (model no. G1367 HIP-ALS). Nitrogen was used as protective gas (60 arbitrary units), and helium served as the collision gas. The separations were performed by using a Luna C18 column (50 × 3 mm, 3 μm particle size; Phenomenex, Aschaffenburg, Germany). The mobile phase consisted of two solvents: 0.1% formic acid in water (solvent A) and acetonitrile with 0.1% formic acid (solvent B). The compounds were eluted with a step gradient as follows: 0 - 1 min (20% B), 6 - 10 min (100% B), and 10 - 15 min (20% B). The flow rate was 0.5 ml/min and the injection volume was 5 μl. Peaks of cannabinoids were detected at 224 nm and identified on the basis of authentic compounds. The spectrometer was operated in positive mode (1 spectrum s⁻¹; mass range: 150 - 450) with nominal mass resolving power of 60,000 at m/z 400 with a scan rate of 1 Hz, with automatic gain control to provide high-accuracy mass measurements within 2 ppm deviation using one internal lock mass; m/z 391.284286; bis-(2-ethylhexyl)- phthalate. MS/MS experiments were performed by CID (collision induced decay, 35 eV) mode. LC/MS² was sufficient for identification THC and CBDA. However, THCA in hairy root samples (time course) were produced in a mount that were two small to be identify by LC/MS². Subsequently, the protonated molecular ion m/z for THCA were used for CID to identify their fragmentation by LC/MS³ with CID (collision induced decay, 40 eV). Following parameters were used for experiments: spray voltage 5 kV, capillary temperature 260˚C, tube lens 70 V.

B5 medium was supplied by Sigma-Aldrich Company (Germany). THCA, THC and CBDA were purchased from THC-Pharm, (Germany). All other plant growth regulators and chemicals involved in this study were reagent grade.

Thermo Electron’s Xcalibur (version 2.2) was used as instrument control and data processing platform for LC- MS data collection, peak identification, and measurement. All other intermediate data manipulation was carried out using. The data were analyzed statistically using Microsoft Excel 2010, and the data of each sample (means ± SE) was analyzed in triplicate.

In general, all the testedauxins (Table 1) did not stimulate root differentiation except full strength B5 medium with 4 mg/l NAA (IHR0). After calli were incubated on IHR0 for 16 weeks in darkness at 25˚C, numerous hairy roots began to emerge vigorously from the surface of calli with 83.3% root formation. Eight weeks later, hairy roots showed the characteristics of the typical adventitious roots, having numerous branching covered with root hairs. However, the ability of callus cultures of other systems to proliferate hairy roots in response to PGRs was studied [26] [27] . Similar results were reported that NAA initiated the growth of hairy roots from callus cultures of C. sativa [19] .

The auxins type influenced the root growth and multiplication (Table 2). Hairy roots on solid media SHR5, and SHR8 gave high significance of numbers of hairy roots with an average (37 ± 4.7 and 27.25 ± 4.9) root per culture, respectively. On the other hand, media SHR2, and SHR8 gave high significance of lateral hairy roots with an average (6.3 ± 0.14 and 5.2 ± 0.28 cm) root length per culture, respectively. The auxin concentration was affected on root phenotypes. While phenotypes of roots hairs formed on SHR0, SHR2, and SHR5 were rather different than on SHR8. From morphologic perspective, hairy roots (Figure 1S) are divided into three zones [28] [29] . The meristematic zone (MZ) where cell division and formation of a lateral branch takes place, the elongation zone (EZ) where cells expand along the longitudinal axis and the differentiation zone (DZ) where root hairs develop. The obtained results are in agreement with those reported that auxins NAA, IBA, and IAA are more efficient in initiation and phenotypic variation of hairy root [30] .

Effects of exogenous auxins on the growth kinetics and cannabinoids formation of hairy root was determined and illustrated in Figure 1S.