# Characterization of Nutrient Disorders of _Camabis sativa_
## 2 Materials and Methods  
Cuttings were taken from a hemp Cannabis sativa 'T1'on 3 July 2018 (Ryes Greenhouses: Sanford, NC, USA) and stuck into 72-cell plug trays filled with a substrate mix of 80:20 (v:v) Canadian sphagnum peat moss (Conrad Fafard, Agawam, MA, USA) and horticultural coarse perlite (Perlite Vermiculite Packaging Industries, Inc., North Bloomfield, OH, USA) amended with dolomite lime at 8.875 kg/m\({}^{3}\) (Rockydale Agricultural, Roanoke, VA, USA) and wetting agent (Aquatrols, Cherry Hill, NJ, USA) at 600.3 g/m\({}^{3}\). After three weeks of rooting, plugs were transplanted (27 July 2018) into 15.24-cm diameter (1.76 L) plastic pots filled with acid-washed silica-sand (Millersville #2 (0.8 to 1.2 mm diameter) from Southern Products and Silica Co., Hoffman, NC, USA). The experiment was conducted in a glass greenhouse in Raleigh, NC, USA (35\({}^{\circ}\)N latitude), with 24\({}^{\circ}\)C/20\({}^{\circ}\)C (D/N) temperature setpoints. Plugs were transplanted, and nutrient treatments started immediately in the automated, recirculating irrigation system made from 10.2-cm diameter PVC pipe (Charlotte Plastics, Charlotte, NC, USA), fit with 12.7-cm diameter openings to hold the pots. Control plants were grown with a complete modified Hoagland's all-nitrate solution consisting of 15 mM nitrate-nitrogen (NO\({}_{3}\)\({}^{-}\)), 1 mM phosphate-phosphorus (H\({}_{2}\)PO\({}_{4}\)\({}^{-}\)), 6 mM potassium (K\({}^{+}\)), 5 mM calcium (Ca\({}^{2+}\)), 2 mM magnesium (Mg\({}^{2+}\)), and 2 mM sulfate-sulfur (SO\({}_{4}\)\({}^{2-}\)) (Hoagland and Arnon, [11]); plus 72 uM iron (Fe\({}^{2+}\)), 18 uM manganese (Mn\({}^{2+}\)), 3 uM copper (Cu\({}^{2+}\)), 3 uM zinc (Zn\({}^{2+}\)), 45 uM boron (BO\({}_{3}\)\({}^{3-}\)), and 0.1 uM molybdenum (MoO\({}_{4}\)\({}^{2-}\)). Nutrient deficiencies began at transplant and were induced by withholding a single nutrient from this solution. Boron (B) and manganese (Mn) toxicities were induced by increasing the concentration tenfold higher than the complete nutrient formula. Reagent grade chemicals and deionized (DI) water (18 M\(\Omega\)) were used to formulate treatment solutions. The plants were drip-irrigated with a sump-pump (model 1A, Little Giant Pump Co., Oklahoma City, OK, USA) system as needed between 6:00 and 18:00 hours. Irrigation solution drained from the pot and was captured for reuse. Nutrient solutions were replaced weekly. The experiment was terminated 9 weeks after treatments began. Plants were tracked daily, and deficiency symptoms were photographed at the initial, intermediate, and advanced stages of symptomatology. Plant anatomy terminology used to describe deficiency symptoms is given in Figure 1. Upon initial symptom development, four plants were selected for sampling. The remaining treatments, which were not symptomatic, were grown until visual symptoms appeared and then were harvested. At the onset of initial visual symptomatology, whole plants (n=4) were destructively harvested, and most recent mature leaves (MRML) were subsampled and rinsed with DI water, washed in a solution of 0.5 M HCl, and again rinsed with DI water. Leaf tissue (MRML) was taken below the meristematic stem regions (apical meristem, axillary meristems, tertiary meristematic regions, etc. ), and only recently matured leaves were sampled. Leaf maturity and morphology were determined based on observational leaf maturity and from indices hybridized from Heslop-Harrison and Heslop-Harrison [12] and [PERSON] et al. [13] and from established standard leaf tissue harvesting protocols ([PERSON] et al., [9]). The remaining plant material after MRML was harvested and was placed in a separate container for aerial tissue biomass determination. Leaf tissues and their respective remaining biomass were dried at 70 \({}^{\circ}\)C for 24 hours.