Only two major positions of the stem were examined because we observed a large variation in the anatomical traits of the hemp stem from the bottom section to the top section during preliminary analyses using one genotype. Immediately after harvest, the stems of each hemp genotype were cut at two stem heights: sections from internodes between nodes 0 –1 and nodes 3–4 positions, starting from the ground level and moving towards the top of the plant. We then determined the cell distribution of each nodal position of each genotype using the manually sectioned transverse sections of stems that were double-stained with methyl green-Congo red . The stained transverse sections were photographed under an Olympus DP73 stereo microscope at 63 . The cortex layer , which contains a high percentage of crystalline cellulose, and the xylem, which contains a high percentage of lignified cellular layers, were visually examined. The cortex layer responsible for containing bast fibres and the xylem layer, which contains hurd fibres, were measured using the digital reticule of the stereomicroscope, and xylem/cortex values were estimated. The diameter of the pith was also measured using the same digital reticule. This procedure was performed in triplicate for each genotype.Fibres from each genotype were extracted using chemical, enzymatic, and microbiological retting methods, as described below. To prepare the stems for retting, the top part of the plant, including inflorescences and leaves, was cut away from the stem. All hemp stems were washed with distilled water, wiped, grow tent for sale and cut into equal pieces using disinfected secateurs. The stem pieces were disinfected with 70% ethanol.
The fresh weight of all stem samples was measured. For the negative control, hemp stems from all genotypes were disinfected with 70% ethanol and immersed in autoclaved distilled water.Samples were chemically retted by pre-treating with 0.3% HCl, treated with 6% NaOH at 70 C for 1 h, and post-treated with 1% acetic acid . This is a chemical method previously shown to be an optimum yielding approach for bast fibres with minimum loss of mechanical properties , and it showed results in the highest yield during our preliminary studies using a series of NaOH concentrations . When easily separable by hand, fibres were washed five times with distilled and deionized water at 50C to remove chemical residues and cellular debris. We performed enzymatic retting using a modified version of the method used by George et al. , as explained below. A mixture of enzymes was used to treat the whole stems. Initially, stems were immersed in 0.1% commercial pectinase at 30C for 30 min at pH 4. The contents were then transferred to 10% cellulase at 50 C for 30 min at pH 6. Finally, they were transferred to 0.05% xylanase and 1% laccase at 70 C for 30 min at pH 7. Then the entire cellulase and fibre mixture was heated at 80 C for 15 min to deactivate enzymes. A pressurized water column was applied to remove the non-fibrous elements, and the separated fibres were collected using tweezers. The fibres were washed five times with distilled and deionized water at 50 C to remove traces of enzymes and cellular impurities. We used an uncharacterised microbial solution obtained from okra Moench, which showed soft rot lesions in its fibrous fruit. About 500 g of rotted A. esculentuswas washed with 5 L of distilled water. The disinfected stems of each hemp genotype were immersed in 100 ml of the microbial solution at room temperature until the fibres separated from the woody core.
A pressurized water column was applied to remove the non-fibrous elements. The fibres were washed five times with distilled water, which was heated at 50 C. The bast and hurd fibres were tested with toluidine blue staining for the purity and evenness of retting. Hurd and bast fibres extracted using all methods were dried naturally at the room temperature. The dry weight of fibres was determined after oven drying them at 80 C for 5 h , until a constant weight was obtained. As the purpose of this is to demonstrate the efficiency of methods for extraction of raw fibres from each genotype, fibres were not subjected to additional treatments. The average bast fibre content in the stem was calculated by dividing the fibre dry weight by the dry weight of stems of three replicates. The bast fibre yield was calculated by multiplying the stem yield by the bast fibre content . Dry weight of hurd and bast fibres obtained from different genotypes under three retting methods were compared.The morphological characters of branch number, node number, stem diameter of position 1, and internodal lengths differed significantly , reflecting the existence of variability among the tested genotypes. This variability can be further used for hemp genotype improvement programs. The genotype Jin Ma showed the lowest number of branches and nodes, while the genotype Tetra showed the highest number of branches and nodes. The number of branches and nodes might interfere with fibre-processing methods. Although the curvature of the stems of the different genotypes did not show a significant difference, it is still an important character to be considered as it might affect the fibre-parallelizing process in large-scale fibre processing via machinery. The internodal length was shown to be the highest in the Jin Ma genotype, whereas it was the lowest in the KLR2020 genotype . Generally, KLR genotypes showed low internodal lengths.
When hemp is retted, it has been observed that tissues in nodes produce more debris, which affects the fibre-refining process. Therefore, genotypes with fewer nodes can be favourable for fibre production. The stem diameter is an important character of fibre production, and the easily separable thin stems are more suitable in the textile industry . The genotype Tetra showed the highest diameter, and Ditch weed showed the lowest diameter, on position 1 of the stem. There is a considerable diversity of height in hemp morphology depending on the genotype, soil, and climate conditions . Although similar environmental conditions were provided in this research, genotypes did not show any significant differences in height.The mean xylem/cortex length of stems of positions 1 and 2 and the mean diameter of the pith layer of position 2 are provided in Table 2. The xylem/cortex value in transverse sections of both positions 1 and 2 hemp stems was not statistically significant . This value may vary depending on the nutrient supply of plants, seeding rate, light conditions, hormonal regulations within plants, etc. . The microscopic images of transverse sections of positions 1 and 2 hemp stems, respectively, are provided in the Mendeley Data repository: Figures S2 and S3. Although the stem samples were collected at the same plant age , their development varied in the secondary bast fibre layer arrangement, lignin deposition, and stem shape . Further, transverse sections were highly variable at position 2 of the stems; however, a distinct pattern was not identified. Transverse sections of all genotypes were different in overall stem shape and pith shape. The variability in the dimension of each cell layer of stems in positions 1 and 2 is shown in Figure 1. Most of the genotypes showed an increase in the dimension of both cortex and xylem layers when they mature. Cortex and xylem characters might not be appropriate to differentiate between hemp genotypes due to their environmental plasticity and variations during different developmental stages. However, the patterns of fibre wedges surrounding the vascular cambium in position 1 were unique in each genotype , which we consistently observed in all three replicates of each genotype. Moreover, all transverse sections between the ground level of the stem and the first node showed a similar pattern. However, the section thickness caused by manual sectioning, the used spatial resolution, stains specific to each cellular type are needed to provide deeper insights into the unique patterns of fibre wedges.
We provided similar conditions to all accessions used in this study at a greenhouse. However, further studies are warranted to examine whether these patterns vary with environmental conditions during field trials.Genotypes significantly affected both bast and hurd fibre yields. This finding agrees with a previous study and is probably due to genotype differences in the formation of stem cellular layers . The Bia- łobrzeskie and Blue genius genotypes resulted in a significantly higher yield of bast and hurd fibres, respectively . Conversely, the Cherry wine genotype showed the lowest bast fibre yield with chemical and enzyme approaches, while the lowest hurd fibre yield was observed with Białobrzeskie in all extraction methods. Although there was no significant difference between the retting approaches in the overall bast yield , most genotypes showed lower weight fibre with the enzymatic method compared with the other two extraction methods . The use of enzymes has been suggested as an attractive future area of focus–particularly, for the improvement of fibre thermal and electrical properties . Images from SEM indicated that the bast fibres existed as bundles and each fibre was composed of a single cell . The SEM micrographs showed that microbial treatment resulted in individual bundles to be exposed with a clean surface. This type of surface was obtained in previous studies that used chemical treatments . Some genotypes showed even thickness throughout the fibres, while others showed uneven thickness and dislocations on their fibre. The diameter of individual bast fibres within one genotype was highly variable. The cell layer that wraps spirally deposited lignin in xylem vessels showed unusual thickness in the genotype Cherry wine , indoor tent grow which might link to the lowest moisture retention in this genotype because thick layer might reduce the permeability. The SEM examination was only carried out for selected microbially treated samples due to the cost and time constraints. However, the SEM micrography of all fibres will be helpful to understand the improvement of surface properties by retting methods and the genetic effect on the surface characteristics.A significant effect of genotype was evident for bast and hurd fibre moisture retention.
Bast fibres of USO31 and Białobrzeskie resulted in the highest and lowest moisture retention, respectively , and hurd fibres of Portland and Cherry wine stains showed the highest and lowest moisture retention, respectively . The interaction of water molecules with fibre may involve several physical phenomena. For instance, the water penetrating the fibre can be taken up into the capillary space between the fibres or absorbed by the fibres via hydrogen bonding . Also, amorphous components such as hemicellulose and lignin content play an important role in water storage by fibres . Further, water retention is governed by the surface properties such as cavities and the pore structure of the fibres . These properties may vary between different genotypes of hemp as they differ in the chemical composition of their stems . Thus, to further explain the results, additional parameters, such as capillarity, porosity, and water sorption of different genotypes, must be considered.The Agricultural Improvement Act of 2018 permits the cultivation and legal trade of industrial hemp in the United States. This act defines hemp as Cannabis sativa and any part or derivative of the plant including seeds, extracts, cannabinoids, isomers, acids, salts, and salts of isomers with a total delta-9 tetrahydrocannabinol concentration below 0.3 % on a dry weight basis. This statute removed hemp cannabis from its schedule I classification by using this definition to separate it from marijuana-type cannabis. Currently, there are no standardized methods to distinguish hemp from marijuana. Most forensic laboratories use chromatographic methods such as Gas Chromatography or High-Performance Liquid Chromatography methods coupled to mass spectrometry to quantitate the THC in suspicious plant materials. Furthermore, colorimetric tests that were once used to presumptively identify cannabis are not able to differentiate between hemp and marijuana, creating the need for an effective field test that can differentiate between hemp-type cannabis and marijuana-type cannabis. Hemp and marijuana are two different strains of the Cannabis plant with the main difference between the two being the concentration of cannabinoids contained in them. The two most important cannabinoids in these plants are THC and cannabidiol . THC is the cannabinoid that causes a psychoactive response in the body giving the person a “high”. It also has anti-inflammatory and analgesic properties, which make it desirable for medical use.