This research was supported by the Institute of Cannabis Research at Colorado State University-Pueblo and the Ministry of Science and ICT (2021-DD-UP-0379), and Chuncheon city (Hemp R&#38;D and industrialization, 2020-2021), The authors also wish to thank Justin Henderson at Summit CBD for the generous donation for &#34;Cherry Wine&#34; seeds.

1. Generation of a mother plant for clonal propagation
<ol>
	<li>Select a healthy, female mother plant that exhibits desirable morphological and chemical characteristics specific to its intended use.</li>
	<li>Allow the mother plant to reach the appropriate size (roughly 25 mature shoots) for clonal propagation (i.e., cuttings).</li>
	<li>Allow the mother plants to remain in the vegetative growth stage (light: dark = 18 h:6 h) to promote shoot growth for future propagation.</li>
</ol>
<p class="jove_ti...

<ol>
	<li>ElSohly, M. A., Radwan, M. M., Gul, W., Chandra, S., Galal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochemistry+of+Cannabis+sativa+L.">Phytochemistry of Cannabis sativa L.</a> Progress in the Chemistry of Organic Natural Products. 103, 1-36 (2017).</li><li>Cunetti, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+pain+treatment+with+cannabidiol+in+kidney+transplant+patients+in+Uruguay.">Chronic pain treatment with cannabidiol in kidney transplant patients in Uruguay.</a> Transplantation Proceedings. 50 (2), 461-464 (2018).</li><li>Hausman-Kedem, M., Menascu, S., Kramer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+CBD-enriched+medical+cannabis+for+treatment+of+refractory+epilepsy+in+children+and+adolescents+-+An+observational,+longitudinal+study.">Efficacy of CBD-enriched medical cannabis for treatment of refractory epilepsy in children and adolescents - An observational, longitudinal study.</a> Brain &amp; Development. 40 (7), 544-551 (2018).</li><li>Linge, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol+induces+rapid-acting+antidepressant-like+effects+and+enhances+cortical+5-HT/glutamate+neurotransmission:+role+of+5-HT1A+receptors.">Cannabidiol induces rapid-acting antidepressant-like effects and enhances cortical 5-HT/glutamate neurotransmission: role of 5-HT1A receptors.</a> Neuropharmacology. 103, 16-26 (2016).</li><li>Lehmann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+cannabidiol+treatment+reduces+early+pancreatic+inflammation+in+type+1+diabetes.">Experimental cannabidiol treatment reduces early pancreatic inflammation in type 1 diabetes.</a> Clinical Hemorheology and Microcirculation. 64 (4), 655-662 (2016).</li><li>Shannon, S., Lewis, N., Lee, H., Hughes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabidiol+in+anxiety+and+sleep:+A+large+case+series.">Cannabidiol in anxiety and sleep: A large case series.</a> The Permanente Journal. 23, 18-41 (2019).</li><li>Russo, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+cannabis+and+its+preparations+in+saga,+science,+and+sobriquet.">History of cannabis and its preparations in saga, science, and sobriquet.</a> Chemistry &amp; Biodiversity. 4 (8), 1614-1648 (2007).</li><li>Vera, C. L., Hanks, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemp+production+in+Western+Canada.">Hemp production in Western Canada.</a> Journal of Industrial Hemp. 9 (2), 79-86 (2004).</li><li>. Hot hemp: How high THC levels can ruin a legal hemp harvest Available from: <a target="_blank" href="https://www.westword.com/marijuana/hot-hemp-how-high-thc-levels-can-ruin-a-legal-hemp-harvest-9963683">https://www.westword.com/marijuana/hot-hemp-how-high-thc-levels-can-ruin-a-legal-hemp-harvest-9963683</a> (2018)</li><li>Lata, H., Chandra, S., Techen, N., Khan, I. A., ElSohly, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+genetic+stability+of+micropropagated+plants+of+Cannabis+sativa+by+ISSR+markers.">Assessment of the genetic stability of micropropagated plants of Cannabis sativa by ISSR markers.</a> Planta Medica. 76 (1), 97-100 (2010).</li><li>Caplan, D., Dixon, M., Zheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+rate+of+organic+fertilizer+during+the+flowering+stage+for+Cannabis+grown+in+two+coir-based+substrates.">Optimal rate of organic fertilizer during the flowering stage for Cannabis grown in two coir-based substrates.</a> HortScience. 52 (12), 1796 (2017).</li><li>Monthony, A. S., Page, S. R., Hesami, M., Jones, A. M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+past,+present+and+future+of+Cannabis+sativa+tissue+culture.">The past, present and future of Cannabis sativa tissue culture.</a> Plants (Basel). 10 (1), 185 (2021).</li><li>Clarke, R. C., Merlin, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabis+domestication,+breeding+history,+present-day+genetic+diversity,+and+future+prospects.">Cannabis domestication, breeding history, present-day genetic diversity, and future prospects.</a> Critical Reviews in Plant Sciences. 35 (5-6), 293-327 (2016).</li></ol>

With the increasing demand for Cannabis plants with consistent cannabinoid content, various clonal propagation methods have been exploited in Cannabis industry. The asexual propagation shows several advantages over sexual methods for large-scale, consistent production. An aeroponic propagation system is a modified version of a hydroponic system that utilizes an aerated nutrient-rich water mist to provide rapid root development. The described aeroponic system is composed of three critical steps, 1) gener...

Cannabis sativa L. is an annual, dioecious, flowering plant classified in the family Cannabaceae. Cannabinoids, produced predominantly within glandular trichomes located on the outer epidermal layer of bract tissues on female inflorescences1, are becoming an increasingly popular research topic, primarily due to their progressively recognized medicinal properties. Cannabidiol (CBD) is the second most prominent cannabinoid found in Cannabis after &#916;9-tetrahydrocannabinol (THC) and is attributed to a host of medicinal benefits, including analgesic properties2, anti-seizure properties3, antidepressant properties4, reducing the risk of diabetes5, and treating various sleep disorders6. Due to the multitude of health benefits associated with the metabolites of the Cannabis plant, there is a growing demand for its commercial-scale production7. To meet this demand, cultivation methods are constantly being improved and reinvented to continuously supply consistent, high-quality plant material to the emerging Cannabis industry.
The propagation of Cannabis can be facilitated in two ways: sexual or asexual reproduction. An example of sexual reproduction is pollinating a female ovule with pollen from a male&#39;s stamen resulting in a seed that can be germinated. Seed germination is a reliable cultivation method that has been used for breeding and cultivation purposes where desirable phenotypical traits are selected in parental lines to improve the quality of the offspring Cannabis plants, including traits such as drought tolerance, insect resistance, increased yield, and increased potency8. However, unintended cross-pollination is an inherent risk when performing sexual reproduction, causing undesirable offspring, which leads to the potential loss of desirable traits or an introduction of unwanted traits. An example of this unintended pollination is highlighted by hemp growers receiving hemp seed pollinated with THC- producing pollen resulting in significant economic loss due to the non-compliant plants (&#62;0.3% total THC w/w)9. Additionally, to generate a crop that consists of only females, a feminized seed must be sown instead of a non-feminized seed, which can lead to hermaphroditism and other undesirable traits leading to economic loss. To overcome the limitation of sexual reproduction of Cannabis, asexual reproduction has been widely practiced in commercial production models of the Cannabis industry10.
Asexual reproduction of Cannabis requires only a single plant, which allows for the multiplication of a single genotype that allows for commercial production of plants carrying desirable agronomic and pharmaceutical traits. A common form of asexual Cannabis reproduction is to cut and insert small portions of a female plant into a soilless substrate11 which is covered by a humidity dome to induce root formation. Although this method has proven successful, a common drawback is the accumulation of a high level of humidity (usually 80% or higher) inside the dome, providing an ideal growth environment for fungal pathogens, which can be detrimental to new, sensitive cuttings. Another form of asexual propagation is micropropagation using tissue culture, where sterile techniques allow for the propagation of insect, microbe, and virus-free Cannabis plant material in limited space12. This process, however, is expensive, time-consuming and requires trained laboratory technicians which are generally inaccessible for large-scale Cannabis facilities.
Very few published research reports exist on the clonal propagation of Cannabis. In order to provide a basis for the understanding of asexual reproduction of Cannabis for research purposes and industrial production, this study aimed to demonstrate the ease and accessibility of employing aeroponic systems for the clonal propagation of Cannabis. Aeroponic systems are ideal for the asexual propagation of Cannabis, consistently supplying nutrient-rich water to the cuttings, inducing early root formation in a timely manner, and allowing for a plant to be maintained indefinitely if needed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-part Fox Farm</td>
			<td>Fox Farm</td>
			<td></td>
			<td>Soil Mix</td>
		</tr>
		<tr>
			<td>1-part Promix</td>
			<td>Promix</td>
			<td></td>
			<td>Soil Mix</td>
		</tr>
		<tr>
			<td>1-part Roots Organic Original</td>
			<td>Auora Innovations</td>
			<td></td>
			<td>Soil Mix</td>
		</tr>
		<tr>
			<td>1-part Wiggle Worm Earth Worm Castings</td>
			<td>UNCO Industries</td>
			<td></td>
			<td>Soil Mix</td>
		</tr>
		<tr>
			<td>Algae and Bacterial Cleaning Solution (Clear Rez)</td>
			<td>EZ Clone</td>
			<td>SKU#: 225</td>
			<td>8 fl. Oz.</td>
		</tr>
		<tr>
			<td>Artificial Lighting</td>
			<td>AgroBrite</td>
			<td>SKU#: 1399</td>
			<td>T5 324W 4&#39; 6-Tube Fixture with Lamps</td>
		</tr>
		<tr>
			<td>Cannabis Mother plant 1 (Cherry Wine)</td>
			<td>Summit CBD</td>
			<td>N/A</td>
			<td>Donated material</td>
		</tr>
		<tr>
			<td>Cannabis Mother Plant 2 (Red Wine)</td>
			<td>Trilogene</td>
			<td>SKU: 0101RR</td>
			<td></td>
		</tr>
		<tr>
			<td>Corresponding Plastic Lid</td>
			<td>Office Depot</td>
			<td>N/A</td>
			<td>38.1 cm x 25.4 cm</td>
		</tr>
		<tr>
			<td>Drill Bit 1</td>
			<td>Dewalt</td>
			<td>DW1586</td>
			<td>38.1 mm spade drill bit</td>
		</tr>
		<tr>
			<td>Drill Bit 2</td>
			<td>Dewalt</td>
			<td>DW1308</td>
			<td>3.175 mm drill bit</td>
		</tr>
		<tr>
			<td>Flora/Bloom (Nutrient Solution)-5 mL</td>
			<td>General Hydroponics</td>
			<td>SKU#: 726</td>
			<td>946 mL (1 Quart) 2.43 lbs. (1.1 kg) (Available Phosphate 5.0%, Soluble Potash 4.0%, Magnesium 1.5%, Sulfur 1.0%)</td>
		</tr>
		<tr>
			<td>FloraGrow (Nutrient Solution)- 5 mL</td>
			<td>General Hydroponics</td>
			<td>SKU#: 724</td>
			<td>946 mL (1 Quart) 2.43 lbs. (1.1 kg) ((Total Nitrogen 2.0% (0.25% Ammoniacal Nitrogen, 1.75% Nitrate Nitrogen), Available Phosphate 1.0%, Soluble Potash 6.0%, Magnesium 0.5%))</td>
		</tr>
		<tr>
			<td>FloraMicro (Nutrient Solution)- 5 mL</td>
			<td>General Hydroponics</td>
			<td>SKU#: 759</td>
			<td>946 mL (1 Quart) 2.43 lbs. (1.1 kg) ((Total Nitrogen 5.0% (0.3% Ammoniacal Nitrogen, 4.7% Nitrate Nitrogen), Soluble Potash 1.0%, Calcium 5.0%, Boron 0.01%, Cobalt 0.0005%, Copper 0.01%, Iron 0.1%, Manganese 0.05%, Molybdenum 0.0008%, Zinc 0.015%))</td>
		</tr>
		<tr>
			<td>Horticultural Scissors</td>
			<td>Shear Perfection</td>
			<td>SKU#: 12620</td>
			<td>Platinum Stainless Steel Bonsai Scissors (2.4&#34;)</td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol</td>
			<td>Equate</td>
			<td>Walmart # 574133562</td>
			<td>70% concentration</td>
		</tr>
		<tr>
			<td>Nutrient Mist Solution (Clonex Mist)</td>
			<td>Growth Technology</td>
			<td>SKU#: 4889</td>
			<td>10.14 fl. Oz (300 ml) (Total Nitrogen: 5.9 &#215; 10-4 %, Available Phosphate: 4.0 &#215; 10-4 %, Soluble Potash: 5.0 &#215; 10-4 %)</td>
		</tr>
		<tr>
			<td>pH Down</td>
			<td>General Hydroponics</td>
			<td>SKU#: 733</td>
			<td>946 ml (1 Quart) 2.43 lbs. (1.1 kg)</td>
		</tr>
		<tr>
			<td>pH Up</td>
			<td>General Hydroponics</td>
			<td>SKU#: 730</td>
			<td>946 ml (1 Quart) 2.43 lbs. (1.1 kg)</td>
		</tr>
		<tr>
			<td>Plastic Container</td>
			<td>Office Depot</td>
			<td>N/A</td>
			<td>38.1 cm x 25.4 cm x 30.48 cm</td>
		</tr>
		<tr>
			<td>Power Drill</td>
			<td>Dewalt</td>
			<td>DCD709B</td>
			<td>20-Volt Max &#189;&#8221; Drill</td>
		</tr>
		<tr>
			<td>Rockwool Cubes</td>
			<td>Grodan</td>
			<td>SKU#: 830</td>
			<td>38.1 mm</td>
		</tr>
		<tr>
			<td>Rooting Solution (Clonex Rooting Gel)</td>
			<td>Growth Technology</td>
			<td>SKU#: 939</td>
			<td>3.4 fl. Oz. (100 ml) (Indolebutyric Acid - 0.31%)</td>
		</tr>
		<tr>
			<td>Statistic Software (Prism)</td>
			<td>GraphPad Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Submersible Water Pump</td>
			<td>ActiveAQUA</td>
			<td>SKU: AAPW250</td>
			<td>Model: AAPW250, Voltage 120V, Power 16W</td>
		</tr>
	</tbody>
</table>

employing aeroponic systems for the clonal propagation of cannabis

This protocol describes the standardization of an efficient clonal propagation technique of hemp by utilizing aeroponic systems. Primary shoot cuttings were excised from two hemp varieties, named &#34;Cherry Wine&#34; and &#34;Red Robin&#34; (17-20% w/w CBD), that served as &#39;mother plant&#39;. An auxin precursor (indole-3-butyric acid) was applied to stimulate root development in the basal portion of the excised cuttings prior to placement in the system. Cuttings were lightly misted with the nutrient mist solution every three days to provide nutritional support as the solution contains the essential macronutrients, including nitrogen, phosphorus, and potassium. The aeroponic system water reservoir maintained a pH range between 5.0-6.0 and a water temperature between 20-22 &#176;C. A submersible water pump was used to deliver water to the cuttings. The shoot tip cuttings were provided with 24 h of light per day for 10 days until root development occurred, upon which the rooted cuttings were transplanted for research purposes. These aeroponic systems have proven to generate desirable results for Cannabis propagation. The method described here alleviates potential time constraints that arise from traditional methods to allow for a more efficient means for the asexual propagation of Cannabis.

To validate the efficiency of the described aeroponic system, a total of 10 and 12 healthy 14 cm long shoots were excised from the mother plants, &#39;Cherry Wine&#39; and &#39;Red Robin&#39;, respectively (Figure 1A,B). After dipping into rooting induction media, the clones were placed into the system (Figure 2A). The construction and operation of an aeroponic system is shown as a schematic diagram in Figure 2A.</p...

Watch this Scientific Journal Video about Employing Aeroponic Systems for the Clonal Propagation of Cannabis at JoVE.com

Employing Aeroponic Systems for the Clonal Propagation of Cannabis

This protocol is designed to provide instructional information for the clonal propagation of Cannabis sativa L. by implementing aeroponic systems. The method described here includes all necessary supplies and protocols to successfully reproduce desirable morphological and chemical properties in the genus Cannabis.

This protocol describes the standardization of an efficient clonal propagation technique of hemp by utilizing aeroponic systems. Primary shoot cuttings ...

This protocol can aid in making an informed decision as to how to proceed with the regeneration of desired cannabis genetics in research or commercial production settings. The main advantage of this technique is inducing early root formation in cannabis cuttings while limiting their susceptibility to potential pathogens. The implications of this technique extend toward the improvement of challenges observed in cannabis propagation by inducing early root formation in cuttings, which can be applied in research or production settings.With a sterilized scalpel or scissor, cut the shoots with several nodes of approximately 10 centimeters in length near the apical meristem at an angle 45 degrees. Remove all foliage except that present on the top three nodes. Dip the newly excised cutting at approximately two to five centimeters up from the base of the stem into the rooting solution containing indole-3-butyric acid for approximately five seconds.Insert the cutting into the center of a rockwool cube til the depth of approximately two centimeters above the cube's base, positioned in the aeroponic system. Spray the unrooted cuttings with a nutrient mist solution every three days. Grow cuttings for about 18 to 24 hours in light per day with a photosynthetic photon flux density of 100 micromoles per second per square meter.Every two to five days, replenish the system with water at a pH between five to six. Lightly mist the cuttings with nutrient mist solution every three days and pour five milliliters of nutrient solution into the reservoir every three to five days. Add 15 milliliters of the algae and bacteria cleaning solution, containing 0.028%hypochlorous acid every five days.Select the cuttings with long, white fibrous roots. Carefully dislodge the rockwool cube from the system and untangle the roots. Transplant the cannabis propagules to a four liter nursery pot filled with a nutritious soil mix.The figure shows clones that developed roots in three to seven days and fully developed roots 37 centimeters in length after 10 to 14 days on the system, planted into a soil-filled pot. The average length of shoots and roots were approximately 24.8 centimeters and 37.8 centimeters for Cherry Wine and approximately 21.4 centimeters and 39.7 centimeters approximately for Red Robin, respectively. The differences between the two varieties were analyzed by two-way ANOVA followed by Tukey's multiple compassions test, showing no significant differences in shoot and root lengths between the two varieties.Selecting healthy cuttings exhibiting vigorous new growth is ideal for propagation. Maintaining the water level, temperature, pH, and nutritional aspects of the system reservoir are key for fast and effective outcomes.

employing-aeroponic-systems-for-the-clonal-propagation-of-cannabis

Research

JoVE Journal

Bioengineering

3.3K vistas.  Colorado State University-Pueblo. Este protocolo puede ayudar a tomar una decisión informada sobre cómo proceder con la regeneración de la genética de cannabis deseada en entornos de investigación o producción comercial. La principal ventaja de esta técnica es inducir la formación temprana de raíces en los esquejes de cannabis al tiempo que limita su susceptibilidad a posibles patógenos. Las implicaciones de esta técnica se extienden hacia la mejora de los desafíos observados en la propagación del cannabis median...