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>
2. Construction and preparation of aeroponic system
<ol>
	<li>Begin by positioning the lid on top of the container (38.1 cm x 25.4 cm x 30.48 cm). Drill the desired number of holes into the lid while providing adequate space (preferably 3 cm) between each.</li>
	<li>Position the water pump (Table of Materials) in the center of the container.</li>
	<li>Pour 7-8 L of distilled water into the container so that the pump nozzle remains roughly 2.5 cm above the waterline. 
	NOTE: This ensures the submersible water pump (Table of Materials) is able to push water with enough force to spread across the container lid. Distilled water is recommended; however, regular tap water may also be used.</li>
	<li>Situate the appropriate amount of Rockwool cubes (3.81 cm) (Table of Materials) or media cubes of choice into each slot. Turn on the pump and allow it to run for 24 h. 
	&#8203;NOTE: Rockwool cubes are preferred due to their &#34;anchoring&#34; ability on the newly rooted cuttings that help keep plants upright following transplant.</li>
</ol>
3. Selecting and excising appropriate shoots
<ol>
	<li>Collect shoots near the apical meristem using a sterilized scalpel or scissor. Cuttings are ~10 cm in length, ideally with several nodes. 
	NOTE: Cut the stem at a 45&#176; angle. Cutting at a 45&#176; angle increases the surface area of the basal portion of the cutting, allowing more space for root development. It&#39;s optional to make a small slit (1-2 cm) in the middle of the 45&#176; cut to further increase surface area.</li>
	<li>Remove all foliage except foliage present on the top three nodes.</li>
	<li>Dip the newly excised cutting into the rooting solution containing indole-3-butyric acid (IBA) (Table of Materials) ~2-5 cm up from the base of the stem for ~5 s.</li>
	<li>Insert the cutting into the center of a Rockwool cube positioned in the aeroponic system. 
	NOTE: The cutting insertion depth is to remain ~1-2 cm from the bottom of the Rockwool cube.</li>
	<li>Spray the unrooted cuttings with the nutrient mist solution (Table of Materials) every 3 days.</li>
	<li>Grow the cuttings with 18-24 h of light per day with a photosynthetic photon flux density (PPFD) of 100 &#181;mol/m2/s at 24-29 &#176;C and 40-60% relative humidity.</li>
</ol>
4. Aeroponic system maintenance and propagule health
<ol>
	<li>Replenish the system with water at a pH between 5.0- 6.0 every 2-5 days.</li>
	<li>Lightly mist the cuttings (one mist per cutting) with the nutrient mist solution (Table of Materials) every 3 days.</li>
	<li>Add 5 mL of each nutrient solution (Table of Materials) to the reservoir every 3-5 days. 
	NOTE: The nutrient addition causes water to be brown and murky.</li>
	<li>Add 15 mL of the algae and bacteria cleaning solution containing hypochlorous acid (0.028%) per 10 L of water every 5 days (Table of Materials).</li>
</ol>
5. Transplanting propagules
<ol>
	<li>Select the cuttings with long, white, fibrous roots. 
	NOTE: Avoid cuttings with brown, slimy, and short root systems as this is an indicator for the presence of root rot and will usually take longer to acclimate to the new growing medium and can bring unwanted diseases.</li>
	<li>Carefully dislodge the Rockwool cube from the system and untangle the roots.</li>
	<li>Transplant the Cannabis propagules to 4 L nursery pot filled with a nutritious soil mix (Table of Materials). 
	&#8203;NOTE: Watering immediately is recommended to prevent the roots from drying out.</li>
</ol>
6. Cleaning and storage of aeroponic system
<ol>
	<li>When the system is no longer in use, wash with water and clean with 70% ethanol or another disinfectant.</li>
	<li>Remove the filter from the water pump and rinse with water to remove debris.</li>
	<li>Dry the system by wiping it down with paper towels or a washcloth.</li>
	<li>Place the pump inside the tub with the lid on and store it until it is needed.</li>
</ol>

<ol><li>ElSohly, M. A., Radwan, M. M., Gul, W., Chandra, S., Galal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phytochemistry+of+Cannabis+sativa+L%5BTitle%5D)+AND+%22Progress+in+the+Chemistry+of+Organic+Natural+Products%22%5BJournal%5D)">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/pubmed?term=((Chronic+pain+treatment+with+cannabidiol+in+kidney+transplant+patients+in+Uruguay%5BTitle%5D)+AND+%22Transplantation+Proceedings%22%5BJournal%5D)">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/pubmed?term=((Efficacy+of+CBD-enriched+medical+cannabis+for+treatment+of+refractory+epilepsy+in+children+and+adolescents+-+An+observational%2C+longitudinal+study%5BTitle%5D)+AND+%22Brain+%26+Development%22%5BJournal%5D)">Efficacy of CBD-enriched medical cannabis for treatment of refractory epilepsy in children and adolescents - An observational, longitudinal study.</a> Brain & Development. 40 (7), 544-551 (2018).</li>
<li>Linge, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cannabidiol+induces+rapid-acting+antidepressant-like+effects+and+enhances+cortical+5-HT%2Fglutamate+neurotransmission%3A+role+of+5-HT1A+receptors%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">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/pubmed?term=((Experimental+cannabidiol+treatment+reduces+early+pancreatic+inflammation+in+type+1+diabetes%5BTitle%5D)+AND+%22Clinical+Hemorheology+and+Microcirculation%22%5BJournal%5D)">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/pubmed?term=((Cannabidiol+in+anxiety+and+sleep%3A+A+large+case+series%5BTitle%5D)+AND+%22The+Permanente+Journal%22%5BJournal%5D)">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/pubmed?term=((History+of+cannabis+and+its+preparations+in+saga%2C+science%2C+and+sobriquet%5BTitle%5D)+AND+%22Chemistry+%26+Biodiversity%22%5BJournal%5D)">History of cannabis and its preparations in saga, science, and sobriquet.</a> Chemistry & Biodiversity. 4 (8), 1614-1648 (2007).</li>
<li>Vera, C. L., Hanks, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hemp+production+in+Western+Canada%5BTitle%5D)+AND+%22Journal+of+Industrial+Hemp%22%5BJournal%5D)">Hemp production in Western Canada.</a> Journal of Industrial Hemp. 9 (2), 79-86 (2004).</li>
<li>Deventer, M. V. 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/pubmed?term=((Assessment+of+the+genetic+stability+of+micropropagated+plants+of+Cannabis+sativa+by+ISSR+markers%5BTitle%5D)+AND+%22Planta+Medica%22%5BJournal%5D)">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/pubmed?term=((Optimal+rate+of+organic+fertilizer+during+the+flowering+stage+for+Cannabis+grown+in+two+coir-based+substrates%5BTitle%5D)+AND+%22HortScience%22%5BJournal%5D)">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/pubmed?term=((The+past%2C+present+and+future+of+Cannabis+sativa+tissue+culture%5BTitle%5D)+AND+%22Plants+%28Basel%29%22%5BJournal%5D)">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/pubmed?term=((Cannabis+domestication%2C+breeding+history%2C+present-day+genetic+diversity%2C+and+future+prospects%5BTitle%5D)+AND+%22Critical+Reviews+in+Plant+Sciences%22%5BJournal%5D)">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>

The authors have no conflicts of interest.

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) generating a healthy female Cannabis &#39;mother&#39; plant as a genetic source for a desired physical/chemical trait, 2) cutting shoots (apical meristems) and treating with root induction media containing the auxin precursor, IBA, and 3) acclimation of the propagule in the desired nursery pot.
The aeroponic propagation system permits the efficient production of hemp clones with clear advantages over different propagation methods. It includes 1) time and cost-saving through less labor-inducing healthy, mature root formation as soon as 5 days; 2) genetic homogeneity - allowing to produce genetically identical clones, excluding any genetic variations, to accommodate reproducible Cannabis research and industry applications; 3) uniformity in large-scale operations; and 4) less vulnerability to microbial pathogens - maintaining a relatively low level of humidity in the upper canopy of plants than the humidity level built inside of the container where root development occurs.
Although the aeroponic system offers a number of advantages, there are a few limitations presented that should be carefully addressed during practices. First, as all the nutrients are provided hydroponically, it can be easily contaminated with algal, fungal, and viral (e.g., hop latent viroid) pathogens leading to the malformation of the root system which is mitigated through regular treatment of anti-microbial/fungal/viral agent(s). Second, the widespread use of a single mother plant can be problematic for production facilities. Having a single genotype clone line as the only plant in a production model leads to a risk of entire crop loss if that genotype is susceptible to a given pest or pathogen that is present in that cultivation facility; thus, it is recommended to maintain multiple mother plants from diverse lineages that are used to propagate production clones with this aeroponic cloning system to limit economic loss from any given pest or pathogen13. It is advised to only vegetatively grow a mother plant for no more than 6 months before cloning a new mother plant to use as the future source for production clones. This prevents overgrown roots that can lead to unhealthy mother plants and lower cloning success rates, as well as overgrown canopies that can harbor pests and pathogens, which may be transmitted to the propagated clones.
The aeroponic system is industrially scalable in a cost-effective manner. Figure 1 shows that at least 20 plant materials can be housed in a single container (38.1 cm x 25.4 cm x 30.48 cm). The system can be readily scaled up to hold more than 50 plants per unit without increasing costs on water, nutrients, and electricity. The advantages described here for the current method provide a reason to implement the aeroponic cloning system into industry practices and research laboratories for the time-efficient and uniform propagation of Cannabis.

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><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&#039; 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&quot;)</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 &amp;times; 10-4 %, Available Phosphate: 4.0 &amp;times; 10-4 %, Soluble Potash: 5.0 &amp;times; 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 &amp;frac12;&amp;rdquo; 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.
After 2 days of acclimation, all clones started to develop roots in 3-7 days and fully developed roots 37 cm in length after 10-14 days on the system, which was sufficient to be planted to a soil-filled pot (Figure 2B,D). Figure 3 shows the average length of shoots and roots of each variety. The shoot length and root length were measured prior to transferring to the soil. The average length of shoots and roots were 24.8 cm &#177; 2.4 cm and 37.8 cm &#177; 2.5 cm for &#39;Cherry Wine&#39; and 21.4 cm &#177; 2.1 cm and 39.7 cm &#177; 5.9 cm for &#39;Red Robin&#39;, respectively (Figure 3A,B). The differences between the two varieties were analyzed by two-way ANOVA, followed by Tukey&#39;s multiple compassions test, showing no significant differences in shoot and root lengths between the two varieties (n = 10-12, p &#60; 0.05).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63117/63117fig01.jpg" /> 
Figure 1: A healthy mother plant that generates multiple shoots for clonal propagation. (A) Mature mother plants, &#34;Cherry Wine&#34; (front) and &#34;Red Robin&#34; (back) at ~4 months of vegetative growth, exhibiting numerous shoots ideal for propagation. (B) Approximate length (14 cm) for proper shoot excision for cloning Cannabis. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63117/63117fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63117/63117fig02.jpg" /> 
Figure 2: Establishment of the aeroponic system for Cannabis clonal propagation. (A) Schematic diagram illustrating the components of the aeroponic system (38.1 cm x 25.4 cm x 30.48 cm). (B) Aeroponic system fully occupied by &#34;Cherry Wine&#34; clones. (C) Inside the aeroponic system with clones exhibiting root growth. (D) Healthy root growth in Rockwool cube after 10 days in the aeroponic system. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63117/63117fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63117/63117fig03.jpg" /> 
Figure 3: Shoot and root length measurements for &#34;Cherry Wine&#34; and &#34;Red Robin&#34; after 10 days in the aeroponic cloning system. (A) A bar-graph and (B) table representing the length of shoot and root in the two hemp varieties. The differences in shoot/root lengths between two varieties were analyzed by two-way ANOVA, followed by Tukey&#39;s multiple comparisons test. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63117/63117fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

3.7K Views.  Colorado State University-Pueblo. 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.

Video: Employing Aeroponic Systems for the Clonal Propagation of Cannabis

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Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Environment

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Culturing Arbuscular Mycorrhizal Fungi Using SAP-AS for Enhanced Observation

Inoculating and Observing Arbuscular Mycorrhizal Cultures on Superabsorbent Polymer-Based Autotrophic Systems

Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the rhizosphere. Typically, AM fungi are cultured under in vivo conditions in pot culture with an autotrophic host or under in vitro conditions with Ri Transfer-DNA transformed roots (heterotrophic host) in a Petri dish. Additionally, the cultivation of AM fungi in pot culture occurs in an opaque and non-sterile environment. In contrast, in vitro culture involves the propagation of AM fungi in a sterile, transparent environment. The superabsorbent polymer-based autotrophic system (SAP-AS) has recently been developed and shown to combine the advantages of both methods while avoiding their respective limitations (opacity and heterotrophic host, sterility). Here, we present a detailed protocol for easy preparation, single spore inoculation, and observation of AM fungi in SAP-AS. By modifying the Petri dishes, high-resolution photographic and video observations were possible on living specimens, which would have been difficult or impossible with current in vivo and in vitro techniques.

Author Spotlight: Enhancing AM Fungi Research with SAP-AS &#8212; A Novel Technique for Single-Spore Cultures

Here, we describe a simple and inexpensive technique for inoculating and observing arbuscular mycorrhizal fungi in superabsorbent polymer-based autotrophic systems.

Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

Immunology and Infection

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Biology

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

Semihydroponic Glass Jar System for Exudate Analysis in Multiple Plant Species

A Versatile Glass Jar System for Semihydroponic Root Exudate Profiling

Root exudates shape the plant-soil interface, are involved in nutrient cycling&#160;and modulate interactions with soil organisms. Root exudates are dynamic and shaped by biological, environmental, and experimental conditions. Due to their wide diversity and low concentrations, accurate exudate profiles are challenging to determine, even more so in natural environments where other organisms are present, turning over plant-derived compounds and producing additional compounds themselves. The semihydroponic glass jar experimental system introduced here allows control over biological, environmental, and experimental factors. It allows the growth of various phylogenetically distinct plant species for up to several months with or without microbes, in a variety of different growth media. The glass-based design offers a low metabolite background for high sensitivity and low environmental impact as it can be reused. Exudates can be sampled nondestructively, and conditions can be altered over the course of an experiment if desired. The setup is compatible with mass spectrometry analytics and other downstream analytical procedures. In summary, we present a versatile growth system suited for sensitive root exudate analysis in a variety of conditions.

Author Spotlight: Exploring Plant-Microbe Interactions Through Root Exudates in a Novel Growth System 

We present a protocol for a glass-based, semihydroponic experimental system supporting the growth of a variety of phylogenetically distinct plants with or without microbes. The system is compatible with different growth media and permits nondestructive root exudate sampling for downstream analysis.

Root exudates shape the plant-soil interface, are involved in nutrient cycling&#160;and modulate interactions with soil organisms. Root exudates are ...

Employing Aeroponic Systems for the Clonal Propagation of Cannabis

Summary

Explore More Videos

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Inoculating and Observing Arbuscular Mycorrhizal Cultures on Superabsorbent Polymer-Based Autotrophic Systems

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

Establishment of a Clonal Culture of Unicellular Conjugating Algae

A Simple Protocol for Mapping the Plant Root System Architecture Traits

A Versatile Glass Jar System for Semihydroponic Root Exudate Profiling