Employing Aeroponic Systems for the Clonal Propagation of Cannabis

Summary

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.

Abstract

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 "Cherry Wine" and "Red Robin" (17-20% w/w CBD), that served as 'mother plant'. 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 °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.

Introduction

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 Δ⁹-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'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 (>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.

Protocol

1. Generation of a mother plant for clonal propagation

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

2. Construction and preparation of aeroponic system

1. 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.
2. Position the water pump (Table of Materials) in the center of the container.
3. 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.
4. 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.
   ​NOTE: Rockwool cubes are preferred due to their "anchoring" ability on the newly rooted cuttings that help keep plants upright following transplant.

3. Selecting and excising appropriate shoots

1. 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° angle. Cutting at a 45° angle increases the surface area of the basal portion of the cutting, allowing more space for root development. It's optional to make a small slit (1-2 cm) in the middle of the 45° cut to further increase surface area.
2. Remove all foliage except foliage present on the top three nodes.
3. 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.
4. 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.
5. Spray the unrooted cuttings with the nutrient mist solution (Table of Materials) every 3 days.
6. Grow the cuttings with 18-24 h of light per day with a photosynthetic photon flux density (PPFD) of 100 µmol/m²/s at 24-29 °C and 40-60% relative humidity.

4. Aeroponic system maintenance and propagule health

1. Replenish the system with water at a pH between 5.0- 6.0 every 2-5 days.
2. Lightly mist the cuttings (one mist per cutting) with the nutrient mist solution (Table of Materials) every 3 days.
3. 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.
4. 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).

5. Transplanting propagules

1. 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.
2. Carefully dislodge the Rockwool cube from the system and untangle the roots.
3. Transplant the Cannabis propagules to 4 L nursery pot filled with a nutritious soil mix (Table of Materials).
   ​NOTE: Watering immediately is recommended to prevent the roots from drying out.

6. Cleaning and storage of aeroponic system

1. When the system is no longer in use, wash with water and clean with 70% ethanol or another disinfectant.
2. Remove the filter from the water pump and rinse with water to remove debris.
3. Dry the system by wiping it down with paper towels or a washcloth.
4. Place the pump inside the tub with the lid on and store it until it is needed.

Representative Results

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, 'Cherry Wine' and 'Red Robin', 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 ± 2.4 cm and 37.8 cm ± 2.5 cm for 'Cherry Wine' and 21.4 cm ± 2.1 cm and 39.7 cm ± 5.9 cm for 'Red Robin', respectively (Figure 3A,B). 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 (n = 10-12, p < 0.05).

Figure 1: A healthy mother plant that generates multiple shoots for clonal propagation. (A) Mature mother plants, "Cherry Wine" (front) and "Red Robin" (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. Please click here to view a larger version of this figure.

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 "Cherry Wine" 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. Please click here to view a larger version of this figure.

Figure 3: Shoot and root length measurements for "Cherry Wine" and "Red Robin" 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's multiple comparisons test. Please click here to view a larger version of this figure.

Discussion

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 'mother' 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.

Materials

Name	Company	Catalog Number	Comments
1-part Fox Farm	Fox Farm		Soil Mix
1-part Promix	Promix		Soil Mix
1-part Roots Organic Original	Auora Innovations		Soil Mix
1-part Wiggle Worm Earth Worm Castings	UNCO Industries		Soil Mix
Algae and Bacterial Cleaning Solution (Clear Rez)	EZ Clone	SKU#: 225	8 fl. Oz.
Artificial Lighting	AgroBrite	SKU#: 1399	T5 324W 4' 6-Tube Fixture with Lamps
Cannabis Mother plant 1 (Cherry Wine)	Summit CBD	N/A	Donated material
Cannabis Mother Plant 2 (Red Wine)	Trilogene	SKU: 0101RR
Corresponding Plastic Lid	Office Depot	N/A	38.1 cm x 25.4 cm
Drill Bit 1	Dewalt	DW1586	38.1 mm spade drill bit
Drill Bit 2	Dewalt	DW1308	3.175 mm drill bit
Flora/Bloom (Nutrient Solution)-5 mL	General Hydroponics	SKU#: 726	946 mL (1 Quart) 2.43 lbs. (1.1 kg) (Available Phosphate 5.0%, Soluble Potash 4.0%, Magnesium 1.5%, Sulfur 1.0%)
FloraGrow (Nutrient Solution)- 5 mL	General Hydroponics	SKU#: 724	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%))
FloraMicro (Nutrient Solution)- 5 mL	General Hydroponics	SKU#: 759	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%))
Horticultural Scissors	Shear Perfection	SKU#: 12620	Platinum Stainless Steel Bonsai Scissors (2.4")
Isopropyl Alcohol	Equate	Walmart # 574133562	70% concentration
Nutrient Mist Solution (Clonex Mist)	Growth Technology	SKU#: 4889	10.14 fl. Oz (300 ml) (Total Nitrogen: 5.9 × 10-4 %, Available Phosphate: 4.0 × 10-4 %, Soluble Potash: 5.0 × 10-4 %)
pH Down	General Hydroponics	SKU#: 733	946 ml (1 Quart) 2.43 lbs. (1.1 kg)
pH Up	General Hydroponics	SKU#: 730	946 ml (1 Quart) 2.43 lbs. (1.1 kg)
Plastic Container	Office Depot	N/A	38.1 cm x 25.4 cm x 30.48 cm
Power Drill	Dewalt	DCD709B	20-Volt Max ½” Drill
Rockwool Cubes	Grodan	SKU#: 830	38.1 mm
Rooting Solution (Clonex Rooting Gel)	Growth Technology	SKU#: 939	3.4 fl. Oz. (100 ml) (Indolebutyric Acid - 0.31%)
Statistic Software (Prism)	GraphPad Inc.
Submersible Water Pump	ActiveAQUA	SKU: AAPW250	Model: AAPW250, Voltage 120V, Power 16W