Tag Archives: environment

Applications for Tissue Culture in Cannabis Growing: Part 3

By Aaron G. Biros

In the first part of this series, we introduced some relevant terms and principles to tissue culture micropropagation and reviewed Dr. Hope Jones’ background in the science of it. In the second part, we went into the advantages and disadvantages of using mother plants to clone and why tissue culture could help growers scale up. In the third part of this series, we are going to examine the five steps that Dr. Jones lays out to successfully micropropagate cannabis plants from tissue cultures.

Cleaning – Stage 0

Explant cuttings are obtained from mother plants. The cuttings are further separated into smaller stem pieces with a single node.

Micropropagation includes 5 stages. “Stage 0 is the preparation of mother plants and harvest of cuttings for the explant material,” says Dr. Jones. “To ensure the best chance of growing well in culture, those ladies [the mom’s] should be cleaned up and at their best. And hopefully not stressed by insects or pathogens.” She says growers should also make sure the plants are properly fertilized and watered before harvesting explants. “Obtaining the explants is done with a clean technique using new disposable blades and gloves,” says Dr. Jones. “Young shoot tips are harvested and placed in labeled, large Ziploc bags with a small amount of dilute bleach and surfactant solution, then placed in a cooler and taken to the lab.” This is a process that could be documented with record keeping and data logs to ensure the same care is taken for every explant. “Once in the lab, working in the sterile environment of the transfer hood, the cuttings are sterilized, typically with bleach and a little surfactant, and then rinsed several times with sterile water,” says Dr. Jones. Once they reach the sterile environment, Dr. Jones removes the leaves and cuts the stem down to individual nodes.

Establishment – Stage 1

Established explants propagating shoots

Establishment essentially means waiting for the shoots to develop. Establishing the culture requires an absolutely sterile environment, which is why the first step is so important. “Proper explant disinfection is equally as important is the control parameters of the facility itself,” says Dr. Jones. Mother plants are not grown in sterile facilities, but in an environment that is invariably contaminated with dust, which harbors micro-organisms, insects and other potential sources of contamination, including human handling. We discussed some of this in Part 2.

Explants, once sterilized and placed in the culture vessel, must establish to the new aseptic conditions. “Basically Stage 0 ends when the explants are cleaned and placed in the vessel. Stage 1 begins on the shelf while we patiently sit, watch and wait for the shoot growth,” says Dr. Jones. “Successful establishment means we properly disinfected the explants because the cultures do not become contaminated with bacteria or fungi and new shoot growth emerges.”

Multiplication – Stage 2

Stage 2 involves subculturing an explant to produce new shoots

This stage is rather self-explanatory as multiplication simplified means generating many more shoots per explant. In order to create a large number of plants needed for meeting the demand of weekly clone orders, Dr. Jones can break up, or subculture, one explant that contains multiple numerous new shoots. “Let’s say one vessel, which originally started with 4 explants each developed four new shoots. Working in the hood, I remove each explant from the vessel and place it on a sterile petri dish. Now I can divide each explant into 4 new explants and then place the four new explant cuttings into their own vessel. In this example, we started with one vessel with 4 explants,” says Dr. Jones. “Which when subcultured 4-6 weeks later, we now have 4 vessels with 16 plants.” This is instrumental in understanding how tissue culture micropropagation can help growers scale without the need for a ton of space and maintenance. From a single explant, you can potentially generate 70,000 plants after 48 weeks, according to Dr. Jones. “Starting with not 1, but 10 or 20 explants would significantly speed up multiplication.” Using tissue culture effectively, one can see how a grower can exponentially increase their production.

Rooting – Stage 3

“When the decision is made to move cultures to the rooting stage, we typically need to subculture the plantlets to a different media formulated to induce rooting,” says Dr. Jones. “In some instances, the media is very dark, and that’s because of the addition of activated charcoal.” Using activated charcoal, according to Dr. Jones, helps darken the rooting environment, which closely mimics a normal rooting environment. “It helps remove high levels of cytokinin and other possible inhibitory compounds,” says Dr. Jones. Cytokinins are a type of plant growth hormone commonly used to promote healthy shoot growth, but it is important to make sure the culture contains the right ratio of hormones, including cytokinin and auxin for maximum root and shoot development. Dr. Jones suggests that growers research their own media formulation to ensure nice, healthy roots develop and that no tissue dies in the process. “With everything I grow in culture, when it comes to media, in any stage and with all new strains, I run some simple experiments in order to refine the media used,” says Dr. Jones. She puts a special focus on the concentrations and ratios of plant hormones in formulating her medias.

After harvesting and multiplying, these explants are ready for rooting

“We commonly think of auxin’s role in rooting, but it’s also important in leaves and acts as a regulator of apical shoot dominance,” says Dr. Jones. “So having no auxin may not be ideal for the shooting media used in Stages 1 and 2.” Auxin is a plant hormone that can help promote the elongation of cells, an important step in any plant’s growth. “And cytokinins are typically synthesized in the root and moves through xylem to shoots to regulate mitosis as well as inducing lateral bud branching, so again finding that nice balance between these two hormones is key.”

Acclimation & Hardening Off – Stage 4

“When plants have developed good looking healthy roots, it’s time to pop the top,” says Dr. Jones. This means opening the vessel, another risk for contamination, which is why having a clean environment is so crucial. “The location of these vessels needs to be tightly controlled for light, relative humidity, temperature and cleanliness.” In the culture, sugar is a main ingredient in the medium, because the growing explants are not very photosynthetically active. “By opening the lid of the vessel, carbon dioxide is introduced to the environment, which promotes and enhances photosynthesis, really getting the plants ready for cultivation.”

Harvesting explant material from mother plants

The very final step in tissue culture micropropagation is hardening, which involves the formation of the waxy cuticle on the leaves of the plant, according to Dr. Jones. This is what preps the plant to actually survive in an unsterile environment. “The rooted plants are removed from the culture vessel, the media washed off and placed in a potting mix/matrix or plug and kept in high humidity and low light,” says Dr. Jones. “Now that there is no sugar, contamination is no longer a threat, and these plants can be moved to the grow facility.” She says conditioning these plants can take one or two weeks. Over that time, growers should gradually increase light intensity and bring down the relative humidity to normal growing conditions.

Overall, this process, if done efficiently, can take roughly eleven weeks from prepping the explants to acclimation and hardening. If growers perform all the steps correctly and with extra care to reduce risks of contamination, one can produce thousands of plants in a matter of weeks.

In the fourth and final part of this series, we are going to dive into implementation. In that piece, we will discuss design principles for tissue culture facilities, equipment and instrumentation and some real-world case studies of tissue culture micropropagation.


How Cannabis Can Positively Impact California’s Drought

By Lukian Kobzeff

As the drought in California persists and quickly becomes the new hydrological norm, many within the state have embraced efforts to find ways and means to live within the drought forced water “budget.” Because of the importance of water conservation, the cannabis industry should embrace its socio-ecological responsibility and seize the opportunity to help shift the perception of cannabis cultivation into that of a sustainable, high-value agricultural crop that can be grown in an environmentally safe manner, while using water efficiently.

The intersection of Prop 64, MCRSA and the drought provides the cannabis industry with a unique opportunity to positively impact water conservation. Because legal cannabis cultivators are just now designing blueprints for grow sites, these cultivators are in a position to build infrastructure and systems specifically designed to achieve permanent, sustainable water conservation.

By embracing and championing water conservation, the cannabis industry will achieve two goals: being a collaborative player in the larger community working towards sustainable water use and enhancing the overall perception of the cannabis industry in the conscious of the general public. For an industry seeking legitimacy, there is no better way to put cannabis in the mainstream conscious than by embracing environmentally responsible philosophies. Here are a few measures the cannabis industry should embrace:


The current drought has generated a state-wide conversation about tracking and recording water usage. Some commentators believe California is suffering from a water data problem. Recently passed AB 1755 is a step by California to address that shortcoming by creating a technology platform to aggregate and share water data. Cannabis cultivators should get onboard with measuring water usage. One method is to install sensitive flow meters in each drip station to precisely measure water used during each grow cycle. First, this provides the cultivator with a precise data set. Precise data sets are extremely important, especially when trying to achieve the two-part-goal of conserving water and maximizing crop yield. Second, having precise data sets allows the cultivator to determine, from harvest-to-harvest, increasingly precise ratios of input (water) to output (flower). Most likely, this input:yield ratio is subject to diminishing returns at the margin; that is, adding additional water will not proportionately increase crop yield. For instance, 50 units of water could produce 50 units of crop, but 75 units of water might only produce 55 units of crop. By measuring the input (water), the cultivator is able to identify the precise threshold where diminishing returns set in and can therefore reduce the “diminishing returns” water usage, saving money and conserving water.


Building on water-usage data collection, cultivators can then collaborate with each other and with water agencies. By sharing data sets, cultivators can quickly develop ideal input:yield ratios, can better understand how water usage fluctuates within each discreet grow cycle and can develop methods such as deficit irrigation and real-time soil moisture measurements. This collective industry knowledge will help each individual cultivator to reduce water-usage. In collaborating with local water boards, the boards will better understand how much water is being used and conserved by the industry. Additionally, if the boards have a more precise understanding of the expected usage per season or per specific period in a grow cycle required by cultivators in their jurisdictions, those boards can better plan for the peaks and troughs in water demand. Besides data sharing, agencies and cultivators can collaborate in developing “fill stations” (offering free, non-potable recycled water for irrigation), or help fund development of direct potable water technologies and other recycled water technologies. Collaboration amongst growers and with water boards will lead to greater water conservation.

Energy Saving

An ancillary benefit to water conservation behaviors is the reduction of energy consumption. It takes an immense amount of energy to pump and transport water to end-users, such as cultivators. Reducing water usage in turn reduces energy consumption, because less water used means less water transported and disposed of. This is one method for indoor cultivators to offset energy consumption. In addition to reducing energy usage by conserving water, cultivators can follow Irvine Ranch Water District’s example of implementing an energy storage system to reduce costs and ease energy demand during peak hours. Indoor cultivators should adopt the same basic structure and mechanics: install Tesla battery packs to store energy for use during peak hours (when electricity is more expensive) and recharge the batteries at night when demand is low (and electricity is cheaper).

Opportunities Abound

This is an exciting time in California’s history, with the pending election of Prop 64, the passage of MCRSA, and the opportunities present in the water-energy nexus. The $6 billion cannabis industry has an incredible opportunity to have a far-reaching impact on water-conservation. By being an active collaborator conserving water, the cannabis industry can position itself as a trendsetter and private sector leader in sustainable and eco-conscious methods, technologies, and processes.


Cannabis, Soil Science and Sustainability

By Drew Plebani

The average commercial cannabis cultivator seems to be following the modern agricultural paradigm. That model is based on questionable and, one might say, ineffective soil systems management.

In the high-yield cannabis world, amidst decades of prohibition, following the lead of the modern agricultural model has resulted in the adoption of cultural practices that go something like this: Use and destroy the soil, then dispose of it once it is rendered lifeless and useless due to repeated heavy applications of chemical fertilizers, pesticides, and other poisons.

(Left) unimproved site soil next to (right) improved site soil. Notice the root mass developing on the right

Certainly conventional agricultural food production and the soil management systems underpinning them are faltering, evidenced by soil systems deteriorating many times faster than they are being improved. This qualifies as a failure in my book.

What will be the fate of profit margins, sustainability and medicine in the cannabis industry if we continue to follow blindly in the footsteps of chemical agriculture? Perhaps it is time to turn over a new leaf.

A little context for the discussion: scientists say the Earth has lost a third of arable land in the past 40 years, and some say soil erosion is the number one challenge facing the world today. Why? How?

Well…world agricultural production accounts for about three-quarters of the soil erosion worldwide. This steep decline in arable soil is occurring during a time when the world’s demand for food is rapidly increasing. It is estimated that the world will need to grow 50% more food by 2050, and it is important to note that, the total volume of food necessary, remains relative to the nutrient density of the food.

Time for a radical solution, and cannabis can lead the way.

Currently, cannabis is the most profitable crop per land area and very likely the most resource-consumptive crop grown (due to the current legal and regulatory climate and thus limited supply vs. demand).

As the cannabis industry continues to grow, now more than ever we have the opportunity, and I believe the responsibility, to cultivate in ecologically mindful ways, improve the end product and it’s positive impacts, increase both short-term and long-term profits, decrease or eliminate waste and lower the carbon footprint of cannabis cultivation operations.

A cover crop under trellis’ with cannabis plants

Most importantly, we have the opportunity to fund, implement and lead the way in research and development of sustainable, medical, phytonutrient-dense crop production methodologies.

Only by implementing more rigorous scientific methods to cannabis cultivation can we hope to provide truly meaningful improvements in and contributions to the fields of agriculture, science, medicine and human health.

While dumpsters of potting soil continue to roll off to the landfill, complex health and human science and the cultivators truly engaged in science will continue to provide meaningful data regarding plant compounds and what factors influence the best outcome for the desired end product.

The same crop pictured above, now two weeks into flowering, using cover crops

I am willing to bet that what is best will not be coming from the business models employing antiquated, wasteful and destructive cultivation strategies, and that in due time these models will fade into distant memories.

This is the first in a series of articles, in which we will explore topics related to the pursuit of high yield, phytonutrient-dense “high brix” cannabis production.

The next article will provide a historical and geologic context to the cannabis plant, as viewed from the scope of soil biology and the progression of ecosystems and soil types, and how maximized genetic expression, through maximized soil and plant health influence the production of high quality cannabis.