Editor’s note: This is the final segment in a four-part series, which you can read on our website by visiting here, on factors associated with plant tissue culture (TC) acclimatization. The prior three articles have demonstrated that ultimate success is heavily impacted by plant quality, plant handling and the substrate matrix. With these factors already in hand, the final aspect is the acclimatization environment.

Tissue culture (TC) plants differ from those in the greenhouse in five fundamental ways.

A plant’s life in TC occurs in nearly 100% relative humidity with no convective air flow, in a very promotive and consistent temperature range, under low light levels that lack strong ultraviolet (UV) radiation, with sugar supplied via the root system in addition to that from photosynthesis, and finally in the absence of potential plant pathogens.

The goal of acclimatization is to gradually transition the plants to withstand and thrive under the much harsher and dynamic conditions in the greenhouse.

Some aspects of a TC plant are “baked in” and can only be corrected as growth and maturation of new roots, leaves, and vascular tissue occur under greenhouse conditions.

Other factors such as the proper opening and closing of stomates, and the gradual formation of a waxy, cuticle layer can be stimulated during acclimatization. Beyond these desiccation resistance alterations, the most critical change during acclimatization is the transition to leaf photosynthesis for sugar production and its efficient transport to root and shoot meristems.

Species and the extent of tissue culture process optimization will determine the time it takes for these changes to occur. Some species such as cherry (Prunus sp.) or Cannabis can acclimatize even as unrooted stage 2 plants. Other species exhibit little or no growth in the first few weeks following transplant before slowly returning to growth. Only as both new root and leaves mature under typical greenhouse conditions can acclimatization be considered complete.

Here are four key factors regarding the plant acclimatization process to keep in mind:

1. Surface sanitization/pathogen and insect suppression

While the plant is undergoing acclimatization, consider it is highly susceptible to attack by insects, pathogenic fungi, and bacteria. Therefore protection against these agents must be a priority.

Early preparation and ongoing vigilance of the acclimatization space is critical. This includes pre-season pressure washing of bench, floor and wall surfaces to remove algae, mycelia and insects, and the washing of all tools, buckets, and fertilizer containers. It is useful to understand the age of the irrigation system and clean or replace components as needed. Where introduction of problem insects occurs, screens should be installed and regularly inspected.

Having sanitized the acclimatization space, frequent plant and facility observation becomes the next most critical activity, particularly for fast spreading diseases such as Botrytis and Fusarium, and for insects such as thrips and white fly. When fungal issues are observed, immediate sanitation of entire affected trays is critical to reduce spread, and sticky traps are advised for early insect detection.

When disease frequently occurs despite best efforts, and particularly during the short photoperiod winter months that favor fungal and algae development, prophylactic application of a rotation of fungicide/algaecide every few weeks has proven useful. As always, remember that rotation of chemicals with different modes of action is critical to avoid resistance development. Also, any chemical should be pre-tested on a small number of acclimatizing plants for phytotoxicity issues before implementation.

2. Temperature, relative humidity (RH) and vapor pressure deficit (VPD)

Temperature optima vary by species, and relates to the temperature at which the plant grows most efficiently.

Temperature additionally impacts both relative humidity and vapor pressure deficit (VPD). As air warms it becomes less dense and thereby can hold more water vapor. This reduces the relative humidity. The combination of temperature with any given relative humidity determines the vapor pressure deficit (VPD).

VPD is most easily thought of as the net drying potential of the air. Warmer temperatures directly reduce relative humidity, and in addition increases VPD. Many tables exist on the web relating VPD values for any given temperature and humidity combination. These are most often shown in a table where “good,” “too dry” or “too moist” have color highlighted backgrounds. These tables are often species specific.

While quite useful in understanding how temperature and RH combine to impact VPD, one should take care in inferring what “good” or “bad” VPD conditions constitute for plants coming from tissue culture.

A lower temperature may at times be one way to reduce plant water stress. This is ideally done via temperature control settings, but it can also be done by misting or fogging.

Misting or fogging does double duty by increasing relative humidity while decreasing leaf and air temperature. Even during the earliest phases of acclimatization, foliage should be allowed to dry off for a short time before the next cycle of misting to avoid disease development and provide a brief drying signal to the plant.

Plastic tunnels can greatly improve RH in simple greenhouses, but care must be taken to provide some ventilation and mist cooling to keep air and leaf temperature moderate.

Instrumentation that logs temperature and RH on an hourly basis are an important aspect of any acclimatization space. This can be a part of an overall greenhouse control system, or as small as pencil-sized loggers that are placed out of direct sunlight and downloaded periodically to a computer via USB connector.

I would also recommend purchase of a hand-held meter that displays both temperature and RH continuously. This enables growers to see how cooling/venting and heating cycles impact greenhouse environment in real time.

Lastly, the presence or absence of air movement or convection is an underappreciated factor in acclimatization. Air turbulence reduces the boundary layer of still air around leaves, increasing evaporative loss. However, air turbulence also decreases leaf temperature, which reduces evaporative loss. For this reason, air flow should be considered a double-edge sword that can be both helpful and detrimental. It should be present to reduce leaf heating as is accomplished by overhead circulation fans, but not in excess as can occur during side wall venting with large exhaust fans.

3. Light levels

Light levels in vitro commonly range from 30-100 µmol/m2/sec-1 while light level outside on a sunny day in mid-summer can reach upwards of 2000 µmol/m2/sec-1. The light saturation point is the point at which plant photosynthesis rates plateau. This light level varies by species and temperature, but for general discussion purposes is typically between 800 and 1000 µmol/m2/sec. Light levels above this does not increase photosynthesis output and can reduce it if the plant is stressed.

The daily light integral (DLI), which is a measure of the net amount of light received during the photoperiod, is an additionally important light consideration. The DLI is calculated by the following equation:

DLI = (Average instantaneous light level (µmol/m2/sec) x photoperiod hours x 3,600) / 1,000,000

Under indoor conditions of 16 hour photoperiod and constant 100 µmol/m2/sec-1 intensity the DLI is 5.8 which is considered at the low end for most growing plants. In greenhouses under cloudy, winter conditions in northern latitudes lacking supplemental illumination, DLI can fall to less than 3.0 which will greatly or completely inhibit growth. As acclimatization proceeds, providing a DLI closer to 10 or greater promotes rapid plant growth.

New transplants in February receive photoperiod extension and DLI supplementation

In addition to reducing net photosynthesis, a short photoperiod under otherwise ideal conditions can result in many perennial species entering a dormant developmental state (or growth cessation). Typically this can only be relieved by extended chilling time at or below 42-4 °F5 (5-7 °C).

Dormancy during and following acclimatization should be avoided at all costs as the lost growing time and resulting non-uniform crop can be highly problematic.

4. Root zone management

Soil matrix considerations were examined in detail in the previous article in this series (link here).

From a management perspective, it is important to consider foliar misting and soil irrigation as separate functions. Foliar misting should occur frequently but only as short, light bursts (1-2 seconds) to reduce VPD and foliage temperature. It should not add appreciable moisture to the soil matrix. Higher volume and longer duration irrigation events to increase soil matrix moisture should occur independently and only as needed.

Of course, there is a natural tendency to over water the soil matrix during the initial days of acclimatization when the leaves often look stressed or dehydrated. Conversely, during later acclimatization insufficient irrigation can occur as increased VPD, and new foliage and root growth increase irrigation frequency need. Attention to substrate drying must be assessed daily and adjusted throughout the acclimatization period.

Nutrition for new transplants are generally low until plants have adjusted physiologically and growth resumes. Premature fertilization, particularly with nitrogen, can also promote pathogen development. The need for a balanced NPK fertilizer between 50 and 100 ppm nitrogen is generally sufficient until acclimatization is complete.

Starting recommendations

There are no universal environmental specifications for acclimation conditions due to the multitude of factors previously discussed in this series. The table below is provided as a list of key parameters and possible starting targets for growers to consider when working with a new TC plant for the first time.

Conclusions

Small quantities of TC plants can generally be managed through acclimatization because they are carefully monitored and slowly transitioned. However, many commercial growers do not have that luxury where large volumes of plants arrive over a limited number of consecutive weeks, and additional physical space for acclimatization is limited.

It is under this production scale situation where plant quality and selection, shipping and storage conditions, substrate matrix, transplanting quality, and environmental conditions must all come together to produce an economically viable crop.

When any of these production factors are not well managed, the result can be widespread mortality and crop failure. Less obvious, but more insidious, is where early survival is not greatly impacted, but where non-uniform plant growth requires costly manual sorting and high cull rates at the end of the season. Bottom line? The keys to achieve TC acclimatization at a commercial scale are 1) understanding through-the-system sensitivities for fall downs, maintaining close and frequent communication between laboratory and greenhouse personnel, acquiring the environmental control and tracking capability required for the species being propagated, and remaining diligent in adjusting to seasonal changes in a timely manner.

The author is a technical sales advisor for Quick Plug, and previously worked as a scientist in tissue culture and greenhouse acclimatization of conifer and horticultural species.

Photo courtesy of Kala Jenkins

K-Coe Isom is AmericanHort’s partner law firm and specializes in agricultural law and consulting. Kala Jenkins, one of the firm’s agricultural specialists, discusses how the recently passed Paycheck Protection Program Flexibility Act of 2020 has changed from previous PPP models and what it offers green industries.

Greenhouse Management: What should business owners know about what this legislation can offer them?

Kala Jenkins: What this is primarily helping business owners do is provide payroll. That’s the biggest thing I consider when I look at PPP. To be able to give yourself that income source to maintain your payroll and labor source for your operation is what this is all about. That’s two-fold too. It’s to keep your doors open, but [it’s] really about helping your employees be able to withstand the economic changes that we are all going through and that are impacting businesses. You’re not only protecting your business, but you are also protecting your employees. The other thing to know about PPP is that [it] allows you to pay a couple of expenses like your mortgage interest, some utilities and rent/leases that are also eligible to be paid via this act.

GM: What are some of the notable changes made to PPP in the recent legislation? 

KJ: Under the old law, your loan terms were a minimum of two years for funds that were not forgiven. Under the new law, that’s now a five-year term. However, within that, you have to look it from your PPP loan approval dates. So if you had a loan prior to June 5, you have that ability to stick to that term or go back and renegotiate with a lender to go out five years for funds that are not forgiven. Any loan approved after June 5 is an automatic five-year window. ... One thing that isn’t clear right now is if transportation costs can be covered by PPP. 

GM: Do growers need a third party to help guide them through this process? 

KJ: I do think it’s good to have a third party walk you through this, especially because of all of the changes that are going on with the PPP program. For a while, we used to get updates from the [Small Business Association] daily or every other day detailing changes. That started with the application process and now it’s extended to the forgiveness process. Instead of you having to know the rules and regulations and what’s required, I think it’d be a good idea to have somebody else who is very knowledgeable with the program and understands its ins and outs so you can take that risk off of your plate. You may also have some lenders who take that third-party help as a good sign and potentially have it result in a quicker process for forgiveness or in the application process. 

Plant handling is a major user of labor in the greenhouse and nursery industry. One to two cents is added anytime you handle a plant. For moving plants from a greenhouse to an outdoor growing area, wagons and trailers can be of considerable help. The following tips should help in making a selection:

Wagons

A wagon is generally considered a four-wheel drawn vehicle for carrying materials. There are many sizes from the small wagon that fits in the aisle between the benches to the tractor pulled farm wagon that is used for order picking in the nursery.

Select a size based on the materials to be carried and the restraints of the aisles or roadways. For retail sales, a small wagon that is easy to maneuver through the aisles of plants is desirable. Larger wagons that hold 10 to 15 flats or 80 to 120 1-gallon containers are convenient for picking plants from closely spaced growing beds.

The farm wagon running gear is standard for many nurseries throughout the U.S. The bed should be sized to fit the roadways and the tractors available. A common size is 8 feet wide by 16 feet long. It will carry about 500 1-gallon containers.

Pneumatic tires are better than solid or semi-pneumatic rubber as they cushion the load. The larger the diameter, the easier it is to move. Flotation tires should be used where the ground is soft.

The tongue can be fitted with either a handle or hitch. A clevis hitch is desirable for positive connection to the drawbar on the tractor. Multiple wagons can be pulled by connecting the tongue of one wagon to a hitch on the rear of the adjacent wagon.

A smooth deck surface such as sheet metal, diamond plate or plywood is best for loading and unloading containers. Be sure to protect any sharp edges.

To be able to carry more plants, especially flats and one-gallon containers, a wagon may be fitted with racks. Keep the height to within easy reach. Removable shelves allow for different size plants.

To eliminate one or more handlings, a wagon works well for transporting large movable production trays from the headhouse or greenhouse to an outdoor growing area (Fig. 1). Trays are rolled on rails onto the trailer and locked in place.

Where wagons will be used in tandem in narrow aisles, tracking wagons should be purchased. Both axles pivot and opposite ends of each axle are connected with a rod so that the trailer will follow the exact track of the towing vehicle. The turning radius is usually specified by the manufacturer.

Trailers

Trailers are usually considered to be a vehicle supported on one end by a towing vehicle, tractor or truck. Trailers are available in load-carrying capacities of 500 pounds to 20 tons or more. They can be designed for handling shipping carts that are loaded in the field and moved to the shipping area for loading onto the truck (Fig. 2). This can eliminate one or more handlings.

The bed should be sized for the equipment or material to be carried. Deck widths from 4 to 8 feet are available. Most manufacturers use steel decking. Diamond plate is good as it gives a rough surface for better traction. Plywood or planking is common on larger trailers. This should be treated for weatherability and have a good wear surface.

Small trailers are usually manufactured with the deck located just above the axle. This makes loading easier. The height may be between 14 to 17 inches, depending on the size of tires. Some trailers with wide decks usually have the wheels under the deck that raises it to 30 inches in height or more. To meet the maximum width requirements for vehicles, the wheels may be placed in wheel wells.

For highway travel, springs are desirable to provide a smooth ride. For slow speed farm use, springs are optional. Lights with flashers and direction signals are required for highway travel.

Trailers under 3 tons usually depend on the towing vehicle’s brakes to stop. Over 3 tons it is desirable to have brakes to assist the truck or tractor. Usually electric brakes are used. Electronic brake control that provides progressive pressure that prevents wheel lockup is available for some trailers.

In an industry where the only constant is change, growers are looking to improve current processes and even set new goals.

Valuable employees remain on the frontlines of cultivation facilities every day, but it often makes the most sense to entrust computers, sensors and smartphones to complete certain tasks, like the daily collecting and correlating of millions of data points, such as room temperature, relative humidity, and nutrient and water uptake. Plus, data collection typically isn’t your or your employees’ primary skillset. Even so, each variable in a cultivation operation generates volumes of data that could take days, even weeks, for a person to analyze and put to use.

This is where smart technologies such as the Internet of Things (IoT), artificial intelligence (AI) and machine learning come into play. IoT has become commonplace in manufacturing to monitor machine performance and output. Likewise, utilities companies are deploying sensing technologies to the grid to balance supply and demand and identify potential outages before they happen. Similar technologies can help transform the cannabis industry.

With IoT, CannabiDOL Consulting owner John Dol says head growers, directors of cultivation and assistant growers can connect their laptops or phones to greenhouses and grows all over the world to remotely monitor and respond to potential issues.

IoT Brings Real-Time Visibility

In short, IoT involves the use of physical objects embedded with sensors that collect and relay information via the internet.

A climate control system in a grow room or greenhouse takes measurements from sensors that measure conditions such as temperature, humidity, lighting, fans/airflow and CO₂ levels—and sometimes substrate pH and electrical conductivity (EC), and vapor pressure deficit (VPD), Dol says. Information transferred via sensors to the climate control system, then to head growers off-site, demonstrates IoT use in cultivation.

Real-time access to data is critical for head growers on the go because it allows them to make remote changes to their climate control systems when certain parameters get out of hand, Dol says. They can set alarm parameters so their systems will alert them if something is wrong. Then, they can log into the systems from their phones, laptops, or either—depending on the systems—using their passwords. The systems log who made changes and when.

“For a grower managing multiple sites, this can be a key differentiator to effectively be ‘on site,’” Dol says.

In expressing the importance of IoT, Dol provides an example where an HVAC company could be servicing a grow room. When they finish, they forget to turn the unit back on. “When the lights come on the next morning, all of a sudden your room is at 100 degrees,” he says. If you are receiving real-time data, this can be prevented.

Cultivators also can give permission to other key decision makers, including consultants, Dol says. “Maybe I can only look at the climate side of things; or maybe I can look at climate and irrigation; or maybe I can look at climate, irrigation and heating,” Dol says. “Or they can set me up so I can also make changes.” However, as making those adjustments is the grower’s responsibility, Dol recommends against relying on consultants to make changes when possible.

Many developments in grow optimization came from the ornamental and produce greenhouse industries, the latter of which Dol worked in from the early 1990s until he joined the cannabis industry about four years ago.

For the most part, head growers have more opportunities to use IoT in greenhouses than grow rooms, Dol says. “In the greenhouses, you have to control more things,” he says. “Therefore, it’s more sophisticated and more widespread throughout the greenhouse. In a greenhouse, you have screens, you have ventilation both on one side of the peak and on the other side of the peak.”

That said, many IoT systems are in use in grow rooms, he says. Some outdoor growers use them, too, to respond to data from weather stations, and monitor and adjust irrigation.

“There are very sophisticated systems, and then there are some systems that only tell you, ‘Okay, it’s this warm, you’ve got this humidity, you’ve got this VPD and your lights are on,’ ” Dol says. “It can be that simple, too.”

As it stands, IoT has its limitations, Dol says, noting that AI and machine learning present future opportunities for cultivation. He points out that teams from around the world are conducting trials to autonomously grow tomatoes using AI. “The teams only have access to the climate computer,” he says. “They can’t physically go into the facility.”

A ‘Single Source of … Truth’

Redbud Roots, a vertically integrated cannabis company that operates more than 50,000 square feet of canopy in Buchanan, Mich., uses multiple types of sensors, including environmental control sensors, HVAC climate sensors and canopy sensors to collect and correlate climate and light data. Employees then manually correlate that data and create rules. “One of the challenges we see is determining a single source of climate truth,” David Frankfater, the company’s chief technology officer, wrote in an email to Cannabis Business Times.

One rule that employees set is the timing of dehumidifiers to react accordingly to separate environmental adjustments. “We have a rule to drive the humidity down in the rooms before the humidity spikes at lights out when the plants release more moisture,” Frankfater says. “This rule engages a second sensor trigger ... two hours before lights out. All of these rules rely on data collected from IoT sensors, and dehumidifiers run until the desired humidity setpoint is reached.”

Redbud Roots uses wireless sensors that provide data to “mitigate microclimates and enable growers to make changes via an app that affect light, temperature and humidity levels,” Frankfater says.

The company’s 88 employees include cultivation technicians who use “traditional farming sensors” to measure the pH of water and soil. In addition, Frankfater says, employees look for “the development of microbials microscopically during the last four weeks of flowering.”

On the cultivation side, Redbud Roots’ technology helps the company grow crops with added precision and improve employee productivity, Frankfater says. Employees also can work shorter days because of it. Information technology has also enabled the business to communicate information to its customers and to Michigan regulators.

When communicating with regulators, Redbud shares a “virtual flow” of its inventory and workflow “from seed to sale.” The cultivator uses a handheld device with a Radio-frequency identification (RFID) reader that utilizes IoT to locate and track plants.

At retail, including at its Williamsburg provisioning center that it opened in January to serve medical patients, Redbud Roots uses various programs to advertise and fulfill orders.

“Information technology’s key deliverable is visibility,” Frankfater says. “Visibility and insight are required to quickly identify and solve problems, to reduce costs and above all, to enhance end-user experience and business productivity.”

AI Enables Smart Growing

The power of AI and machine learning is playing a more significant role in cultivation. AI allows machines to learn from data to perform specific tasks. Machine learning is a subset of AI that allows computers to learn from past trends. An example of machine learning in the consumer world could be a streaming music or video service that recommends certain songs or movies based on past listening or viewing experience.

In the cannabis industry, AI and machine learning technology are becoming more common in cultivation facilities, says Andrew Lange, president of Ascendant Management and chief technical officer at Agrios Global Holdings. Agrios provides management and consulting services, equipment and facility leasing to licensed growers, while Ascendant provides buildouts, and engineering and design services.

“Depending on what you’re attempting to analyze, AI has the ability to detect and identify patterns and outliers in real time,” Lange says. “The computer will start to learn from your data. That’s machine learning. It can look at all these data points over the course of a harvest, and then input all of your yield metrics, and it can learn from the outcomes. You can do it again and again and again, and the computer is monitoring it each time.”

In addition, AI and machine learning can improve pest and disease scouting by putting “eyes” on plants all the time, Lange says, rather than cultivators having to rely on the subjectivity of employees and trusting that they will scout as diligently as they should. Another process that Lange currently doesn’t use, but is exploring, is using AI and “volumetric cameras.” Although these cameras largely resemble 2D cameras, growers can combine three or more of them to provide 3D imaging that can measure plant volume and predict yields. “It does always require multiple sensors to [create] that 3D image that the data is based on,” Lange says.

Using AI, cultivation facilities can sort through data they otherwise wouldn’t be able to, Lange says. For example, AI helps him manage multiple grow rooms in a 70,000-square-foot Washington facility. Every day at the operation, sensors used in cultivation and to monitor mechanical systems collect about 100 million data points—including 32 environmental points measured every two seconds. These points measure levels of temperature, relative humidity, VPD, CO₂, airflow, pressure and more.

“But data points [alone aren’t] really useful,” Lange says. “You have to be able to correlate that data. You have to be able to analyze it effectively. It used to be you would utilize data analysts … then we’re getting into trending software. Now comes AI. AI is really taking that data, looking at it in real time and giving you real-time answers, instead of having to fully analyze everything every time you do a grow. You’re getting faster results, faster data.”

AI then takes information—humidity measurements, for instance—and quickly adjusts levels to create better growing conditions, Lange says.

“It’s able to comprehend the data, analyze it and make changes in real time to affect our environmental outcomes—[it] could be more toward [the] target with less spikes,” he says. “Essentially, you’re just trying to lower any curves inside that data set, so you can utilize the technology to do things like that.”

AI can “identify patterns or anomalies” in data that sensors and IoT technology provide to growers, he says, adding, “This ability can help reduce downtime, lower operational costs and increase production.”

Patrick Williams is senior editor for Cannabis Business Times and Cannabis Dispensary magazines.