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.
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.
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.