Key factors in the ex-vitro acclimatization of tissue culture in plants

Features - Production

What you need to know about transplant and substrate matrices: Part 3.

March 30, 2020

Orchid plants acclimatized in stabilized molded-open matrix

Once a tissue culture (TC) protocol and transplant specification are established as discussed in part one here, and procedures are in place for transport and temporary storage as stated in part two here, the next critical factor for TC plant acclimatization is the soilless-substrate matrix the plants will be transplanted into.

Key roles of the substrate matrix

There are four critical roles that any substrate matrix provides plants: water, oxygen, physical support and nutrient delivery. Water, oxygen and physical support are largely determined by the physical texture and structure of the matrix, while nutrient delivery is impacted by the type and sourcing of substrate materials included.

Matrix “texture” and “structure” are fundamental

Texture refers to the particle-size distribution of the matrix, while structure relates to how those particles are spatially arranged and distributed in the container. Together, the texture and structure influence all four critical matrix roles, but perhaps most directly affect oxygenation and water-holding capacity.


Pores are the spaces or cavities between the “solids” (peat, perlite, coir, rockwool, wood fiber, bark, compost). The size and distribution of these spaces determines the amount of air and water the matrix can hold, referred to as the total percentage porosity, on a volume basis.

Pore space is not all the same. Following irrigation, some pores will immediately drain, while others will retain water. Large pores tend to be “drainable,” while smaller pores tend to be “water-holding.” These measures are calculated either as a percentage of total matrix volume or the total pore-space volume. Make sure you know which one is being presented!

The volume, depth, width and hole size at the base of the container greatly influences drainage characteristics. Low volume, shallow trays (less than 2 inches deep) tend to have a lower drainable pore space due to reduced gravity forces and a “perched” (or hanging) water table at the base of the container that represents a higher percentage of container volume than in larger finish containers.

There is a need for a standardized methodology that accurately measures porosity characteristics in the shallow containers that are commonly used for tissue-culture plant acclimatization. Measurement standardization across commercial labs and academia will help growers make more informed purchasing and irrigation decisions. I have seen measures for total pore space on a total-volume basis in shallow propagation trays that range from 75% to 95%, with drainable pore space on a total-volume basis from 5% to 20%.

Water and oxygen

While water-holding naturally comes to mind when properties of a substrate are discussed, oxygen is at least as important, if not more so. Oxygen transport happens via passive diffusion. Therefore, the surface-area-to-volume ratio of the matrix exposed to air is critical. This ratio is influenced by the depth, width and height of the matrix, and whether there is a gap between the container and the matrix.

TC plants, during early acclimatization, have limited water-uptake requirements and ability to transport water from root to leaf. Only when root growth resumes and new foliage surface area is created does matrix-moisture management become a significant issue.

It is important to appreciate that the density and porosity of a matrix will be dynamic over time. Porosity generally decreases with time due to particles settling from watering and growth of roots into pore spaces. Matrix texture and structure, and the extent of plant root growth, determine the extent of this change.

Physical support

Tissue culture plants, by virtue of their in vitro origin, require insertion into a matrix. The ability of that matrix to keep the plant oriented vertically and in contact with roots is critical. Strongly associated with these outcomes are the insertion hole and the gap that remains following transplant.

Molded-open plugs are the result of a need by the orchid industry in which large, delicate TC roots had to be transplanted rapidly. These plugs are shaped like the old video game “PacMan”, permitting the precise, vertically oriented placement of stage 3 roots with minimal stress and damage. Such plugs are finding broader application across the tissue-culture industry for species or cultivars that have long or brittle roots, or do not perform well when planted in loose fill or pre-dibbled holes.

The physical support required for a TC plant is largely determined by the size (foliage area and height) of the above-ground portion of the plant. While larger explants usually perform better when conditions are otherwise promotive, these bigger plants can also pose challenges remaining upright and inserted, even when the matrix opening is a perfect fit for the plant stem.

Lupine TC plant acclimatized in stabilized molded-open matrix

Nutrient delivery/pH/microbiome

Most commercially available substrates for tissue culture plants contain peat and coconut coir. Other frequently used components include compost, wood fiber, perlite or rockwool. The need for the matrix to “hold” nutrients during acclimatization is usually not critical due to frequent fertilizer application and the inherent cation-exchange capacity (CEC) of these materials; the exception is rock wool, which lacks any CEC. Some manufacturers may also include a starting charge of NPK or micronutrient fertilizer.

The “availability” of nutrients is determined by the nutrient solution concentration and pH, and the pH of the matrix. The pH optimum for a substrate varies by species, but in general a value between 5.5 and 6.0 works well for most plants and can be modified as needed by altering the pH of the fertilizer solution. For peat and coir-based substrates, the addition of some lime is common to achieve this pH range. For rock wools, pH must be carefully managed throughout crop culture to avoid wide swings.

Growers and matrix manufacturers are increasingly adding beneficial bacteria and fungi for both pathogen suppression and as a growth promotion agent (or probiotic). These approaches recognize the key role the soil microbiome plays as “the second genome of the plant.”

Ingredient quality

Substrate ingredients can vary substantially in critical physical, chemical and microbiological attributes. The most important aspects are particle size distribution, pH and absence of plant pathogens. For substrate containing coir, salt level as measured by electrical conductivity (EC) can also be important.

When purchasing prepared matrix or individual components, periodically perform a careful inspection of the product, ask for sub-supplier identities, and request quality-control specifications and documentation. While some information may be considered proprietary by manufacturers for valid competitive reasons, the more information and context a vendor is willing to supply and discuss, the more likely that you will receive a consistent product over time.

Matrix options for TC plants

Compressed plugs

Compressed plugs come dry as a disc, requiring hydration to expand to full size. They can be shipped quite efficiently and inexpensively. These products, however, tend to lack an opening appropriate to receive most TC plants and can exhibit irreversible shrinkage with time if significantly dried.

Loose fill

Loose fill is the simplest and most cost-effective because it can be formulated on site or purchased from a vendor. The medium works well for creating quality plants with high success rates and logistical efficiency, and it can be the most cost-effective option. Manual creation of a planting hole is required with loose fill, which for well-rooted or delicate-tissue-culture plants can pose a planting challenge and also slow transplanting rates.

Douglas fir TC plants in manually slit, stabilized matrix

Paper pots

Paper pots use loose fill that is wrapped in a paper sleeve with an open bottom. These plugs sit loose in a tray and allow early sorting to occur. The quality of the materials being used to fill the paper pot is critical, and manufacturer specifications should always be followed to avoid low drainable pore space issues. As with loose fill, paper pots require an opening to receive the plant.

Mineral wool

Mineral or rock wool provides a planting hole or slit for planting, has a fixed structure, permits early sorting and handling, and can be sterilized. However, it’s inorganic origin and more labile and elevated pH can at times reduce plant performance in the absence of careful monitoring and active chemical management.

Stabilized soilless plugs

Stabilized matrix uses a binder to fix the peat and coir matrix in place and, in some products, through use of a foaming binder to actively create pore spaces. The principal advantages of a stabilized matrix is the combination of (1) a fixed structure that is less prone to reduction in pore space over time, (2) the presence of a consistent opening in the plug by dibbling, cutting or forming the plug into a molded-open configuration that permits rapid and precise transplanting,(3) the ability to be used right out of the box without any pre-wetting, and (4) the ability to sort plants before they are rooted into the matrix. Stabilized plugs generally arrive at 75% to 80% moisture content with a six to 12-month shelf life and should be kept from environmental extremes such as direct sunlight.


The selection of planting matrix for tissue culture plants requires careful consideration of many factors. These include the plant morphology and quality being transplanted, the ergonomics and transplanting efficiency required and the overall cost sensitivity to losses or quality issues. This requires a through-the-system understanding of how acclimatization period impacts overall greenhouse cost and profitability of final marketable product.

Saving pennies per cell position by purchasing the lowest cost matrix method or supplier for TC plant acclimatization may appear initially attractive.

However, where there is history of species or cultivar mortality, slow growth, lack of uniformity or where transplant rate is a bottleneck to meet production goals, a change to a higher quality matrix that impacts those issues can usually be economically justified.

This is in part due to the level of upstream financial investment for any tissue culture plant. Increasingly consideration of the ability to partially or completely automate TC transplant into the matrix is being included in the decision-making process.

Having first done your homework on your system’s needs and challenges, an efficient vendor search can then be done that weighs the cost, characteristics, ingredient inputs and quality required for your acclimatization situation.

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.