A plant cell suspension culture is a liquid culture where cells from a soft or friable callus divide and multiply.
Plant cell suspension cultures are used to obtain secondary metabolites in pharmaceuticals, cosmetics, or used in the food industry.
Researchers use plant cell suspension cultures to analyze metabolic pathways for basic science and even knockdown experiments.
Five steps are required to establish a plant cell suspension: Plant material preparation, explant sterilization and isolation, initial cell suspension stage, growth of the cell suspension, and metabolite production.
We also provide the methods to measure the growth of cell suspensions including fresh weight, dry weight, packed cell volume and medium conductivity/osmolality.
In this article
Plant Cell Suspension Culture: What Is It and How to Establish It
What is a plant cell suspension culture?
How to establish a cell suspension
Step one: Prepare the plant material
Step two: Sterilize and isolate explants
Step three: Initial stage of cell suspension
Step four: Grow the cell suspension
Step five: Metabolite’s production
Methods for measurement of growth in cell suspensions
Medium conductivity/osmolality
What is a plant cell suspension culture?
A plant cell suspension is a culture developed by transferring the relatively friable portion of the callus into a liquid medium and is maintained under suitable conditions of aeration, agitation, light, temperature, and other physical parameters.
In a plant cell suspension, the cells divide, multiply and grow.
To clarify what we mean by a friable callus – there are two types of plant calli, calli used for plant regeneration, and calli that do not produce plants, which are used for plant cell suspensions.
Within the group of calli that are not used for plant regeneration, there are two subcategories: compact calli and friable calli.
To understand both, you might want to look at our article detailing the different types of explants and calli. But for now, we'll briefly explain what a friable callus is.
A friable callus is a callus characterized by loosely attached and watery cells. Usually, friable calli are associated with weaker cell walls and a soft texture. A friable callus is the starting plant material to establish a plant cell suspension.
The purpose of plant cell suspensions is to produce massively bioactive secondary metabolites that run under controlled conditions, independently from climate and soil conditions and continuous production. Other cell suspensions are established to study proteins or metabolites pathways.
How to establish a cell suspension
There are five steps to establishing a cell suspension: plant material preparation, explant sterilization and isolation, initial cell suspension stage, growing the cell suspension, and metabolite production.
Five steps to establish a cell suspension culture
- Plant material preparation
- Explant sterilization and isolation
- Initial cell suspension stage
- Cell suspension growth
- Metabolite production
Below, we highlight the steps to establish a plant cell suspension. We also have another helpful article with considerations and tips to help you improve your plant cell suspension culture. It's a pretty quick read and you can look at it here.
Step one: Prepare the plant material
Plant material is critical when it comes to establishing a cell suspension. Cell suspensions can be raised from leaf discs, seed fragments, meristems, stem pieces, mature or immature embryos, anthers, pollen, microspores and ovules, flowers, or root sections.
Generally speaking, young tissues work better for plant cell suspensions.
The plant fragments to induce tissue cultures are known as explants.
Step two: Sterilize and isolate explants
Once the plant material (or explant) has been selected, the tissue needs superficial disinfection to remove contaminants that can be introduced into the plant tissue culture.
The contaminant removal is essential because microbes grow faster than the plant tissues in a culture medium, exhausting the nutrients of the media.
Thus, cell suspensions cannot be established if explants are not well disinfected. The disinfection can be done with commercial disinfectants like sodium hypochlorite, calcium hypochlorite, ethanol, and mercuric chloride (HgCl2) (Hussain et al. 2012).
Once the explant is disinfected, it is generally cut to induce wounding and transferred into a solid medium to induce callus production.
Step three: Initial stage of cell suspension
The calli formed over plant tissues are generally compact. After successive passages (or subcultures) of the calli from old solid media to fresh media, the calli become friable.
The friable calli are ideal for establishing a cell suspension because their texture favors nutrient uptake. Also, cells can be easily distributed over the liquid medium. After calli become friable, they are transferred from a solid medium to a liquid.
Step four: Grow the cell suspension
In this step, the cells from the calli in liquid media follow a growth curve, where there are lags, exponential and stationary phases.
The friable calli is adapted to the new liquid environment in the lag phase. In the exponential phase, cells grow and multiply until they reach the stationary phase, when the cell growth stops.
Step five: Metabolite's production
This step is adjusted according to the research goals. Cell suspensions can be used for basic science, such as studying the behavior of a given metabolite or protein in a liquid medium or for massive production of metabolites.
The metabolite's production in cell suspensions is commonly a two-step process. In the first step, the cell suspension needs to grow enough to provide mature cells that can produce metabolites.
Then, the metabolite production is induced in a second step. Modifying the liquid media with specific reagents (e.g., coconut water, jasmonic acid) or changing environmental conditions (such as light and temperature) is typically used to stimulate metabolite production.
Methods for measurement of growth in cell suspensions
A plant cell suspension's growth can be measured by fresh weight, dry weight, packed cell volume (PCV), and medium conductivity/osmolality.
Fresh weight
Fresh weight is a measurement of growth for a cell suspension and requires you harvest cells from a cell suspension every so often to determine the fresh weight of cells per volume of cell suspension.
How to calculate the fresh weight on a cell suspension?
First, mix your cell suspension culture and collect cells of your cell suspensions with the help of a pipette daily or every two days, according to your experimental setup and volume of cell suspension culture.
Proceed to place the volume on a pre-weighted container and place the container on a bench scale and take the value. Then, calculate the difference between the data for the pre-weighted container and the container plus the fresh sample.
The resulting data will be your measure of fresh weight in the cell suspension.
Tip: Some researchers prefer to sacrifice one flask per sampling to improve the accuracy of the measurement.
Dry weight
Dry weight is a measurement of growth on a cell suspension where dried cells are weighed every so often to determine the dry weight of cells per volume of cell suspension.
How to calculate dry weight on a cell suspension?
First, mix your cell suspension culture and collect cells of your cell suspensions with the help of a pipette daily or every two days, according to your experimental setup and volume of cell suspension culture.
Proceed to dry the cells in an oven to low temperature (~45°C) or use a freeze drier. Place the dried cells on a pre-weighed container, and put the container on a bench scale, and take the value.
Then, calculate the difference between the data for the pre-weighed container and the container plus the dry sample.
The resulting data will be your measure of dry weight in the cell suspension.
Packed cell volume (PCV)
The Packed cell volume (PCV) is a measurement of the growth non-invasive in cell culture. It consists in decanting the cells within a flask, marking the top of the decanted cells and proceeding with a rule to measure how high is the packed cell volume.
How to calculate the packed cell volume on a cell suspension?
First, allow the cells to decant within the flask. Slightly tilt the flask and make a mark with a marker pen. Then use a ruler to measure the cell’s height and write down the data in your notebook.
Repeat the process every sampling day. At the end of the sampling, you will notice how much your cell suspension grew.
Medium conductivity/osmolality
The medium conductivity is a measurement of the growth in plant cell suspensions.
Conductivity is a measure of the ability of water to pass an electrical current. Cells absorb nutrients (present in the form of salts) from the medium, and then as the cells take nutrients from the medium, the electrical conductivity decreases.
How to calculate the medium conductivity on a cell suspension?
The medium conductivity is calculated using a conductivity meter. Introduce the conductivity meter in the cell suspension, take note of the value and record the data every sampling day.
In the end, you can plot the data and expect a decreasing conductivity curve which indicates the cells are absorbing the nutrients of the medium and growing.
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Keywords
Plant cell suspension culture, calli, callus, friable callus, cell suspension establishment, plant research.
References
Chandran, H., Meena, M., Barupal, T., & Sharma, K. (2020). Plant tissue culture as a perpetual source for production of industrially important bioactive compounds. Biotechnology Reports, 26, e00450. https://doi.org/10.1016/j.btre.2020.e00450
Chimdessa, E., & Agricultural, O. (2020). Composition and Preparation of Plant Tissue Culture Medium. Journal of Tissue Culture and Bioengineering, 3(01). https://doi.org/10.29011/2688-6502.000020
Espinosa-Leal, C. A., Puente-Garza, C. A., & García-Lara, S. (2018). In vitro plant tissue culture: means for production of biological active compounds. Planta, 248(1), 1–18. https://doi.org/10.1007/s00425-018-2910-1
