New research is using plant cell suspensions as biofactories to produce commercially important products like pharmaceutical molecules, pigments, fragrances, flavors, and others.
However, optimizing a plant cell suspension protocol is not always easy, and there are three important considerations to improve your plant cell suspension cultures: explant type, culture media composition, and environmental factors.
A plant cell suspension is a liquid culture where plant cells are multiplied to obtain products such as secondary metabolites used in the pharmaceutical, cosmetic, and food industries.
In this article, we present three important considerations as well as tips to improve plant cell suspension cultures.
In this article
The first consideration when improving your cell suspension is the explant choice.
Recall, an explant is the fragment of the plant tissue used as starting material in plant tissue culture protocols. Explants can be fragments such as a section of a flower, roots, shoots, leaves, and seeds.
Once transferred to a suitable medium, an explant may produce a callus.
Remember, a callus (calli – plural) is a mass of unorganized, undifferentiated cells that grow over the explants. They appear due to wounding or the addition of plant growth regulators.
A callus can give rise to shoot-buds and somatic embryos that form complete plants or can be transferred into a liquid medium to grow cells that produce metabolites.
So, the explant is critical to obtain a specific type of callus that may regenerate plants or massively produce metabolites.
Unfortunately, it is impossible to predict the explant response to the tissue culture in advance. However, we present some tips to guide you in your explant choice.
Keynote: To better understand explants and calli, we have another article with some basic concepts. It’s a short read that you can find here.
The tissue has a determined amount of internal growth regulators and may have specific metabolic capabilities (e.g., production of antioxidants). These internal factors associated with the explant selection affect the success of the cell suspension establishment.
Below we present some tips on how to choose an explant.
- Some explants are easier to isolate and disinfect compared to others. Some explants, like the root tip, are hard to isolate and are contaminated with soil microflora that becomes problematic during the tissue culture process.
- Some explants oxidize faster than others due to their high polyphenol content. Examples include seeds or colored tissues like floral sections. You may prefer leaf, shoot or root fragments to avoid excessive oxidation and death of the tissues.
- If it is unavoidable to use highly oxidative explants (e.g., seeds), try to use antioxidant solutions for washing or in the culture media such as citric or ascorbic acid in a concentration less than 3%.
- Young tissues are preferred over mature ones because they may grow faster and be more resistant to in vitro stress.
- Among different explants, leaf sections are commonly used for their abundance, ease of collection, and disinfection.
- Meristems are preferred for micropropagation because they can differentiate into other tissues. Tissues already developed like floral sections, roots, or shoots need to de-differentiate (return to a juvenile stage like meristem) and then differentiate into calli for plant regeneration or metabolite production.
Culture media comprises macro and micronutrients and additional components such as vitamins and plant growth regulators.
The composition is critical for optimization. Unfortunately, there is no magic formula to know what medium or component will work better for a given explant.
In tissue culture, many media are optimized by trial-and-error experiments, consuming considerable time.
However, new data mining techniques, such as Neuro-Fuzzy logic technology (NLT), have been proposed.
The NLT uses algorithms to identify relationships between several factors (cultivars, mineral nutrients, and plant growth regulators) and growth parameters (productivity), extracting biologically helpful information from each database and combining them to create a model which is used for media optimization (Gago et al., 2011). To test NLT, a lot of data to train the model is necessary, so the spread of NLT is currently limited.
We know that if you’re doing trial-and-error, this will be a bit of a pain. So here are some of our best tips for improving the culture media:
- Organize your literature and protocol research to identify specific regulators, additives, and observations when using them as it relates to your study.
- Keep meticulous notes about your process.
- Plan the experimental design carefully before running the experiments. A professor used to say 80% planning and 20% of experimentation is the key to success in science.
- Don’t forget to include positive and negative controls to have a reference for comparison.
- Perform the experiments in small batches. You will be able to evaluate a large set of variables in an organized way. A variable is a measurable factor such as the concentration of a component in the medium, which may affect your tissue culture positively, negatively, or neutral.
- Adding elicitors to the culture media is a common practice to induce metabolite production. For instance, jasmonate is a common elicitor to induce metabolite production like flavonoids.
- Analyze the statistical significance of independent and co-related variables in your experiment carefully. This analysis will help you to discard irrelevant variables and save additional time.
- If you succeed in a protocol, reproduce it and validate the results.
Factors such as light intensity, photoperiod, darkness, humidity, oxygen, and shake speed are important and affect the performance of a cell suspension.
Using these factors to promote stress favors metabolite production, as most secondary metabolites are produced in response to environmental stress.
When it comes to environmental stress, you do not want to make your plants go crazy or die. There is a fine line. But there are a few things you can do to bump up the stress in a healthy way so that you are working more efficiently on your tissue cultures:
- Review relevant protocols associated with your study to help you better understand appropriate environmental stressors.
- Be very organized in your note-taking when reviewing relevant literature. Tables and lists are your friends.
- A 16-hour dark and 8-hour day ratio for photoperiod is a good start. From there, you can explore 12 hours dark to 12 hours day, and so on.
- UV light is commonly used to induce in vitro secondary metabolites.
- Be gradual when you introduce stress, so you don’t kill your plants.
- Altering temperature depends on whether you have the ease to modify this variable. To introduce this stress, start at room temperature of 25 °C + 2 °C, then increase or decrease depending on the stress you want to test. Heat and cold stresses are well studied in plant models like Arabidopsis thaliana.
- Your suspension can be sensitive to shake speed, so be gradual as you increase the speed to increase the stress level. Start with a moderate shake speed to move all the cell suspension content within the flask.
Understanding the biology of your target plant is key to adapting in vivo conditions to in vitro systems and favors the plant cell suspension growth and metabolite yield.
Plant cell suspension, tissue culture, type of callus, callus.
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