30 Sep 2022

Coffee Chat with our Lab Director

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplifying intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

You Had Questions… Francisco Has Your Answers!

Francisco Palacios is Plant Cell Technology’s Lab Director. He holds a degree in Plant Molecular Genetics with a concentration in crop genetics, plant biotechnology, and plant nursery research from the University of Tennessee. Francisco also serves as a Research Specialist for the Variety Testing and Agroecology program of the University of Tennessee. He has worked with carnivorous, indoor, and aquarium plants as a plant tissue culture specialist.

At Plant Cell Technology, Francisco tests the capabilities of PPM™, various gelling agents, along with a selection of complimentary products that will be added to our line of products soon.

In addition to all this great work, Francisco manages PCT’s YouTube channel, where he teaches everything from beginner courses to the expert aspects of plant tissue culture, including best practices for an aseptic environment, sterilization methods, media preparations ways, explant cutting, equipment operations, and many other topics, including his latest series on finding the best media for philodendrons.

You have sent us many questions for Francisco across our social media channels. And, we thought to answer some of the commonly asked questions in Francisco’s words. So, over a nice cup of virtual coffee, we brought your question to Francisco’s attention, and here’s what he had to say.

Oh! And if you are a beginner, do not miss questions 10 and 12; these are just for you!

1. What are some practices to follow to maintain a sterile environment in home-based labs?

Here’re some best tips to keep your home-based labs clean and sterile:

• Clean everything as clean as possible
• Try to disinfect all surfaces. For example, regularly sweep your room (including the shell and walls).
• Keep a HEPA filter in your working space
• Disinfect your hood, containers, and whatever you use for the tissue culture process.
• Use 70% alcohol to disinfect your culture area, rather than 90% because it evaporates faster.
• Wear gloves and masks to prevent any contaminant transfer from your hands or mouth.

“The primary thing to do to avoid contamination in a home-based lab is limiting the airflow and keeping the environment as clean and aseptic as possible.”

2. Can we perform tissue culture without a laminar hood?

Although it is recommended, the answer is yes! You just need to minimize the airflow in your culture room to reduce contamination. Keep the sterile environment by reducing movement in the area, wear gloves, wipe everything using 70% alcohol, and if you want, keep a small affordable air filter in your room.

3. When should you adjust the pH of the tissue culture medium, before sterilization or after sterilization?

Before sterilizing, the pH of the medium should be adjusted. What I do is I first add sucrose to the distilled water as it takes longer to dissolve, followed by MS media, plant growth regulators, and Plant Preservative Mixture (PPM).

Then, calibrate the pH using NaOH (because the media is usually acidic) to increase the pH or HCL or distilled vinegar to decrease the pH.

Then, add agar and sterilize your media in the autoclave at 121 ℃, 15psi, for 20 minutes.

“Some people say there might be chances of change in pH of the media after adding agar. But, this is not the case as pH is temperature dependent. So, when you sterilize the media, the pH may go down, but as soon as it starts cooling, the pH returns to what you set before.”

4. Precautions must be taken while cultivating carnivorous plants, especially during the surface sterilization stage.

Carnivorous plants’ leaves are fragile, delicate, sensitive, and fragile. So, any harsh chemicals used on the leaves can kill the tissues. Thus, a low bleach concentration of 1-2% will be suitable for working with such plants. This gives a longer window for the sterilization of tissues and reduces the chances of contamination.

However, in the end, the protocol depends on the plant species, explant source, type of explant, and explant size.

5. Between other commercial temporary immersion bioreactors and Biocoupler(TM), which one is easier to manage and worth every penny?

First, if you talk about prices, BioCouplers(TM) will be more affordable than the traditional temporary immersion bioreactors (TIB). It’s mainly because while working with TIB, you need pumps, expensive filters, and more space for such systems. However, Biocoupler(TM) saves you a lot more space, its jar is a standard mason jar that comes in less than a dollar, and the filter is cheap and readily available online. Even if the filter is not found, it can be easily replaced with medical tapes (only as a temporary solution)”

When you talk about the easy operation, then TIB is easy to operate as once you set them up, you can just forget about them. However, in the case of Biocoupler(TM), you need manual work. You need to tilt the bottle manually at a regular time interval, which is super easy to do, but, yes, it requires your attention.

“The good news is manufacturers are working on bringing an automated Biocoupler(TM) system for such customers who want to get rid of the manual work. So, soon, you might get to see a futuristic BioCoupler(TM) system to ease your tissue culture process.”

6. Can you use PPM to surface sterilize explants? If yes, how?

Yes. You definitely can! I use 5% of PPM (prepared using 5 ml PPM and 95 ml sterile water). For 100 ml f this solution, I used 1.18 grams of MS media to add to the PPM solution. The exposure time of explants to the sterilizing solution is based on the source, thickness, and size of the explant. For example, Mexican Pinguicula plants have thin leaves, so exposing them for 4-6 hours to PPM solution works well based on their size.

7. Can you sterilize plant growth regulators (PGRs) in the microwave? Is it as effective as sterilization in an autoclave?

Yes, you can sterilize PGRs in the microwave. However, the autoclave sterilization better ensures that the media (or any other material or equipment) is wholly sterilized. An autoclave can also set a specific pressure and temperature based on the amount of media you sterilize.

Whereas, the microwave has many variables, such as the brand, size, and power of the microwave, and media amount you need to sterilize, and it becomes difficult to determine a single time or set of conditions for sterilization of media. So, the autoclave is a much better technique for tissue culture media than microwave sterilization.

8. How long can you store prepared and sterilized tissue culture medium, and at what temperature?

Though using the prepared media in the next 2-4 weeks is highly recommended, you can store it in the fridge at 4℃. However, the storage period of media also depends on what components it contains.

For example, MS media without plant hormones can be used up to 3-6 months. However, antibiotics or plant hormones should be used between 1-2 months as they start degrading.

For research purposes, it's recommended to prepare and use new media. However, when growing your plants on your own, you can use these a bit older media if stored adequately at 4℃.

9. Is it possible to make $2K - $4K using tissue culture? How can we exactly do it? What are some potential places to sell these plants?

Yes, it’s possible to make that much money selling your tissue-cultured plants. However, it depends on many factors, such as what plants you sell, their demands in your area, your selling skills, etc. For example, there’s a lot of competition for aquarium plants, and a single cup has a price between $10 - $20. But, the sellers might only give you anything between $3 - $5. So, depending on the number of plants you sell, you will make that much money.

“Once, I made $800 in 2 hours. But that could be because the seller knew me, and he trusts me and my work on plants.”

You can either sell your plants by yourself or work in partnership with nurseries. It’s entirely up to you!

10. Is tissue culture expensive for hobby growers?

Well, you need approximately $200 - 500 to get started in tissue culture with a basic setup, equipment, and the required chemicals. You don’t need fancy equipment when you start. Just stay cautious with your experiments, and only perform these in a sterile environment with sanitized hands using sterilized tools.

The two most expensive pieces of equipment in the tissue culture lab are a laminar flow hood, which comes at around $5K- $10K, and an autoclave/big pressure cooker. The small pressure cooker won’t be as effective for your process.

“There are ways, like auctions, through which you can acquire this equipment at a meager cost. For example, I got my laminar hood for $36 (an unbelievable price) in an auction.”

You can always level up your lab as you move forward and earn some money from your plants. For example, in the beginning, a small bunsen burner and alcohol will work to sterilize your forceps, but late you can upgrade it glass bead sterilizer.

11. What kind of equipment do you use in your tissue culture setup?

I use a laminar flow hood, fridge, pressure cooker, LED lights, HEPA filter, cleaning equipment, and tools to perform the culture process.

12. What’s the difference between learning tissue culture at home vs. school? Can both give similar results?

This is a great question. Yes, there will be some differences in both cases. Let’s start with the case when you must learn by yourself. This will be a slow learning process as you will be learning by yourselves from online procedures or protocols or theories or any other resource you get.

Learning from your experience helps you retain what you have learned, and you can test out any theory you like. You can experiment with whatever plant you want.

“I have started learning tissue culture on my own. I loved carnivorous plants, so I spent a lot of time sterilizing and culturing them and seeing them grow. I have learned a lot in this process.”

Though it might be expensive and frustrating initially because you will face many challenges, like contamination, improper surface sterilization, killing plant tissues, plants not working well in acclimation, no callus formation, and many more. But, when you start getting the idea of tissue culture, you will enjoy the process.

Learning tissue culture from classes will follow a traditional pathway. You will get to know all theories with a little experiment. You will get to work with plants.

The good thing is as you will be learning from an experienced instructor. The instructor will teach you the right way to experiment with tissue culture, from how to obtain explant in the right way, how surface sterilization effectively works, how you should decide which explant, and which chemicals to choose for your plants. So, the tips and tricks you learn in the process come in handy when you apply them to the experiment. But, it's of no use when you do nothing with it.

Tissue culture is all about basic theory and a lot of practice. And to start on your own, you need that foundation; if you don’t have it, it’s better to join a short-term course to start getting the hang of this idea.

When you learn from the experts, more than anything, you are learning the right way to do things. You are learning experience, so you avoid all the trial and error that they went through on your behalf. So you get it right the first time.

We have started a Tissue Culture Master Class Series at Plant Cell Technology to help you in your tissue culture journey. The next Master Class is happening on October 13-16, 2022, in Washington, DC, with Tissue Culture Expert David Critzer. I will also teach upcoming Master Classes, so stay tuned for forthcoming details!

“You must understand what's right for you based on your situation. For example, college is an excellent choice if you are interested in research. And, as I wasn’t that big a fan of research, I didn’t find college very useful. I gained much-applied knowledge in the area on my own and more short-term master classes, which turned out excellent for me!”

13. What is your favorite plant to tissue culture and why?

Because I started tissue culture with Carnivorous plants, they have been my favorite. I have worked a lot with them and still do.

However, recently I started working on many indoor plants, as you know they are currently in trend in the market.

So, among all those house plants I have worked with, Mexican Pinguicula is the one I like the most. The main reason is that it’s easy to work with and sterilize and multiply fast. Thus, you can quickly obtain hundreds of these plants in a few days.