30 Aug 2022

5 Reasons You May Be Failing at Tissue Culture

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

Introduction to Tissue Culture

Tissue culture is a collection of techniques used to produce new plants from a small piece of plant tissue or a single cell. It’s performed in an artificially controlled environment under complete aseptic or sterilized conditions.

The piece of the plant introduced in cultures is known as explant (you might also hear other culturists calling it the “plant material”). The medium in which the explants are grown is known as culture media, growth media, or nutrient media.

While introducing a plant into tissue culture, you collect the explant from the mother plant or your main source of the plant material, surface sterilize it, followed by introducing it to the sterilized tissue culture medium. After this, the cultured plants are incubated in a suitable environment (with optimum temperature, light intensity, and humidity) to allow them to grow and develop.

As a whole, the tissue culture process is divided into 4 stages:

Establishment Stage: Establish the plant in tissue culture.
Multiplication Stage and Shooting: Divide the obtained tissues from the plant and sub-culture them in a medium containing cytokinin or shoot hormone.
Rooting Stage: Transfer plants to the media containing auxin (rooting hormone).
Acclimatization Stage: Transfer plants to a peat moss mixture and keep them in the greenhouse before transferring to the outside environment.

Tissue culture offers several advantages over the conventional approach:

Grow plants on a large scale in a short amount of time.
Produce diseases free plants.
Produce thousands of plants from just a small piece of plant tissue.
Produce plants throughout the year, irrespective of seasons
Require only a small space to keep your plants
Introduce new varieties into the plants.
Develop transgenic plants.
Conserve endangered plant species.
And, much more!

However, the only limitation of the technique is that it’s a bit more expensive than the traditional plant propagation techniques.

Thus, it’s recommended to try the procedure only when you growing plants for commercial purposes (on a large scale), want to achieve the above-mentioned benefits in your plant propagation system, or have sufficient cash on hand to try this at your home as a hobby.

However, you might extend your cash runway and reduce the overall cost of tissue culture by avoiding some mistakes—which especially some beginner culturists commonly ignore or do not know about.

What are they?

Find out in this article.

Moreover, also learn the factors that can affect the productivity of your tissue cultured plants in addition to five reasons why you aren’t getting any success with them.

3 Factors Affecting The Tissue Culture Plants

The performance of plants introduced in tissue culture is based on several factors:

1. Explant Source and Type

An explant is the starting material you use to grow plants and further multiply them on large scale. Thus, it’s essential to be careful while choosing the plant material for your cultures. The factors that affect an explant’s response in tissue culture include:

Physiological stage of the donor plant
Size of the explant
Age of the explant source
Explant position in donor plants
Explant density
The genotype of the donor plant

An explant can either be a stem, root, leaf, embryo, or flower of plants. Also, based on the explant you choose to carry out your tissue culture experiment, further techniques like sterilization and culture procedure change accordingly.

2. Surface Sterilization Technique

Explant health is an essential factor that influences how plants respond to tissue culture. Further, the viability of the explant is necessary to obtain high-frequency shoot regeneration.

The tissue culture conditions provide a favorable environment for microbes to grow and thrive. And, the first treatment provided to plants for their optimal response in tissue culture is surface sterilization of explants.

Thus, all sterilization processes need to be performed efficiently to maintain the complete aseptic condition and allow plants to progress into tissue culture.

Additionally, ensure the chemicals you use and their exposure time to your cultures are optimum and not too much to kill plant tissues.

3. Culture media

This is most important as culture media are the source of nutrients and elements required for the growth and development of plants.

A plant tissue culture medium is composed of macronutrients, micronutrients, vitamins, amino acids, salts, and other organic compounds supporting plant development.

A variety of media are available and used in tissue culture based on the plant species needed to be grown. It includes: MS (Murasheige and Skoog Media), Nitsch and Nitsch (NN) media, B5 medium, N6 medium, and Driver/Kuniyuki walnut medium (DKW).

All these mediums differ in their composition and have different effects on plants. Thus, before moving to your large-scale experiments, determine which medium helps you to obtain more shoot regeneration percentage and shoot number per explant.

4 Environmental Factors

The environmental factors affecting tissue culture plants include:

Light: Three characteristics of light influence plant development in tissue culture: wavelength, flux density, and photoperiod. Cultured plants are primarily affected by these factors when it comes to stem elongation, leaf size, and plant anatomy. During micropropagation, white fluorescent light is the primary source of light.
Temperature: It affects many physiological processes of plants, such as respiration and photosynthesis. Thus, an optimum temperature is necessary for the growth of plants.
Humidity: The typical relative humidity inside a tightly-sealed culture vessel ranges from 95% - 100%. It has a role in the germination of in vitro somatic embryos.
Gas exchange: Proper gas exchange between the cultured plants prevents ethylene accumulation, high humidity, and CO2 depletion that negatively affect the plants.

5 Possible Reasons For Your Failure in Tissue Culture

The main challenges faced by culturists in tissue culture are the contamination issue, media browning, vitrification, reduced yield, and plant death during acclimation. Here are five possible reasons for these problems that explain why your plants are dying in tissue culture.

Improper Plant Selection

The correct choice of explant is necessary when it comes to tissue culture. The factors that need to be considered while collecting explant for your tissue culture procedure are the health of the plant, its size, age, position, and source.

If you aren’t getting better or see contamination at a very early stage of your tissue culture, chances are you have completely ignored these factors.
Using chemicals improperly or using poor-quality chemicals

DO NOT use low-grade chemicals for your tissue culture procedure. They might contain some impurities that can affect the growth of your plant and might cause contamination as well.

Also, even if you are using high-grade chemicals ensure they are good for your explant. You must know that the chemicals you use at the surface sterilization stage are at an optimum concentration based on the size and thickness of your explants. Furthermore, the exposure time to chemicals is another factor that should be considered.

For example, while surface sterilization of Darlingtonia californicaexplants, the small tissues are only exposed for 3-5 minutes to H2O2 solution, whereas the large and thick tissues are exposed for 5-7 minutes.

If you see no response in your tissue culture plants, chances are you’ve killed or damaged the plant tissues using too much of a surfactant or other chemical or exposed the tissue for much longer than it was optimally required.
Plant hormones Imbalance

Not all plant hormones are suitable to grow all plant species. Thus, you need to make sure which plant hormone is inducing a better growth response in your plants and at what concentration.

If ignores, the imbalance of the plant hormones in your culture media can affect the shoot induction, development, cell division, root development, elongation, and proliferation of the cultured explant.

Some common auxins used in tissue culture include: indole-3-acetic acid, naphthalene acetic acid, 2, 4, indole-3-butyric acid, 5-trichlorophenoxyacetic acid, 2, 4 dichlorophenoxyacetic acid, and naphthoxyacetic acid.

Some common cytokinin used in tissue culture: amino purine, isopentenyl adenine, 4-hydroxyl-3-methyl-trans-2-butenylaminopurine, and kinetin.
Avoiding Tissue Culture Contamination Prevention Best Practices

Contamination is one of the major challenges in tissue culture. So, if you are getting frequent contamination in your cultures, chances are either your lab is contaminated, you are using contaminated explant, or you are not following best practices to prevent contamination.

To prevent this, deep clean your lab by following the sterilization procedures, following tissue culture best practices like wearing a PPE kit and sterilizing the workbench and transfer area, and keeping your workspace organized and uncrowded.

Also, though the use of the Plant preservative Mixture (PPMTM) is optional, however, adding the chemical to your culture media helps you to prevent all kinds of contamination, whether it's airborne, waterborne, or comes from your unclean hands.
Making Mistakes at Acclimatization Stage

According to the NIH, acclimatization is the capacity for plants to adapt to environmental changes, and is an essential and final stage of tissue culture. And, even if you have managed to maintain your plants at their best, any mistake at this stage can spoil all your effort and money you have put into the process.

And, if your plants are dying at this stage or getting thin and unhealthy, chances are you have not been careful while transferring your plants to the greenhouse. Or, you have given a sudden shock to the plants by exposing them instantly to the outside environment after taking them out of the lab.

Remember your tissue culture plants are clumsy. They have grown in a controlled environment. Thus, they are more prone to attack by pathogens at this stage.

So, if you aren’t following the best practices of acclimating plants, you can’t blame the process, plant, or chemicals you are using for obtaining a decreased yield, the death of plants, or observing more unhealthy plants.

How Can Plant Cell Technology Help You Tackle Your Tissue Culture Challenges?

Now, you might have several questions regarding the mentioned challenges:

How to know which explant is perfect and how to choose them? What characteristic should you observe?
How can one differentiate between low-grade and high-grade chemicals?
How to know which surface sterilization technique is suitable for your plant? And, how to learn about their effect on your chosen explant?
How to know among all the available hormones, which will best suit your plant and in what concentration?
How should you transition your plants from the lab to the greenhouse and acclimate them? What practices or steps to follow?

You can get answers to all these questions and more using our one-on-one phone consultation service.

The service allows you to live talk to a tissue culture exert and ask as many questions as you have related to your tissue culture process.

Is this something you're interested in?

Book your appointment now: PCT Consultation Services.

Furthermore, you can also choose a physical-visit consultation if you need help with establishing your lab or training your staff in tissue culture.

And, if you have any tissue culture requirements for your lab, you can always visit the PCT Store and shop for products ranging from MS media, agar, gellan gum, Plant Preservative Mixture (PPM), culture vessels, Biocoupler (TM), and masks that best suit your tissue culture experiments.