7 Jan 2025

Callus Culture: From Undifferentiated Cells to Regenerated Plants

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

Plant tissue culture holds immense potential in horticulture and agriculture, offering transformative applications. While many recognize its benefits, fewer are aware of its variations—this field encompasses various specialized techniques for propagating plants, including the fascinating callus culture.

Micropropagation, or plant tissue culture, has gained immense popularity, not just among commercial growers but also among small-scale plant propagators. Thanks to advancements in technology and companies like Plant Cell Technology, knowledge about this incredible technique has become widely accessible, allowing more people to witness its magic firsthand.

However, there’s a common misconception: plant tissue culture is often viewed as a single technique. In reality, it’s a broad field with multiple approaches, including:

• Somatic embryogenesis
• Organogenesis
• Ovary culture
• Anther culture
• Callus culture
• Suspension culture
  …and more.

The key difference lies in the type of explants—the starting tissues or materials—used to grow plants.

In this article, we’ll delve deeper into callus culture, exploring what a callus is, the process involved, and how to determine when to use this versatile technique.

What is Callus?

Callus is an undifferentiated mass of cells—or, to put it simply, an unstructured cluster of tissue with the potential to form any plant organ since its fate hasn’t been determined yet. 

Fascinating, isn’t it?

These cells are totipotent, meaning they have the potential to develop into any type of plant tissue or organ under the right conditions. The "fate" of these cells is influenced by the plant growth regulators (like auxins and cytokinins) and environmental conditions provided during the culture process.

In scientific terms—Callus is a disorganized, proliferated mass of thin-walled parenchyma cells that actively divide. It often originates from the wound response in plants, a natural process triggered by physical damage or stress.. 

What is Callus Culture?

In vitro, callus is induced from plant explants under sterile conditions. While carrot (Daucus carota) and tobacco (Nicotiana tabacum) have historically served as model systems, callus culture is applicable to a wide range of plant species.

The undifferentiated nature of callus makes it a valuable tool for various plant biotechnology applications, including plant regeneration, genetic transformation, and secondary metabolite production.

It's important to differentiate callus from other plant tissues; it lacks the organized structure of differentiated tissues like xylem, phloem, or epidermis.

Callus Induction and Maintenance:

Callus induction and subsequent development are heavily influenced by the composition of the culture medium, particularly the balance of plant growth regulators (PGRs).

• Auxins: Primarily responsible for cell elongation and callus induction. 2,4-D (2,4-Dichlorophenoxyacetic acid) is a commonly used synthetic auxin, known for its strong callus-inducing properties. Other auxins like NAA (1-Naphthaleneacetic acid) and IAA (Indole-3-acetic acid) are also used, often in combination with cytokinins.
• Cytokinins: Primarily promote cell division and shoot differentiation. Common cytokinins include BAP (6-Benzylaminopurine), kinetin, and zeatin.

The ratio of auxins to cytokinins is crucial:

• High auxin/low cytokinin: Favors root formation.
• Low auxin/high cytokinin: Favors shoot formation.
• Balanced auxin/cytokinin: Maintains undifferentiated callus growth.

Beyond PGRs, the culture medium also contains:

• Macronutrients and Micronutrients: Essential mineral elements required for plant growth.
• Carbon Source: Usually sucrose, providing energy for cell metabolism.
• Vitamins: Organic compounds that act as cofactors for enzymes.
• Other Additives: Sometimes, other substances like activated charcoal, polyvinylpyrrolidone (PVP), or specific amino acids are added to the medium to improve callus growth or reduce browning.

Historical Background:

The concept of plant cell totipotency, the ability of a single plant cell to regenerate into a whole plant is fundamental to callus culture. Haberlandt's early experiments in the late 19th and early 20th centuries laid the groundwork, but it was the work of Gautheret, White, and Skoog in the 1930s and 1940s that truly established callus culture as a viable technique. Skoog's work on the role of auxins and cytokinins in controlling cell differentiation was particularly significant.

How Callus is Formed:

1. Explant Selection and Preparation: The choice of explant (the starting plant tissue) is critical and depends on the plant species and the objective of the culture. Considerations include:
• Source: Leaf, stem, root, petiole, embryo, or other tissues.
• Physiological State: The age and health of the donor plant.
• Pre-treatment: Surface sterilization is crucial to prevent microbial contamination. This usually involves a multi-step process using ethanol, sodium hypochlorite, and sterile rinses. Explant size also plays a role in callus induction.
2. Culture Initiation: Explants are placed on the sterile culture medium in controlled environmental conditions (temperature, light, humidity).
3. Callus Induction: Cells at the cut edges of the explant begin to divide and proliferate, forming the undifferentiated callus mass. This process is influenced by the PGRs in the medium.
4. Callus Maintenance and Subculture: To maintain callus viability and prevent nutrient depletion and accumulation of toxic metabolites, callus is regularly subcultured onto fresh medium. The subculture interval depends on the growth rate of the callus.

Types and Characteristics of Callus:

• Friable Callus: Composed of loosely aggregated cells, often used for cell suspension cultures.
• Compact Callus: Densely packed cells, often preferred for regeneration studies.
• Nodular Callus: Contains organized structures resembling meristematic regions, indicating a tendency towards differentiation.
• Color: Can vary from white to green, yellow, brown, or even reddish, depending on the plant species, medium composition, and culture conditions. Browning can be due to phenolic compound oxidation.
• Texture: Can range from soft and watery to hard and dry.

Applications of Callus Culture:

• Plant Regeneration and Micropropagation: Callus culture is used for clonal propagation of elite genotypes, especially those difficult to propagate by conventional methods. Somatic embryogenesis (formation of embryos from somatic cells) and organogenesis (formation of organs from callus) are key processes in plant regeneration.
• Secondary Metabolite Production: Callus cultures can be used to produce valuable secondary metabolites, such as pharmaceuticals, flavors, and fragrances. This offers a sustainable alternative to extraction from wild plants.
• Genetic Transformation: Callus is a target tissue for genetic engineering techniques like Agrobacterium-mediated transformation or gene gun bombardment. The transformed callus can then be regenerated into genetically modified plants.
• Germplasm Conservation: Callus cultures can be cryopreserved (frozen) for long-term storage, providing a valuable tool for conserving endangered plant species and valuable germplasm.
• Disease Elimination: Callus culture can be used to eliminate viruses and other pathogens from infected plants.

Challenges in Callus Culture:

• Contamination: Microbial contamination (bacteria, fungi, yeasts) is a major challenge. Strict aseptic techniques, including sterilization of media, equipment, and working environment, are essential. The use of antibiotics or fungicides in the medium can be considered, but it can also have negative effects on plant cells.
• Genetic Instability (Somaclonal Variation): Prolonged callus culture can lead to genetic and epigenetic changes, resulting in somaclonal variation. This can be both a disadvantage (producing off-type plants) and an advantage (creating new genetic variability). Strategies to minimize somaclonal variation include limiting subculture duration and using specific culture conditions.
• Phenolic Browning: Oxidation of phenolic compounds released from damaged plant tissues can cause browning of the medium and inhibit callus growth. Adding antioxidants like ascorbic acid or activated charcoal to the medium can help mitigate this problem.
• Optimization of Culture Conditions: Determining the optimal combination of PGRs, nutrients, and environmental conditions for callus induction and maintenance can be challenging and often requires extensive experimentation.

Callus culture remains a fundamental technique in plant biotechnology, offering a powerful tool for plant propagation, genetic improvement, and the production of valuable compounds. Continued research and technological advancements are expanding its applications and addressing its limitations.

Tissue Culture Masterclass: Achieve Your Production Goals

Every cultivation method has its pros and cons, but the right one aligns with your goals and resources. Tissue culture is quickly becoming the future of plant propagation, and now is the perfect time to explore its benefits.

Join our Tissue Culture Masterclass to learn the ins and outs of tissue culture, including protocols, sterilization, gender screening, and preservation techniques. This is your chance to level up your business, even if you're already experienced in tissue culture.

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Happy Tissue Culturing!