Tissue Culture: The Basics
Tissue culture is a technique that utilizes the fragments of plants and cultures them in nutrient medias under suitable artificial environmental conditions. The idea of totipotency, or the ability of plant cells to regenerate into a whole plant, was first introduced by Haberlandt. The development of a plant in tissue culture depends on two fundamental principles of cells which include totipotency and plasticity. These two principles decide the growth of a cell in in-vitro conditions. Plasticity is the ability of plants to alter their growth, metabolism, and development depending on their best-suited environment.
In tissue culture, segments of different plant parts (called explants) are cultured. The explants that are used in tissue culture include leaf, stem, root, petiole, hypocotyl, cotyledon, embryo, or meristem. In this article, you are going to learn different types of tissue culture processes depending on the cultured explants.
A callus is an unspecialized, unorganized, and dedifferentiated mass of cells arising from explants under in vitro conditions. Scientifically, it is a loosely arranged group of parenchyma cells that originated from proliferating cells of parent tissue. Under favorable conditions, the mass of cells (callus) is organized into leaf primordia or somatic embryos.
The structure and growth habit of callus differs depending on the species being cultured. The growth of the calli depends on several factors including the age of the explant, plant part to be used as an explant, species of the plant, growth conditions, etc. After germination of callus, it is subcultured and maintained by transferring it to fresh media every 28 days. It prevents nutrient depletion and the production of toxic metabolites in cultures that eventually trigger the healthy growth of the plants.
In this type of culture, the embryo is isolated and cultured under in vitro conditions. Embryo culture can be done either by using mature or immature embryos. The mature embryos are obtained from ripe seeds and the immature embryos are obtained from the unripened or hybrid seeds that failed to grow and couldn’t produce viable plants.
The best thing about this culture technique is the absence of any surface or treatment stress to the explant being cultured. This is because the whole ovule, seed, or fruit is surface sterilized before obtaining the embryo. This protects the embryo from any damages that can occur during the process of surface sterilization.
In seed culture, the explants are obtained from the plants that are already cultured and grown under in vitro conditions. This type of culturing is best for plants that are sterile. For the optimum regeneration of the plants and to achieve higher output from your cultures, the surface sterilization of the explant or starting materials should be properly and efficiently performed. This culture method is popular to culture orchids under in vitro conditions.
A cell without a cell wall is called a protoplast. They are also known as naked cells and the term is used for the fungal, bacterial, and plant cells. Protoplasts can be isolated by mechanical and enzymatic methods. The methods used to perform the protoplast culture include:
- Hanging-drop cultures
- Micro culture chambers
- Soft agars matrix
The cultured protoplast goes through several phases that include the development of cell wall, dell division, and regeneration of the whole plant. After cell division, the cells form a callus which is then subcultured for continued growth. The application of protoplast culture include:
- Development of hybrids
- Cell cloning
- Develop genetically transformed plants
- Membrane studies
In organ culture, any part of the plant (root, stem, leaf, and flower) is used as an explant for culturing purposes. This technique is extensively used worldwide to retain or preserve the original (as it is, in its natural environment) structure and function of the particular part of the plant itself. It also helps to study the growth, differentiation, and development of the plant part.
The methods used to perform the organ culture include:
- Plasma Clot Method:This method involves the use of a clot that is composed of plasma and any extract (like chick embryo extract) in a watch glass.
- Raft Method: In this method, the explant is kept on a raft of lens paper or rayon acetate and floated on a serum in a watch glass.
- Agar Gel Method: In this method, the explant is subcultured every 5 to 7 days in the medium composed of salt solution, serum, embryo extract, vitamin, and 1% agar.
- Grid Method: In this method, explants are placed in a perforated stainless steel sheet before they are placed in a culture chamber containing a fluid medium.
Other types of Culture
Some other types of tissue culture include:
- Single-cell culture
- Suspension culture
- Anther Culture
- Pollen culture
Tissue culture has many applications including gene conservation, genetic transformation, development of hybrid plants, production of viable plants, etc. There are several techniques to perform tissue culture under artificial conditions and several different kinds of explants are used for the culturing processes. The advantage of tissue culture is enhancing its popularity among culturing of lower scale to a higher scale. However, the most important point is to understand what works best for your plant of interest. Then, the rest is easy and output will be great if every step is done carefully and aseptically!