15th Jun 2021
TOTIPOTENCY: THE BASICS
Totipotency is one of the magical features of plant cells. It’s an inherited characteristic in a plant cell to develop into a whole plant. The discovery of plant growth hormones and MS media revolutionized the research and development areas of plant research. Tissue culture has been identified as one of the most crucial techniques in labs.
Today, the plant tissue culture technology branches into several other culture techniques including organ culture, callus culture, meristem culture, cell-suspension culture, etc. All these techniques have their own advantages and applications depending on the purpose of culturing.
This article briefs about the callus and organ culture and the five main factors that decide the regeneration of tissues or organs into a whole plant.
What is Callus and Organ Culture?
Have you ever observed tumor-cell outgrowth at wounded places in plants? That’s exactly how calluses look when a few tissues of plants are placed in the culture media. Even after some similarities, the natural wound callus and in vitro callus differ from each other in morphology, growth, and cell metabolism. A callus is defined as an unspecialized, unorganized, dividing the mass of cells.
Callus culture was first introduced in the late 1920s. As a result, Callus culture's became a crucial part of almost every biochemical, physiological, and genetic experiment. Callus culture is an excellent way to achieve genetic variability in plants. Some other different significances of callus culture include initiation of cell suspension culture, in vitro development of somatic embryos, in vitro organ development, and obtaining secondary metabolites for commercial use.
As the name suggests, organ culture is in vitro generation of plants using plant organs such as the leaf, node, internode, shoot, root, axillary bud, and seedling.
If you aim to avoid somaclonal variation in your experiments, organ culture is the preferable technique. Some other applications of the technique include the production of secondary metabolites; the study of the pattern of growth, differentiation, and development of organisms; and biochemical and molecular functions of an organ or tissue.
The best advantage of organ culture is the maintenance of organ structure and function even after culturing. It preserves the originality of the plant and shows the same characteristics as it would show in vivo.
The two main types of organ cultures include: Root culture and Shoot culture.
1. ROOT CULTURE
Hairy root or root culture involves the infection of explant by gram-negative soil bacterium Agrobacterium rhizogenes. The hairy root is induced by integrating root-inducing (Ri) plasmid into the plant’s genome followed by infection with Agrobacterium rhizogenes.
After 2-3 days, explants are transferred to a solid medium containing antibiotics such as Cefatoxime or Ampicillin to kill redundant bacteria. The induced hairy root will be observed between one week to over a month depending on the plant species.
Hairy roots are genetically and biosynthetically stable and are perfect means to produce secondary metabolites at the commercial level. The hairy root cultures do not require any plant hormones and conditioning of the medium.
Apart from the production of secondary metabolites, hairy root cultures are used to enhance the concentration of secondary compounds, produce those compounds that are not found in non-transformed roots or regenerate whole plants.
Image: An illustration of hairy root induction steps and their application in the regeneration of the whole transformed plant.
Source: Hairy Root Culture for In Vitro Production of Secondary Metabolites: A Promising Biotechnological Approach. Biotechnological Approaches for Medicinal and Aromatic Plants.
2. SHOOT CULTURE
Shoot culture or shoot tip culture is the culture of the terminal portion of shoots with primordia, developing shoot, and adjacent stem tissue on B5 or MS media. The most popular application of shoot culture is virus elimination in plants. The shoot tip is supposed to be virus-free because of the absence of cell differentiation and vascular system, and intense metabolic activity.
Some other applications of shoot tip culture include international transportation of plants and storage of genetic material of plants producing recalcitrant seeds.
Related: Meristem and Shoot tip culture
Image: An illustration of shoot tip culture.
FACTORS AFFECTING CALLUS AND ORGAN CULTURE
1. SOURCE OF MATERIAL
The source of the material should be chosen properly. It’s better if the plants are disease-free. Successful in vitro culture has been observed by using vascular cambia, storage organs, pericycle of roots, endosperm, cotyledons, leaf mesophyll, and provascular tissue. Also, the choice of explant depends on the purpose of culturing.
2. STERILIZATION OF SOURCE MATERIAL
The surface sterilization of the explant is an essential step. The explant collected from the outside source may be infested with spores or other microbial cells. And, if explants are not sterilized properly before culturing it will cause contamination in all your cultures and lead to heavy culture loss. Also, if there are any damaged or dead tissues, remove them completely before culturing the explant on the culture media.
3. EXPLANT PREPARATION
Size and shape of the explant is a critical factor in callus and organ culture. If the size of the explant is not optimum, it may lead to failure in callus induction and culture loss as well. Culturists majorly prefer the use of large sizes of explant to obtain viable cultures.
4. CULTURE MEDIUM
The type and composition of the culture medium are some of the essential factors affecting callus and organ culture. The cultures are more affected by the level of plant hormones—both endogenous and exogenous—in the medium. The callus or organ cultures are generally grown on solid media but sometimes liquid media is also preferred. They sometimes use of liquid media refers to the condition when a plant is unable to grow in the solid media. It can be because of uneven distribution of nutrients, improper exchange of gases, and accumulation of toxic waste products that may develop between the callus and the medium.
The culture should be transferred to fresh media after some time. Keeping cultures on one media for a longer time leads to waste buildup, exhaustion of nutrients from the media, and media dryness. So, it's recommended that cultures should be subcultured every 4-6 weeks when incubated at 25°C.
- Torres, K. C. (1988). Tissue Culture Techniques for Horticultural Crops. doi:10.1007/978-1-4615-9756-8
- Gulzar, B., Mujib, A., Qadir Malik, M., Mamgain, J., Syeed, R., & Zafar, N. (2020). Plant tissue culture: agriculture and industrial applications. Transgenic Technology-Based Value Addition in Plant Biotechnology, 25–49. doi:10.1016/b978-0-12-818632-9.00002-2
- Singh, R. S., Chattopadhyay, T., Thakur, D., Kumar, N., Kumar, T., & Singh, P. K. (2018). Hairy Root Culture for In Vitro Production of Secondary Metabolites: A Promising Biotechnological Approach. Biotechnological Approaches for Medicinal and Aromatic Plants, 235–250. doi:10.1007/978-981-13-0535-1_10
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