Lecture 8: Callus Culture In Plant Tissue Culture PDF

Lecture 8 a Callus Culture in Plant Tissue Culture Prepared and presented by : Dr. Abdelaziz Mohamed Nasr Lesson Objectives Understand the concept and significance of callus culture. Learn the process of callus induction and organ formation....

Lecture 8 a Callus Culture in Plant Tissue Culture Prepared and presented by : Dr. Abdelaziz Mohamed Nasr Lesson Objectives Understand the concept and significance of callus culture. Learn the process of callus induction and organ formation. Discuss the applications and challenges of callus culture. Callus culture Callus is a coherent and amorphous tissue, formed when plant cells multiply in a disorganized way. It is often induced in or upon parts of an intact plant by wounding, by the presence of insects or microorganisms, or as a result of stress. Callus can be initiated in vitro by placing small pieces of the whole plant (explants) onto a growth-supporting medium under sterile conditions. During this process, cell differentiation and specialization, which may have been occurring in the intact plant, are reversed, and the explant gives rise to new tissue, which is composed of meristematic and unspecialized cell types. Pale yellow (p-y) callus developed from cambium of root discs, Dark orange (d-o) callus line established from p-y callus by visual selection and subsequent subculture. Callus culture During dedifferentiation, storage products typically found in resting cells tend to disappear. Although most experiments have been conducted with the tissues of higher plants, callus cultures can be established from gymnosperms, ferns, and mosses. Callus cultures are more easily established from some organs than others. Young meristematic tissues are most suitable, but meristematic areas in older parts of a plant, such as the cambium, can give rise to callus. Callus culture The choice of tissues from which cultures can be started is greatest in dicotyledonous species. In most cereals, for example, callus growth can only be obtained from organs such as zygotic embryos, germinating seeds, seed endosperm or the seedling mesocotyl, and very young leaves, but so far never from mature leaf tissue. The callus formed on an original explant is called ‘primary callus’. Secondary callus cultures are initiated from pieces of tissue dissected from primary callus. Callus culture is often performed in the dark as the light can encourage differentiation of the callus. Monocot seedling Callus culture Callus tissue is not of one single kind. Strains of callus differing in appearance, color, degree of compaction and morphogenetic potential commonly arise from a single explant. Sometimes the type of callus obtained, its degree of cellular differentiation and its capacity to regenerate new plants, depend upon the origin and age of the tissue chosen as an explant. Variability is more likely when callus is derived from an explant composed of more than one kind of cell. Types of callus Compact callus Shows as large, densely aggregated cells. Friable callus Shows as loosely associated cells, and the callus becomes soft and breaks apart easily Mostly used to start cell suspension cultures. Stages of callus culture Induction phase Division phase Differentiation phase Induction Cells in explant dedifferentiate and begin to divide. Division phase Rapid cell division. In case of long-term culture, the requirements of auxin and/or cytokinin are reduced, which is called habituation. Differentiation phase Differentiation and formation of organized structure. Organogenesis Somatic embryogenesis Notes on callus culture Explants grown in agar medium with appropriate nutrients with suitable proportion of auxin and cytokinin exhibit callusing at cut ends, which gradually extends to the entire surface of the tissue. Callus cultures need to be subcultured every 3 – 5 weeks. Repeated subculture on an agar medium improves the friability of the callus. Notes on callus culture Subculture is the transfer of cultures with or without dilution from one culture vessel to another containing fresh culture medium. It is also known as passage. The callus tissue shows an eventual loss of organogenic response as subculture proceeds. Callus cultures may show some changes of chromosomal structure or number such as aneuploidy, polyploidy. Current applications Manipulation of the auxin to cytokinin ratio in the medium can lead to the development of shoots, roots or somatic embryos from which whole plants can subsequently be produced. Callus cultures can also be used to initiate cell suspensions. To study nutrition requirement of plants. Current application To study cell and organ differentiation and morphogenesis. Somaclonal variations and its exploitation. Genetic transformation The production of secondary metabolites. What is the take-home message from today’s lecture. Let’s discuss. Any more questions?