INITIATION AND MAINTENANCE OF CALLUS INTRODUCTION 1 A

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INITIATION AND MAINTENANCE OF CALLUS

INITIATION AND MAINTENANCE OF CALLUS

INTRODUCTION 1. A callus consists of an amorphous mass of loosely arranged thin walled

INTRODUCTION 1. A callus consists of an amorphous mass of loosely arranged thin walled parenchyma cells arising from the proliferating cells of the cultured explants. 2. Frequently, as a result of wounding, a callus is formed at the cut end of a stem or root. 3. The term “callus” should not be confused with “callose”, another botanical term. The latter refers to a polysaccharide associated primarily with sieve elements. 4. The stimuli involved in the initiation of wound callus are the endogenous hormones auxin and cytokinin.

5. In addition to mechanical injury, callus may be produced in plant tissues following

5. In addition to mechanical injury, callus may be produced in plant tissues following an invasion by certain microorganisms or by insect feeding. 6. Using tissue culture techniques, callus formation can be induced in numerous plant tissues and organs that do not usually develop callus in response to an injury. 7. Plant material typically cultured includes vascular cambia, storage parenchyma, pericycle of roots, cotyledons, leaf mesophyll, and provascular tissue. In fact, all multicellular plants are potential sources of explants for callus initiation.

FIRST SUCCESSFUL CULTURE 1. In 1939 the first successful prolonged cultures of experimentally induced

FIRST SUCCESSFUL CULTURE 1. In 1939 the first successful prolonged cultures of experimentally induced callus were achieved almost simultaneously at the research laboratories of Gautheret in Paris, Nobecourt in Grenoble, and White in Princeton. 2. These cultures were originally derived from explants of cambial tissue of carrot and tobacco. 3. The term “tissue culture”, as applied to such cultures, is a misnomer: A cultured tissue does not maintain its unique characteristics as a plant tissue, but reverts to a disorganized callus. 4. The most important characteristics of callus, from a functional viewpoint, is that it has the potential to develop normal roots, shoots, and embryoids that can form plants and, in addition, can be used to initiate a suspension culture.

ESTABLISHMENT OF A CALLUS 1. Establishment of a callus from an explant can be

ESTABLISHMENT OF A CALLUS 1. Establishment of a callus from an explant can be divided roughly into 3 developmental stages: A. Induction B. Cell division C. Differentiation 2. During the initial induction phase metabolism is stimulated prior to mitotic activity. The length of this phase depends on the physiological status of the explants cells as well as the cultural conditions. 3. Afterward, there is a phase of active cell division as the explants cells revert to a meristematic state. 4. The third phase involves the appearance of cellular differentiation and the expression of certain metabolic pathways that lead to the formation of secondary products.

GROWTH CHARACTERISTICS OF A CALLUS 1. Growth of a callus involve a complex relationship

GROWTH CHARACTERISTICS OF A CALLUS 1. Growth of a callus involve a complex relationship among A. The plant material used to initiate the callus B. The compostion of the medium C. The environmental conditions during the incubation period 2. Some callus growths are heavily lignified and hard in texture, whereas others break easily into small fragments. 3. The fragile growths that crumble readily are termed “friable cultures”.

PIGMENTATION 1. Callus may appear yellowish, white, green, or pigmented with anthocyanin. 2. Pigmentation

PIGMENTATION 1. Callus may appear yellowish, white, green, or pigmented with anthocyanin. 2. Pigmentation may be uniform throughout the callus or some regions may remain un pigmented. 3. Anthocyanin synthesizing and – nonsynthesizing cell lines have been isolated from carrot cultures and a stable pigment producing strain of cultured Euphorbia sp. cells was isolated after 24 clonal selections and subcultures.

ANATOMY OF CALLUS CULTURES 1. A homogenous callus consisting entirely of parenchyma cells is

ANATOMY OF CALLUS CULTURES 1. A homogenous callus consisting entirely of parenchyma cells is rarely found. 2. Cytodifferentiation (The morphological development of undifferentiated cells into more specialised ones) occurs in the form of tracheary elements, sieve elements, suberized cells, secretory cells, and trichomes. 3. Small nests of dividing cells form vascular nodules (meristemoids “A small, triangular stomatal precursor cell that functions temporarily as a stem cell in a meristem”) that may become centers for the formation of shoot apices, root primordial, or incipient embryos.

4. Vascular nodules typically consist of discrete zones of xylem and phloem separated by

4. Vascular nodules typically consist of discrete zones of xylem and phloem separated by a cambium. 5. The orientation of the xylem and phloem with respect to the cambial zone is influenced by the nature of the original tissue. 6. The location of the nodules within the callus can be modified by altering the composition of the medium. 7. Vascular differentiation may also take the form of somewhat randomly arranged strands of tracheary elements.

HORMONAL REQUIREMENTS 1. The hormonal requirements for the initiation of callus depend on the

HORMONAL REQUIREMENTS 1. The hormonal requirements for the initiation of callus depend on the origin of the explants tissue. 2. Juice vesicle from lemon fruits, and explants containing cambial cells, exhibit callus growth without the addition of any exogenous growth regulators. 3. Most excised tissues, however, require the addition of one or more growth regulators in order to initiate callus formation. 4. Explants can be classified according to their exogenous requirements, in the following manner: A. Auxin B. Cytokinin C. Auxin and Cytokinin D. Complex natural extracts.

SUBCULTURING OF THE CALLUS 1. After the callus has been grown for a while

SUBCULTURING OF THE CALLUS 1. After the callus has been grown for a while in association with the original tissue, it becomes necessary to subculture the callus to a fresh medium. 2. Growth on the same medium for an extended period will lead to a depletion of essential nutrients and to a gradual desiccation of the gelling agent. 3. Metabolites secreted by the growing callus may accumulate to toxic levels in the medium. 4. The transferred fragment of callus must be of a sufficient size to ensure renewed growth on the fresh medium. 5. If the transferred inoculum is too small, it may exhibit a very slow rate of growth or none at all. 6. Street (1969) recommended that the inoculum be 5 -10 mm in diameter and weigh 20 -100 mg.

7. Successive subcultures are usually performed every four to six weeks with cultures maintained

7. Successive subcultures are usually performed every four to six weeks with cultures maintained on an agar medium at 25 o. C or above. 8. Passage time, however, is somewhat variable and depends on the rate of growth of the callus. 9. A friable callus can be subdivided with a thin spatula or scalpel, transferred directly to the surface of a sterile Petri dish, and sliced into fragments with a scalpel. 10. Only healthy tissue should be transferred, and brown or necrotic tissue must be discarded. 11. Interest has been shown in developing alternative methods for long term maintenance of tissue cultures, for example, freeze preservation.

WHAT ARE SOME OF THE BEST PLANT MATERIALS FOR THE INITIATION OF A CALLUS

WHAT ARE SOME OF THE BEST PLANT MATERIALS FOR THE INITIATION OF A CALLUS CULTURE? 1. Young healthy tissue that are rich in nutrients, and possibly endogenous hormones, are the best choices for the induction of cell division; for example, storage organs and cotyledons of seeds. 2. These include tissues from potato tuber (Solanum tuberosum), storage roots of turnip (Brassica rapa), sweet potato (Ipomea batatas), and carrot (Daucus carota). 3. Callus is easily started from the cotyledons of soyabean (Glycine max). 4. Stem pith parenchyma from lettuce (Latuca sativa) and tobacco (Nicotiana tabacum) readily divides in the presence of auxin and cytokinin.