Organogenesis

Organogenesis

Fundamental Aspects, Techniques and Utility of

Organogenesis

Callus Cultures

If organized tissue diverted into an unorganized proliferation mass of cells they form callus tissue. Some times deep, large wounds in branches and crumps of the intact plants results in formation of soft mass of unorganized parenchymatous tissue which are rapidly form on or below the injured surface of the organ concern.

Such callus tissue is known as wound callus and is formed by division of cambial tissues. Secondly sometimes unorganized, compact, white, light outgrowth or callus like masses on stem, leaf, root and formed by stimulus of cell division in fully differentiated cell due to some diseases.

Definition: Callus tissue means an unorganized, proliferative mass of cells produced from isolated plant cell, tissues organs. When grown aseptically or artificial nutrient media under cultured conditions.

Principle- For successful initiation of callus culture 3 important criteria should be accomplished.

1. Aseptic preparation of plant material: First washed with liquid detergent with generally 5% made by Teepol) than surface sterilize by 0.1% made by volume MgCl2, 0.8% to 1.6% sodium hypochloride.

2. Selection of suitable media supplement with appropriate ratio of auxins and cytokinin.

3. Incubation of culture under controlled physical condition of light,

temperature and humidity.

 Temperature 25±2°C

 Light duration – Totally dark for 16 hrs.

 Light intensity – Cool white light 2000 to 3000 lux approximately.

 Humidity – 60%

Once the growth of the callus tissue is well established, portions of callus tissue can be removed and transferred directly on to fresh media.

Callus Formation

Formation of callus is outcome of cell division of cells of explants. During formation of callus tissue explants losses its original characteristics. 

For initiation of callus culture, tissue from young seedlings and juvenile part of a mature plant are generally taken. As the explant absorbs exogenously supplied hormones along with other nutrients, it makes at continuous nutrient gradient among the different cell of the explants on the basis of their location. 

As a result, cell divide asynchronously depended upon the availability of nutrients and hormones. Both auxins and cytokinins required for indefinite growth and cell division in callus culture. Sometimes only 2,4-D is sufficient as auxins promote growth and cytokinins promote cell division. After formation of visible unorganized mass of cells at cut end, gradually the old tissue is involved to form callus.

Caullogenesis– formation of shoots induction or proliferation

Rhizogenesis – Formation of Root..

Organogenesis – The development of adventitious organ or primodia from undifferentiated cell mass in tissue culture by the process of differentiation. When periphery cell start dividing it forms abnormal growth and it will degenerates after some time.

Some anomolous abnormal structure which are structurally similar to the organ between the dermal vascular and ground tissue present in plant tissue. But they differ from the true organ because they are obtained directly from peripheral cells of the callus and not from the organized meristemoid (patches of actively dividing cells of the callus).

Characteristics of Organogenesis

Unlimited growth of callus should be formed for proper differentiation.

Initiation of shoot buds prior to rooting between rooting potential persist for a longer time and the shooting potential is for smaller time therefore rooting is done prior than shooting it is very difficult to initiate shooting.

Once Rhizogenesis starts callus obtained is yellow in colour and it turns green when caulogenesis starts.

Organogenetic capacity is a capacity of callus to differentiate into different organ.

The use of the growth regulators – when we use it the cell may undergo some mutation (aneuploidy, polyploidy if we subculture the callus, the callus formed may undergo some change in genetic makeup and hence they may loose their organogenetic potential.

Habituated callus – It shows the undifferentiated growth this habituated callus gives organogenetic potential.

Factors Affecting Organogenesis

Following factors affects process of organogenesis -

1. Size of explants.

2. Age of the explants. - A younger leaf produces roots and older leaves produce shoots after organogenesis.

3. Seasonal explants - Explants excised during summer and winter do not produce shoot at all.

4. Oxygen gradient

Low – caulogenesis

High – Rhizogenesis

5. Colour spectrum or quality or intensity of light. -Blue colour of spectrum Red colour promotes the rooting, light duration is 16 hrs and light intensity is 2000-3000 lux and temperature is 25°C.

6. Ploidy level. - Large number of plant species including economically important medicinal plants, horticultural important plant etc.have been successfully regenerated from the callus.

 Regeneration of whole plants from explant is of special interest in mutagenic studies.

 Regeneration of whole plants from somatically mutant cell types new strains of mutant plant are obtained through organogenesis.

 New source of genetic variability is also available in plant regenerated for cell culture. This somaclonal variation is a useful source of variability.

 A chromosomal variation is associated with phenotypic variation including agriculture by use of characters such as disease resistance.

Morphology of Callus

Callus tissue proliferates as an amorphous mass of cell having no regular shape.

All calluses derived from different plants look alike externally but can be distinguish on basis of internal structure.

Internal Structure of Callus

Cellular composition of callus tissue is extremely heterogenous ranging from cell with dense cytoplasm, plant cells with vacuolated cytoplasm, shape of cells very strong spherical to elongated plant elongated cells are usually non dividing having large central vacuole while small cells are actually dividing cells which have reduced vacuole size and dense cytoplasm, formation of xylem and phloem with in callus which is known as cytodifferentiation.

Soft callus is friable in nature and is made of heterogeneous mass of cells having minimal content. Hard callus consist of giant cell which closely packed that is compact in nature.

Colouration

Generally creamish yellow in colour.Sometimes callus tissue may be pigmented; pigmentation may be uniform or patchy – some time Callus Tissue grows in dark and turn green after transferring in light condition due to development of chloroplast.

Yellow - Carotinoids

Purple - Xanthocyanin

Brown - Phenolic

Habituation

Sometimes, after repeated subculturing the callus tissue gains ability to grow on a std. basal medium which is devoid of growth hormones. This property is called as habituation and callus tissue is known as or habituated callus tissue.

Cells in habituated callus tissue appear to have developed a capacity to synthesize adequate amount of auxins and or cytokinins. These can't be distinguish from normal callus accept in their hormonal requirement.

Chromosomal Variation in Callus Tissues

1. Genetic basis of variation in callus tissue

Endoreduplication is of frequent occurrence in differentiated tissue of

higher plants and such cells remain in mitotic state. Therefore callus tissue may get such genomic heterogeneity possibly due to non selective induce of asynchronous division of both diploid and endoreduplicated cells. Variation of chromosomes no ranges from aneuploidy to different level of polyploidy.

2. Epigenetic basis

Highly meristematic cells are expected to be diploid but callus derived from meristem also shows variation in chromosome no. it is also found that prolong sub culture may read to establishment of one karyotype and other are gradually eliminated. Sometimes structure changes of chromosome like deletion, translocation etc. may occur in culture. Ideal callus culture is characterized by the passion of numerical or structural stability in long term culture. But it is very rare that cells of callus tissue may be haploid if it is derived from microspore culture.

Significance of Callus

1. Whole plant can be regenerate in large number from callus tissue which manipulation of the nutrient and hormonal consequence in culture medium.

This phenomenon is known as plant regeneration, organogenesis or

morphogenesis.

2. Callus tissue is good source of genetic or karyotyping variability, so regenerate the plant from genetically variable cells of callus tissue

3. Cell suspension culture is moving liquid medium and can be initiated from callus culture.

We use only tissue culture technique and it is part of the clonal propagation and in clonal we use conventional and tissue culture also.

Clonal propagation through tissue culture popularly known as micropropagation and can be achieved in a short time and space. Thus, it is possible to produce plants in large number starting from a single individual – use of tissue culture for micropropagation was inflated by G. Morel (1960). Products of this rapid vegetative propagation can be regarded as done only when it is established that the cell, they comprise are genetically identical.