tissue culture

in-vitri culture of medicinal plant, types of media

1. IN- VITRO CULTURE OF MEDICINAL PLANT NAME: HEMA CMAP: 301 M.SC : 3 RD SEM

2. INTRODUCTION: Tissue culture is a special type of laboratory mediated propagation where a very small piece of tissue (shoot apex or even individual cell) is placed under sterile condition culture in a test tube. It was 1 st done in 1885 by Wilhelm roux, he extracted small part of medullary plate embryo of chicken.

3. DEFINITION OF PLANT TISSUE CULTURE ( PTC) : PTC is a collection of technique used to maintain or grow plant cells, tissue or organ, under sterile condition on a nutrient culture medium of known composition. In this process, explants from healthy and young part of the plant is used to regenerate the whole plant.

4. HISTORY OF PLANT TISSUE CULTURE 1902- the idea of totipotence of plant cell was given by Haberlandt. 1937 - White first time established successful root culture of tomato. 1941- J.Van overbeck used coconut milk for growth and development of young Datura embryos 1957- Skoog and miller demonstrated the role of auxins and cytokinin on root and shoot formation in tobacco -tissue. 1962- Murashige and Skoog introduced the medium for tobacco culture. 1987- isolation of Bt. Gene from bacterium Bacillus thuringiensis.

5. IMPORTANT FEATURES OF IN-VITRO TECHNIQUES : The In- vitro works are carried out in a test tube, dish or flask i.e. outside the living organism. Hence in vitro techniques are also referred to as plant tissue culture techniques. Tissue culture work is confined to plant biology because plants are totipotent. The tissues culture are grown in suitable nutrient medium like MS agar media, Gamborg media etc. Tissues grow from the parent organism i.e, explant from which tissues have been taken.

6. CONCEPT OF PLANT TISSUE CULTRE : The basic concept of plant tissue culture is: Totipotency: It is the ability of plant cell to regenerate into a whole plant Differentiation: It is the process by which meristematic cells undergo changes in structure, shape, to perform the specific function. Dedifferentiation: Process in which living differentiated cell regain their capacity to divide. Redifferentiation: Process in which differentiated cell that have lost their ability to divide are reformed from dedifferentiated cells and have the ability to perform specific functions.

7. General steps of plant tissue culture: Preparation of suitable nutrient medium Selection of explant Sterilization of explant Inoculation of explant in culture medium Incubation of culture Transfer of callus and regeneration Hardening Transfer of plantlet to the field

8. Type of plant tissue culture: Cell culture: Cell culture is the culture of isolated individual cell, obtained from an explant tissue or callus, these culture are carried out in dispersion medium and are referred to as cell suspension culture . Protoplast culture: The cell in which the cell wall has been removed. In this isolated protoplasts are culture on a suitable medium under the aseptic condition. Aimed to develop genetically transformed plant, and somatic hybridization of two plant species through protoplast fusion in done by protoplast culture.

9. Callus culture: Callus is the unorganized and undifferentiated dividing mass of cells derived from an explant(stem, root, cotyledon, leaf) in vitro on a suitable medium . Seed culture: seed may be culture in vitro to generate seedling or plants. It is the best method for raising the sterile seedling .

10. Embryo culture: Excision of embryos regardless of age, size and developmental stage from their natural environment and growing them under artificial environmental condition. Anther/Pollen culture: Produced haploid plant from the pollen grains under the suitable medium. This technique was 1 st used in India to produced haploid of Datura.

11. Meristem culture: The apical meristem of shoots of is cultured to get the disease-free plants even if the plant is infected .

12. Plant tissue culture techniques : Micro- propagation: This techniques is used for developing high quality clonal plants. this has the potential to provide rapid and large scale propagation of new genotypes. Somatic cell genetics: It is used mostly in terms of haploid production and somatic hybridization. Transgenic plants: It is used for expression of mammalian genes or plant genes for various species. It is helpful to give resistance against insects, virus and other pathogens as well as the herbicide.

13. Sterilization method use in tissue culture laboratory : The material e.g.- vessels, instrument, medium, plant material etc., culture work must be freed from microbes. Dry heat treatment Autoclaving Flame sterilization Filter sterilization Wiping with 70% ethanol Surface sterilization

14. Application of plant tissue culture : A large number of plant having identical features to the parents can be produced by this method. To conserve rare or endangered plant species. For the production of highly valuable transgenic plant. Many plantlets can be produced without seed. Germplasm conservation. Healthy and disease free plants can be propagated by this technique. Biotransformation. Production of somatic hybrid. Rapid multiplication of rare and elite genotypes such as Aromatic and Medicinal plants. Immobilization of plant cell. Production of secondary metabolites. e.g. Caffine from Coffee.

15. Production of secondary metabolite: Cultured plant cells often produce reduced quantities and different profiles of secondary metabolites when compared with the intact plant and these quantitative and qualitative features may change with time. This method of production called as phytoproduction. This extraction of metabolites can be possible from both callus and suspension culture. Plant species Compound Culture type Catharanthus roseus Ajmalicine Suspension Digitalis Cardinolides Suspension/callus Coffee Arabica Caffine Callus

16. Role of growth regulators in tissue culture: Auxins (Indole 3-acetic acid) = Auxins containing medium which help in shoot development Cytokinins ( Benzyl aminopurine ) = promote cell division- in embryos/germinating seed portion(root development) Gibberelins (GA3) = Used in regulating plant cell elongation.

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