This document discusses plant tissue culture techniques. It defines plant tissue culture as the maintenance or growth of plant cells, tissues, or organs under sterile conditions on a nutrient medium. The key steps are explained as preparation of a sterile medium, selection and sterilization of an explant, inoculation, incubation, and regeneration. Different types of culture are described, including callus culture and the use of tissue culture in micropropagation, genetic modification, and production of secondary metabolites. Growth regulators like auxins, cytokinins, and gibberellins are also discussed.

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

9. Type of plant tissue culture:
1) 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.
2) 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.

10. 3) 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.
4) Seed culture:
seed may be culture in vitro to generate seedling or plants. It is the best method for
raising the sterile seedling.

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

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

13. 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.

14. • 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

15. 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.

16. 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

17. 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.