plant tissue culture technique used to produce plant in vitro condition

1. Praveen Garg
VITS College, Satna
Plant Tissue Culture

2. INTRODUCTION
• Tissue culture involves the use of small pieces of plant tissue (explants) which
are cultured in a nutrient medium under sterile conditions.
• Using the appropriate growing conditions for each explant type, plants can be
induced to rapidly produce new shoot and new roots with the addition of suitable
hormones.
• Plant tissue culture is a collection of techniques used to maintain or grow plant
cells, tissues or organs under sterile conditions on a nutrient culture medium of
known composition.
• It is widely used to produce clones of a plant in a method known
as micropropagation.
• Many plant cells have the ability to regenerate a whole plant (totipotency).
• Single cells, plant cells without cell walls (protoplasts), pieces of leaves, stems or
roots can often be used to generate a new plant on culture media given the
required nutrients and plant hormones.

3. Medium for Tissue Culture
• Solid and Liquid sterile medium used for tissue culture techniques.
• Solid and liquid media are generally composed of inorganic salts plus a few
organic nutrients, vitamins and plant hormones.
• Solid media are prepared from liquid media with the addition of a gelling agent,
usually purified agar.
• The plant hormones and the nitrogen source (nitrate versus ammonium salts or
amino acids) have profound effects on the morphology of the tissues that grow
from the initial explant.
• For example, an excess of auxin will often result in a proliferation of roots, while
an excess of cytokinin may yield shoots.
• A balance of both auxin and cytokinin will produce an unorganised growth of
cells, or callus, but the morphology of the outgrowth will depend on the plant
species as well as the medium composition.

4. Techniques of Tissue Culture
• Preparation of plant tissue for tissue culture is performed under aseptic conditions
under HEPA filtered air provided by a laminar flow cabinet.
• The tissue is grown in sterile containers, such as Petri dishes or flasks in a growth
room with controlled temperature and light intensity.
• Living plant materials from the environment are naturally contaminated on their
surfaces (and sometimes interiors) with microorganisms, so their surfaces are
sterilized in chemical solutions (usually alcohol and sodium or calcium
hypochlorite) before suitable samples (known as explants) are taken.
• The sterile explants are then usually placed on the surface of a sterile solid culture
medium but are sometimes placed directly into a sterile liquid medium,
particularly when cell suspension cultures are desired.
• As shoots emerge from a culture, they may be sliced off and rooted with auxin to
produce plantlets, when mature, can be transferred to potting soil for further
growth in the greenhouse as normal plants

5. Types of plant tissue culture

6. Advantages of Tissue Culture
• The production of exact copies of plants that produce particularly good flowers,
fruits, or have other desirable traits.
• To quickly produce mature plants.
• The production of multiples of plants in the absence of seeds or necessary
pollinators to produce seeds.
• The regeneration of whole plants from plant cells that have been genetically
modified.
• The production of plants in sterile containers that allows them to be moved with
greatly reduced chances of transmitting diseases, pests, and pathogens.
• The production of plants from seeds have very low chances of germinating and
growing, i.e. orchids and Nepenthes.
• To clean particular plants of viral and other infections and to quickly multiply
these plants as 'cleaned stock' for horticulture and agriculture.

7. Application of Plant Tissue Culture
• Plant tissue culture is used widely in the plant sciences, forestry, and in horticulture.
• The commercial production of plants used as potting, landscape, and florist subjects,
which uses meristem and shoot culture to produce large numbers of identical
individuals.
• To conserve rare or endangered plant species.
• Large-scale growth of plant cells in liquid culture in bioreactors for production of
valuable compounds, like plant-derived secondary metabolites and recombinant
proteins used as biopharmaceuticals.
• To cross distantly related species by protoplast fusion and regeneration of the
novel hybrid.
• To rapidly study the molecular basis for physiological, biochemical, and reproductive
mechanisms in plants, for example in vitro selection for stress tolerant plants.

8. • For chromosome doubling and induction of polyploidy, for example doubled
haploids, tetraploids, and other forms of polyploids. This is achieved by application
of antimitotic agents such as colchicine or oryzalin.
• Regeneration of transgenic plants by transfer of specific tissue into another plants.
• Certain techniques such as meristem tip culture can be used to produce clean plant
material from virused stock, such as sugarcane, potatoes and many species of soft
fruit.
• Production of identical sterile hybrid species can be obtained.
• Large scale production of artificial seeds through somatic embryogenesis.
• Synthetic seeds - A somatic embryo is encapsulated by artificial endosperm and
artificial seed coat.

9. Nutritional requirement
• The plant cell and tissue require a proper nutrient medium for their growth and
development.
• The nutrient medium contains both major and minor nutrients to assist the
growing cell or tissue.
• Each element offers certain physiological control over the growing cells or tissue
in the medium.
• The Deficiency of there element cause the deficiency symptoms on the growing
tissue.
• Inorganic Nutrients require for tissue culture techniques.
Macro Nutrients
Micro Nutrients

10. Macronutrients and their function
Nitrogen
Component of proteins, nucleic acids and some co-
enzymes
Potassium
Regulates osmotic potential, principal inorganic
cation
Calcium
Cell wall synthesis, membrane function, cell
signaling
Magnesium Enzyme cofactor, component of chlorophyll
Phosphorus Component of nucleic acid, energy transfer
Sulphur Component of some amino acid and some cofactor

11. Copper Enzyme co-factor , electron transfer factor
Zinc Enzyme co-factor , chlorophyll biosynthesis
Molybdenum Enzyme co-factor , component of nitrate reductase
Chlorine Required for photosynthesis
Iron Electron transfer as a component of cytochromes
Manganese Enzyme co-factor
Micronutrients and their function

12. Other Nutrients
Amino Acids
• Amino acid are added for substitution for augmentation of the nitrogen supply. If
is noted that threonine, glycine and valine reduce aminoium utilisation by
inactiving glumate synthase located in chloroplasts and cytoplasm.
Vitamins
• Plant synthesis required vitamin
• Essential for many biochemical reaction
• Cultured cell are capable to produce vitamins at some level
• They require an exogenous supply vitamin at some level
• They require an exogenous supply of different vitamin for optimum growth
• Most Usable Vitamin are thiamine pyridoxine nicotinic acid vitamin B complex

13. Plant Growth Regulation
• The growth and the development of higher plant tissue in Vitro is controlled by
Endogenous plant growth regulators.
• There are 5 known groups of these growth regulators namely auxins, cytokinins,
gibberellins, unsaturated hydrocarbon gases and growth inhibitor.
• The previous two group are of great importance for culture media preparation
while the letter to group are represented by ethylene and abscisic acid
Organic Substance
• In order to stimulate the growth of the cell organic supplements are sometimes
added to the medium.
• The supplement include pattern yeast extracts, malt extract and coconut milk.
• Malt extract and coconut milk is also known as a supplier of growth regulator.

14. In Vitro Culture
• In plant cell culture, plant tissues and organs are grown in vitro on artificial media,
under aseptic and controlled environment.
• The technique depends mainly on the concept of totipotentiality of plant cells, which
refers to the ability of a single cell to express the full genome by cell division.
• In vitro plant regeneration is a process in which explants, after undergoing cell
division and differentiation, form organs and tissues throughout their growth period.
• Organogenesis is the process by which new organs and even whole plants are usually
formed in response to wounds from other parts of the organs.
• A test performed in vitro ("in the glass") means that it is done outside of a living
organism and it usually involves isolated tissues, organs or cells.
• Micropropagation refers to the in vitro multiplication and/or regeneration of plant
material under aseptic and controlled environmental conditions to produce thousands
or millions of plants for transfer to the field.

15. THANK YOU