tissue culture hybridization

jaisreenivasan

PLANT TISSUE CULTURE

An IntroductionPlant tissue culture is:A tool in biotechnology, used in micropropagation, secondary metabolic production(taxol), hybridisation etc.It refers to the growth of plant parts(organs, tissues etc.) in a sterile environment in a nutrient medium.

Why Plant T.C.?? Saves time & labour in comparison to

conventional hybridization techniques. Useful when other techniques are not

useful. Eg.: Apical meristems which are free of

viruses(present in shoot tips) can be harvested & nurtured to grow into virus-free plants in times of viral attack.

Uses• To save species from extinction• To isolate disease from plants • To produce plants with enhanced

stress or pest resistance• To create new plant varieties • To make money

Speed of multiplication in T.C.

Totipotency Plant tissue culture is based on

totipotency Plant cells have the ability to produce

the whole plant from single cells. This is called totipotency.

Plants have the ability to reproduce asexually through a type of cell division- mitosis.

Mitosis

• Daughter cells of identical chromosome numbers are produced.

Beginning of T.C. In 1902, a German physiologist, Gottlieb

Haberlandt proposed that single plant cells could be cultured.

He isolated single fully differentiated individual plant cells from different plant species and was first to culture them in Knop’s salt solution enriched with glucose.

History-1930s In the 1930s, importance of vitamins

was determined for shoot and root culturing

Indole-Acetic Acid(IAA), a natural auxin, and the most important, was discovered during the 1930s.

Many more synthetic auxins were discovered. Eg.: 2,4d & NAA(Napthaleneacetic acid)

History-1957-58 Miller and Skoog discovered kinetin, a

synthetic cytokinin, in the University of Wisconsin – Madison.

Kinetin plays active role in organogenesis.

Steward developed somatic embryo from carrot cells

History-1958-60 Morel cultured orchids and dahlias & freed them

from a viral disease

History-1962• Murashige and Skoog published recipe for MS

Medium for T.C.

T.C. Medium Functions: provide H2O, provide mineral &

vitamin nutritional needs. H2O is usually distilled minerals must provide 17 essential elements energy source - sucrose is preferred provide access to atmosphere for gas

exchange serve as a dumping ground for plant

metabolites

T.C. medium Macronutrients-eg: K, Ca, Mg, N, P, O

etc. Micronutrients-eg: Fe, Mn, Mo, etc. Vitamins & Amino acids:thiamine,

pyridoxin, nicotinic acid, biotin, citric acid, ascorbic acid, Inositol, Glycine etc.

Growth Regulators: auxins and cytokinins

Carbon Source:Sucrose pH usually 5.0-5.7

T.C. Steps1. Explant collection2. Callus generation3. Organogenesis (shoot and root

induction)4. Hardening Micropropagation does not involve the

second step. There is direct organogenesis.

Explant collection

portion of plant removed and used for T.C.

Important features size source - some tissues

are better than others species dependent physiological age -

young portions of plant are most successful

Explant collection degree of contamination external infestation - soak plant

in sodium hypochlorite solution internal infection - isolate cell

that is not infected roots - especially difficult

because of soil contact herbaceous plants soft stem easier to culture than woody

plants

Callus Generation•The dedifferentiation of a plant cell(explant) produces callus. •Callus is then expanded into a larger mass of undifferentiated cells.•Callus is then activated, by selective use of plant hormones to redifferentiate to produce, shoots, roots and ultimately, plantlets

Organogenesis Usually induced by changes in hormonal

environment Shooting: Higher cytokinin

concentration & lower auxin concentration(cytokinins promote shoot growth).

Rooting: lower cytokinin concentration and increase auxin(auxins promote shoot growth)

Hardening Hardening involves the formation of the

waxy cuticle on the leaves of the plant. Plants in T.C. do not have cuticle usually done in greenhouse with high

relative humidity(RH). gradually increase light intensity and

lower RH after rooting occurs allows plants to harden and helps plants

form cuticle

CREDITS TO The inventors of tissue culture Google images The internet’s information Microsoft Office 2010

THANK YOU!!