PREPARED BY

UNDER SUPERVISION OF:Prof. BENALI D.

PLAN

•Definition of plant tissus culture•Advantages•Organogenesis•Stages•What is needed?•Conclusion

the in vitro aseptic culture of cells, tissues, organs or whole plant under controlled nutritional and environmental conditions often to produce the clones of plants.

Tissue culture is..

DEFINITION

This technology relies on two main concepts:

TOTIPOTENCY DIFFERENTIATION

It implies that every cell of theplant contains all the informationnecessary for growth and it iscapable of developing into anentire plant if suitably stimulated.(the meristemic cells are best ableto express this ability)

The ability of mature cells toreturn to the meristematiccondition and reorganizeinto new organs.

DEFINITION

•To quickly produce mature plants.

•The production of multiples of plants in the absence of seed or necessary pollinators to produce seeds.

•The regeneration of whole plants from plant cells that have been genetically modified.

ADVANTAGES

•The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens.

•To clean particular plant of viral and other infections and to quickly multiply these plants as'cleaned stoc' for horticulture and agriculture.

ADVANTAGES

ORGANOGENESIS

Refers to the production ofplant organs. roots, shoots andleaves that may arise directlyfrom the meristem or indirectlyfrom the undifferentiated cellmasses (callus).

Plant regeneration via organogenesisinvolves the callus production anddifferentiation of adventitious meristemsinto organs by altering the concentrationof plant growth hormones in a nutrientmedium.

ORGANOGENESIS

starts with the selection of plant tissues (explant) from a healthy, vigorous mother plant. We use the young offshoots for the preservation of true-to-typeness of multiplied genotypes.

PREPARATION OF DONOR PLANT

STAGES

Extracting the offshoot to be ready for disinfection

Removing

the offshoot

In this stage an explant (from shoot tip) is surface sterilized (by chemical solutions) to remove contaminants, and transferred into nutrient medium.

STAGES

INITIATION STAGE

Explants are incubated for 3–6 months in the dark condition, (to reduce oxidation of phenolic secretions) and should be transferred to fresh media each month..

STAGES

The aim of this step is toincrease the number ofpropagules.This number is multipliedby repeated subculturesuntil the desired number ofplants is attained.

THE MULTIPLICATION STAGE

STAGES

In this stage, it is necessary to change media, including nutritional modification and growth regulators composition to induce rooting and the development of strong root growth.

THE ROOTING STAGE

STAGES

Major differences between the environment of plants growing intissue culture and those in the field; particularly, differences inlight, both quantity and quality, relative humidity, nutrients andother growth promoters..

THE ACCLIMATIZATION STAGE (IN VIVO)

STAGES

At this stage, the in vitro plants are weaned and hardened. (Hardening is done gradually from high to low humidity and from low light intensity to high light intensity). The plants are then transferred to an appropriate substrate and gradually hardened under a greenhouse.

STAGES

STAGES

WHAT IS NEEDED?

A Suitable Growth Medium

MS medium (Murashige and Skoog medium)

MS is a plant growth medium used in thelaboratories for cultivation of plant cell culture.

Ingredients:

•Mineral elements

1- The macroelements2- The microelements

•Vitamins

•Growth Regulators

1-Auxins2-Cytokinins

•Sugars

•Agar (Gelling Agent)

WHAT IS NEEDED?

Aseptic (Sterile) Conditions

Oven

Laminar flow hoodAlcohol (70%)Autoclave

the microorganisms grow much more quickly than plant tissue and can

overrun the culture... so:

•Media and apparatus are sterile by autoclaving (120C for 20 minutes)

•Using sterile instruments and culture media.

•During excision and culture, procedures are carried out in sterile laminar

flow hood.

Kind of ORGANOGENISIS is controled by relative concentrations of AUXINs and CYTOKININs

Hormonal balance

AUXIN

CYTOKININ

Growth Regulators

WHAT IS NEEDED?

Initialexplant

Nutrient agar medium

Callus Roots Shoots

More cytokinin / low auxinratio regenerated to shoot part

Low cytokinin / more auxinregenerated to only root part.

Medium cytokinin / medium auxinonly regenerated to callus.

WHAT IS NEEDED?

WHAT IS NEEDED?

Frequent Subculturing

Subculturing is transferring explant from a previous culture to fresh growth medium..

Subculturing is used to ensure adequate nutrition and to avoid the build up of waste metabolites

Warmth and good light are essential

WHAT IS NEEDED?

The culture chambers

Temperature (about 27 ° C)

Light

The onset of growth requires a low

light intensity (500 to 1000 lux) with

a photoperiod of 16 hours of light

and 8 hours of darkness.

is a means of preserving the biodiversity of rare or difficult

species to be multiplied naturally

thanks for

your attention