INTRODUCTION TO PLANT TISSUE CULTURE

TECHNOLOGY

PLANT TISSUE CULTURE ?

Any technique of growing cells/tissues/organs/seeds/

embryos or other plant parts in a controlled environment on a nutrient medium

Cultural techniques for regeneration of functional plants from different tissues/organs/cells/protoplasts

Also known as micropropagation

The growth or maintenance of plant cells, tissues, organs or whole plants in vitro.

the science of growing plant cells/tissues/organs isolated from the mother plant on artificial media.

• Faster propagation reduce the time

• Speed up the selection process for crop improvement save time

• Disease free plants

• Save space reduce cost

• Pharmaceutical properties

• Conservation of endangered species

• Management of genetic resources

WHY?

WHY?

Mass propagation/production

Plant breeding selection of somaclonal variants mutation selection haploid/dihaploid production

In-vitro hybridization – protoplast fusion

Genetic manipulation

Secondary product synthesis

HISTORY – Chronology of Important Development

1902 (Gottlieb Haberlandt) culture isolated, differentiated cells from leaves of various

plants on salt solution (Knop’s) with sucrose. maintain cells alive for a period but not divided (lack of plant hormones).

continued cell division in explanted tissues on artificial medium.

1922 (Robbins & Kotte) culture of isolated root tips.

1934 (White) culture of tomato roots (long period).

culture tomato meristematic cells on medium containing

salts, yeast extract, sucrose, vitamin B (pyridoxine, thiamine, nicotinic acid)

1939 (Gautheret & Nobecourt) 1st long term

callus culture involving cambial tissues of carrot.

1939 (White) Callus culture of tobacco tumor

tissues from interspecific hybrid of Nicotina glaucum x N.longsdorffi.

1941 (Van Overbeek) Discovery of nutritional

value

1942 (White & Braun) Experiments on crown-

gall and tumor formation in plants, growth of bacteria free crown-gall tissues.

1948 (Caplan & Steward) Use of coconut milk +

2,4-D for proliferation of carrot and potato.

HISTORY-(cont.)

1950 (Morel) Culture of monocot tissues using

coconut milk.

1953 (Muir) Innoculation of callus in liquid medium.

Development of technique for single isolated cell culture.

1953 (Tulecke) Haploid culture from pollen

gymnosperm (Ginkgo).

1955 (Miller et al.,) Discovery of cytokinins

(kinetin).

1955 (Ball) Culture of gymnosperm tissues (Sequoia).

1957 (Skoog & Miller) Hypothesis that shoot and

root initiation in cultured callus is regulated by the proportion of auzin and cytokinins.

HISTORY-(cont.)

1960 (Cocking) Enzymatic isolation and culture of

protoplast.

1960 (Morel) Development of shoot apex culture

technique.

1964 (Morel) Use of modified shoot apex

technique for orchid proportion.

1966 (Guha & Maheshwari) Cultured anthers

and pollen and produce haploid embryos.

1974 (Nitsch) Culture of microspores of Datura

and Nicotina to double up the chromosome number.

HISTORY-(cont.)

1978 (Melchers) Production of somatic hybrids

from attached to plasmid vectors into naked plant protoplast.

1983 (Barton et al.,) Insertion of foreign genes

attached to plasmid vectors into naked plant protoplast.

1983 (Chilton) Production of transformed tobacco

plants following single cell transformation or gene insertion.

HISTORY-(cont.)

BASIS

• Plant hormones

Auxin

Cytokinin

The ratio can determine plant growth and development

Auxin > Cytokinin

Auxin < Cytokinin

auxin = Cytokinin

Totipotency of plant cells

ability to develop into whole plants/plant organs in vitro

not all cells are totipotent

Differentiated cells can be dedifferentiated

callus

callus can be redifferentiated back to somatic embryo which later regenerate the entire plant

BASIS/PRINCIPLE

Growth Media

minerals, C source, hormones, etc.

Environmental

light, T, photoperiod, etc.

Explant Source

age, type of organ, etc.

Genetics

diff species or genotype have diFf response

FACTORS

• Adventitious - Developing from unusual points of origin, such as shoot or root tissues, from callus or embryos, from sources other than zygotes.

• Agar - a polysaccharide powder derived from algae used to solidify a medium.

• Aseptic - Free of microorganisms.

• Aseptic Technique - Procedures used to prevent the introduction of microorganisms into cultures.

• Autoclave - An equipment that provides heat under high steam pressure for sterilization.

• Axillary bud/shoot - bud/shoot arises from the axil of leaves or normal origin.

• Callus - An unorganized, proliferate mass of differentiated plant cells, a natural response of wound.

TERMINOLOGY

• Chemically Defined Medium - A nutritive solution for culturing cells in which each component is specifiable and ideally of known chemical structure.

• Clone - Plants produced asexually from a single source plant.

• Clonal Propagation - Asexual reproduction of plants that are considered to be genetically uniform and originated from a single individual or explant.

• Contamination - Being infested with unwanted microorganisms.

• Culture - plant growing in vitro.

• Differentiated - Cells that maintain, in culture, all or much of the specialized structure and function typical of the cell type in vivo. Modifications of new cells to form tissues/organs with a specific function.

• Epigenetic changes - persistent changes in phenotype that involve the expression of particular genes.

TERMINOLOGY

• Explant - Tissue taken from its original site and transferred to an artificial medium for growth or maintenance.

• Hormones - Growth regulators, generally synthetic in occurrence, that strongly affects growth (i.e. cytokinins, auxins, and gibberellins).

• Internode - The space between two nodes on a stem.

• In vitro - isolated from living organism and artificially maintained.

• Media - Plural of medium.

• Medium - A nutritive solution, solid or liquid, for culturing cells.

• Micropropagation - In vitro clonal propagation of plants from shoot tips or nodal explants, usually with an accelerated proliferation of shoots during subcultures.

• Organ - a distinct and visibly differentiated part of an organism.

TERMINOLOGY

• Organogenesis - refers to forming organs, in particular both roots and shoots from callus cells.

• Node - A part of the plant stem from which a leaf, shoot or flower originates.

• Passage - Transfer or transplantation of cells/tissues with or without dilution/division, form one culture vessel to another.

• Passage Number - The number of times the cells or tissues in culture have been subcultured or passaged.

• Pathogen - A disease-causing organism.

• Pathogenic - Capable of causing a disease.

• Petiole - A leaf stalk; the portion of the plant that attaches the leaf blade to the node of the stem.

• Plantlets - small complete plants that were produced via tissue culture.

TERMINOLOGY

• Propagules - tissue that is divided and used for further multiplication.

• Regeneration - a morphogenetic response to a stimulus that results in the products of organs, embryos, or whole plants.

• Somaclonal Variation - Phenotypic variation, either genetic or epigenetic in origin, displayed among somaclones.

• Somaclones - Plants derived from any form of cell culture involving the use of somatic plant cells.

• Somatic - cells/tissues that are vegetative and have the complete genetic material of the organism (plant).

• Sterile - (A) Without life. (B) Inability of an organism to produce functional gametes. (C) A culture that is free of viable microorganisms.

• Sterile Techniques - The practice of working with cultures in an environment free from microorganisms.

TERMINOLOGY

• Subculture - See “Passage”. The process by which the tissue or explant is first subdivide, then transferred into fresh culture medium.

• Tissue Culture - The maintenance or growth of tissue, in vitro, in a way that may allow differentiation and preservation of their function.

• Totipotency - A cell characteristic in which the potential for forming all the cell types in the adult organism are retained.

• Undifferentiated - With plant cells, existing in a state of cell development characterized by isodiametric cell shape, very little or no vacuole, a large nucleus, and exemplified by cells comprising an apical meristem or embryo.

TERMINOLOGY

ADVANTAGES & APPLICATION Can grow plant cells in liquid culture on a large

scale

Shortens the time taken to achieve uniform homozygous lines and varieties

Protoplast isolation and somatic fusion of distantly related species increases the possibility for the transfer and expression of novel variation in domestic crops

Increases the potential number of individuals in a screening program

Allows the production of large numbers of uniform individuals of species from limited starting material Enables very specific information to be introduced into single cells which can then be regenerated

Overcomes seasonal restriction for germination or cultivation

Enable conservation, preservation, international exchange of materials

ADVANTAGES

DISADVANTAGES?