Lecture 4

By

Dr. Ahmed Metwaly

TISSUE CULTURE

Objectives:

Sterlization

Applications of plant tissue culture

■ Breeding

■ Genetics

■ Model system

■ production of secondary metabolites

Sterilization methods■ Medium, tools, vessels

Autoclaving

Filtering

Ethylene oxide gas

UV radiation

Dry heat

Microwave

■ plant materials Surface-sterilizing

Alcohol

Sodium /Calcium hypochlorite

Mercuric chloride

Hydrogen peroxide

Autoclaving

■ In order to eliminate bacterial and fungal

contaminants, media must be submitted to heat and

high pressure. Fungal spores may survive if only heat is

used. Therefore, media is sterilized by heating to 121

˚C at 103.5kPa for 15-20 min (15 lb in-2)

Dry Sterilization

■ Glassware can be sterilized in an oven by placing them

at 200 ˚C for 1-4 hours. Be sure to cover glassware with

aluminum foil to maintain aseptic conditions after

removing the glassware from the oven. Avoid the use of

any plastic caps, paper (i.e. labeling tape), or other

flammable materials as they are fire hazards.

Filter Sterilization (for heat labile compounds)

■ Certain media components are

susceptible to heat denaturation and

therefore must be added to the media

after autoclaving. To do so, you must

filter the components using a 0.22μm

pore size filter that is appropriate to the

solvent used

Applications

Breeding

■ Micropropagation – using meristem and shoot culture to produce

large numbers of identical individuals; Examples for

micropropagation; dwarfing sweet cherry, Shade trees, Ornamental

shrubs, Roses, Clematis, Lilacs, Saskatoon berries, Nutraceutical

Plants, Rhododendron, Azalea, mustard, corn, soybeans, wheat, rice,

cotton, tomato, potato, citrus, turf, legumes

■ crossing distantly related species and regeneration of the novel

hybrid by protoplast fusion

Protoplast fusion products are presently grown on approximately 42% of

the fluecured tobacco acreage in Ontario, Canada. This represents a

value of approx. US$199,000,000.

■ production of dihaploid plants from haploid cultures

■ Removal of viruses by propagation from meristematic tissues for ex;

virus-free potatoes, produced by culture in vitro, gave higher yields

than the normal field plants, with increases up to 150%. As only

about 10% of viruses are transmitted through seeds

Genetics

■ Transformation, followed by either short-term testing of

genetic constructs or regeneration of transgenic plants.

For example; Flowering in aspen is generally observed after 8–20

years. One of homeotic genes (LFY) has been expressed in

transgenic aspen and was able to produce flowers after 7 months

of vegetative Growth (early flowering and faster growth)

The transfer of herbicide resistant genes in tree species as the

transgenic for herbicide resistance gene in Populus tree.

Model system

■ For study of plant cell genetics, physiology, biochemistry, and

pathology

Production of secondary metabolites(Mechanisms)

■ Selection of high-producing strains

■ Precursor feeding

■ Elicitation

Selection of high-producing strains

■ Plant cell cultures produces a heterogeneous population of

cells in which physiological characteristics are different.

■ The aim is to select cell lines yielding the higher levels of the

desired product.

■ Cell suspension culture of Coptis japonica , which grew faster

and produced a higher amount of alkaloids. Selected cell line

increased growth about 6-fold in 3 weeks and the highest

amount of alkaloid was produced 1.2 g/L of the medium and

the strain was very stable, producing a high level of berberine

even after 27 generations.

Precursor feeding

■ exogenous supply of a biosynthetic precursor to culture

medium may also increase the yield of the desired product.

■ This approach is useful when the precursors are inexpensive.

■ The concept is based on the idea that any compound, which

is an intermediate, in or at the beginning of a secondary

metabolite biosynthetic route, stands a good chance of

increasing the yield of the final product.

■ Feeding ferulic acid to cultures of Vanilla planifolia resulted in

increase in vanillin accumulation

Elicitation

■ plants produce secondary metabolites in nature as a defense

mechanism against attack by pathogens.

■ Elicitors are signals inducing the formation of secondary

metabolites.

■ Biotic and abiotic elicitors which are classified on their origin

are used to stimulate secondary metabolite formation in plant

cell cultures.

Biotic elicitors. They include:

■ Enzymes, cell wall fragments of microorganisms, polysaccharides

derived from microorganisms (chitin or glucans), and glycoproteins;

■ phytochemicals produced by plants in response to physical

damage, fungi or bacteria attack, polysaccharides derived from

plant cell walls (pectinor cellulose), fragments of pectin formed by

action of microorganisms on plant cell wall.

Abiotic elicitors are the substances of non-biological origin. The causes

of the abiotic stress can be of chemical or physical nature; among

them are:

■ Chemicals such as inorganic salts, heavy metals, some chemicals

that disturb membrane integrity,

■ physical factors like mechanical wounding, ultraviolet irradiation,

high salinity, high or low osmolarity, extreme temperature (freezing,

thawing), high pressure

Production of secondary metabolites(Examples)

■ Taxol: Taxol (plaxitaxol), a complex diterpene alkaloid These found

in the bark of the Taxus tree, is one of the most promising

anticancer agents known due to its unique mode of action on the

microtubular cell system .

■ A century old tree yields an average of 3 kg of bark, corresponding

to 300 mg of taxol, which is approximately a single dose in the

course of a cancer treatment.

■ Taxus mairei calli were induced from needle and stem explants on

B5 medium [Gamborg's et al., 1968] supplemented with 2 mg/l

2,4-D or NAA. Different cell lines were established using stem and

needle-derived callus. One of the cell lines, after precursor feeding

and 6 weeks of

incubation, produced 200 mg taxol per liter cell suspension cultures.

Food additives from tissue culture sources

Summary:

Applications of plant tissue culture

■ Breeding

■ Genetics

■ Model system

■ production of secondary metabolites