4 th lecture about plant tissue culture applications

1. Lecture 4
By
Dr. Ahmed Metwaly
TISSUE CULTURE

2. Objectives:
Sterlization
Applications of plant tissue culture
■ Breeding
■ Genetics
■ Model system
■ production of secondary metabolites

3. 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

4. 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)

5. 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.

6. 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

8. Applications

9. 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

10. 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.

11. Model system
■ For study of plant cell genetics, physiology, biochemistry, and
pathology

12. Production of secondary metabolites
(Mechanisms)
■ Selection of high-producing strains
■ Precursor feeding
■ Elicitation

13. 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.

14. 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

15. 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.

16. 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

20. Production of secondary metabolites
(Examples)

21. ■ 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.

23. Food additives from tissue culture sources

25. Summary:
Applications of plant tissue culture
■ Breeding
■ Genetics
■ Model system
■ production of secondary metabolites