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Biotechnology
Lecture 1
Applications on Plants
Tissue culture or In vitro culture
Plant tissue culture:
tissue culture means the culturing of pieces , tissues or cells of a plant in
glass or plastic vessels containing specific nutritional medium (consists
of macro and micro nutrients, hormones, energy source, ..).
All growing materials (vessels, media, plant tissues, …etc.) must be
sterilized, then the culture is then placed under suitable light and
temperature for the tissues to grow and differentiate.
Components of tissue culture lab:
1- Preparation room: The first room where the plant materials, media
and vessels are prepared, washed and sanitized.
Instruments: Autoclave, Balance, water distiller, Fridge, hot plate stirrer,
pH meter, glassware (Petri dishes, pipettes, flasks, …etc.)
Components of tissue culture lab:
2- Transfer room: It is a sanitized room where the plants are planted on
a sanitized media.
Instruments: Tissue culture hood, Bunsen flame or electric heater for
sanitizing blades and forceps, microscope.
3- Culture room: a room equipped with suitable temperature (25 oC) and
light (16 hrs light and 8 hrs darkness) used for growing the tissues,
contains plastic or aluminum racks where the vessels are placed.
1- Autoclave 2- sinks 3- Hood 4- Racks 5- Benches 6- Fridge
water distiller 8- 9- 10 – pH meter, hot plate stirrer , balance
7-
Components of growing media
1- Macro and micro
nutrients:
Non organic matter,
important for the life
and survival of all
plants
Macro nut.: N, P, K,
Ca, Mg, S, Na. needed
in quantities more then
0.5 mil mol / lit.
Micro nut.: Mn, Ni, Al,
Co, Mo, B, Fe, I, Cu,
needed in quantities less
then 0.5 mil mol / lit.
2- Source of
carbohydrates and
sugars:
3- Vitamins:
Added to the media in
a very small quantities.
Organic matter
It works as a catalyst,
needed as a source of regulator or as a
energy, the most
cofactor in the
important
enzymatic reactions
carbohydrate used is
and the metabolic
the sucrose (Glucose pathways.
+ Fructose).
Ex: vit. B, C, D, E
Sucrose is added to
the media in a conc.
Of 2 – 6%
Components of growing media
4- Hormones and growth regulators:
5- Gelling agents:
They are added to the growing media in a
very small quantities and they are added to
regulate the growth and differentiation of the
tissues, they are categorized in 3 groups:
It is used to give a solid
texture (Gelatin texture)
to the growing media. Ex
Agar, Agarose, Gelrite.
1- Auxins: a group of compounds
responsible for the elongation of cells, plant
apical dominance and promote root
formation. Ex: (IAA, IBA, NAA, 2,4-D)
5-9 gms/lit. of the Gelling
agent is added to the
media.
2- Cytokinin: a group of compounds responsible
for the cell division and promote the formation of
buds and lateral shoots Ex: Kinetin, Zeatin , BA,
TDZ.
3- Gibberellins: growth regulators used in
breaking the dormancy of buds and seeds and
cell elongation and growth Ex: GA3
If the gelling agent is not
added to the media, the
media becomes liquid
and it must be placed on
a shaker after planting
and is called in this case
(liquid media).
Tissues are placed first on a medium containing
cytokinins (at conc. 0.5-10 mg/lit) for the
production of shoots then the formed shoots are
then transferred to a new medium containing
Auxins (at a conc. of 1-20 mg/lit) for the
production of roots.
If both Auxins and cytokinins are added to the
medium, the tissues will grow to undifferentiated
cells called callus , the callus differentiate to
shoots or roots after that if placed on a medium
containing the suitable growth regulator.
The explants used in tissue culture:
1- Meristem culture: The cells at the growing tip of
the plant are used, and it is used if a free virus plant
is needed to be obtained.
2- Shoot tip culture: The cells at the growing tip of the
plant are used + the leaf primordia at the growing tip.
3- Organ culture: if a while organ from a plant is used Ex: leaf culture, root
culture, stem culture, bud culture …etc.
4- Embryo culture: used to grow the mature or immature embryos, it is used
with tiny seeds or if the seed doesn’t contain enough endosperm to feed the
embryo during seed germination Ex: Orchids
5- Pollen and anther culture: if 1n plant is needed for the heredity studies or
genetics program.
6- Ovary culture: if 1n plant is needed for the heredity studies or genetics program.
7- Protoplast culture: which is a cell without a cell wall, used in protoplast fusion and
in plant transformation programs.
8- Single cell culture: One cell only is placed on a medium, but because of the
difficulty of one cell to grow by its self and differentiate a nursing cells must be used.
Factors affecting the growth and differentiation in in-vitro culture:
A- External factors:
1- Chemical factors: - The Auxin to cytokinin concentration A:C if the A>C
(Root formation), A<C (Shoot formation), A=C (Callus).
- Sugar concentration and type: example fructose sugar if used instead of
sucrose will cause the production of secondary products (Taxol), also if the
concentration of sucrose is increased in the medium this will lead to the
production of antioxidants and phenolic compounds.
- 2,4-D if added in increased conc will lead to the formation of mutations in the
growing tissues
2- Physical factors: - Light: Total darkness and very high light intensity cause
the stopping of differentiation and tissues form callus only.
It was found that light at wave length of 400-450 nanometer enhance the
formation of flowers, blue wave length enhance antioxidant formation while the
red light enhance the cytokinin therefore increase the bud and lateral shoot
formation.
- Temperature: The optimum temperature for the tissues to grow and
differentiate is 25 oC.
-pH: The best pH for the tissues to grow is between 5-6 any fluctuation will
cause the change in membrane permeability and the absorption of nutrients.
B- Internal Factors:
-Species of stock or mother plant: plants are different in their rate of growth
and differentiation on the media for example Nicotiana plant and family
Solanaceae grow and differentiate very fast on the growing media while
woody plants are slower in their rate of growth.
- Physiological age of the explants: as the explant ages its rate of growth
and differentiation decreases, also the possibility of genetic distortion
increases.
- Cell to cell or Tissue to tissue: it was found that the relationship between cells in
the same tissue or the tissues in the same organ had a great influence on the growth
and differentiation of the tissue
Sanitation of utilities, glasses, media and explants:
-All utilities (papers, Aluminum foil, blades, forceps…) glasses (Petri
dishes, flasks, beakers….) are sanitized in autoclave for 30 min. at 121 oC
and pressure 15 pound/inch2.
-Media are autoclaved for 20 min. at the same temp. and pressure.
- Explants are sanitized using Clorox bleach at 10% for 10 min. , 2 drops
of tween 20 are added to reduce the water surface tension.
- Tissue culture hood and hands are sanitized by alcohol 75%.
Practical uses of tissue culture:
1- Protoplast fusion:
Protoplast fusion is used in the cases of sexual incompatibility between
parents or in the cases of male sterility.
Cell wall of cells are removed and 2 parental cells are fused together, after
fusion the cell wall is formed once again. The cell wall is removed by the
use of cellulase enzyme for 4-12 hrs at 25-30oC and pH 5-6.
The osmotic of the solution used pressure is very important because if the
osmotic pressure increases it might cause plasmolisis to the protoplast
leading to the death of the cells. The osmotic pressure is stabilized by the
use of Ca or K chloride or Mg sulfate.
In 1978 was the first successful trial to make a fusion between 2 cells from
different species which is known as somatic hybrid plants this fusion was
done between tomato cells and potato cells forming a new plant called
pomato or potomato.
The fusion is done by two ways:
1- Chemical fusion
2- Electro fusion
Depends on the
presence of Ca++ ions
and PEG in the
solution. The PEG
causes the coagulation
and gathering of cells
together.
The protoplasts are
placed in an electric
circuit, the –ve charges
on the cell membrane
causes the cells to
migrate to the +ve pole
and the coagulation of
the cells together.
This way is not widely
used because the PEG
is toxic substance and
if not washed quickly it
might cause the death
of the cells.
Selection of the cells fused
successfully is done by the use of a
microscope and a micro pipette.
The 2 parental cells must have 2
different colors or shapes so that the
selection of the fused cells become
easier .
After selecting the successfully fused
cells, the cells must be transferred to
a suitable growing media to form
callus then the plant.
Benefits of protoplast fusion:
-Transferring characteristics (drought tolerance, salinity tolerance, …) from
one species to another
- making a hybrid of 2 parents even if they haven't reach their maturity
stages (orchids).
-Overcoming the male sterility in some species
Problems of protoplast fusion:
-Difficult to select the successfully fused cells.
- most hybrids are sterile and have deformations in their genome due to
the lost or addition of organelles.
- the successfully fused cells might not differentiate to a full plant.
Practical uses of tissue culture:
2- Micropropagation:
Means the production of numerous number pf plants from a single tissue,
this is done by placing the tissue on a medium containing cytokinins in
order to enhance the formation of lateral shoots, then the shoots are
removed and placed once again on medium containing cytokinin to
enhance more shoot formation (Sub-culturing). After a large number of
shoots are formed, the shoots are transferred to rooting medium (contains
auxins) .
After root formation the small plants are transferred to trays containing peat
moss + fungicides and covered for 15 days , the covered is then removed
gradually and the plants are transferred to the green house.
2- Production of secondary products: Benefits
1- Saving the land for food crops.
2- The production of secondary products in small areas in its final form.
3- Stress can be induced properly without the interfere of external factors.
4- No contamination and no pesticides are used.
5- Production at any time of the year (no seasonal variation).
6- Producing constant amount that can meet with market demand
(Bioreactors).
Practical uses of tissue culture:
3- Saving endangered plants:
Wild and endangered plants can be propagated by the use of tissue
culture in large numbers and replanted once again in the wild or its place
of origin.
4- genetic improvement:
-Somaclonal variation or the use of chemicals causing mutation might
lead to the formation of a plant that caries a desired characteristics which
might enter after that in a genetics program.
5- production of virus free plants:
-Using meristem culture leads to the production of a virus free plants and
it is very important in fruits and vegetable production
6- production of roots or tubers:
- By the use of tissue culture potato tubers or tuberous roots can be formed
directly from the explant without the need of a whole plant for the production of
roots