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PLANT TISSUE CULTURE.ppt
1. Pharmacognosy and Phytochemistry I
(BP405T)
Course outcome
• C.O.-1
• Students would be able to know the basics & scope of pharmacognosy
• C.O-2
• Students would be acquainted with the techniques in the cultivation and
production of crude drugs available in the healthcare system
• C.O-3
• Students would be able to know the various aspects of crude drugs, such as
their uses and chemical nature that are obtained from natural products
• C.O-4
• Students would be able to understand the evaluation techniques for the
herbal drugs
• C.O-5
• Students would learn importance of various techniques in microscopy which
is important for quality evaluation of herbal drugs and to carry out the
microscopic and morphological evaluation of crude drugs
3. Content
• Historical development of plant tissue
culture
• Types of cultures
• Nutritional requirements, growth and their
maintenance
• Applications of plant tissue culture in
pharmacognosy.
4. • Introduction: Tissue culture
• A method of biological research in which fragments of tissues
from an animal or plant are grown in vitro in artificial medium
under aseptic conditions and continue to survive and function.
• The aseptic culture of plant protoplasts, cells, tissues or organs
under aseptic conditions which lead to cell multiplication or
regeneration of organs or whole plants.
• Tissue culture is in vitro cultivation of plant cell or tissue under
aseptic and controlled environmental conditions, in liquid or on
semisolid well defined nutrient medium for the production of
primary and secondary metabolites or to regenerate plant.
5. • Basic concepts of plant tissue culture(PTC)
• Two concepts, are central to understanding plant cell, tissue,
organ culture and regeneration.
• Plasticity
• ability to initiate cell division from almost any tissue of the
plant.
• ability to regenerate lost organs or undergo developmental
pathways in response to particular stimuli.
• Totipotency
• each cell has the capacity to regenerate the entire plant.
6. • Features of plant tissue culture
• Cells lines differentiate to form specialized tissues and
organs.
• Unlike animal cells, living plant cells re-differentiate.
• Therefore, tissue can be regenerated from explants such as
cotyledons, hypocotyls, leaf, ovary, protoplast, petiole, root,
anthers, etc.
7. • Short history of Plant Tissue Culture
• A German plant physiologist Gottlieb Haberlandt (1902)
cultured isolated single palisade cells from leaves in
Knop's salt solution enriched with sucrose. Haberlandt is
regarded as the father of plant tissue culture.
• Embryo culture was carried by Hanning.
• Kotte cultured excise root tips of pea.
• Van Ovetbeek used coconut milk for development of callus
formation.
• Loo developed whole plant from stem tips of asparagus.
• Skoog explained role of auxin & gibberlin.
• Steward raised plantlets from carrot.
8. Micropropagation vs Tissue Culture
• The fundamental difference between micropropagation and tissue
culture is that the micropropagation is a method of tissue culture.
Tissue culture is a technique that is used to propagate plants in
large quantities in relatively short period. Micropropagation is a
method that comes under tissue culture and it is used to produce
clones of mother plants.
• Starting material for Micropropagation
• (Explant)
• Propagation from plant cells, tissues or organs under aseptic
conditions in synthetic medium in vitro.
• Many different explants can be used for micropropagation, but
axillary buds and meristems are most commonly used
9. • Tissue culture can create a plant directly, whereas
micropropagation must use tissue cultures to create a new
plant.
• Both tissue cultures and micropropagation are forms of
asexual reproduction and are found in the category of
vegetative propagation , which is why they are commonly
used synonymously. Both methods can be used to create
thousands of identical plants in a small amount of time.
• Micropropagation
• “… the art and science of multiplying plants in vitro
• It implies
• - regeneration
• - multiplication
• - uniformity
10. • Applications of plant tissue culture
• Agriculture
• Anther & pollen culture-Production of haploid plants.
• Production of virus-free plants for safe germplasm transfer.
• Screening of in vitro lines for stress and disease resistance.
• Somaclonal Variations has been used in plant breeding programmes
where the genetic variations with desired or improved characters are
introduced into the plants.
• Horticulture and Forestry:
• Micropropagation method is used for rapid multiplication of
ornamental plants as well as important trees yielding high fuel, pulp,
fruits or oil at a large scale.
11. • Today's high yielding oil palms on plantations in Malaysia were
generated in the 1960s by tissue culture methods.
• These palms produce 30% more oil than normally cultured palms.
• Improvement of economically important forest trees is being done
through genetic transformation and rapid Micropropagation
• .e.g in vitro regeneration and genetic transformation of conifers
12. • Industries:
• Plant cell culture is used for biotransformation (modification of
functional groups of organic compounds by living cells).
• Food and agricultural biotechnologists are involved in using tools of
molecular biology to enhance the quality and quantity of foods and
economic crops.
• For example, Golden Rice was genetically enhanced with added
beta carotene, which is a precursor to Vitamin A in the human body.
• Plant cells can be cultured in fermenters for the industrial production
of secondary Metabolites using cell culture.
14. • Plant growth regulators
• Two major hormones affect Plant Differentiation:–
• Auxins: Stimulates Root Development
• Cytokinin: Stimulates Shoot Development
15. ADVANTAGES OF PTC:
• Availability of raw materials.
• Fluctuation in supplies & quantity.
• Patent rights.
• Political reasons.
• Easy purification of the compound.
• Modifications of chemical structure.
• Disease free & desired propagule.
• Crop improvement.
• Bio-synthetic pathway.
• Immobilization of cell.
.
16. FACTORS AFFECTING PTC
• Growth Media– Minerals, Growth factors, Carbon source,
Hormones
• Environmental Factors– Light, Temperature, Photoperiod.
• Explant Source– Usually, the younger, less differentiated the
explant, the better for tissue culture
• Genetics– Different species show differences in amenability to
tissue culture. In many cases, different genotypes within a
species will have variable responses to tissue culture.
17. Critical requirements of Plant tissue culture:
• Appropriate tissue.
• A suitable growth medium which can be liquid or
semisolid.
• Aseptic (sterile) conditions, as microorganisms grow
much more quickly than plant and animal tissue and can
over run a culture.
18. • BASIC REQUIREMENT FOR A TISSUE CULTURE LABORATORY
• For the successful achievement, the following general basic
facilities are required:
• Equipment & apparatus
• Washing and storage facilities
• Media preparation room
• Sterilization room
• Aseptic chamber for culture
• Culture rooms or incubators fully equipped with temperature, light
• and humidity control devices
• Observation or recording area well equipped with computer for
data processing
19. • Equipment & apparatus
• Vessels & glass ware :
• All the glassware should be neat & clean.
• Large test tubes,flasks,graduated pipettes etc.. are used.
• Equipment :
• • Scissors,scapels,foreceps are used for explants preparation.
• • A spirit burner for flame sterilization.
• • Hot air oven.
• • A PH meter.
• • A BOD incubator.
• • Laminar air flow chamber.
• • A balance to weigh nutrients.
• • Data collection and recording room.
20. • Establishment of plant tissue culture
• In vitro culturing of plant tissue culture involves the following
steps.
• Collecting & sterilization of glassware tools/vessels.
• Preparation of explant.
• Surface sterilization of Explant.
• Production of callus from explant.
• Proliferation of culture.
• Sub culturing of callus.
• Suspension culture
21. • Explant preparation
• Explant: It is defined as a portion of plant body, which
has been taken from the plant to establish a culture.
• Explant may be taken from any part of the plant like root,
stem, leaf or meristematic tissue like cambium, floral parts like
anthers, stamens etc..
• Age of the explant should be considered.
22. • The correct choice of explant material can have an
important effect on the success of tissue culture. The
choice of explants depends on:
• the kind of culture to be initiated;
• the purpose of the proposed culture;
• the plant species to be used.
23. • The explant size has an effect on the response of the
tissue. Generally, the smaller the explant, the harder it is
to culture. The culture medium usually has to have
additional components. The larger explants probably
contain more nutrient reserves and plant growth
regulators to sustain the culture.
24. • Surface sterilisation of explant
• For surface sterilization chromic acid, Hgcl (0.11%),calcium
hypochlorite, sodium hypochlorite(1-2%),alcohal(70%) are
used.
• Process depends on the type of explant.
• SEED : Absolute ethyl alcohol calcium hypochlorite bromine
water sterile water
• FRUIT : Ethyl alcohol sodium hypochlorite sterile water
• STEM : Running water sodium hypochlorite sterile water
• LEAF : Surface clean Hgcl2 sterile water dried
25. • Production of callus from explant
• Sterilized explant is transferred aseptically onto defined
medium.
• Transfer to BOD incubator.
• Temperature and light is necessary for callus production.
• Callus produced with in 3-8 days.
26. • Proliferation of culture
• If callus is well developed, it should cut into small pieces &
transferred to another fresh medium containing hormones,
which supports growth.
• The medium used for production of more amount of callus is
called proliferation medium.
27. • Subculturing of callus
• After sufficient growth of callus it should be periodically
transferred to fresh medium to maintain viability of cells.
• There is change in the culture medium from previous to fresh
growth medium.
• This subculture will be done at the interval of 4-6 weeks.
• After a maximum growth transfer into soil under required
condition.
• Subculture is used to prolong the life and/or expand the
number of cells or microorganisms in the culture.
28. • Suspension culture
• It is a type of culture in which single cells or small aggregates
of cells multiply while suspended in agitated liquid medium.
• It contains a uniform suspension of separate cells in a liquid
medium.
• Callus taken in a liquid medium agitated continuously, finally
cells separated, sub-culture the cells.
• This can be achieved by rotary shaker attached within the
incubator at a rate of 50-150 rpm.
• Widely used for the analysis of complex physiological
processes at cellular & molecular level.
29. • Growth medium
• The growth medium used depends on the plant species
being grown. The medium contains the following components:
• all of the minerals and vitamins needed for plant growth and
differentiation.
• a carbon/energy source
• various growth regulators to encourage cell enlargement,
division and differentiation;
• agar which is used to solidify the medium.
30. • The success of plant tissue culture as a means of plant
propagation is greatly influenced by the nature of the culture
medium used.Each plant tissue culture medium must contain
the following essential components to support in vitro plant
growth.
• These are as follows
1) Macro inorganic nutrients.
2) Micro inorganic nutrients
3) Iron (as chelating agent)
4) Vitamins
5) Carbon sources
6) Amino acids
7) Plant growth regulators
8) Agar (as gelling substance)
31. • Macro nutrients
• Need of macro nutrients is higher.
• It is present in milli molar (mM) quantities (more than 30
ppm/1 or mg/1)
• Macro nutrients provide both anions and cations for the
plant cells.
32. • Macro nutrients are nitrogen (as NO3 and NH4),
phosphorus (PO4), potassium (K), sulphur (as SO4),
magnesium (Mg), and calcium (Ca).
• Macro nutrients have structural and functional role in
protein synthesis, cell wall synthesis, enzyme, Co-factors
and membrane integrity.
33. • Nitrogen
• In organic form used as amino acids, different organic
acids and casein hydrolysate.
• In inorganic form used as Nitrate or ammonia.
• Nitrogen is major component of all plant tissue culture
media.
• Nitrogen helps to synthesis complex organic molecule.
34. • Potassium
• K ion is present in high concentration in the cytoplasm
(100-200 mM) and in chloroplast(20-200 mM).
• K+ is essential for maintaining the ion balancing
activation of many enzymes.
• Maintaining osmotic pressure and osmotic regulation of
cells.
35. • Micro Nutrients
• Boron(B),Manganese(Mn),Zinc(Zn),Molybdenum(Mo),
• Copper(Cu),Cobalt(Co) .
• Used in less amount less than 30ppm.(mg/l).
• Concentration is always in uM.
• Zinc
• Zn plays an active role in protein synthesis and in the
synthesis of tryptophan.
• Supplied as Zinc Sulphate.
36. • Iron
• Iron is an essential micronutrient for plant tissue culture
media and can be provided from either ferrous or ferric
salts.
• The presence of iron is particularly important for
adventitious shoot and root formation.
• Its addition to tissue culture media can help to make
macro and micro-nutrients more accessible to plant cells.
37. • Vitamins
• Plant synthesis requires vitamins.
• Essential for many biochemical reaction.
• PTC requires an exogenous supply of different vitamins
for optimum growth.
• Most usable vitamins are Thiamine, Pyridoxine nicotinic
acid Vitamin B Complex.
38. • Hexitols
• Most tissue culture media require this compound called as
myo-inositol.
• Essential for seed germination, sugar transport,
carbohydrate metabolism, membrane structure and cell wall
formation.
• Also aids as a growth enhancer, carbohydrate source,
germination of seeds, mineral nutrition, hormonal
homeostasis & cell wall formation.
39. • Carbon Sources
• These are required to maintain the osmotic potential, as
well as to serve as energy and carbon sources for
developmental processes including shoot proliferation,
root induction as well as emission, embryogenesis and
organogenesis, which are highly energy demanding
developmental processes in plant biology. A variety of
carbon sources (both reducing and non-reducing) are
used in culture media depending upon genotypes and
specific stages.
40. • The exogenously added carbon sources to the medium
supplies energy and also to uphold the osmotic potential.
The effect of the suitable carbon source and
concentration also determines the health and viability of
the in vitro plants.
• Sucrose 3% was commonly used as a source of
carbohydrate followed by Fructose, Maltose and Glucose
.
41. • Amino Acids
• Glycine is the most common Amino Acid used in
different culture media.
• They play a role in enhance growth and plant
regeneration.
42. • Plant growth regulators
• Plant growth regulators, often called phytohormones,
regulate growth, development, and responses to stimuli.
In tissue culture the concentration of growth regulators in
the medium must be optimized and may change
according to the desired result.
• For example, the synthetic auxin 2,4-D is very often the
primary phytohormone to support callus formation and
maintenance. Cytokinins promote shoot formation, but
need auxins as well to promote mitosis.
43. • Gelling Agent
• PTC is conducted on some stationary support &
a gelling agent is required.
• Agar – Agar/ gellan gum
• Agar is a natural product of seaweeds prone to
impurities so washed or purified is preferred.
• Since 1658 agar-agar is obtain from red algae
(Gelidium Gracilaria)
• With water it melts at 100’C and solidify at 45’C
44. • PH of tissue culture media
• Plant culture media are usually started at pH 5.4-5.8.
However, in one containing both nitrate and ammonium
ions, a rapid uptake of ammonium into plant tissue
causes the pH to fall to 4.2-4.6. As this happens, further
ammonium uptake is inhibited, but uptake of nitrate ion is
stimulated, causing the pH to rise again.
• pH is adjusted between 5 & 5.8 before gelling and
sterilization with the help of dilute NaOH, KOH or HCL.
• PH below 5 will not gel properly.
• PH above 6 may be too hard.
