1st lecture in Tissue culture for Pharmacy students

1. Lecture 1
By
Dr. Ahmed Metwaly
TISSUE CULTURE

2. Objectives:
■ Introductory definitions
■ History
■ Factors affecting tissue culture
■ Tissue culture and agriculture
■ Tissue culture and pharmacy
■ Factors necessitate the development of tissue culture
■ Advantages of Plant Cell Cultures
■ Disadvantages

3. Objectives:
Types of tissue culture
■ Callus
■ Suspension
■ Organs
■ Protoplast

4. Definitions:
■ Biotechnology
Application of techniques to modify the fundamental processes of growth and
reproduction in plants, animals or microorganisms, to enhance productivity or
produce new products.
■ Culture
Growing cells, tissues, plant organs, or whole plants in nutrient medium, under
aseptic conditions, e.g. cell culture, embryo culture, shoot-tip culture, anther
culture, etc.
■ Cell culture
Culture of single cells or small groups of similar cells
■ Plant Tissue culture
is the in vitro aseptic culture of cells, tissues, organs or whole plant under
controlled nutritional and environmental conditions
■ Organ culture
Aseptic culture of organized structures, e.g. root tip, shoot tip, shoot segments,
embryo, etc.
■ Micropropagation
Asexual or vegetative propagation of plants in vitro.

5. Some Basics;
The Architecture of Plants

6. Totipotency
The total potential of a plant cell to develop into an entire plant if
suitably stimulated.
Plasticity
Allows plants to alter their metabolism, growth and development to
best suit their environment
Dedifferentiation
Reversal of organized structures into an undifferentiated state.
Capacity of mature cells to return to meristematic condition and
development of a new growing point, follow by redifferentiation which is the
ability to reorganize into new organ.
Explant
The tissue obtained from a plant to be cultured.

7. Callus
■ A proliferating mass of undifferentiated plant parenchyma cells derived
from plant tissue (explants) for use in biological research.
■ This type of cells can be produced by plants as a result of a wound.
■ A tissue arising from disorganized proliferation of cells either in cultures
or in nature. Plural: calluses/ calli.

8. ■ Clone
A population of cells derived from a single cell by mitotic divisions. It is
also commonly used to denote a population of plants derived from a
single individual through vegetative propagation
■ Cloning
Asexual multiplication starting from a single cell or an organism. In
molecular biotechnology it refers to the replication of a small DNA
molecule or a gene as in cloning a vector
■ Contaminants
In the present context, refers to microorganisms, which may inhibit the
growth of cells or tissues in culture

9. Organogenesis
A process of differentiation by which plant organs are formed from tissue or
callus or de novo differentiation of organs as separate entities, i.e., roots and shoots.
Protoplast
A single plant cell from which the cell wall has been removed (usually by use cell
wall degrading enzymes).
Single cells with their walls stripped off.
Protoplast

10. ■ Somatic embryogenesis
In plant tissue culture, the process of embryo initiation and
development from vegetative or non-gametic cells
An in vitro plant regeneration process from somatic cells that involves
differentiation via a somatic embryo which mimics a zygotic embryo.

11. ■ Nutrient medium
A combination of nutrients and water, usually including several salts, a
carbohydrate (e.g. sucrose), and vitamins. Such a medium, liquid or
gelled, is often referred to as a basal medium and may be supplemented
with growth hormones and, occasionally, with other defined and
undefined substances; plural: Nutrient media.
■ Growth regulators
Organic compounds other than nutrients that, in small amounts, influence
growth, differentiation and multiplication, such as auxins, cytokinins,
ethylene and gibberellin.
■ Cryopreservation
Preservation and storage of cells, tissues and organs at temperatures
around -196~ or by immersion into liquid nitrogen.

12. History of plant tissue culture:
1838-39 cellular theory (Cell is totipotent) Schleiden-
Schwann
1902 First attempt of plant tissue culture Haberlandt
1939 Continuously growing callus culture White
1946 Whole plant developed from shoot tip Ball
1950 Organs regenerated on callus Ball
1954 Plant from single cell Muir
1960 Protoplast isolation Cocking

13. 1962 MS media Murashige -
Skoog
1964 Clonal propagation of orchids Morel
1964 Haploids from pollen Guha
1970 Fusion of protoplasts Power
1971 Plants from protoplasts Takebe
1981 Somaclonal variation Larkin
Haberlandt Earnest A. Ball

14. Factors affecting tissue culture
■ Growth Media
– Minerals, Growth factors, Carbon source,
Hormones (Two Hormones Affect Plant
Differentiation):
– Auxin: Stimulates Root Development
– Cytokinin: Stimulates Shoot Development
–  Auxin ↓Cytokinin = Root Development
–  Cytokinin ↓Auxin = Shoot Development
– Auxin = Cytokinin = Callus Development
■ Environmental Factors
– Light, Temperature, Photoperiod, Sterility.
– Explant Source
Usually, the younger, less differentiated the
explant, the better for tissue culture

15. Tissue culture in agriculture
■ Production of improved crop varieties
■ Production of disease-free plants (virus)
■ Genetic transformation
■ Production of varieties tolerant to salinity, drought and heat
stresses

16. Tissue culture in pharmacy
■ The elucidation of the biosynthetic pathways of secondary metabolites
with isolation of corresponding enzymes.
■ Discovery of new secondary metabolites in vitro.
■ The commercial production of expensive secondary metabolites .
■ They have also been used for metabolic and genetic studies.
■ The selection of superior strains of medicinal plants.

17. Factors necessitate the
development of tissue culture
1. Availability of raw material:
Some plants cannot be produced in economically sufficient quantity to
satisfy demand, e.g. Taxus species, the principal source of a diterpene
alkaloid; taxol.
2. Variation and fluctuation of supplies and quality:
The production as well as the quality of crude drugs is affected by
climatic variability, crop diseases and various methods of collection and
drying. In addition, variation in the active constituents may arise in
plants of the same species having different genetical characteristics

18. . On contrary, plant tissue culture techniques would permit a steady
growth of tissues or cells away from the effect of the above variables.
• Growing plant cells or tissues under optimum environmental
conditions, i.e. better control can be attained of light, temperature
and nutrition.
• Production of active constituents could be continuous at all times
and at a standard quality.
• Growing plant cells or tissues yield no artifacts or stress
metabolites attributed to bacteria, fungi, algae, viruses and
insects.

19. Advantages of Plant Cell Cultures
1. it is independent of geographical and seasonal variations and
environmental
factors – the synthesis of bioactive secondary metabolites runs in
controlled environments and the negative biological influences that affect
secondary metabolites production in nature are eliminated (microorganisms
and insects);
2. it offers a defined production system which ensures the continuous supply
of products, uniform quality, and yield;
3. it is possible to select cell lines with higher production of secondary
metabolites;
4. it is possible to produce novel compounds that are not normally found in
parent plant;
5. plant cell can perform stereo- and regio-specific biotransformations for
the production of novel compounds from cheap precursors;
6. with automatization of cell growth control and regulation of metabolic
processes, cost price can decrease and productivity increase.

20. Disadvantages
• Specialized equipment required
Laminar flow cabinets
Autoclave
Water purification systems
Glassware etc…
 High labor cost is the most limiting factor
 Skilled labor required
• Contamination risks
Maintenance of aseptic (sterile) environment difficult.
Rapid spread of contaminants = widespread loss.
• Risk of mutation arising
•  Artificial environment induces mutations.
•  Responses to tissue culture conditions varies
•  Trial and error to determine optimum media or
conditions

21. Types of Cell Cultures
■ Plant TC includes a set of different techniques to manipulate
cells. Among the different PTC that can be obtained are
callus, suspension cultures, protoplasts, anther and ovule
cultures and somatic embryos.

22. Callus culture
What is callus culture?
■ Callus is a largely unorganized, proliferating mass of parenchyma cells.
■ A first step in many tissue culture experiments, it is necessary to induce
callus formation from the primary explant.
■ Calluses are slow growing, small, and convenient to handle, and hence are
a useful means tissue culturing.

23. ■ The growth rate and friability of callus produced can vary widely between
explants and even within replicates of the same medium .
■ This heterogeneity is seen in established calluses as differences in color,
morphology, structure, growth, and metabolism.
■ The cells although undifferentiated, contain all the genetic information
present in the normal plant.

24. ■ The level of plant growth regulators is a major factor that controls callus
formation in the culture medium.
■ Culture conditions (temperature, type of jellification agent, light, etc.) are
also important in callus formation and development.
■ A wide variety of media compositions have been used with success to
induce calluses.

25. How callus culture can be prepared?
■ How ?????????

26. Why ?
■ A first step in many tissue culture experiments.
■ Can be used in biochemical and pharmaceutical researches.

27. Suspension Cultures
What is Suspension Culture?
■ A rapidly dividing, homogeneous
suspension of cells ???? .
■ Suspension cultures should
ideally consist of single cells, but
this is rarely the case and usually
small aggregates of 20–100 cells
(100–1,000 μm) are found. The
suspension cultures grow faster
than callus cultures and they are
more homogenous.

28. ■ However, the rate of
variability in suspension
culture also increases
producing variability and
instability of the cultures.
To avoid the problem of
instability, the cultures
are subcultured when
the cells are at the end
of the exponential growth
phase.

29. How?
■ There is not a standard
method to produce a
suitable suspension
culture. However, in most
of the cases the
transference of friable
callus to a liquid media
under agitation during
incubation (50–200
rpm), can produce the
dispersion of the cells

30. Why?
These cultures can be used in;
■ biochemical research
■ Study of growth metabolism
■ Molecular biology and genetic engineering experiments.
■ large scale secondary metabolites production.

31. Types of suspension culture:
I. Batch suspension culture:
■ In this technique the cells
multiply and grown in a fixed
volume of a liquid nutrient
medium, which is being
continuously agitated to
break up any cell
aggregates, to maintain
uniform distribution of cells
in the medium and to allow
gaseous exchange between
the culture medium and
culture air.

32. A hypothetical growth curve of cell cultures

33. II. Semi-continuous culture:
■ In this type of culture the
inflow of fresh medium is
manually controlled at
infrequent intervals by a “drain
and refill” process, such that
the volume of culture removed
is always replaced by an
equivalent volume of fresh
medium, by this means the
growth of the culture is
continuously maintained.

34. III. Continuous culture:
■ A fresh medium
and culture are
continuously added
and withdrawn,
respectively, in
which the volume
of culture remains
constant and cell
proliferation takes
place under
constant
conditions.

36. Organ Culture
What ???
■ In 1934 Phillip White, one of the pioneers of PTC, developed
the first system that allowed indefinite proliferation of roots
tips.

37. How?
■ Root cultures can
be established by
cultivating roots
isolated from
aseptic plant
cultivate in vitro.

38. ■ shoot cultures also have
been established.
■ These cultures can be
used to produce natural
products in which
biosynthetic pathway is
located in the aerial part
of the plant

39. Why?
■ Root and shoot cultures have emerged as powerful tools to
study the biochemistry and molecular biology of secondary
metabolite biosynthetic pathways.
■ Production of secondary metabolites.

40. ■ Flores and Filner were capable to demonstrate that Hyosciamus
muticus hairy roots are able to synthesize hyoscyamine at levels
equal to or greater than the roots in plant.

41. Protoplasts culture(Protoplast
fusion)
■ Protoplasts are basically
plant cells without the
cell wall.
■ The removal of the cell
wall makes it necessary
to include osmotic
stabilizers into the
medium and additional
nutritional ingredients to
preserve the protoplast
and ensure their viability.

42. How
■ The isolation of plant cell wall happen using enzymatic
degradation or by mechanical procedures.
■ The technique involves the fusion of protoplasts of two different
genomes followed by the selection of desired somatic hybrid cells
and regeneration of hybrid plants.

43. Why
■ In vitro fusion of protoplast opens a way of developing unique
hybrid plants by overcoming the barriers of sexual incompatibility.
■ Also it can be used to introduce a specific genes into the plant
nucleus.

45. Summary:
■ Introductory definitions
■ History
■ Factors affecting tissue culture
■ Tissue culture and agriculture
■ Tissue culture and pharmacy
■ Factors necessitate the development of tissue culture
■ Advantages of Plant Cell Cultures
■ Diadvantages

46. Summary:
Types of tissue culture
■ Callus
■ Suspension
■ Organs
■ Protoplast