Plant Tissue Culture, principles and methods

1. Prepared By
Dr. Rehab AlMousa
Researcher, Biotechnology
Department, GCSAR
Email:
bebo_moussa13@yahoo.com

2. Plant Tissue culture
It is a technique of
1. growing cells, tissues and organs in vitro (in glass)
2. on artificial culture medium
3. under aseptic and controlled conditions.
Plasticity Totipotency

3. Plasticity
• Many of the processes involved in plant growth and
development adapt to environmental conditions.
•This plasticity allows plants to alter their metabolism, growth
and development to best suit their environment.
•Particularly important aspects of this adaptation are the
abilities to initiate cell division from almost any tissue of
the plant and to regenerate lost organs or undergo different
developmental pathways in response to particular stimuli.
•When plant cells and tissues are cultured in vitro they generally
exhibit a very high degree of plasticity which allows one type of
tissue or organ to be initiated from another type. In this
way, whole plants can be subsequently regenerated.

4. Totipotency
• the regeneration of whole organisms depends upon the
concept that all plant cells can , given the correct stimuli,
express the total genetic potential of the parent plants. The
maintenance of genetic potential is called “Totipotency”
Cell
division
Cell elongation (enlargement) Cell maturation (differentiation)

5. Differentiated
cells
Dedifferentiation
Redifferentiation
•Mature cell reverts to meristematic state and
formed undifferentiated callus
•Can divide and produce new cells
• Conversion of component cells of callus tissue to
whole plant or plant organ
•Loose the power of division
• Cell that has developed a specialized form
(morphology) and/or function (physiology)
• Cannot divide
Regeneration
• genesis of an entire plant from cultured explants
directly or via callus indirectly

7. Factors affecting explant’s regeneration
capacity
1. Plant material
• Genotype
• Physiological stage of donor plant
• Explant source
• Explant age
• Explant size
• Explant position in donor plant
• Explant density
2. Surface sterilization process
3. Culture medium
4. Culture condition
• Light
• Temperature
• Composition of gas phase

8. Media Components
1. Inorganic supplements
 Macronutrients
 Micronutrients
2. Organic supplements
 Vitamins
 Amino acids
3. Carbon source
4. Gelling agent
5. Plant growth regulators
 Growth promoters (auxins, cytokinins, gibberellins)
 Growth inhibitors (abscisic acid, ethylene)
6. pH

9. Hormonal balance
Auxin
Cytokinin
High Low
Root formation on cuttings
Embryogenesis
Adventitious root formation in callus
Callus initiation
Adventitious shoot formation
Axillary shoot growth
Low High

11. A section of a culture room
Timer switch to
control
photoperiod
Fluorescent tube
placed above shelf
for lighting
A level of shelf
containing in vitro
cultures

12. Tissue Culture
CULTURES OF ORGANISED
STRUCTURES
CULTURES OF UNORGANISED
TISSUES
• Meristem cultures (shoot apices)
• shoot cultures (shoot tips, or buds)
• Node cultures (lateral buds)
• Isolated root cultures
• Embryo cultures (zygotic (seed)
embryos)
•Callus (or tissue) cultures
• Suspension (or cell) cultures
• Protoplast cultures
• Anther cultures

13. Meristem cultures:
• shoot apex (apical meristematic
dome with or without one or two leaf
primordia)
• give one single shoot.
shoot cultures:
• shoot tips, or buds (larger than the
shoot apices having several leaf
primordia).
• Usually produce multiple shoots.
Node cultures:
• stem pieces carrying either
single or multiple nodes.
• Each bud is grown to provide
a single
shoot.

14. Root cultures:
• can be established from
root tips taken from
primary or lateral roots of
many plants
Embryo and ovule cultures:
• embryos are dissected from seeds,
individually isolated and
germinated in vitro to provide one
plant per explant.
•in some plant, it has been possible
to excise and culture pollinated
ovaries and immature ovules.

15. Callus cultures:
An amorphous mass of loosely arranged thin-walled parenchyma
cells arising from the proliferating cells of the parent tissue cultured
on agar medium.
Suspension cultures:
• Tissues and cells cultured in a agitated liquid medium
produce a suspension of single cells and cells clumps of few to
may cell, these are called suspension cultures.
• Batch culture: A batch culture is a cell suspension culture
grown in a fixed volume of nutrient culture medium.
• Continuous culture:
1. Open: Both cells and the used medium are taken out from
open continuously cultures and replaced by equal volume
of fresh medium.
2. Closed: The cells separated from used medium taken out
for replacement and added back to the suspension culture,
So that the cell biomass keeps on increasing.

17. Protoplast cultures:
Isolated protoplasts have been described as "naked" cells because
the cell wall has been removed by either a mechanical or an
enzymatic process.
Protoplasts can be induced to reform a cell and divide if placed in
a suitable nutrient medium than form callus.

18. Embryo Culture
Embryo culture is usually done from the need to rescue embryo
from wide crosses where fertilization occurred, but not the
embryo development.
 Production of haploid plants
 A common explant for the initiation of somatic embryogenic cultures
 Overcoming abortion of embryos of wide hybrids at very early stages
of development due to incompatibility barriers
 In vitro fertilization for the production of distant hybrids avoiding
style and stigmatic incompatibility that inhibits pollen germination
and pollen tube growth

19. Anther culture/Pollen culture
Anther culture is a technique
by which the developing
anthers from unopened flower
bud are cultured on a nutrient
medium where the microspores
within the cultured anther
develop into callus tissue or
embryoids that give rise to
haploid plant.
• Production of haploid plants
• Production of homozygous diploid lines through chromosome
doubling, thus reducing the time required to produce inbred
lines
• Uncovering mutations or recessive phenotypes

21. Organogenesis:
The process of initiation and development of a structure
that shows natural organ form and /or function
Embryogenesis:
The process of initiation and development of embryos or
embryo-like structures from somatic cells (somatic
embryogenesis)

22. OrganogenesisSomatic
embryogenesis

23. 1. Contamination
2.Hyperhydricity
3. Phenolic exudation
4. Shoot-tip necrosis
5. Tissue proliferation
6. Somaclonal variation
Problems encountered in tissue culture

24. Contamination:
Surface contaminants:
•Surface sterilization
Internal Contaminants:
•Antibiotics
•Thermotherapy
•Culture of explants free of organisms
Maintenance of asepsis:
•Procedure are carried out in sterile
laminar flow hood

25. Associated the chlorophyll deficiency, poor
lignifications, and excessive hydration of tissue.
Symptoms:
Tissue becomes water-soaked and translucent, have
reduced or retarded growth, thickened and
malformed stem and leaves.
Causes:
•Genotype
•Low potassium concentration
•High NH+4 concentration
•High levels of cytokinin
•Low concentration of solidifying agent
•Low concentration of sucrose
•Inadequate aeration
•An excess of Cl-
Hyperhydricity

26. Many plants, especially medicinal and aromatic plants, are
naturally rich in polyphenolic compounds. When explants are
cut and placed on the in vitro culture medium, these phenolic
compounds are released from cut surface of the explants and
oxidize to form phytotoxic products. As a result of this event,
the media and explants turn brown and the explants are unable
to grow further and eventually die.
•Genotype
•Explant age
•Light intensity
•Sterilization
•Planting date
Phenolic oxidation (Browning)
No additive Ascorbic acid Activated charcoal

27. Overcome:
• choice of juvenile explants or new growth flushes during the
active growth period.
• culture in darkness.
• transfer to fresh medium at short intervals.
• culture in liquid medium.
•Inclusion of antioxidants (ascorbic acid, citric acid) in the
culture medium.
•Use of adsorbing agents (AC, PVP).
• soaking explants in water or solutions containing
antioxidants prior to inoculation.
• choice of a low salt medium and proper growth regulators.

28. Symptoms:
the apical shoot becomes brown
and later dies (browning of buds
and youngest leaves).
Shoot tip necrosis
Causes:
•deficiency of Ca or B
•Ca/B balance
•High Ca in ½ MS medium
•Diluting medium salt concentration
•Excessive subculture
•Absence of cytokinin
•pH fluctuation
•Genotype
•Nature of explant: cuttings with large leave
area

29. Symptoms:
Tissue proliferation is an abnormal tumor-
like growth produced at or near the crown of
the plant, but may also be found on aerial
plant parts of some genotypes.
Causes:
It is most likely caused in sensitive plants by
in vitro conditions and not a pathogen or
genetic mutation
Tissue proliferation

30. Plant tissue culture involves asexual process and, in theory,
should result in clonal propagation of true-to-type
regenerants.
1. Genetic or chimeral variation (Somaclonal variation).
Changes in the DNA sequence.
2. Epigenetic (rejuvenation).
Long-lasting changes in the expression of the information
in the genome.
Variation in micropropagated plants

31. Somaclonal variation involves irreversible
genetic changes originating in cell and tissue
culture.
Somaclonal variation occurs because of genetic
mutation caused by in vitro conditions or by
chimeral separation.
Somaclonal variation
The occurrence of somaclonal variation can
be reduced by:
1. Avoid long term cultures.
2. Use axillary shoot induction systems
where possible.
3. Propagate chimeras by other clonal
systems.

32. •Habituation is now defined more
generally as a stable heritable loss in the
requirement of cultured plant cells for
growth factors.
•Habituation is when a culture continues
to develop in the absence of auxin or
cytokinin.
Habituation

33. Applications of plant tissue culture in crop
improvement
 Micropropagation
1. Clonal reproduction ( generation of genetically identical
plants)
2. Rapid multiplication of rare and elite genotypes such as
Aromatic and Medicinal plants.
3. Mass multiplication of plants which are difficult to propagate
through conventional methods (produced unlimited number
of clones in less time).
4. Easy to manipulate production cycles
5. Production of disease-free plants
6. Development of genetically uniform plants in cross pollinated
crops is possible through tissue culture .

34. •Plant breeding
1. Somaclonal variation and mutation breeding
2. Haploid and dihploid production (anther and pollen
culture)
3. triploids can be produced through endosperm culture.
4. Somatic hybrids and cybrids can be produced through
protoplast fusion (or) somatic hybridization
5. Inter specific and inter generic hybrids can be produced
through embryo rescue technique which is not possible
through conventional method.
6. Development of transgenic plants.
7. Screening of large number of cells in small space.

35. • Germplasm preservation
1. Short-term preservation
2. Medium-term preservation
3. Long-term preservation (Cryopreservation)
•Plant physiology
1. Ovary culture is helpful to know the physiology of fruit
development.
2. Plant tissue culture can also be used for studying the
biochemical pathways and gene regulation
•Production of secondary metabolites.
1. Secondary metabolites are those cell constituents which are not
essential for survival.
2. In vitro production of secondary metabolites is much higher
from differentiated tissues compared to non-differentiated
tissues.
Eg:- Caffine from coffea arabica, Nicotine from Nicotiana rustica.

36. Prepared By
Dr. Rehab AlMousa
Researcher, Biotechnology
Department, GCSAR
Email:
bebo_moussa13@yahoo.com