PLANT TISSUE CULTURE

‫ هديل مكي المؤمن‬.‫د‬.‫م‬.‫أ‬

 What is plant tissue culture
Plant tissue culture is a technique of growing plant
cells, tissues, organs, seeds or other plant parts in a
sterile environment on a nutrient medium
 Advantage of plant tissue culture
• The production of clones of plants that
produce particularly good flowers, fruits, or
have other desirable traits.

• To quickly produce mature plants.

• The production of multiples of plants in the

absence of seeds or necessary pollinators to
produce seeds.

• The regeneration of whole plants from plant

cells that have been genetically modified.
 Advantage of plant tissue culture
• The production of plants in sterile
containers reduces disease transmission

• Allows production of plants from seeds

that otherwise have very low chances
of germinating and growing,
i.e.: orchids and Nepenthes.

• To clean particular plants of viral and other

infections and to quickly multiply these
plants as 'cleaned stock'
for horticulture and agriculture.
 How
Adult plant cells are totipotent
totipotent meaning they have the ability to give rise to a fully
differentiated plant. Because of this, it is possible to collect cells
from a mature plant and use those cells to produce clones of that
plant.
 Plant tissue Culture Basics
• Modern plant tissue culture is performed under
aseptic conditions

• Living plant materials from the environment are

naturally contaminated on their surfaces (and
sometimes interiors) with microorganisms, so
surface sterilization of starting material (explants)
in chemical solutions (usually alcohol and sodium
or calcium hypochlorite is required).
 Plant tissue Culture Basics
• Explants are then usually placed on the
surface of a solid culture medium, but are
sometimes placed directly into a liquid
medium, when cell suspension cultures are
desired.

• Culture media are generally composed of

inorganic salts plus a few organic nutrients,
vitamins and plant hormones.
 Plant tissue Culture Basics
• As cultures grow, pieces are typically sliced off
and transferred to new media (subcultured) to
allow for growth or to alter the morphology of
the culture.
 Plant Tissue Culture Applications
• The commercial production of plants used as potting,
landscape, and florist subjects

• To conserve rare or endangered plant species.

• To screen cells rather than plants for advantageous

characters, e.g. herbicide resistance/tolerance.

• Large-scale growth of plant cells in liquid culture

in bioreactors for production of valuable compounds, like
plant-derived secondary metabolites and recombinant
proteins used as biopharmaceuticals.
 Plant Tissue Culture Applications
• To cross distantly related species by protoplast
fusion and regeneration of the novel hybrid.

• To produce clean plant material from stock

infected by viruses or other pathogens.

• Production of identical sterile hybrid species

can be obtained
 ‫ هديل مكي حبيب‬.‫ د‬.‫م‬.‫أ‬ ‫ المرحلة الثالثة‬/ ‫ نظري‬/ ‫زراعة انسجة نباتية‬

The following points highlight the top ten applications of plant cell and tissue culture.

The applications are:

1. Clonal Propagation and Micro-Propagation

2. Biomass Energy 3. Secondary Metabolites 4. Genetic Variability 5. Somatic

Embryogenesis and Synthetic Seed 6. Breaking Dormancy 7. Haploid Plants 8. Somatic
Hybrids 9. Transgenic Plants 10. Germplasm Conservation

clonal propagation: the rapid multiplication of genetic stocks, including procedures for
isolation of pathogen-free plant materials and freeze-preservation of germplasm;

• embryo culture: the rescue and propagation of immature embryos following wide
hybridization;

• anther culture: for isolation and production of haploid and doubled haploid plants;

• somaclonal variation: genetic variation occurring in somatic cells cultured in vitro;

• somatic cell hybridization: fusion of protoplasts from genetically diverse germplasms;

• genetic engineering: cellular transformation or gene splicing through recombinant

DNA technology
 ‫المحاضرة الرابعة‬

‫‪Callus culture‬‬
Plant callus (plural calluses or calli):- is a mass of
unorganized parenchyma cells derived from plant
tissue (explants) for use in biological research and
biotechnology. In plant biology, callus cells are those
cells that cover a plant wound.
Callus cells are not necessarily genetically
homogeneous because a callus is often made from
structural tissue , not individual cells
Callus formation is induced from plant tissues after
surface sterilization and plating onto in vitro tissue
culture medium. Plant growth regulators, such as
auxins, cytokinins, and gibberellins, are
supplemented into the medium to initiate callus
formation or somatic embryogenesis.
Callus:
• Definition: It is an unspecialized and
unorganized, growing and dividing mass
of cells,.
• During callus formation there is some
degree of dedifferentiation both in
morphology and metabolism, resulting in
the lose the ability to photosynthesis.
• Compact callus
• Friable callus
Plant callus is usually derived from somatic
tissues. The tissues used to initiate callus
formation depends on plant species and
which tissues are available for explant
culture.

The cells that give rise to callus and somatic

embryos usually undergo rapid division or
are partially undifferentiated such as
meristematic tissue.

Plant hormones are used to initiate callus

growth.
Morphology
Specific auxin to cytokinin ratios in plant tissue
culture medium give rise to an unorganized
growing and dividing mass of callus cells.

Callus cultures are often broadly classified as

being either compact or friable. Friable calluses
fall apart easily, and can be used to generate cell
suspension cultures. Callus can directly undergo
direct organogenesis and/or embryogenesis
where the cells will form an entirely new plant.
• The explant is commonly cultured on a
nutrient medium solidified in agar. Explants
from most species of plants may be induced to
divide in an unorganized manner on
specifically formulated nutrient media
• An undifferentiated mass of cells, known as
callus (plural, calli), is formed within 4 to 8
weeks.
• The callus may be divided, with clusters of
cells transferred to fresh agar media to form
subcultures. Repeated subculturing of the
callus permits rapid multiplication of the
cultured material.
• Plant regenerability may decline, and genetic
stability of the plant material may be altered, with
successive subculturing.
• Callus cultures are incubated under aseptic
conditions, normally in dim light, with
temperatures around 25°C.
Callus induction
A callus cell culture is usually sustained on gel
medium. Callus induction medium consists of agar
and a mixture of macronutrients and
micronutrients for the given cell type.
Murashige and Skoog medium,White's medium
(woody plant medium).
Vitamins are also provided to enhance growth such
as B5 vitamins.For plant cells, enrichment with
nitrogen, phosphorus, and potassium is especially
important.
Stages of initiation callus
- Induction stage
- Cell division stage
- Differentiation stage
Callus cells deaths
Callus can brown and die during culture, but
the causes for callus browning are not well
understood.
Browning has also been associated with
oxidation and phenolic compounds in both
explant tissues and explant secretions.
Uses
Nevertheless, callus cells are often
considered similar enough for standard
scientific analysis to be performed as if on a
single subject. For example, an experiment
may have half a callus undergo a treatment
as the experimental group, while the other
half undergoes a similar but non-active
treatment as the control group
Plant calli can differentiate into a whole
plant, a process called regeneration, through
addition of plant hormones in culture
medium. This ability is known as totipotency.
Regeneration of a whole plant from a single
cell allows researchers to recover whole
plants that have a copy of the transgene in
every cell. Regeneration of a whole plant that
has some genetically transformed cells and
some untransformed cells is called a chimera.
In general, chimeras are not useful for
genetic research or agricultural applications.
• Genes can be inserted into callus cells using
biolistic bombardment, also known as a gene
gun, or Agrobacterium tumefaciens. Cells that
receive the gene of interest can then be
recovered into whole plants using a
combination of plant hormones. The whole
plants that are recovered can be used to
experimentally determine gene function(s), or
to enhance crop plant traits for modern
agriculture.
• Callus is of particular use in micropropagation
where it can be used to grow genetically
identical copies of plants with desirable
characteristics
Application of Callus Culture
1 . The whole plant can be regenerated in
large number from callus tissue through
manipulation of the nutrient and hormonal
constituents in the culture medium which is
called as organogenesis or morphogenesis.
Similarly, callus can be induce to form
somatic embryo which can gives rise to
whole plant.
2 . Callus tissue is good source of genetic or
karyotypic variability, so it may be possible to
regenerate a plant from genetically variable
cells of the callus tissue.
3 . Cell suspension culture in moving liquid
medium can be initiated from callus culture.
4 . Callus culture is very useful to obtain
commercially important secondary
metabolites. If a bit tissue from a medicinally
important plant is grown in vitro and
produced callus culture, then secondary
metabolites or drugs can be directly
extracted from the callus tissues without
sacrfting the whole plant.
5 . Several biochemical assays can be
performed from callus culture.
Micropropagation
 Micropropagat
• Micropropagation is the practice of rapidly
multiplying stock plant material to produce a
large number of progeny plants, using modern
plant tissue culture methods
 Introduction
• In nature plants propagate either
• Sexually (seeds generation) results heterogeneity
Or
• Asexually (vegetative multiplication) produce
genetically identical plants.
Multiplication of genetically identical copies of a
cultivar by asexual reproduction is called clonal
propagation.
• Via tissue culture called micropropagation,
Micropropagation
 Advantages
• Micropropagation has a number of advantages over traditional plant propagation
techniques:
• The main advantage of micropropagation is the production of many plants that
are clones of each other.
• used to produce disease-free plants.
• It can have an extraordinarily high fecundity rate, producing thousands of
propagules while conventional techniques might only produce a fraction of this
number.
• regenerating genetically modified cells or cells after protoplast fusion.
• It is useful in multiplying plants which produce seeds in uneconomical amounts,
or when plants are sterile and do not produce viable seeds or when seed cannot be
stored (see recalcitrant seeds).
• Some plants with very small seeds, including most orchids, are most reliably
grown from seed in sterile culture.
• A greater number of plants can be produced per square meter and the propagules
can be stored longer and in a smaller area
 Disadvantages
• Micropropagation is not always the perfect means of
multiplying plants. Conditions that limits its use
include:
• It is very expensive, and can have a labour cost of
more than 70%.
• An infected plant sample can produce infected
progeny.
• Not all plants can be successfully tissue cultured,
often because the proper medium for growth is not
known or the plants produce secondary metabolic
chemicals that kill the explant.
• Some plants are very difficult to disinfect of fungal
organism
 Benefits of Micropropagation
• Many genetically identical plants can be created
from one parent plant
• Because plants are clones, the uniformity assures
quality
• Allows many plants to growing in a small place
in a short time
• In some species this method will produce
healthier plants
 Micropropagation (contin)

• Positives and negatives of micropropagation

– positives
• rapid multiplication rates
• low space requirement
– negatives
• labor costs
• expensive (equipment, facilities, supplies)
• loss by contamination
• danger of variation
Methods of micropropagation

• Axillary branching >95% of all micropropagation

Genetically stable
Simple and straightforward

• Adventitious shoot Efficient but prone to genetic

formation instability

• Somatic Little used, but potentially

embryogenesis phenomenally efficient
 Axillary shoot proliferation
Growth of axillary buds stimulated by cytokinin treatment; shoots
arise mostly from pre-existing meristems
 Clonal in vitro propagation by
repeated enhanced formation
of axillary shoots from shoot-
tips or lateral meristems
cultured on media
supplemented with plant
growth regulators, usually
cytokinins.

 Shoots produced are either

rooted first in vitro or rooted
and acclimatized ex vitro
 Disinfestation

• Stock plant preparation

• Washing in water
• Disinfecting solution
• Internal contaminants
• Screening
 Stage I - Sterilization
Pre-treatments
• Bacteria and fungi will • Transfer plants to a
overgrow the explant greenhouse to reduce
on the medium unless endemic contaminants
they are removed • Force outgrowth of
• Pre-treatments to clean axillary buds
up the explant • Washing removes
• Detergents endemic surface
• Sterilants and contaminants
Antibiotics • Antibiotics,
fungicides, Admire,
others
STAGE II: Shoot Production
•
Stage II selection of cytokinin type and
concentration determined by:

•Shoot multiplication rate

•Length of shoot produced
•Frequency of genetic variability
•Cytokinin effects on rooting and survival
 STAGE II: Shoot Production

•Subculture shoot clusters at 4 -5 week

intervals
•3 -8 fold increase in shoot numbers
• Number of subcultures possible is
species/cultivar dependent
STAGE II: Shoot Production
STAGE III: Pretransplant (rooting)

Goals:

•Preparation of Stage II shoots/shoot clusters

for transfer to soil (prehardening)
•Elongation of shoots prior to ex vitro rooting
•Fulfilling dormancy requirements
 Root initiation ...
Auxins
Co-factors
C : N ratio
Light / darkness
Initiation vs growth
Juvenility / rejuvenation
Genotype
 Shoot elongation ...

• Basal ‘hormone free’ medium

• Gibberellins
• Carry-over of hormones
 Root initiation
• Auxins
• Charcoal
• C : N ratio
• Light / darkness
• Initiation vs growth
• Juvenility / rejuvenation
• Genotype
STAGE III: Pretransplant (rooting)
Acclimatization (hardening)

- survival of the new plant when removed

from the in vitro environment
 Micropropagation of almost all the
fruit crops and vegetables is possible

• Some examples: 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
Production of virus Free Plants
„ Tissue culture is an excellent tool for multiplying,
maintaining, storing and distributing plants.

„ Maintaining unique plants for breeding,

propagation, or distribution can be expensive in
terms of the time, space and labor required.

„ In-vitro propagation of apical meristems is an

important part of virus-elimination therapy for
improving the health of plant collections.

„ Distribution of tissue cultured germplasm often

assists breeders and nurseries in meeting
quarantine regulations.
 Production of virus Free Plants
„ Germplasm may be a source of genes for improving such traits
as insect and disease resistance, improved quality, drought or
cold tolerance, increased yields, low chilling requirements,
adaptation to mechanical harvesting or longer shelf life in
distribution and retail channels.

„ Foreign pathogens continue to pose a considerable threat to

crops. Prevent introduction of quarantine pathogens in
imported prohibited germplasm using a range of diagnostic
techniques to intercept them

„ Develop improved methods of detecting quarantine pathogens

and investigate the etiology of poorly described diseases and
pathogens of quarantine significance

„ Eliminate quarantine pathogens from valuable plant

germplasm

„ Complete therapy on sweet potatoes infected with

geminiviruses.

„ Provide tissues from in vitro therapy for PCR detection of

geminiviruses in sweet potatoes
 WHY DEVELOP CERTIFICATION AND
INDEXING PROGRAM
„ To stop the distribution of viruses in propagative
plant material

„ To avoid losses due to viruses

„ An insurance against distribution of exotic and

destructive viruses

„ To lower the inoculum potential of viruses with in

the country

„ To determine the type of viruses present in a state

or country
 CERTIFICATION AND INDEXING
PROGRAM DEPENDS ON
„ Proper identification and detection of virus

„ Development of sensitive and reliable indexing

method(s)

„ Proper selection of plant material and detection

method

„ Knowledge of host, pathogen and environment

„ Maintenance and multiplication of virus free

material

„ Distribution and Propagation of pathogen free

material under high hygiene
 Meristem tip culture

„ Apical meristem tips (domes with 1-2 leaf

primordia) were excised in sterile conditions either
from in vivo or in vitro plants or highly proliferating
meristems
„ Transferred to glass tubes on 10 ml of solid MS
medium.
„ Tubes were maintained in a growth cabinet (culture
room) at 24 ± 1°C in dark conditions for 3 days,
and then under standard illuminated conditions
Mechanism of virus Elimination in meristems
„ Failure to invade meristem is due to:
1. High auxin concentration in meristematic cells
2. Competition for nutrients enzymes for virus
replication
3. Active metabolic process which is not suitable for
virus multiplication
4. Action of growth regulators (Cytokinins)
5. Presence of inhibitors (Phenolamines)
6. Metabolic disruption of enzymes necessary for viral
replication, RNA degradation
Characteristics of meristem tissues
„ Apical meristem-during embryo development, is a dome of
actively dividing cells located at the apex of shoots and
roots.
„ Plantlets derived from meristem-tip culture usually retain
the genetic characteristics of mother plants
„ Virus elimination from selected plants-do not differ
phenotypically from mother plants
„ Variants comparable to traditional methods of propagation
„ Genetic stability of the germ line is prerequisite
„ Genetic stability of meristems under the strict control of
DNA synthesis.
„ Avoids Spontaneous chromosomal structural changes
„ Undifferentiated tissues such as apical meristems
uniformity of diploid state of cells is maintained
 Micrografting
„ Meritem tip can be grafted onto a
rootstock
„ Meristem (0.1 to 0.4mm) excised
from the infected cultivars
„ Aseptically grafted onto the
vascular ring of a decapitated
virus free rootstock
„ Culture of grafted plantlets in
vitro
„ Transfer plants to soil and
maintain
„ Suitable for woody species (fruit
crops)
„ Indexing for viroid, viruses, and
phytoplasma
 Virus elimination through heat treatment
Preparation for therapy: surface sterilization & culture establishment

Temperature treatment: Three weeks after subculture in

vitro shoots are transferred to a special growth chamber and
grown at an increased temperature regime for 14 to 21 days

Regeneration and indexing: Regeneration of small

plantlets occurs within a few weeks. After indexing virus free
plants selected and multiplied further
Acclimatization: Plantlets are rooted in vitro and
transferred to the greenhouse. After careful hardening potted
plants are re-tested for their pathogen-free status
Multiplication of Virus Free Plants:
For budwood production plants are transferred to an
insect proof screenhouse.
 Thermotherapy
• High temperature treatment has been widely used in production of
virus free plants (30 to 40oC)

Host Virus eliminated Temp.

Chrysanthemum Chrysanthemum B virus 35 to 38oC

Carnation Carnation ringspot virus 35 to 40oC

Carnation vein mottle
virus
Banana Cucumber mosaic virus 35 to 43oC

Goose berry Gooseberry vein banding 35oC

virus
Potato Potato virus Y, S, X 33 to 38oC
 Chemotherapy
„ The use of chemicals to suppress virus symptoms
and multiplication in infected plants
„ Use of antiviral compounds- Ribavirin/Virazole, DTH
„ Growth promoting chemicals-cytokinins
„ Antimetabolite chemicals-Azaguanine, Thiouracil
 Electrotherapy
„ The application of electrical pulses to eliminate viruses from plant
tissue has recently received much attention.
„ Quacquerelli et al. [1980] obtained symptomless almond plants and
Lozoya-Saldana et al. [1996] reported on the elimination of PVX from
different clones of potato.
„ Using an electrotherapy apparatus developed in Cuba (Patent Cuba
37/95 AO 1C/08 1524/97),
„ Hernandez et al. [1995] treated garlic (Allium sativum L), sugar cane
(Saccharum sp. hibrido L.), potatoes (Solanum tuberosum L.) and
araceas (Xanthosomas and Colocasia) for Potyvirus, Luteovirus and
Carlavirus elimination respectively.
„ For banana (cv. W. Bungulan (AAA)), Hernandez et al. [1996]
reported BSV elimination in approximately 40-80% of regenerated
plants.
Virus elimination in onion-Chemotherapy
 Virus Elimination
„ Virus elimination
depends on:
„ Meristem size
„ Type of virus/Phytoplasma
„ Virus strain
„ Plant species
„ Cultivar type
„ Physiological condition of
mother plants and position
of meristem
 Virus indexing

„ Biological assays:
A. Indicator hosts
B. Transmission by graft
inoculation
C. Transmission by Dodder
D. Vector transmission
 Virus indexing
Physical assays: A. Electron microscope
 Virus indexing
„ Serological assays:
A. Immuno Electron microscopy
B. Enzyme linked immunosorbent assay (ELISA)
C. Dot immunobinding assay (DIBA)
D. Tissue blot immuno assay (TIBA)
 Virus indexing
„ Molecular assays:
A. Double stranded RNA (dsRNA) analysis
B. Molecular hybridization analysis:
1. Dot blot assay/ Squash blot
2. Southern blot assay
3. Northern blot assay
C. Microarrays
D. Polymerase Chain reaction (PCR)
1. Multiplex PCR
2. Immunocapture PCR (IC-PCR)
3. Nested PCR
4.Competitive fluorescence PCR (CF-PCR)
Development of Membrane based nucleic
acid Protocol for PCR detection of BBTV
1. Blotting of tissue on membrane

2. Dissolving of membrane or mixing

with PCR reaction mixture
12 3 4 5 M

3. PCR amplification

4. Visualization of bands

PCR detection
 Development of nucleic acid probes for
the Diagnosis of Banana Streak Virus

H 1 2 3 4 5 6 7

Colorimetric detection by dot hybridization

M 1 2

Chemiluminesnce detection
by Dot Blot Assay
PCR Amplification and labeling of DNA
Virus indexing by PCR
Citrus Yellow corky vein Viroid
Diagnosis of Citrus Witches Broom

PCR Amplification of Phytoplasma

ITS rDNA
C CB H N D d n h b

0.5 0.5
Tissue culture indexing
 Detection of Viruses in Tissue Culture
Banana From M/S Cadilla, Ahamdabad

Crop Virus No Detection of Samples

Test by
ed EM ELIS NAP PCR
A
Banana BBTV 50 NT NT 12 12
BBrMV 56 0 22 NT 12
BSV 50 NT NT 0 1
CMV 53 0 2 NT 2
Unknown 10 3 3 NT NT
Total 209 3 27 12 27
 Detection of Viruses in TC Plants from
Shri Ramco Biotech, Bangalore
Crop Viruses No Method of Testing by
Teste EM ELIS NAP PCR
d
Banana BBTV 70
70 NT A NT 4 8
BBrMV 115 6 4 NT 2
BSV 70 4 NT 0 8
CMV 70 8 12 NT 8
Unknow 20 10 6 NT NT
Caladiu n
DsMV 12 10 4 NT NT
m Poyvirus 12 10 6 NT 4
INSV 12 NT 0 NT NT
Total 381 48 32 4 30
 IIHR

IIHR
Valuable Chemical Production
 1. Plants produce secondary
metabolites
• Primary metabolites run $1 to $2 per pound
• Secondary metabolites run up to several
hundred thousand dollars per pound
 Primary Metabolites
• are substances that are widely distributed in
nature, occurring virtually in all organisms. In
higher plants these substances would be
concentrated in seeds and vegetable storage
organs. There are needed for general growth
and development. Primary metabolites are low
value-high bulk commodity items from plants
(e.g. amino acids, starch, sugars, vegetable
oils, etc.)
 Secondary Metabolites
• are biosynthetically derived from primary
metabolites. They are more limited in
distribution being found usually in specific
families. They are not necessary for growth
and development, but may serve as pollination
attractants, environmental adaptations, or
protection.
 Kinds of Secondary Metabolites
• alkaloids
• phenolics (including polyphenols and tannins)
• terpenoids
2. Establishing a plant cell culture
for secondary metabolite production
is a complex problem
 Not all cell types produce the desired
metabolite
• Within a specific cultivar of Catharanthus
roseus, 62% of the clones produced the desired
metabolite
• whereas in another only 0.3% produced the
metabolite
 Culture conditions must be optimized
• e.g. concentrations of sugar, hormones, and
vitamins
• light
• temperature
 Metabolite production is frequently
higher in cell cultures
• Berberine production from Coptis japonica is
about 5% of dry weight after 5 years of root
growth, which equals 0.17 mg/g per week.
• Whereas in selected cell lines it can be 13.2%
of the dry weight in cell culture after 3 weeks,
which is about 44 mg/g/week or about 250
times higher
3. Metabolites can be produced in
root cultures
 Many secondary metabolites are
produced in roots
• Scientists have developed a form of root culture
using Agrobacterium rhizogenes, the cause of
hairy root disease. (Show Fig 14.3)
• Cells transformed with some of the bacteria’s
DNA, causes the cells to be more sensitive to the
hormones they produce. The cells form into roots.
These roots grow very fast and produce the
secondary metabolites that ordinary roots
produce.
Root cultures are often better than cell
cultures
• Roots often secrete the metabolites into the
surrounding medium, making it easy for
collection.
• Charcoal can be added to the medium, the
metabolites are absorbed by the charcoal, and
this stimulates even higher production of the
metabolite.
Biochemical pathways of secondary
metabolites can be quite long

(sometimes up to 12 steps)

Precursors can be fed to either cell culture or

roots to produce the metabolite in question.

In addition, cells can be genetically engineered to

over-produce the metabolite, but this may be
more difficult with pathways that have many
enzymes.
Some secondary metabolites produced
in cell and root culture
• L-DOPA: a precursor of catecholamines, an
important neurotransmitter used in the treatment
of Parkinson’s disease
• Shikonin: used as an anti-bacterial and anti-ulcer
agent
• Anthraquinone: used for dyes and medicinal
purposes
• Opiate alkaloids: particularly codeine and
morphine for medical purposes
• Berberine: an alkaloid with medicinal uses for
cholera and bacterial dysenterry
• Rosmarinic acid: for antiviral, suppression of
endotoxin shock and other medicinal purposes
• Quinine: for malaria
• Cardenolides or Cardioactive glycosides: for
treatment of heart disease
Some goals are to eliminate secondary
metabolites
• Caffeine: to produce caffeine-free plants
 Taxol: an example
• Taxol is a unique anticancer drug from the
bark of the Pacific Yew (Taxus breviola)
 Taxol Facts
• Very effective treatment against ovarian
cancer, breast cancer, melanoma, and colon
cancer
• Stops cell division, thus blocking cancer. It
does this by interfering with microtubule
function. Microtubules are responsible for
pulling apart the sets of chromosomes in
mitosis.
 Taxol is a very good target for
biotechnology
• a) tissue culture of bark cells
• b) fungus produces taxol
• c) alternative species
• d) genetic engineering
• e) chemical synthesis
 a) tissue culture of bark cells
• Many cells from different bark tissues from
different trees were screened.
• There are at least 25 fold differences in
production. It was found to be secreted into
the medium thus facilitating collection.
• So far 1 to 3 mg of taxol are produced per liter
of cell culture. This is equivalent to about 25 g
of bark.
 b) fungus produces taxol
• It was found that a fungus that colonizes yew
trees also produces taxol
• Fungal culture technology which is better
developed than plant cell culture technology
could be an important source for taxol
production
 c) alternative species
• Some researchers found that the European Yew
(Taxus baccata) produces a precursor to taxol.
• This precursor can then be converted to an
analog of taxol in the laboratory.
• The precursor is used for chemical synthesis of
taxol.
 d) genetic engineering
• Other scientists are trying to identify and clone
the genes which produce taxol
• This will enable them to scale up production in
cell culture
 e) Chemical synthesis
• Until 1994, chemical synthesis was formidable
• 3 different ways to synthesize taxol are now
known
• Some take up to 13 steps
• Cost per patient still expensive; about $20,000
4. The economics of large-scale plant cell
culture favor only a few products at the
present time
This is because it usually takes 10 years of research to
produce a product. This requires that a product sell for at
least $400 per kg to make it economically worthwhile.
 5. Producing secondary metabolites in tissue
culture may have a negative impact on the
economics of the Third World countries
Many of these Third World countries may lose
market share to superior, more efficient production
of secondary metabolites in industrial countries.
Is this right? Is it fair? Are third world countries
capable of competing? What should they do?
• Genetic variations in plants that have been
produced by plant tissue culture and can be
detected as genetic or phenotypic traits

Basic Features of Somaclonal Variations

• Variations for Karyotype, isozyme

characteristics and morphology in
somaclones may also observed.
• Calliclone (clones of callus), mericlone (clones
of meristem) and protoclone (clones of
Protoplast) were produced.
• Generally heritable mutation and persist in plant
population even after plantation into the field
 Mechanism of Somaclonal Variations
1. Genetic (Heritable Variations)
• Pre-existing variations in the somatic cells of
explant
• Caused by mutations and other DNA changes
• Occur at high frequency

2. Epigenetic (Non-heritable Variations)

• Variations generated during tissue culture
• Caused by temporary phenotypic changes
• Occur at low frequency
 Callus Tissue

Organogenesis Somaclonal Variants

Regenerated plants Hardening and Selfing

Steps involved in induction and selection of Somaclonal Variations

 Causes of Somaclonal
Variations

Biochemical
Physiological
Cause
Cause

Genetic Cause
 Physiological Cause

• Exposure of culture to plant growth

regulators.

• Culture conditions
 Genetic Cause

1. Change in chromosome number

• Euploidy: Changes chromosome Sets
• Aneuploidy: Changes in parts of chromosome Sets
– Polyploidy: Organisms with more than two chromosome
sets
– Monoploidy: Organism with one chromasomes set

2. Change in chromosome structure

• Deletion
• Inversion
• Duplication
• Translocation
 Genetic Cause

3. Gene Mutation
• Tansition
• Transversion
• Insertion
• Deletion
4. Plasmagene Mutation

5. Transposable element activation

 Genetic Cause

6. DNA sequence

 Change in DNA
 Detection of altered fragment size by using Restriction enzyme

 Change in Protein
 Loss or gain in protein band
 Alteration in level of specific protein

 Methylation of DNA
 Methylation inactivates transcription process.
 Biochemical Cause

• Lack of photosynthetic ability due to alteration

in carbon metabolism

• Biosynthesis of starch via carotenoid pathway

• Nitrogen metabolism

• Antibiotic resistance.
Detection and Isolation of Somaclonal Variants

1. Analysis of morphological characters

• Qualitative characters: Plant height, maturity date, flowering
date and leaf size
• Quantitative characters: yield of flower, seeds and wax contents
in different plant parts

2. Variant detection by cytological Studies

• Staining of meristematic tissues like root tip, leaf tip with
feulgen and acetocarmine provide the number and morphology
of chromosomes.

3. Variant detection by DNA contents

• Cytophotometer detection of feulgen stained nuclei can be
used to measure the DNA contents
 Detection and Isolation of Somaclonal Variants

4. Variant detection by gel electrophoresis

• Change in concentration of enzymes, proteins and hemical products
like pigments, alkaloids and amino acids can be detected by their
electrophoretic pattern
5. Detection of disease resistance variant
• Pathogen or toxin responsible for disease resistance can be used as
selection agent during culture.
6. Detection of herbicide resistance variant
• Plantlets generated by the addition of herbicide to the cell culture
system can be used as herbicide resistance plant.
 Detection and Isolation of Somaclonal Variants

7. Detection of environmental stress tolerant variant

• Selection of high salt tolerant cell lines in tobacco
• Selection of water-logging and drought resistance cell lines in
tomato
• Selection of temperature stress tolerant in cell lines in pear.
• Selection of mineral toxicities tolerant in sorghum plant (mainly for
aluminium toxicity)
 Advantages of Somaclonal Variations

• Help in crop improvement

• Creation of additional genetic varitions
• Increased and improved production of
secondary metabolites
• Selection of plants resistant to various toxins,
herbicides, high salt concentration and
mineral toxicity
• Suitable for breeding of tree species
 Disadvantages of Somaclonal Variations

• A serious disadvantage occurs in operations which require

clonal uniformity, as in the horticulture and forestry
industries where tissue culture is employed for rapid
propagation of elite genotypes
• Sometime leads to undesirable results
• Selected variants are random and genetically unstable
• Require extensive and extended field trials
• Not suitable for complex agronomic traits like yield, quality
etc.
• May develop variants with pleiotropic effects which are not
true.
Protoplast culture
Somatic fusion, also called protoplast fusion, is a
type of genetic modification in plants by which two
distinct species of plants are fused together to form a
new hybrid plant with the characteristics of both, a
somatic hybrid. Hybrids have been produced either
between different varieties of the same species (e.g.
between non-flowering potato plants and flowering
potato plants) or between two different species (e.g.
between wheat triticum and rye secale to produce
Triticale).
Uses of somatic fusion include making potato plants
resistant to potato leaf roll disease.[1] Through
somatic fusion, the crop potato plant Solanum
tuberosum – the yield of which is severely reduced by
a viral disease transmitted on by the aphid vector – is
fused with the wild, non-tuber-bearing potato
Solanum brevidens, which is resistant to the disease.
The resulting hybrid has the chromosomes of both
plants and is thus similar to polyploid plants. Somatic
Hybridization was first introduced by Carlson
The somatic fusion process occurs in four steps:
• The removal of the cell wall of one cell of each
type of plant using cellulase enzyme to produce a
somatic cell called a protoplast
• The cells are then fused using electric shock
(electrofusion) or chemical treatment to join the
cells and fuse together the nuclei. The resulting
fused nucleus is called heterokaryon.
• The somatic hybrid cell then has its cell wall
induced to form using hormones
• The cells are then grown into calluses which then
are further grown to plantlets and finally to a full
plant, known as a somatic hybrid
 Somatic hybridization technique

1. isolation of protoplast from suitable plants

2. Fusion of the protoplasts of desired species/varieties

3. Identification and Selection of somatic hybrid cells

4. Culture of the hybrid cells

5. Regeneration of hybrid plants

‫ هديل مكي المؤمن‬.‫ د‬.‫م‬.‫أ‬ ‫ المرحلة الثالثة‬/ ‫ نظري‬/ ‫زراعة انسجة نباتية‬

History of Plant Tissue Culture

Plant tissue culture, or the aseptic culture of cells, tissues, organs, and their
components under defined physical and chemical conditions in vitro, is an
important tool in both basic and applied studies as well as in commercial
application. It owes its origin to the ideas of the German scientist, Haberlandt, at
the beginning of the 20th century

1- Perhaps the earliest step towards plant tissue culture was made by Henri-
Lous Duhumel du Monceau in 1756, who during studies on wound healing
in plants, observed callus formation
2- Scheilden and Schwann put forward the co-called totipotency theory, which
states that cells are autonomic, and in principle, are capable of regenerating
to give a complete plant. Their theory was in fact the foundation of plant cell
and tissue culture.(proposed that cell is the basic unit of organisms. They
visualized that cell is capable of autonomy and therefore it should be
possible for each cell if given an environment to regenerate into whole
plant.).
3- in 1902, a German physiologist, Gottlieb Haberlandt developed the concept
of in vitro cell culture. He isolated single fully differentiated individual plant
cells from different plant species like palisade cells from leaves of Laminum
purpureum, glandular hair of Pulmonaria and pith cells from petioles of
Eicchornia crassiples etc and was first to culture them in Knop’s salt
solution enriched with glucose. But the effort failed because the cells do not
have cleavage, it was allegedfailures because they do not use growth
regulator substances needed for cell division, proliferation and induction of
the embryo. Haberlandt made several predictions about the requirements in
media in experimental conditions which could possibly induce cell division,
 proliferation and embryo induction. G Haberlandt is thus regarded as father
of tissue culture. But totipotency term coined by Steward in 1968.
4- Hannig (1904) chose embryogenic tissue to culture. He excised nearly
mature embryos from seeds of several species of crucifers and successfully
grew them to maturity on mineral salts and sugar solution.
5- In 1908, Simon regenerated callus, buds and roots from Poplar stem
segments and established the basis for callus culture
6- Following the unsuccessful in vitro cultivation of root tips by Kotte, a
student of Haberlandt in Germany and Robins in 1912,
7- White.. In the early 1920s workers again attempted to grow plant tissues and
organ in vitro , then White in (1934) developed the first permanent root and
meristem cultures of Lycopersicon esculentum. White generated
continuously growing culture of meristematic cells of tomato on medium
containing inorganic salts, yeast extract and sucrose and 3 vitamins B
(pyridoxine, thiamine, nicotinic acid) – established the importance of
additives.
8- White, 1930’s White worked on T.C. discovery of plant growth regulators,
published independently studies on the successful cultivation for prolonged
periods of cambial tissues of carrot root (Gautheret, 1939) tobacco (White,
1939) and carrot (Nobecourt, 1939).

Then having achieved success and expertise in growth of callus cultures from
explants under in vitro conditions and focus now shifted to preparation of single
cell cultures

Haberlandt’s objectives was development of whole plant from the proliferated

tissue of these cells. Vasil and Hilderbrandt were first to regenerate plantlets
from colonies of isolated cells of hybrid Nicotiana glutinosa x N tabacum.

* In 1962 Murashige and Skoog published recipe for MS Medium

* Important discoveries in the history of plant tissue culture.

* 1838 Totipotency theory (Schwann and Scheilden) – cells are autonomic,
and in principle, are capable of regenerating to give a complete plant
* 1902 First attempt at plant tissue culture (Haberlandt)
* 1904 First attempt at embryo culture of selected crucifers (Hannig)
* 1909 Fusion of plant protoplasts, although the products failed to survive
(Kuster)
* 1922 Asymbiotic germination of orchid seeds in vitro (Knudson) In vitro
culture of root tips (Robbins)
* 1925 Embryo culture applied in interspecific crosses of Linum (Laibach)
* 1929 Embryo culture of Linum to avoid cross incompatibility (Laibach)
* 1934 In vitro culture of the cambium of a few trees and shrubs failed to be
sustained since auxin had not yet been discovered (Gautheret) Successful
culture of tomato roots (White)
* 1939 Successful continuously growing callus culture (Gautheret, Nobecourt
and White)
* 1940 In vitro culture of cambial tissues of Ulmus to study adventitious shoot
formation (Gautheret)
* 1941 Coconut milk (containing a cell division factor) was the first time used
for the culture of Datura embryos (van Overbeek). In vitro culture of crown-
gall tissues (Braun)
* 1944 First in vitro cultures of tobacco used to study adventitious shoot
formation (Skoog)
* 1948 Formation of adventitious shoots and roots of tobacco determined by
the ratio of auxin/adenin (Skoog and Tsui)
* 1950 Organs regenerated from callus tissue of Sequoia sempervirens (Ball)
* 1952 Virus-free dahlias obtained by meristem culture (Morel and Martin).
First application of micro-grafting (Morel and Martin)
* 1953 Haploid callus of Gingko biloba produced from pollen (Tulecke)
* 1954 Monitoring of changes in karyology and in chromosome behavior of
endosperm cultures of maize (Strauss)
* 1955 Discovery of kinetin, a cell division hormone (Miller et al.)
* 1956 Realization of growth cultures in multi-litre suspension systems to
produce secondary products by Tulecke and Nickell (Staba, 1985)
* 1957 Discovery of the regulation of organ formation (roots and shoots) by
changing the ratio of cytokinin/auxin (Skoog and Miller)
* 1958 Regeneration of somatic embryos in vitro from the nucellus of Citrus
ovules (Maheshwari and Rangaswamy). Regeneration of pro-embryos from
callus clumps and cell suspensions of Daucus carota (Reinert, Steward)
* 1960 First successful test tube fertilization in Papavar rhoeas (Kanta)
Enzymatic degradation of the cell walls to obtain large numbers of
protoplasts (Cocking). Vegetative propagation of orchids by meristem
culture (Morel) Filtration of cell suspensions and isolation of single cells by
plating (Bergmann).
* 1962 The development of the famous Murashige and Skoog medium
(Murashige and Skoog).
* 1964 First haploid Datura plants produced from pollen grains (Guha and
Maheshwari). Regeneration of roots and shoots on callus tissue of Populus
tremuloides (Mathes
* 1965 Induction of flowering in tobacco tissue in vitro (Aghion-Prat).
Differentiation of tobacco plants from single isolated cells in micro-culture
(Vasil and Hilderbrandt)

* 1971 First plants regenerated from protoplasts (Takebe et al.)

* 1972 Interspecific hybridization through protoplast fusion in two Nicotina
species (Carlson et.al.)