Plant Tissue Culture

Plant Tissue Culture
Dr. Isam Fattash

INTRODUCTION
One of the most interesting areas of biotechnology is
tissue culture and micropropagation.
Tissue culture is the ability to establish and maintain

plant organs (embryos, shoots, roots, and flowers) and
plant tissues (cells, callus, and protoplasts) in aseptic
culture.
Plasticity and Totipotency
 Plasticity allows plants to alter their metabolism, growth, and

development to best suit their environment.
Totipotency: 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.
The culture environment
Both chemical (see Table 2.1) and physical, have to met by the culture
vessel, the growth medium, and the external environment (light,
temperature, etc.).
The growth medium has to supply all the essential mineral ions
required for growth and development.
 It must also supply additional organic supplements such as amino
acids and vitamins.
Addition of a fixed carbon source in the form of a sugar (most often
sucrose).
Physical factors, such as temperature, pH, the gaseous environment,
light (quality and duration), and osmotic pressure
Plant cell culture media
Culture media used for the cultivation of plant cells in vitro are
composed of three basic components:
1. Essential elements, or mineral ions, supplied as a complex mixture of
salts.
2. Organic supplement supplying vitamins and/or amino acids.
3. Source of fixed carbon; usually supplied as the sugar sucrose.

Plant cell culture media
For practical purposes, the essential elements are further divided into
the following categories:
1. Macroelements (or macronutrients).
2. Microelements (or micronutrients).
3. Iron source.
Media components
Macroelements:
• Nitrogen, phosphorus, potassium, magnesium, calcium, and sulphur
(and carbon, which is added separately) are usually regarded as
macroelements.
• Nitrogen is most commonly supplied as a mixture of nitrate ions
(from KNO3) and ammonium ions (from NH4NO3).
• High concentrations, ammonium ions can be toxic to plant cell
cultures and uptake of ammonium ions from the medium causes
acidification of the medium.
• High concentrations of ammonium ions can also cause culture
problems by increasing the frequency of vitrification.
Media components
• Phosphorus is usually supplied as the phosphate ion of ammonium,
sodium, or potassium salts.
• High concentrations of phosphate can lead to the precipitation of
medium elements as insoluble phosphates.
Media components
Microelements:
• Manganese, iodine, copper, cobalt, boron, molybdenum, iron, and
zinc usually comprise the microelements.
• Iron is usually added as iron sulphate, although iron citrate can also
be used.
Media components
Organic supplements:
• Only two vitamins, thiamine (vitamin B1) and myoinositol (considered
a B vitamin), are considered essential for the culture of plant cells in
vitro.
• Amino acids; The most frequently used is glycine (arginine,
asparagine, aspartic acid, alanine, glutamic acid, glutamine, and
proline are also used).
• Amino acids provide a source of reduced nitrogen and, like
ammonium ions, uptake causes acidification of the medium. Casein
hydrolysate can be used as a relatively cheap source of a mix of amino
acids.
Media components
Carbon source
Sucrose is cheap, easily available, readily assimilated, and relatively
stable, and is therefore the most commonly used carbon source.
Gelling agents
Agar, produced from seaweed, is the most common type of gelling
agent.
Plant growth regulators
Classes of plant growth regulator
(1) Auxins
(2) Cytokinins
(3) Gibberellins
(4) abscisic acid
(5) Ethylene.
Auxins
• Auxins promote both cell division and cell growth.
• Naturally occurring auxin is indole-3-acetic acid (IAA), but its use in

plant cell culture media is limited because it is unstable to both heat
and light.
• 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used

auxin and is extremely effective in most circumstances.
Cytokinins
• Cytokinins promote cell division.
• Naturally occurring cytokinins; zeatin and N6-(2-isopentyl)adenine

(2iP). They are expensive (particularly zeatin) and relatively unstable.
• The synthetic analogues kinetin and 6-benzylaminopurine (BAP) are

therefore used more frequently.
Gibberellins
Involved in regulating cell elongation, and are agronomically important
in determining plant height and fruit-set. GA3 being the most common.
Abscisic acid
Abscisic acid (ABA) inhibits cell division.
Ethylene
Ethylene is a gaseous, naturally occurring and controlling fruit ripening
in climacteric fruits.
Plant growth regulators and tissue culture
Figure 17–2 The relationship between the ratio of cytokinin to auxin on organogenesis in culture. A high
cytokinin to auxin ratio tends to promote shoot organogenesis, while a low cytokinin to auxin ratio promotes
rooting. More equal concentrations tend to promote both shoots and roots from the same culture or
undifferentiated callus.
Culture types
Callus
• Unorganized, growing, and dividing mass of cells
• Composed of unspecialized parenchyma cells
• Lose the ability to photosynthesize
• Habituation; longterm culture where the culture may lose the

requirement for auxin and/or cytokinin.
Figure 17–28 Callus developed on a
stationary medium showing nicely that
different parts of the callus have different
cellular characteristics.
Culture types
Cell-suspension cultures
• Callus cultures fall into one of two categories: compact or friable.
• In compact callus, the cells are densely aggregated.
• In friable callus, the cells are only loosely associated with each other
and the callus becomes soft and breaks apart easily.
• Friable callus provides the inoculum to form cell-suspension cultures.
• After subculture, the cells divide and the biomass of the culture
increases in a characteristic fashion, until nutrients in the medium are
exhausted and/or toxic byproducts build up to inhibitory levels: this is
called the stationary phase. If cells are left in the stationary phase for
too long, they will die and the culture will be lost.
Figure 17–29 Suspension culture of
soybean (Glycine) containing
proembryogenic masses.
Culture types
Protoplasts
Figure 17–30 Protoplasts from

tulip leaves and flower petals. (a)
In leaf protoplasts, epidermal cells
lack chloroplasts (red arrow) while
those from palisade and
mesophyll cells have chloroplasts
(white arrow). (b) In flower petal
protoplasts, the red and blue cells
contain anthocyanins that give the
flower petals their color.
TYPES OF TISSUE CULTURE SYSTEMS
• Micropropagation of Plantlets from Tissue Culture

• Developmental Stages in Micropropagation
– Stage I: Establishment and Stabilization of Explants in Culture
– Stage II: Shoot Multiplication
– Stage III: Root Formation
– Stage IV: Acclimatization
• Systems Used to Regenerate Plantlets by Micropropagation
– Axillary Shoot Formation
– Adventitious Shoot Formation
Hartmann and Kester’s Plant Propagation Principles and Practices 8e Copyright ©2011, 2002, 1997, 1990, 1983, 1975, 1968, 1959 by Pearson Education, Inc.
Hudson Hartman, Dale Kester, Fred Davies and Robert Geneve Upper Saddle River, New Jersey 07458 • All rights reserved.
Figure 17–4 Typical microbial growth in a tissue culture from (a) fungal and yeast and (b)
internal bacterial contamination.
Figure 17–5 Kentucky coffee tree (Gymnocladus dioicus) fails to stabilize when cultured from explants
collected from mature trees. (a) Short shoots are produced that fail to elongate. (b) However, explants
from juvenile explants (69) and explants from stump sprouts of mature trees will stabilize and form
elongated shoots for micropropagation.
Figure 17–6 Generalized scheme of the three important phases in the microculture period through
which a shoot must progress to be successfully microcultured. The second period is rooting and
acclimatization of shoots.
Figure 17–7 Illustration of shoot induction and morphology in tobacco leaf discs with increasing
cytokinin concentration. (a) An insufficient concentration of cytokinin leads to little growth. (b)
Suboptimal amounts induce few shoots and some callus. (c) An optimal concentration induces
numerous well-formed elongating shoots. (d) At superoptimal concentrations, shoots are initiated but
become malformed and fail to elongate.
Figure 17–8 Although cytokinin promotes shoot formation, high

concentrations also inhibit shoot elongation. The culture of gas plant
(Dictamnus albus) on the left was treated with 5 μM benzyladenine
(BA), while the culture on the right was treated with 20 μM BA (123).
Figure 17–9 Relationship between cytokinin

(BA) concentration and shoot initiation in shoot
cultures of eastern redbud (Cercis canadensis)
(275). The number of shoots per explant
increases as the cytokinin level increases. This
response is consistent between subcultures,
but the overall number of shoots per explant
increases until the cultures stabilize.
 Root cultures
Figure 17–12 Root formation in
microcuttings of eastern redbud (Cercis
canadensis). (a) Root formation in vitro.
It is common that roots on NAA-treated
microcuttings (left) are shorter and
thicker than roots on IBA-treated
microcuttings (right). IBA is used most
often to root microcuttings in a wide
variety of species. (b) Three cultivars of
redbud microcuttings rooted ex vitro. (c)
Anatomy of in vitro and (d) ex vitro
developed roots. Observe the swollen
cortical cells and the less developed
vascular system on in vitro roots.
Figure 17–13 Scanning electron

micrograph of stomata from the lower
leaf surface of carnation plants grown in
the greenhouse (top) or in vitro
(bottom). Note lack of wax on in vitro–
grown plants.
 Shoot tip and meristem culture
Figure 17–14 Patterns of plantlet development by micropropagation: (a) Axillary

shoot formation directly from existing buds from nodal explants. (b) Adventitious
shoots formed directly from tissue without pre-existing buds. (c) Adventitious
shoots formed indirectly after callus develops from the initial explant.
Figure 17–15 Shoot tip of carnation stem with outer leaves removed,
showing the apical and lateral meristems (growing points). Part of the
shoot tip to be excised for culturing is indicated in lines.
Figure 17–16 Multiple shoots formed by

an axillary branching type of culture. (a)
An explant with several nodes. (b) Shoots
proliferating from axillary buds.
Figure 17–17 Shoots formed by a nodal type

of culture. (a) Explants show strong apical
dominance with very little axillary branching.
The elongated shoot will be subcultured by
being cut into several single-node explants. (b)
A cotyledonary node explant showing
elongation of shoot buds in the axils of the pair
of cotyledons.
Figure 17–18 Stool shoots formed by

horizontally placed explants. (a) Multiple
node explants without leaves or an apical
meristem are placed horizontally on the
medium. (b) Single shoots arise from
existing buds at each node.
Figure 17–19 Shoot formation via pseudocorms produced by orchids. (a) Proliferating pseudocorms
on a charcoal medium. (b) Numerous pseudocorms with the medium washed away from the roots,
ready for transplanting to the greenhouse.
Figure 17–20 A specialty cactus being

micrografted.
Figure 17–21 A leaf explant showing direct formation of

adventitious shoots.
Figure 17–22 Isolated root explant from

Kentucky coffee tree (Gymnocladus
dioicus) showing both (a) direct and (b)
indirect shoot formation.
Figure 17–23 Thin layer explants of

epidermal tissue showing organ initiation
potential relative to location on the mother
plant where the explant was taken (257).
Figure 17–24 Hosta showing a progression from (a) initial flower stem explant to (b
and c) multiple adventitious shoot formation.
Figure 17–25 The formation of lily bulbs in tissue culture. (a) Bulbs initiating from leaf scales. (b)
Vegetative leaf growth from regenerated bulbs.
Culture types
Embryo culture
• Embryos can be used as explants to generate callus cultures or
somatic embryos. Both immature and mature embryos can be used
as explants
Microspore culture
• Haploid tissue can be cultured in vitro by using pollen or anthers as an
explant.
• Both callus and embryos can be produced from pollen.
Culture types
 Two main approaches can be taken to produce cultures in vitro from
haploid tissue.
1. Anthers (somatic tissue that surrounds and contains the pollen)
can be cultured on solid medium. Pollen-derived embryos are
subsequently produced via dehiscence of the mature anthers.
2. Anthers can also be cultured in liquid medium, and pollen released
from the anthers can be induced to form embryos, although the
efficiency of plant regeneration is often very low.
• Plants obtained from haploid cultures may not be haploid. This can be
a consequence of chromosome doubling during the culture period.
Such plants are often referred to as di-haploids, because they contain
two copies of the same haploid genome.
Plant regeneration
1. Somatic embryogenesis
• In somatic (asexual) embryogenesis, embryo-like structures, which
can develop into whole plants in a way analogous to zygotic embryos,
are formed from somatic tissues (Figure 2.2).
• These somatic embryos can be produced either directly or indirectly.
• In direct somatic embryogenesis, the embryo is formed directly from
a cell or small group of cells without the production of an intervening
callus.
• In indirect somatic embryogenesis, callus is first produced from the
explant.
• Embryos can then be produced from the callus tissue or from a cell
suspension produced from that callus.
Plant regeneration
• Somatic embryogenesis usually proceeds in two distinct stages.
1. In the initial stage (embryo initiation), a high concentration of 2,4-D is used.
2. Embryos are produced in a medium with no or very low levels of 2,4-D.
2. Organogenesis
• Organogenesis relies on the production of organs, either directly from
an explant or from a callus culture.
• There are three methods of plant regeneration via organogenesis.
• The first two methods depend on adventitious organs arising either
from a callus culture or directly from an explant.
• The third method is by axillary bud formation and growth, which can
also be used to regenerate whole plants from some types of tissue
culture.

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Plant Tissue Culture

Dr. Isam Fattash

Tissue culture is the ability to establish and maintain

 Plasticity allows plants to alter their metabolism, growth, and

Totipotency: the abilities to initiate cell division from almost any

2. Organic supplement supplying vitamins and/or amino acids.

3. Source of ﬁxed carbon; usually supplied as the sugar sucrose.

2. Microelements (or micronutrients).

• Naturally occurring auxin is indole-3-acetic acid (IAA), but its use in

• 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used

• Naturally occurring cytokinins; zeatin and N6-(2-isopentyl)adenine

• The synthetic analogues kinetin and 6-benzylaminopurine (BAP) are

• Unorganized, growing, and dividing mass of cells

• Composed of unspecialized parenchyma cells

• Lose the ability to photosynthesize

• Habituation; longterm culture where the culture may lose the

Figure 17–30 Protoplasts from

• Micropropagation of Plantlets from Tissue Culture

Figure 17–8 Although cytokinin promotes shoot formation, high

Figure 17–9 Relationship between cytokinin

Figure 17–13 Scanning electron

Figure 17–14 Patterns of plantlet development by micropropagation: (a) Axillary

Figure 17–16 Multiple shoots formed by

Figure 17–17 Shoots formed by a nodal type

Figure 17–18 Stool shoots formed by

Figure 17–20 A specialty cactus being

Figure 17–21 A leaf explant showing direct formation of

Figure 17–22 Isolated root explant from

Figure 17–23 Thin layer explants of

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