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2012 Plant Tissue Media
2012 Plant Tissue Media
Media
1
Logical Basis
For healthy and vigorous growth, intact plants need to take
up from soil of an essential elements.
2
Essential element
Macro element/major plant nutrition:
Relatively large amount required
a. Carbon (C) d. Nitrogen (N) g. Potassium (K)
b. Hydrogen (H) e. Calcium (Ca) h. Phosphorus (P)
c. Oxygen (O) f. Magnesium (Mg) i. Sulphur (S)
Micro element/ minor plant nutrient/trace elements:
Small quantities required
a. Iron (Fe) f. Sodium (Na)
b. Chlorine (Cl) g. Manganese (Mn)
c. Zinc (Zn) h. Boron (B)
d. Copper (Cu) i. Molybdenum (Mo)
e. Nickel (Ni)
3
Why plant in vitro culture needs
media?
Functions of media:
Provide water
Provide mineral nutritional needs
Provide vitamins
Provide growth regulators
Provide amino acids
Provide sugars
Access to atmosphere for gas exchange
Removal of plant metabolite waste
Plant tissue culture media
1. Macronutrients (always employed)
2. Micronutrients (nearly always employed, although sometimes just
one element, iron, has been used)
3. Vitamins (generally incorporated , although the actual number of
compounds added, varies greatly)
4. Amino acids and other nitrogen supplements (usually omitted, but
sometimes used with advantage)
5. Sugar (nearly always added, but omitted for some special purposes)
6. Undefined supplements (which, when used, contribute some above
components, and also plant growth substances or regulants)
7. Buffers (have seldom been used in the past, but recently suggest
that the additions of organic acids or buffers could be beneficial in
some circumstances)
8. A solidifying agent (used when a semi solid medium is required)
5
Macronutrient
1. Macronutrients for plant tissue culture are provided from
salt, however plant absorb entirely as ions
2. Nitrogen is mainly absorbed in the form of ammonium or
nitrate
3. Phosphorus as the phosphate ions
4. Sulphur as sulphate ions
5. The most important step in deriving medium is the selection
of macronutrient ions in the correct concentration and
balanced
6. The salts normally used to provide macroelements also
provide sodium and chlorine, however, plant cell tolerate
high concentration of both ions without injury, these ions are
frequently given little importance when contemplating media
6
changes
Quantity of the Macronutrient
Nitrogen
1. It is essential to plant life
2. Both growth and morphogenesis is markedly influenced by
the availability of nitrogen and the form in which it is
presented
3. Most media contain more nitrate than ammonium ions. Most
intact plants, tissues and organ taken up nitrogen effectively,
and grow more rapidly on nutrient solutions containing both
nitrate and ammonium ions
4. Nitrate has to be reduced to ammonium before being utilized
biosynthetically
5. Ammonium in high concentration is latent toxic
6. For most type of culture, nitrate needs to be presented
together with the reduced form of nitrogen and tissue will
usually fail to grow on a medium with nitrate as the only
nitrogen source 8
NH4+ and NO3- Regulate Medium pH
and Root Morphogenesis of Rose Shoots
Amino acids
1. Amino acids can be added to satisfy the requirement for
reduced nitrogen, but as they are expensive to purchase, they
will only be used on media for mass propagation where this
results in improved result
2. A casein hydrolysate, yeast extract which mainly consist of a
mixture of amino acids substantially increased the yield of
callus
3. Organic supplements have been especially beneficial for
growth or morphogenesis when cells were cultured on media
which do not contain ammonium ions
4. Glycine os an ingredient of many media. It is difficult to
find hard evidence that glycine is really essential for so many
tissue culture, but possible it helps to protect cell membranes
from osmotic and temperature stress 10
Amino Acids
The most common sources of organic nitrogen used in
culture media are amino acid mixtures.
Its uptake more rapidly than in organic amino acids
14
Sodium
1. It is taken up into plant but in most cases it is not required
for growth and development
2. Many plants actively secret it from their roots to maintain a
low internal concentration
3. It is only appeared to be essential to salt tolerance plant
15
Magnesium
1. It is an essential component of the chlorophyll molecules
2. It is also required non-specifically for the activity of many
enzymes, especially in the transfer of phosphate
3. ATP synthesis has an absolute requirement for magnesium
and it is a bridging element in the aggregation of ribosome
sub-unit
4. It is the central atom in the phorphyrin structure of the
chlorophyll molecules
16
Sulfur
1. It is mainly absorbed as sulfate
2. Its uptake is coupled to nitrogen assimilation
3. It is incorporated into chemical compounds mainly as
reduced –SH, -S_ or –S-S groups
4. It is used in lipid synthesis and in regulating the structure of
proline through the formation of S-S bridges
5. It acts as a ligand joining ion of iron, zinc, copper to
metalloportein and enzymes
17
Calcium
1. It helps to balance anion within the plant
2. It is not readily mobile
3. It is involved in the structure and physiologically properties
of cell membranes and the middle lamella of the cell walls
4. The enzyme -(1-3)-glucan synthase depends on calcium
ions
5. It is a cofactor in the enzymes responsible for the hydrolisis
of ATP
18
Chlorine
1. It has been found to be essential for plant growth
2. It is sometimes considered as micro nutrient, because it is
required in a small amount
3. It is required for water – splitting protein complex of
photosystem II
4. It can function in osmoregulation in particular stomata guard
cell
19
Micronutrients
1. Plant requirement for microelement have only been elucidated in
the 19th century
2. In the early of 20th century, uncertainty still existed over the
nature of the essential microelements
3. many tissue undoubtedly grown successfully because they were
cultured on media prepared from impure chemicals or solidified
with agar which acted as a micronutrient source
4. In the first instance, the advantage of adding micronutrients was
mainly evaluated by their capability to improve the callus growth
or root culture
5. Knudson (1922) incorporated Fe and Mn on very successful
orchid seed media
6. Heller (1953) was first well demonstrated the advantages of
microelement on tissue culture media
20
Why in the first development
many tissue were undoubtedly
grown successfully in tissue
culture media without
micronutrient?
21
Quantity of the Micronutrient
25
Copper (Cu)
1. Plant only contains a few part of million of Cu
2. It becomes attached to enzymes, many of which bind to and
reach with oxygen
3. It occurs in plastocynain, a pigment participating in electron
transport
4. Highly concentration of Cu can be toxic
26
Molybdenum (Mo)
1. It is utilized in the form of hexavalent Mo
2. It is absorbed as the molybdate ions
3. It is a component of several plant enzymes, two being nitrate
reductase and nitrogenase, in which it is a cofactor together
with iron
27
Cobalt (Co)
1. It is sometimes not regarded as an essential elements
2. It might have a role in regulating morhogenesis of higher
plants
3. It is the metal component of vitamin B12 which is
concerned with nucleic acid synthesis, though evidence that it
has any marked stimulatory effect on growth and
morphogenesis is hard to find
4. It can have a protective action against metal chelate toxicity
and it is able to inhibit oxidative reaction catalyzed by copper
and iron
5. Cobalt can inhibit ethylene biosynthesis
28
Nickel (Ni)
1. It is a component of urease enzyme which convert urea to
ammonia
2. It has been shown to be an essential micronutrient for some
legumes
3. The presence of Ni strongly stimulate the cell growth in a
medium containing urea as a nitrogen source
4. Agar contains relatively high levels of nickel and the
possibility of urea toxicity may have been avoided because in
tissue culture media, urea diffuses into the medium
29
Iodine (I)
1. It is not recognized as a essential element for plants, although
it may be necessary for the growth of some algae and small
amount was accumulated in higher plant
2. It has been added to many tissue culture media
3. In improve the in vitro root growth
4. It prevent the explant browning
5. It enhance the destruction and/or the lateral transport of
auxin
30
Iron (Fe)
1. A key properties of iron is its capacity to be oxidized easily from
the ferrous (Fe(II)) to the ferric (Fe(III)) state and for ferric
compounds to be readily reduced back to the ferrous form
2. Iron is primarily used in the chloroplasts, mitochondria and
peroxisomes for effecting oxidation/reduction reaction
3. It is a component of ferredoxin proteins which function as
electron carriers in photosynthesis
4. Iron is an essential micronutrient for plant tissue culture and can
be taken up as either ferrous or ferric ions
5. Iron may not be available to plant cells, unless the pH falls
sufficiently to bring free ions back to solutions
6. Iron can be chelated with EDTA
7. The addition of Fe-EDTA chelate greatly improved the
availability of the element
31
Chelating agent
1. Some organic compounds are capable of forming complexes with
metal cations, in which the metal is held with fairly tight chemical
bonds
2. Metal can be bound (sequestered) by a chelating agent and held in
solution under conditions where free ions would react with anions
to form insoluble compounds, and some complexes can be more
chemically reactive than the metals themselves
3. Chelating agents vary in their sequestering capacity according to
chemical structure and their degree of ionisation, which changes
with pH of the solution
4. Naturally –occurring compounds can act as chelating agents such
as proteins, peptides, carboxylic acids and amino acids
5. There are also synthetic chelating agents with high avidity for
divalent and trivalent ions
32
Chelating agents
No. Chelating agents Chemical names
1. EDTA Ethylenediamine tetra acetic acid
2. EGTA Ethyleneglycol-bis(2-
aminoethylether) tetra acetic acid
3. EDDHA Ethylenediamine-di(o-
hydroxyphenyl) acetic acid
4. DTPA Diethylenetriaminepentaacetic acid
5. DHPTA 1,3 diamino-2-hydroxypropane-
tetra acetic acid
33
Carbon Source
Most plant tissue cultures are not highly autotrophic due to limitation
of CO2. Therefore, sugar is added to the medium as an energy source.
Sucrose is the most common sugar added, although glucose, fructose,
manitol and sorbitol are also used in certain instances.
The concentration of sugars in nutrient media generally ranges from 20
to 40 g/l.
Sugars also contribute to the osmotic potential in the culture
The presence of sucrose specifically inhibits chlorophyll formation and
photosynthesis, making autotrophic growth less feasible
Sucrose in the culture media is usually hydrolyzed totally or partially
into the component monosaccharides glucose and fructose
The general superiority of sucrose over glucose may be on account of
the more effective translocation of sucrose to apical meristems
Organic supplement
a) Vitamins:
Only thiamine (vitamin B1) is essential for most
plant cultures, it is required for carbohydrate
metabolism and the biosynthesis of some amino
acids
c) Complex organics
Such as coconut milk, coconut water, yeast extract, fruit juices
and fruit pulps.
Physical support agents
A. Gelling agents
When semi-solid or solid culture media are required,
gelling agents are used.
An example:
Agar, agarose, gelrite, phytagel
B. Structural supports
Filter paper bridges, liquid permeable membrane
support systems
Agar
Agar is the most commonly used gelling agent
It is a natural product extracted from species of red algae,
especially Gelidium amansii
It is synthetic polysaccharide gelling agents
prevent clumping.
Commercial Media Formulations
• Murashige and Skoog (MS)
• White Medium
• Gamborg medium
• Knudson’s medium
Hormone
(the Greek word hormaein, meaning "to excite").
Growth-inhibiting chemicals
Growth-promoting chemicals
Root-promoting chemicals
Auxin
Cytokinin
Gibberelin
Classification of
PGRs
Jasmonates
Abscisic acid
Salicylic acid
Ethylene
Other Brassinosteroids
Plant hormones as
“Chemical Messengers”
Auxins
Cytokinins
Gibberellins
Ethylene
Auxins
Auxin
Arpad Paál (1919) - Asymmetrical placement of cut tips on
coleoptiles resulted in a bending of the coleoptile away from the
side onto which the tips were placed (response mimicked the
response seen in phototropism).
Frits Went (1926) determined auxin enhanced cell elongation.
Auxins
Absolutely essential (no mutants known)
One compound: Indole-3-acetic acid.
Many synthetic analogues:
NAA, IBA, 2,4-D, 2,4,5-T, Picloram
Cheaper & more stable
Generally growth stimulatory.
Promote rooting
Stimulate cell elongation
Increase the rate of transcription
Mediate the response of bending in response to gravity or light
Produced in meristems, especially shoot meristem and transported
through the plant in special cells in vascular bundles.
Cytokinins
Cytokinin
Cytokinins
Discovery of cytokinins
Gottlieb Haberlandt in 1913 reported an unknown compound
that stimulated cellular division.
In the 1940s, Johannes van Overbeek, noted that plant embryos
grew faster when they were supplied with coconut milk (liquid
endosperm), which is rich in nucleic acids.
In the 1950s, Folke Skoog and Carlos Miller studying the
influence of auxin on the growth of tobacco in tissue culture.
When auxin was added to artificial medium, the cells enlarged but
did not divide. Miller took herring-sperm DNA. Miller knew of
Overbeek's work, and decided to add this to the culture medium,
the tobacco cells started dividing. He repeated this experiment
with fresh herring-sperm DNA, but the results were not repeated.
Only old DNA seemed to work. Miller later discovered that
adding the purine base of DNA (adenine) would cause the cells to
divide.
Discovery of cytokinins
Adenine or adenine-like compounds induce cell division in plant
tissue culture. Miller, Skoog and their coworkers isolated the
growth facto responsible for cellular division from a DNA
preparation calling it kinetin which belongs to a class of
compounds called cytokinins.
H H
\ /
C = C
/ \
H H
Discovery of ethylene
In the 1800s, it was recognized that street lights that
burned gas, could cause neighboring plants to develop
short, thick stems and cause the leaves to fall off. In
1901, Dimitry Neljubow identified that a byproduct of
gas combustion was ethylene gas and that this gas could
affect plant growth.
In R. Gane showed that this same gas was naturally
produced by plants and that it caused faster ripening of
many fruits.
Synthesis of ethylene is inhibited by carbon dioxide and
requires oxygen.
Ethylene
Gas - diffuses through tissues
Stimulates abscission and fruit ripening
Used in commercial ripening for bananas & green picked fruit
Involved in leaf abscission & flower senescence
Primarily synthesized in response to stress
Regulate cell death programming
Brassinosteroids
Promote shoot elongating
Inhibit root growth
herbicides
Salicylic acid
Promote flowering
production
Jasmonate
Play an important role in plant defence
mechanisms
Jasmonate
Play an important role in plant defence mechanisms