Transcript Plant Tissue Culture Media Logical Basis For healthy and vigorous growth, intact plants need to take up from soil of an essential elements. Essential.

Plant Tissue Culture
Media
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Logical Basis
For healthy and vigorous growth, intact plants need to take
up from soil of an essential elements.
Essential elements (Epstein, 1971):
1. A plant grown in a medium adequately purged of
that elements, failed to grow properly or to complete
its life cycle
2. It is a constituent of a molecule that is known to be
an essential metabolite
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Essential element
 Macro element/major plant nutrition:
Relatively large amount required
a. Carbon (C)
b. Hydrogen (H)
c. Oxygen (O)
d. Nitrogen (N)
e. Calcium (Ca)
f. Magnesium (Mg)
g. Potassium (K)
h. Phosphorus (P)
i. Sulphur (S)
 Micro element/ minor plant nutrient/trace elements:
Small quantities required
a. Iron (Fe)
b. Chlorine (Cl)
c. Zinc (Zn)
d. Copper (Cu)
e. Nickel (Ni)
f. Sodium (Na)
g. Manganese (Mn)
h. Boron (B)
i. Molybdenum (Mo)
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Why plant in vitro culture needs
media?
Functions of media:
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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)
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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
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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
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nitrogen source
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
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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
. (e.g., casein hydrolysate), L-glutamine, L-asparagine,
and adenine.
When amino acids are added alone, they can be inhibitory
to cell growth.
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Beneficial effects of amino
acids
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Rapid growth
Protoplast cell division
Conservation of ATP
AS chelating agent
Enhanced nitrogen assimilation
Not toxic as ammonium
As buffer
Phosphorous
1. It is a vital element in plant biochemistry
2. It occurs in numerous macromolecules such as nucleic acids,
phospholipids and co-enzymes
3. It functions in energy transfer via pyrophosphate bond I ATP
4. Phosphate groups attached to different sugar provide energy in
respiration and photosynthesis and phosphate bound to proteins
regulate their activity
5. Phosphorous is absorbed into plants in the form of the primary
or secondary orthophosphate anions by an active process which
requires the expenditure of respiratory energy
6. Phosphate in not reduced in plants, but it remains in the oxydised
form
7. It is used in plant as the fully oxydised orthophosphate form
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Potassium
1.
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4.
It is not metabolized
It is a major cation within the plants
It contributes significantly to the osmotic potential of cells
It is transported quickly across cell membrane and two of its
major role is regulating the pH and osmotic environment
within the cells
5. Many protein show a high specificity for potassium which
acting as a cofactor, alters their configuration so that it
become active enzyme
6. It is also neutralize organic anions produce in the cytoplasm
and so stabilize the pH and osmotic potential of the cells
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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
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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
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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
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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
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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
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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
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Why in the first development
many tissue were undoubtedly
grown successfully in tissue
culture media without
micronutrient?
 Media is solidified with agar which acted as a micronutrient
source
 Plant cells are more demanding for micronutrients when
undergoing morphogenesis
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Quantity of the Micronutrient
MS medium was formulated from the ash content of tobacco callus.
The higher concentration of salts substantially enhanced cell division
Boron (B)
1. It is involved in plasma membrane integrity and function,
probably by influencing membrane protein and cell wall
intactness
2. It is required for the metabolism of phenolic acids, and for
lignin biosynthesis
3. It is probably a component, or co-factor of the enzyme
which converts p-coumaric acid to 5-hydroxyferulate
4. It is necessary for the maintenance of meristematic activity,
most likely because it is involved in the synthesis of N-bases
5. It is also thought to be involved in the maintenance of
membrane structure and function, possibly by stabilizing
natural metal chelates, which are important in wall and
membrane structure and function
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Manganese (Mn)
1. It is the most important micro nutrients
2. It has similar properties to Magnesium, it is apparently
able to replace magnesium is some enzyme systems
3. It is involved in respiration and photosynthesis as
metalloprotein structure
4. It is known to be required for the activity of several
enzymes
5. It is necessary for the maintenance of chloroplast ultra
structure
6. It is involved in regulation of enzymes and growth
hormones.
7. It assists in photosynthesis and respiration.
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Zinc (Zn)
1. It is a component of stable metallo enzymes with many
diverse function
2. It is required in more than 300 enzymes
3. Its deficient plants will suffer from reduced enzyme activities
and as a consequent will diminute in protein, nucleic acid and
chlorophyl synthesis
4. There is a close relationship between zinc concentration of
plants and their auxin content
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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
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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
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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
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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
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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
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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
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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
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Chelating agents
No. Chelating agents Chemical names
1.
EDTA
Ethylenediamine tetra acetic acid
2.
EGTA
Ethyleneglycol-bis(2aminoethylether) tetra acetic acid
3.
EDDHA
Ethylenediamine-di(ohydroxyphenyl) acetic acid
4.
DTPA
Diethylenetriaminepentaacetic acid
5.
DHPTA
1,3 diamino-2-hydroxypropanetetra acetic acid
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Carbon Source
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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
 Thiamine
(vitamin B1)
Essential as a coenzyme in the citric acid cycle
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Nicotinic acid (niacin) and pyridoxine (B6)
Organic supplement
b)
Myo-inositol
Although it is not essential for growth of many plant species, its
effect on growth is significant.
Part of the B complex, in phosphate form is part of cell
membranes, organelles and is not essential to growth but
beneficial
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
support systems
membrane
Agar
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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
Agar consists of 2 components
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Agarose is an alternating D-galactose and 3,6-anhydro-L-galactose
with side chains of 6-methyl-D-galactose residues (50 -90%).
Agaropectin is like agarose but additionally contains sulfate ester side
chains and D-glucuronic acid.
Agar tertiary structure is a double helix the central cavity of
which can accommodate water molecules
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Advantages:
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Agar is an inert component, form a gel in water that melt at
100 ° C and solidify at nearly 45 ° C
Concentrations commonly used in plant culture media range
between 0.5% and 1%
If necessary, agar can be washed to remove inhibitory organic
and inorganic impurities.
Gels are not digested by plant enzymes
Agar does not strongly react with media constituent
Disadvantages:
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Agar does not gel well under acidic conditions (pH <4.5).
The inclusion of activated charcoal in media may also inhibit
gelling of agar.
Agarose
It is extracted from agar
leaving
behind
agaropectin and its
sulfate groups.
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is used when the
impurities of agar are a
major disadvantage.
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Gelrite™
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Gelrite consists of a polysaccharide
produced
by
the
bacterium
Pseudomonas elodea.
It gives clear-solidified medium that
leads to detection of contamination at
an early stage.
Gelrite requires more stirring than
agar.
Concentration of divalent cations
such as calcium and magnesium must
be within the range of 4-8 mM/L or
the medium will not solidify
Phytagel™
It is an agar substitute produced from a bacterial
substrate composed of glucuronic acid,
rhamnose and glucose.
 It produces a clear, colorless, high-strength gel,
which aids in detection of microbial
contamination.
 It is used at a concentration of 1.5-2.5 g/L.
 It should be prepared with rapid stirring to
prevent clumping.
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Commercial Media Formulations
• Murashige and Skoog (MS)
• Linsmaier and Skoog (LS)
• White Medium
• Gamborg medium
• Schenk and Hildebrandt medium
• Nitsch and Nitsch Medium
• Lloyd and McCown Woody plant medium
• Knudson’s medium
Hormone
(the Greek word hormaein, meaning "to excite").
Small organic molecule that elicits a physiological response
at very low concentrations
Chemical signals that coordinate different parts of the
organism
Internal and external signals that regulate growth are
mediated, at least in part, by growth-regulating
substances, or hormones
Plant Hormone
 A natural substance which produced by plant and acts to
control plant activities.
 Chemical messengers influencing many patterns of plant
development
 Naturally occurring or synthetic compounds that affect plant
growth and development
Plant hormones differ from animal hormones in that:
 No evidence that the fundamental actions of plant and animal
hormones are the same.
 Unlike animal hormones, plant hormones are not made in tissues
specialized for hormone production. (e.g., sex hormones made in the
gonads, human growth hormone - pituitary gland)
 Unlike animal hormones, plant hormones do not have definite target
areas (e.g., auxins can stimulate adventitious root development in a
cut shoot, or shoot elongation or apical dominance, or differentiation
of vascular tissue).
Characteristics
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2.
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5.
Synthesized by plants.
Show specific activity at very low concentrations
Display multiple functions in plants.
Play a role in regulating physiological phenomena in
vivo in a dose-dependent manner
They may interact, either synergistically or
antagonistically, to produce a particular effect.
Synthetic plant hormone
Plant growth regulators
Growth-inhibiting chemicals
 Growth-promoting chemicals
 Root-promoting chemicals
Auxin
Cytokinin
Gibberelin
Classification of
PGRs
Abscisic acid
Ethylene
Other
Jasmonates
Salicylic acid
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
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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.
 In 1964, the first naturally occurring cytokinin was isolated from
corn called zeatin. Zeatin and zeatin riboside are found in coconut
milk. All cytokinins (artificial or natural) are chemically similar to
adenine.
 Cytokinins move nonpolarly in xylem, phloem, and parenchyma
cells.
 Cytokinins are found in angiosperms, gymnosperms, mosses, and
ferns. In angiosperms, cytokinins are produced in the roots, seeds,
fruits, and young leaves
Cytokinins
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Absolutely essential (no mutants known)
Natural compound: Zeatin, 2-isopentyl adenine (2iP)
Synthetic analogues: Benyzladenine (BA), Kinetin.
Stimulate cell division (with auxins).
Promotes formation of adventitious shoots
Stimulate cell division
Stimulate dark germination
Stimulate leaf expansion
Produced in the root meristem and transported throughout the
plant as the Zeatin-riboside in the phloem.
Auxin and Cytokinin Ratio
Interaction of cytokinin and auxin in
tobacco callus (undifferentiated plant
cells) tissue
Organogenesis: Cytokinins and auxin affect organogenesis
High cytokinin/auxin ratios favor the formation of shoots
Low cytokinin/auxin ratios favor the formation of roots.
Gibberellin(GA’s)
Discovered of Gibbereline
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In 1930's, Ewiti Kurosawa and colleagues
were studying plants suffering from bakanae,
or "foolish seedling" disease in rice.
Disease caused by fungus called, Gibberella
fujikuroi, which was stimulating cell elongation
and division.
Compound secreted by fungus could cause
bakanae disease in uninfected plants.
Kurosawa named this compound gibberellin.
 Gibberella fujikuroi also causes stalk rot in
corn, sorghum and other plants.
 Secondary metabolites produced by the
fungus include mycotoxins, like
fumonisin, which when ingested by
horses can cause equine
leukoencephalomalacia - necrotic brain or
crazy horse or hole in the head disease.
 Fumonisin is considered to be a
carcinogen.
Gibberellins
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A family of over 70 related compounds, all forms of Gibberellic
acid and named as GA1, GA2.... GA110.
Commercially, GA3 and GA4+9 available.
Stimulate etiolation of stems.
Help break bud and seed dormancy.
Stimulate stem elongation by stimulation cell division and
elongation
Stimulate germination of pollen
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Produced in young leaves
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Abscisic acid (ABA)
Discovery of abscisic acid
 In 1940s, scientists started searching for hormones that
would inhibit growth and development, what Hemberg
called dormins.
 In the early 1960s, Philip Wareing confirmed that
application of a dormin to a bud would induce dormancy.
 F.T. Addicott discovered that this substance stimulated
abscission of cotton fruit. he named this substance
abscisin. (Subsequent research showed that ethylene and
not abscisin controls abscission).
 Abscisin is made from carotenoids and moves nonpolarly
through plant tissue.
Abscisic Acid
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Only one natural compound.
Promotes leaf abscission and seed dormancy.
Plays a dominant role in closing stomata in response to water
stress
Involved in the abscission of buds, flower and fruits
Inhibit cell division and elongation
Has an important role in embryogenesis in preparing embryos
for desiccation.
Helps ensure ‘normal’ embryos.
Ethylene
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
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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
 Promote ethylene biosynthesis
 Enhance resistance to chilling, disease and
herbicides
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Salicylic acid
Promote flowering
Stimulate plant pathogenesis protein
production
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Jasmonate
Play an important role in plant defence
mechanisms
Jasmonate
Play an important role in plant defence mechanisms