Chapter 3 The Components of Plant Tissue Culture Media I:

Chapter 3 The Components of Plant Tissue Culture Media I:
Chapter 3
The Components of Plant Tissue Culture Media I:
Macro- and Micro-Nutrients
1. INORGANIC MEDIUM COMPONENTS
formulation. These media result often in a much
improved growth (Rugini, 1984; El Badaoui et al.,
1996; Pullman et al., 2003; Bouman and Tiekstra,
2005; Nas and Read, 2004; Gonçalves et al., 2005).
A major problem in changing the mineral composition of a medium is precipitation, which may often
occur only after autoclaving because of the
endothermic nature of the process.
Plant tissue culture media provide not only these
inorganic nutrients, but usually a carbohydrate
(sucrose is most common) to replace the carbon
which the plant normally fixes from the atmosphere
by photosynthesis. To improve growth, many media
also include trace amounts of certain organic
compounds, notably vitamins, and plant growth
regulators.
In early media, ‘undefined’ components such as
fruit juices, yeast extracts and protein hydrolysates,
were frequently used in place of defined vitamins or
amino acids, or even as further supplements. As it is
important that a medium should be the same each
time it is prepared, materials, which can vary in their
composition are best avoided if at all possible,
although improved results are sometimes obtained by
their addition. Coconut milk, for instance, is still
frequently used, and banana homogenate has been a
popular addition to media for orchid culture.
Plant tissue culture media are therefore made up
from solutions of the following components:
• macronutrients (always employed);
• micronutrients (nearly always employed but
occasionally just one element, iron, has been used);
• sugar (nearly always added, but omitted for some
specialised purposes);
• plant growth substances (nearly always added)
• vitamins (generally incorporated, although the
actual number of compounds added, varies greatly);
• a solidifying agent (used when a semi-solid
medium is required. Agar or a gellan gum are the
most common choices).
• amino acids and other nitrogen supplements
(usually omitted, but sometimes used with
advantage);
Plant tissues and organs are grown in vitro on
artificial media, which supply the nutrients necessary
for growth. The success of plant tissue culture as a
means of plant propagation is greatly influenced by
the nature of the culture medium used. For healthy
and vigorous growth, intact plants need to take up
from the soil:
• relatively large amounts of some inorganic
elements (the so-called major plant nutrients): ions of
nitrogen (N), potassium (K), calcium (Ca),
phosphorus (P), magnesium (Mg) and sulphur (S);
and,
• small quantities of other elements (minor plant
nutrients or trace elements): iron (Fe), nickel (Ni),
chlorine (Cl), manganese (Mn), zinc (Zn), boron (B),
copper (Cu), and molybdenum (Mo).
According to Epstein (1971), an element can be
considered to be essential for plant growth if:
1. a plant fails to complete its life cycle without it;
2. its action is specific and cannot be replaced
completely by any other element;
3. its effect on the organism is direct, not indirect on
the environment;
4. it is a constituent of a molecule that is known to
be essential.
The elements listed above are - together with
carbon (C), oxygen (O) and hydrogen (H) - the 17
essential elements. Certain others, such as cobalt
(Co), aluminium (Al), sodium (Na) and iodine (I), are
essential or beneficial for some species but their
widespread essentiality has still to be established.
The most commonly used medium is the
formulation of Murashige and Skoog (1962). This
medium was developed for optimal growth of
tobacco callus and the development involved a large
number of dose-response curves for the various
essential minerals. Table 3.1 shows the composition
of MS compared to the elementary composition of
normal, well-growing plants. From this table, the
relatively low levels of Ca, P and Mg in MS are
evident. The most striking differences are the high
levels of Cl and Mo and the low level of Cu. Each
plant species has its own characteristic elementary
composition which can be used to adapt the medium
65
E. F. George et al. (eds.), Plant Propagation by Tissue Culture 3rd Edition, 65–113.
© 2008 Springer.
66
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
Table 3.1 A comparison between the average concentrations of elements in plant shoots (dry weight basis) considered sufficient
for adequate growth [from Epstein (1972), content of Ni is according to Brown et al. (1987)] and in MS. The elements that show
striking differences between MS and ‘plants’ are indicated. For Na, no data were found, but in glycophytes grown in 1 mM Na,
the endogenous level is 10 – 1000 mmol.kg-1 (Subbarao et al., 2003)
N
K
Ca
Mg
P
S
Cl
Fe
Mn
B
Zn
Cu
Mo
Ni
Na
In tissue mmol
kg-1
In MS
mmol l-1
In tissue
mol%
In MS
mol%
1000
250
125
80
60
30
3
2
1
2
0.3
0.1
0.001
0.001
60
20
3
1.5
1.25
1.5
6
0.1
0.1
0.1
0.03
0.0001
0.001
0
0.1
64.4
16.1
8.0
5.1
3.9
1.9
0.19
0.13
0.06
0.13
0.02
0.0060
0.0001
0.0001
0.0000
64.0
21.3
3.2
1.6
1.3
1.6
6.4
0.11
0.11
0.11
0.03
0.0001
0.0011
0.0000
0.1067
• undefined supplements such as coconut milk etc.
(which, when used, contribute some of the five
components above and also plant growth substances
or regulants);
• buffers (have seldom been used, but the addition
of organic acids or buffers could be beneficial in
some circumstances).
Finally, it should be noted that minerals may also
have a signalling role altering developmental patterns. This is most obvious in root architecture
(Lopez-Bucio et al., 2003) which is logical as roots
have a principal function in ion uptake and the root
system should be such that uptake is optimal. So
growth and branching of roots should be affected by
mineral concentrations in the soil. Ramage and
Williams (2002) also argue that minerals appear to
have an important role in the regulation of plant
morphogenesis as opposed to just growth. Some
reviews of whole plant mineral nutrition will be
found in Grusak (2001), Leiffert et al., (1995),
Mengel and Kirkby (1982), Hewitt and Smith (1975)
and Epstein (1971).
1.1. UPTAKE OF INORGANIC NUTRIENTS
Plants absorb the inorganic nutrients they require
from soils almost entirely as ions. An ion is an atom,
or a group of atoms, which has gained either a
positive charge (a cation) or a negative charge (an
anion). Inorganic nutrients are added to plant culture
media as salts. In weak aqueous solutions, such as
plant media, salts dissociate into cations and anions.
Thus calcium, magnesium and potassium are
absorbed by plant cells (normally those of the root) as
the respective cations Ca2+, Mg2+ and K+; nitrogen is
mainly absorbed in the form nitrate (the anion, NO3-)
although uptake of ammonium (the cation, NH4+)
may also occur, phosphorus as the phosphate ions
HPO42- and H2PO4-; and sulphur as the sulphate ion
In tissue culture, uptake is generally
SO42-.
proportional to the medium concentration up to a
concentration of twice MS (Williams, 1993). For
specific elements this may be different. For example,
Leiffert et al, (1995) found only a small increase in
Zn uptake with increasing medium concentration
indicating that the concentration of Zn in the cultured
tissues was adequate, not requiring further uptake.
Selective uptake also suggests active uptake.
67
Chapter 3
Table 3.2 Content (mmol/kg) of elements in various agar brands.
[agar 1-7: Scholten and Pierik (1998); agar 8 and gelrite: Scherer et al.(1988)].
Na = not analysed, nd = not detected. It should be noted that some elements present in agar are not present in MS. This is
particularly relevant for Ni which is an essential element
Agar 1
53
2
68
28
202
1
184
47
0.015
0.073
0.510
0.352
0.040
0.013
0.092
1.896
0.037
na
na
N
K
Ca
Mg
Na
P
S
Cl
Cu
Mn
Fe
Al
Cr
Cd
Zn
Sn
Ni
B
Co
Agar 2
1
1
41
24
56
18
78
33
0.034
1.093
5.376
12.444
0.098
0.069
0.107
1.542
0.045
na
na
Agar 3
178
16
34
31
552
1
232
220
0.018
0.036
0.564
1.333
0.029
0.008
0.054
nd
nd
na
na
Agar 4
100
9
137
29
330
5
296
113
0.024
0.173
2.987
4.944
0.026
0.025
0.933
3.572
0.037
na
na
Agar 5
74
6
66
48
427
1
204
197
0.004
0.036
0.859
0.352
0.054
0.015
0.046
1.862
0.025
na
na
Agar 6
54
13
1
3
634
40
262
95
0.016
0.027
0.599
0.963
0.025
nd
0.038
nd
nd
na
na
Agar 7
2
2
5
3
114
1
66
12
nd
0.055
0.528
0.685
0.009
nd
0.015
nd
0.007
na
na
Agar 8
na
51
2.8
2.6
52
42
184
na
0.005
0.01
0.6
0.3
0.002
0.0002
0.02
0.003
0.005
2
0.0005
gelrite
na
718
123
64
296
68
6
na
0.05
0.1
5
6.8
0.01
0.002
0.3
0.003
0.004
0.13
1.0
Table 3.3 Increase of the content of Na, S and Cu relative to MS caused by adding agar (0.6%) or gelrite (0.2%) to the medium.
Increases are shown as percentages. The proportional increase in other elements is maximally 20%
Agar 1 Agar 2 Agar 3 Agar 4 Agar 5 Agar 6 Agar 7 Agar 8 gelrite
Na
1212
336
3312
1980
2562
3804
684
313
591
S
69
29
87
111
77
98
25
69
0.8
Cu
90
204
108
144
24
96
nd
28
91
In the whole plant, nutrients are either taken up
passively, or through active mechanisms involving
the expenditure of energy. Active uptake is generally
less dependent on ionic concentration than passive
uptake. Both systems are however influenced by the
concentration of other elements, pH, temperature, and
the biochemical or physiological status of the plant
tissues. These factors can in turn be controlled by the
solution presented to the roots, or they may dictate
the ionic balance of an ideal solution. For example,
Mg2+ competes with other cations for uptake. Under
conditions of high K+ or Ca2+ concentrations, Mg
deficiency can result, and vice versa. Active uptake
of phosphate falls off if the pH of the solution should
become slightly alkaline when the (H2PO4)- ion
becomes changed to (HPO4)2-. There is some
evidence that ammonium is utilised more readily than
nitrate at low temperatures and that uptake may be
enhanced by high carbohydrate levels within plant
cells. Calcium is not absorbed efficiently and
concentrations within plant tissues tend to be
proportional to those in the soil.
Plants are
comparatively insensitive to sulphate ions, but high
concentrations of dissolved phosphate can depress
growth, probably through competitively reducing the
uptake of the minor elements Zn, Fe and Cu.
Although the biochemistry and physiology of nutrient
uptake in tissue cultures may be similar, it is unlikely
to be identical.
In vivo, plants take up mineral ions with their
roots. No studies have been made on how uptake of
nutrients occurs in shoot cultures. For IAA, it has
been shown that most uptake is via the cut surface
and that only a small fraction is taken up via the
68
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
epidermis (Guan and De Klerk, 2000). The same
likely holds for minerals. It should be noted, though,
that in tissue culture the stomata are always open in
the portion of the explant exposed to the gaseous
phase (De Klerk and Wijnhoven, 2005) and the same
may apply for tissues that are exposed to semi-solid
or liquid medium. Uptake via the stomata is well
possible.
Once taken up, transport within the plant occurs in
the mass flow via the xylem. In in vivo plants there
are two mechanisms for driving the water flow, root
pressure and water potential gradient between at one
end the soil and at the other end the atmosphere.
Under normal conditions, the latter is the far more
important, but water flow resulting from root pressure
is in itself sufficient for long-distance mineral supply
(Tanner and Beevers, 2001). Plants without roots are
often cultured in vitro where the atmosphere is very
humid, and the flow driven by a difference in water
potential consequently reduced. In spite of this, in
tissue culture there still seems to be sufficient water
flow (Beruto et al., 1999) which may be favoured by
the stomata being continuously open (De Klerk and
Wijnhoven, 2005). There are no indications that the
structure of the xylem is altered in such a way as to
reduce transport of ions.
When explants are first placed onto a nutrient
medium, there may be an initial leakage of ions from
damaged cells, especially metallic cations (Na+, Ca2+,
K+, Mg2+) for the first 1-2 days, so that the
concentration in the plant tissues actually decreases
(Chaillou and Chaussat, 1986). Cells then commence
active absorption and the internal concentration
slowly rises. Phosphate and nitrogen (particularly
ammonium) are absorbed more rapidly than other
ions. In liquid medium, almost all phosphorus and
ammonium are taken up in the first two weeks of
culture (e.g. by 5 microshoots of Dahlia in 50 ml
stationary liquid medium; G. de Klerk, unpublished
results).
After uptake, phophorus is massively
redistributed to tissues that are formed after the initial
two weeks. Nitrate in a medium very similar to that
of Wood and Braun (1961) B medium, was reduced
by Catharanthus roseus suspensions from 24 mM to
5 mM in 15 days, while Na+, K+, and SO42-, fell to
only just over half the original concentrations in the
same time (MacCarthy et al., 1980). Carnation shoot
cultures were found to use 31-75% Mg2+, and 2941% Ca2+ in MS medium during a 4 week period
(Dencso, 1987).
1.2. UNINTENDED ALTERATIONS
Nutrients, and especially micronutrients, may also be
added via impurities, and especially via agar. Such
impurities may well be beneficial.
This is
particularly true of Ni, which has recently been
shown to be an essential element (Gerendás et al.,
1999) but was not known to be when most medium
formulations were established. This element is
usually not included in the inorganic constituents but
can be provided by impurities. Tables 3.2 and 3.3
show impurities of various agar brands and their
relative contribution to MS. Agar provides a large
addition of sodium but levels of sulphur and copper
are also significantly increased. Increases in the other
elements in MS, are less than 20 %. Gelrite contains
fewer organic impurities but inorganic ones occur at
high concentrations (Table 3.2). It should be noted
that the data in Table 3.2 are from determinations
done more than 15 years ago and that the production
process of gelrite has been improved ever since.
Gelrite is being used in medicines as an ophthalmic
vehicle. Furthermore, minerals are absorbed to a
significant percentage by agar (Scholten and Pierik,
1998 Leiffert et al., 1995) and by activated charcoal
(Van Winkle et al., 2003) but whether this has a
significant effect has not been examined.
2. MACRONUTRIENTS
2.1. NITROGEN
2.1.1. Forms of nitrogen
Nitrogen is essential to plant life. It is a
constituent of both proteins and nucleic acids and
also occurs in chlorophyll. Most animals cannot
assimilate inorganic nitrogen or synthesize many of
the amino acids unless assisted by bacteria (e.g. in the
rumen of cattle). Nitrogen is available in the
atmosphere as N2 but only legumes have the capacity
to utilize this nitrogen using Rhizobium bacteria in
the root nodules. In most plants, nitrate (NO3-) is the
sole source of nitrogen. After uptake, NO3- is
reduced to NH4+ prior to incorporation into organic
molecules. (The removal of oxygen from a chemical
compound and its replacement by hydrogen, is
termed reduction.) The relevance of nitrogen is
illustrated by the vast amounts of nitrogen reserves in
seeds (as storage proteins).
Both growth and morphogenesis in tissue cultures
are markedly influenced by the availability of
nitrogen and the form in which it is presented.
Compared to the nitrate ion, NO3- (which is a highly
Chapter 3
oxidized form of nitrogen), the ammonium ion, NH4+,
is the most highly ‘reduced’ form. Plants utilise
reduced nitrogen for their metabolism and internally,
nitrogen exists almost entirely in the reduced form.
As a source of reduced nitrogen, plant cultures are
especially able to use primary amines:
R-NH2 and amides: R-CO-NH-R- (where R and
R- are functional groups)
The primary amines which are most commonly
employed in culture media are ammonia (NH4+) and,
occasionally, amino acids.
Amides are less commonly added to culture
media: those which can be used by plants are
particularly
NH2-CO-NH2 (urea)
and ureides, which include allantoin and allantoic
acid (Kirby, 1982) (Fig. 3.1). Allantoin or allantoic
acid are sometimes more efficient nitrogen sources
than urea (Lea et al., 1979).
an active (energy-dependent) process (Heimer and
Filner, 1971) and is dependent on a supply of oxygen
(Buwalda and Greenway, 1989).
Plant culture media are usually started at pH 5.45.8. However, in one containing both nitrate and
ammonium ions, a rapid uptake of ammonium into
plant tissue causes the pH to fall to ca. 4.2-4.6. As
this happens, further ammonium uptake is inhibited,
but uptake of nitrate ion is stimulated, causing the pH
to rise again. In unbuffered media, efficient nitrogen
uptake can therefore depend on the presence of both
ions. Unless otherwise stated, comments in this
section on the roles of nitrate and ammonium refer to
observations on unbuffered media.
There is generally a close correlation in tissue
cultures between uptake of nitrogen, cell growth and
the conversion of nitrogen to organic materials. A
readily available supply of nitrogen seems to be
important to maintain cultured cells in an
undifferentiated state. The depletion of nitrogen in
batch cultures, triggers an increase in the metabolism
of some nitrogen-free compounds based on
phenylpropanes (such as lignin), which are associated
with the differentiation of secondarily-thickened cells
(Hahlbrock, 1974). However, the growth in culture
of differentiated cotton fibres composed largely of
cellulose, is nitrate-dependent; the presence of some
reduced nitrogen in the culture medium decreased the
proportion of cultured embryos which produced
fibres, and particularly in the absence of boron,
promoted the cells of the embryos to revert to callus
formation (Birnbaum et al., 1974).
2.1.2. Nitrate ions (NO3-)
Fig. 3.1 The structures of allantoin and allantoic acid.
Most media contain more nitrate than ammonium
ions, but as plant tissue culture media are usually not
deliberately buffered, the adopted concentrations of
ammonium and nitrate ions have probably been more
due to practical pH control, than to the requirement of
the plant tissues for one form of nitrogen or another
(see chapter 4). Uptake of nitrate only takes place
effectively in an acid pH, but is accompanied by
extrusion of anions from the plant, leading to the
medium gradually becoming less acid. By contrast,
uptake of ammonium results in the cells excreting
protons (H+) into the medium, making it more acid.
The exchange of ions preserves the charge balance of
the tissues and may also assist in the disposal of an
excess of protons or hydroxyl (OH–) ions generated
during metabolism (Raven, 1986). Uptake of nitrogen
by cell suspension cultures of Nicotiana tabacum is
69
Nitrate ions are an important source of nitrogen
for most plant cultures, and nearly all published
media provide the majority of their available nitrogen
in this form. However, once within the cell, nitrate
has to be reduced to ammonium before being utilised
biosynthetically. Why not simply supply nitrogen as
NH4+ and avoid the use of NO3- altogether? The
reason lies in the latent toxicity of the ammonium ion
in high concentration, and in the need to control the
pH of the medium.
Conversion of nitrate to ammonium is brought
about firstly by one, or possibly two, nitrate reductase
enzymes, which reduce NO3- to nitrite (NO2-). One
nitrate reductase enzyme is thought to be located in
the cytoplasm, while the second may be bound to
membranes (Nato et al., 1990). The NO2- produced
by the action of nitrate reductase is reduced to NH4+
by a nitrite reductase enzyme located in plastids (Fig.
3.2). Reduction of nitrate to ammonia requires the
70
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
cell to expend energy. The ammonium ions produced
are incorporated into amino acids and other nitrogencontaining compounds. Nitrate and nitrite reductase
enzymes are substrate induced, and their activity is
regulated directly by the level of nitrate-nitrite ions
within cultured cells (Chroboczek-Kelker and Filner,
1971; Hahlbrock, 1974), but also apparently by the
products of the assimilation of reduced nitrogen (see
below).
Fig. 3.2 The metabolism of nitrate and ammonium ions.
Unlike the ammonium ion, nitrate is not toxic and,
in many plants, much is transported to the shoots for
assimilation. On the other hand, the nitrite ion can
become toxic should it accumulate within plant
tissues or in the medium, for example when growth
conditions are not favourable to high nitrite reductase
activity and when nitrate is the only nitrogen source
(Jordan and Fletcher, 1979; Grimes and Hodges,
1990). In Pinus pinaster, nitrate reductase is induced
by the presence of KNO3, and plants regenerated in
vitro exhibit an ability to reduce nitrate similar to that
of seedlings (Faye et al., 1986).
For most types of culture, the nitrate ion needs to
be presented together with a reduced form of nitrogen
(usually the NH4+ ion), and tissues may fail to grow
on a medium with NO3- as the only nitrogen source
(Hunault, 1985).
2.1.3. Reduced nitrogen
In the natural environment and under most
cropping conditions, plant roots usually encounter
little reduced nitrogen, because bacteria rapidly
oxidize available sources (Hiatt, 1978). An exception
is forest soils in mountainous regions of the northern
hemisphere, where nitrates are not usually available
(Durzan, 1976). If NH4+, and other reduced nitrogen
compounds are available, (and this is particularly the
case in the aseptic in vitro environment), they can be
taken up and effectively utilized by plants. In fact the
uptake of reduced nitrogen gives a plant an
Chapter 3
ergonomic advantage because the conversion of
nitrate to ammonium ions (an energy-requiring
process) is not necessary. The free ammonium ion
can cause toxicity, which, at least in whole plants,
can lead to an increase in ethylene evolution (Barker
and Corey, 1987; Corey and Barker, 1987). Shoots
grown on an unbuffered medium containing a high
proportion of ammonium ions may become stunted or
hyperhydric. These effects can sometimes be reversed
by transfer to a medium containing a high proportion
of NO3⎯ or to one where NO3⎯ is the only N source
(Mott et al., 1985). Hyperhydricity is the in vitro
formation of abnormal organs, which are brittle and
have a water-soaked appearance.
Growth of plant cultures may also be impaired in
media containing high concentrations of NH4+ even
when high concentrations of NO3⎯ are present at the
same time. Growth inhibition may not only be due to
depressed pH (Mott et al., 1985), but may reflect a
toxicity induced by the accumulation of excess
ammonium ions. In normal circumstances the toxic
effect of ammonium is avoided by conversion of the
ion into amino acids. There are two routes by which
this takes place (Fig. 3.2), the most important of
which, under normal circumstances, is that by which
L-glutamic acid is produced from glutamine through
the action of glutamine synthetase (GS) and
glutamate
synthetase
(GOGAT)
enzymes.
Compounds, which block the action of GS can be
used as herbicides (De Greef et al., 1989). The
reaction of α-ketoglutaric acid with NH4+ is usually
less important, but seems to have increased
significance when there is an excess of ammonium
ions
(Furuhashi
and
Takahashi,
1982).
Detoxification and ammonium assimilation may then
be limited by the availabilty of α-ketoglutaric acid,
but this may be increased in vitro by adding to the
medium one or more acids which are Krebs’ (tricarboxylic acid) cycle intermediates. Their addition
can stimulate growth of some cultures on media
containing high levels of NH4+ (Gamborg, 1970).
In comparison with media having only nitrate as
the nitrogen source, the presence of the ammonium
ion in media usually leads to rapid amino acid and
protein synthesis, and this takes place at the expense
of the synthesis of carbohydrate compounds. This
diversion of cellular metabolism can be
disadvantageous in some shoot cultures, and can
contribute towards the formation of hyperhydric
shoots. Hyperhydricity no longer occurs when NH4+
is eliminated from the medium or greatly reduced. It
71
is possible that adding an organic acid to the medium
might also alleviate the symptoms on some plants.
A supply of reduced nitrogen in addition to
nitrate, appears to be beneficial for at least two
processes involved with cell division:
• the formation of a cell wall. Without a complete
cell wall, protoplasts require a factor capable of
inducing wall formation. Freshly-isolated protoplasts
may contain sufficient of this substance to promote
wall formation for just a few divisions. The wallforming factor is only effective when NH4+ is present
in the medium (Meyer and Abel, 1975a,b): glutamine
does not substitute for NH4+:
• the activity of growth regulators. (see below)
There are several reports in the literature that,
with constant amounts of NO3-, ammonium sulphate
has not provided such a good source of NH4+ as
ammonium nitrate or ammonium chloride (De Jong
et al., 1974; Steward and Hsu, 1977; Singh, 1978;
Kamada and Harada, 1979). Possibly the reason is
that a medium containing ammonium sulphate has a
greater tendency to become acid (Harris, 1956), than
one containing less sulphate ions. This would result
if the presence of sulphate ions accelerated the uptake
of NH4+, or slowed the uptake of NO3-. Ammonium
sulphate has been used as the only source of the
ammonium ion in some media used for the culture of
legumes, including B5 (Gamborg et al., 1968).
2.1.4. Ammonium as the sole nitrogen source
pH adjustment. If plant tissues are presented
with a medium containing only NH4+ nitrogen, the
pH falls steadily as the ion is taken up (for example, a
decrease of 0.9 pH units in 15 days in Asparagus
callus – Hunault (1985) or 0.7 pH units with potato
shoots – Avila et al., (1998). Growth and
morphogenesis is possible in suspension cultures
containing only NH4+ ions, providing the pH of the
medium is frequently adjusted by the addition of a
base (Martin et al., 1977), or the medium is buffered
(see below). In wild carrot, the induction of
embryogenesis required the medium to be adjusted to
pH 5.4 at 8 hourly intervals (Dougall and Verma,
1978).
Without adjustment, the pH of media
containing only NH4+ falls rapidly to a point where
cells cannot grow (Dougall, 1981).
Buffering. Ammonium can also serve as the only
nitrogen source when the medium is buffered (see the
section on pH, below). Tobacco cells could be grown
on a medium containing NH4+ nitrogen if the organic
acid ion, succinate, was added to the medium.
Gamborg and Shyluk (1970) found that cultured cells
72
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
could be grown without frequent pH adjustment on a
medium containing only NH4+ nitrogen, when a
carboxylic acid was present. The organic acids
appeared to minimize the acidification of the medium
through NH4+ uptake. Similarly Asparagus internode
callus grew just as well on NH4+ as the only nitrogen
source as on a medium containing both NH4+ and
NO3-, but only when organic acids (such as citrate, or
malate) or MES buffer were added to the medium.
When media were buffered with MES, the best callus
growth occurred when the pH was 5.5 (Hunault,
1985).
The additional effect of organic acids.
Although Krebs’ cycle organic acids can act as
buffers, they may also act as substrates for amino
acid synthesis from NH4+. To be assimilated into
amino acids via the GDH enzyme, the ammonium ion
must react with α-ketoglutaric acid, which is
produced by the Krebs’ cycle (Fig 3.2).
Its
availability may govern the rate at which ammonium
can be metabolised by this route. The rate of
assimilation might be expected to be improved by
supplying the plant with α-ketoglutarate directly, or
by supplying acids which are intermediates in the
Krebs’ cycle (citrate, iso-citrate, succinate, fumarate
or malate), for then the natural production of
α-ketoglutarate should increase (Gamborg, 1970).
This hypothesis was confirmed by Behrend and
Mateles (1976) who concluded that succinate, or
other Krebs’ cycle acids, acted mainly as a nutrient,
replacing α-ketoglutarate as it was withdrawn from
the cycle during NH4+ metabolism and amino acid
synthesis. Depletion of α-ketoglutarate causes the
cycle to cease unless it, or another intermediate, is
replaced. The optimum molar ratio of NH4+ to
succinate, was 1.5 (e.g. 10 mM NH4+: 15 mM
succinate). Chaleff (1983) thought that the growth of
rice callus on Chaleff (loc. cit.) R3 (NH4) medium,
containing 34 mM of only ammonium nitrogen,
[Chaleff (1983) R3 NH4 medium] was enabled by the
presence of 20 mM succinate or a-ketoglutarate,
partly by the buffering capacity of the acids, and
partly by their metabolism within the plant, where
they may serve as substrate for amino acid synthesis.
Similar conclusions have been reached by other
workers (e.g. Fukunaga et al., (1978); Dougall and
Weyrauch (1980); Hunault (1985); Molnar (1988b),
who have found that compounds such as ammonium
malate and ammonium citrate are effective nitrogen
sources.
Orange juice promotes the growth of Citrus
callus. Einset (1978) thought that this was not due to
the effect of citric acid, but Erner and Reuveni (1981)
showed that citric acid, particularly at concentrations
above the 5.2 mM found in the juice used by Einset,
does indeed promote the growth of Citrus callus; it
had a more pronounced effect than other Krebs’ cycle
acids, perhaps due to the distinctive biochemistry of
the genus.
Organic acids not only enhance ammonium
assimilation when NH4+ provides the only source of
nitrogen, but may sometimes also do so when nitrate
ions are in attendance. The weight of rice anther
callus was increased on Chaleff (1983) R3 medium, if
20 mM succinate [Chaleff (1983) R3 Succ. medium]
or α-ketoglutarate was added (Chaleff, 1983).
Similarly the rate of growth of Brassica nigra
suspensions on MS medium, was improved either by
adding amino acids, or 15 mM succinate. An
equivalent improvement (apparently due entirely to
buffering) only occurred through adding 300 mM
MES buffer (Molnar, 1988b). However, the presence
of organic acids may be detrimental to
morphogenesis.
In Chaleff’s experiment, the
presence of succinate in R3 medium markedly
decreased the frequency of anther callus formation.
Photosynthesis.
Although plants grown on
nutrient solutions containing only NH4+ nitrogen have
been found to possess abnormally high levels of PEP
enzyme (Arnozis et al., 1988) (the enzyme
facilitating CO2 fixation in photosynthesis), media
containing high levels of NH 4+ tend to inhibit
chlorophyll synthesis (Yoshida and Kohno, 1982) and
photosynthesis.
2.1.5. Urea
Plants are able to absorb urea, but like the
ammonium ion, it is not a substance that is normally
available in soils in the natural environment. It is
however produced as a by-product of nitrogen
metabolism; small quantities are found in many
higher plants, which are able to utilise urea as a
source of nitrogen, providing it is first converted to
ammonium ions by the enzyme urease. In legumes
and potato, urease requires the microelement nickel
for activity (see below). In conifers, the epidermal
cells of cotyledons and cotyledons are capable of
urease induction and ammonium ion formation
(Durzan, 1987).
Urea can be used as the sole nitrogen source for
cultures, but growth is less rapid than when
ammonium and nitrate ions are supplied (Kirkby
et al., 1987); urease enzyme increases after cultures
have been maintained for several passages on a ureabased medium (King, 1977; Skokut and Filner,
Chapter 3
1980). Although the metabolism of urea, like that of
other reduced nitrogen compounds, causes the
production of excess hydrogen ions, less are
predicted to be secreted into the medium than during
the utilisation of NH4+ (Raven, 1986), so that urea is
less suitable than ammonium to balance the pH of
media containing NO3-. Nitrate ions are utilized in
preference to urea when both nitrogen sources are
available (King, 1977). Urea is able to serve as a
reduced nitrogen source during embryogenesis
(Durzan, 1987), but has been used in relatively few
culture media, and of these, none has been widely
adopted (George et al., 1987).
2.1.6. Media with nitrate and ammonium ions
Most intact plants, tissues and organs take up
nitrogen more effectively, and grow more rapidly, on
nutrient solutions containing both nitrate and
ammonium ions, than they do on solutions containing
just one of these sources. Although in most media,
reduced nitrogen is present in lower concentration
than nitrate, some morphogenic events depend on its
presence, and it can be used in plant cultures in a
regulatory role.
Adventitious organs may also
develop abnormally if NH4+ is missing (Drew, 1987).
Possible explanations, which have been put
forward for the regulatory effect of NH4+ are:
• that the reduction and assimilation of NO3⎯ is
assisted by the presence of of NH4+ or the products of
its assimilation (Bayley et al., 1972a,b; Mohanty and
Fletcher, 1978; 1980). When grown on a medium
containing a small amount of NH4+ nitrogen in
addition to nitrate, suspension cultured cells of
‘Paul’s Scarlet’ rose accumulated twice as much
protein as when grown on a medium containing only
nitrate, even though ammonium finally accounted for
only 10% of the total protein nitrogen (Mohanty and
Fletcher, 1980). Dougall (1977) considered this to be
an oversimplified interpretation, moreover nitrate
reductase activity is effectively increased by the
presence of NO3⎯ (Müller and Mendel, 1982) and in
some plants, a high concentration of NH4+ inhibits
nitrate reductase activity (see below).
• that ammonium ions effectively buffer plant
nutrient media in the presence of nitrate and so
enhance nitrate uptake (see the section on pH).
Cultures of some plants are capable of growing
with only NO3⎯ nitrogen (e.g. cell cultures of Reseda
luteoli, soybean, wheat, flax and horse radish –
Gamborg (1970); callus of Medicago sativa - Walker
and Sato (1981), although yields are generally better
when the medium is supplemented with NH4+.
73
Craven et al., (1972) with carrot, and Mohanty and
Fletcher (1978) with Rosa ‘Paul’s Scarlet’, found that
the presence of NH4+ was particularly important
during the first few days of a suspension culture.
After that cells increase in cell number and dry
weight more rapidly on NO3- nitrogen alone.
The response of plant cultures to nitrate and
ammonium ions depends to a large extent on the
enzymes shown in Fig 3.2, and the manner in which
their activities are increased or inhibited in different
tissues by the presence of the ions. These factors
vary according to the degree of differentiation of the
tissue (Suzuki and Nato, 1982), its physiological age,
and its genotype. For example, the high level of
NH4+ in MS medium inhibited the activity of
glutamate synthetase enzyme in soybean suspension
cultures (Gamborg and Shyluk, 1970), while in N.
tabacum, a peak of glutamate dehydrogenase (GDH)
appeared to exist at 10 mM NH4+ (Lazar and Collins,
1981). The activity of GDH and NADH-dependent
GOGAT developed rapidly in cultured tobacco cells,
while nitrate reductase and ferridoxin-dependent
GOGAT activity increased more slowly during
growth. By contrast, in sunflower cultures, the
specific activity of GDH and ferridoxin-dependent
GOGAT only reached a maximum at the end of
growth, and the presence of 15 mM NH4+ inhibited
the activity of nitrate reductase, indicating that the
cells were entirely dependent on the reduced nitrogen
in the medium (Lenee and Chupeau, 1989).
In consequence of such variation, the relative
concentrations of ammonium and nitrate in media
may need to be altered for different cultures.
2.1.7. The correct balance of ions
When trying to find media formulations suitable
for different plant species and different kinds of
cultures, two important factors to be considered are:
• the total concentration of nitrogen in the medium;
• the ratio of nitrate to ammomium ions.
There is a high proportion of NH4+ nitrogen in MS
medium [ratio of NO3⎯ to NH4+, 66:34] and the
quantity of total nitrogen is much higher than that in
the majority of other media. For some cultures, the
total amount of nitrogen is too high and the balance
between the two forms in the medium is not optimal.
It is noticeable that in several media used for
legume culture, there is a greater proportion of NO3⎯
to NH4+ ions than in MS medium. Evans et al.,
(1976) found that soybean leaf callus grew more
rapidly and formed more adventitious roots when the
ammonium nitrate in MS medium was replaced with
74
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
500 mg/l ammonium sulphate. A reduction in the
ammonium level of their medium for the callus
culture of red clover, was also found to be necessary
by Phillips and Collins (1979, 1980) to obtain
optimum growth rates of suspension cultured cells. A
similar adjustment can be beneficial in other genera.
Eriksson (1965) was able to enhance the growth rate
of cell cultures of Haplopappus gracilis when he
reduced the ammonium nitrate concentration to 75%
of that in MS medium, and doubled the potassium
dihydrogen phosphate level.
Table 3.4 lists examples of where changes to the
nitrogen content of MS medium resulted in improved
in vitro growth or morphogenesis. It will be seen that
the balance between NO3⎯ and NH4+ in these different
experiments has varied widely. This implies that the
ratio between the two ions either needs to be
specifically adjusted for each plant species, or that the
total nitrogen content of the medium is the most
important determinant of growth or morphogenesis.
Only occasionally is an even higher concentration of
nitrate than that in MS medium beneficial. Trolinder
and Goodin (1988) found that the best growth of
globular somatic embryos of Gossypium was on MS
medium with an extra 1.9 g/l KNO3.
There are reports that adjustments to the nitrogen
content and ratio of NO3⎯ to NH4+, can be
advantageous in media containing low concentrations
of salts (Table 3.5). Biedermann (1987) found that
even quite small adjustments could be made
advantageously to the NO3⎯ content of a low salt
medium [that of Biedermann (1987)] for the shoot
culture of different Magnolia species and hybrids, but
too great a proportion of NO3⎯ was toxic.
2.1.8.
The nitrate-ammonium ratio for various
purposes
Root growth. Root growth is often depressed by
NH4+ and promoted by NO3⎯. Media for isolated root
culture contain no NH4+, or very little. Although
roots are able to take up nitrate ions from solutions,
which become progressively more alkaline as
assimilation proceeds, the same may not be true of
cells, tissues and organs in vitro.
Shoot cultures. Media containing only nitrate
nitrogen are used for the shoot culture of some plants,
for example strawberry (Boxus, 1974), which can be
cultured with 10.9 mM NO3⎯ alone; supplementing
the medium with 6 mM NH4+ causes phytotoxicity
(Damiano, 1980).
However, shoot cultures of
strawberry grown without NH4+ can become
chlorotic: adding a small amount of NH4NO3 (or
another source of reduced nitrogen) to the medium at
the last proliferation stage, or to the rooting medium,
may then give more fully developed plants with green
leaves (Zimmerman, 1981; Piagnani and Eccher,
1988). On some occasions it is necessary to
eliminate or reduce NH4+ from the medium for shoot
cultures to prevent hyperhydricity.
2.1.9. Nitrogen supply and morphogenesis
Morphogenesis is influenced by the total amount
of nitrogen provided in the medium and, for most
purposes, a supply of both reduced nitrogen and
nitrate seems to be necessary. The requirement for
both forms of nitrogen in a particular plant species
can only be determined by a carefully controlled
experiment: simply leaving out one component of a
normal medium gives an incomplete picture. For
example, cotyledons of lettuce failed to initiate buds
when NH4NO3 was omitted from Miller (1961) salts
and instead formed masses of callus (Doerschug and
Miller, 1967): was this result due to the elimination
of NH4+, or to reducing the total nitrogen content of
the medium to one third of its original value?
Importance of the nitrate/ammonium balance.
The importance of the relative proportions of NO3⎯
and NH4+ has been demonstrated during indirect
morphogenesis and the growth of regenerated plants.
Grimes and Hodges (1990) found that although the
initial cellular events which led to plant regeneration
from embryo callus of indica rice, were supported in
media in which total nitrogen ranged from 25 to 45
mM and the NO3⎯ to NH4+ ratio varied from 50:50 to
85:15 (Table 3.5), differentiation and growth were
affected by very small alterations to the NO3⎯ to NH4+
ratio. Changing it from 80:20 (N6) medium, to
75:20, brought about a 3-fold increase in plant height
and root growth, whereas lowering it below 75:25,
resulted in short shoots with thick roots. Atypical
growth, resulting from an unsuitable balance of
nitrate and ammonium, has also been noted in other
plants. It gave rise to abnormal leaves in Adiantum
capillus-veneris (Pais and Casal, 1987); the absence
of ammonium in the medium caused newly initiated
roots of Carica papaya to be abnormally thickened,
and to have few lateral branches (Drew, 1987). Shoot
cultures may survive low temperature storage more
effectively when maintained on a medium containing
less NH4NO3 than in MS medium (Moriguchi and
Yamaki, 1989).
By comparing different strengths of Heller (1953;
1955) and MS media, and varying the NH4NO3 and
NaNO3 levels in both, David (1972) was led to the
conclusion that the principal ingredient in MS
favouring differentiation in Maritime pine explants is
75
Chapter 3
NH4NO3. However, in embryonic explants of Pinus
strobus, adventitious shoot formation was better on
Schenk and Hildebrandt (1972) medium than on MS
(which induced more callus formation).
The
difference in the ammonium level of the two media
was mainly responsible (Flinn and Webb, 1986).
Table 3.4 Examples of adjustments of the nitrogen content of MS medium, which resulted in improved growth or morphogenesis.
In each case, only NO3⎯ and NH4+ were changed, the rest of the medium being the same except for K+ and Cl⎯
Plant species
Type of
culture
Nicotiana
tabacum
Nicotiana
tabacum
Callus
Nicotiana
tabacum
Dioscorea spp.
Diospyros kaki
Rubus ideaus
Prunus avium
Castanea
sativa and
Castanea
hybrids
Peltophorum
pterocarpum
Results
Optimum callus
growth
Callus
Equal callus
growth and shoot
formation
Callus
Callus growth
and root
formation
Shoot
Optimal number
regeneration
of shoots
from leaf
disks
Callus
Callus growth
and adventitious
plantlets
Shoot
Shoot
proliferation
Shoot
Shoot
proliferation
Shoot
Shoot
proliferation
Shoot
Shoot
proliferation
Anther
Euphorbia
esula
Haplopappus
gracillis
Oryza sativa
Suspension
Allium sativum
Shoot
Solanum
tuberosum
Shoot
Suspension
Protoplast
Callus formation
and shoot
regeneration
Plant
regeneration
Improved growth
rate
Cell division
Shoot
proliferation
Shoot
proliferation
NO3⎯
(mM)
NH4+
(mM)
39.4
Total N
(mM)
Reference
20.61
Ratio of
NO3⎯ to
NH4+
1.91
60.01
40.0
48.0
52.0
24.0
20.0
12.0
8.0
12.0
2.00
4.00
6.50
2.00
60.0
60.0
60.0
36.0
Murashige and
Skoog (1962)
Behki and
Lesley (1980)
40
20
2.3
60
Ramage and
Williams (2002)
6.25
6.25
1.00
15.0
Asokan et al.,
(1983)
19.7
20.61
0.96
40.31
19.7
10.30
1.91
30.0
Sugiura et al.,
(1986)
Welander (1987)
29.09
10.3
2.82
39.40
18.00
3.00
6.00
21.0
34.7
9.99
3.47
44.6
Lakshmana Rao
and De (1987)
18.00
8.00
2.25
27
33.78
14.99
2.25
8.77
Davis et al.,
(1988)
Eriksson (1965)
18.79
1.01
18.53
19.80
35.0
8.0
4.4
43.0
41.3
17.7
2.3
59.0
Righetti et al.,
(1988)
Piagnani and
Eccher (1988)
Yamada et al.,
(1986)
Luciani et al.,
(2001)
Avila et al.,
(1998)
76
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
Table 3.5 Examples of beneficial and harmful adjustments to the total nitrogen and the NO3⎯ and NH4+ in low salt media
Plant Type of culture and
Basic
results
Medium species
or
in which
variety
N
modified
Biederman (1987)
Magnolia stellata
Shoot culture:
maximum
proliferation
Magnolia
Shoot culture:
‘Elizabeth’
maximum
proliferation
Magnolia ‘Yellow Shoot culture:
Bird’ and ‘#149’
maximum
proliferation
Magnolia (all vars.) Shoot culture: death
of all cultures
Chu et al., (1975) N6
Oryza sativa
Callus induction
from immature
embryos
Plant regneration
after callus
induction with
2,4-D
Optimum number
of plants per
zygotic embryo
Poor regeneration
Optimum plantlet
growth
NO3⎯
(mM)
NH4+
(mM)
Ratio of
NO3⎯ to
NH4+
Total N
(mM)
8.62
10.99
6.25
6.25
58:42(1.38)
1.76
14.87
17.24
8.6210.99
6.25
1.38 – 1.76
14.87 –
17.24
7.43
6.25
1.19
13.68
25.04
6.25
4.01
31.29
28.00
28.00
7.00
7.00
80:20(4.00)
4.00
35.00
35.00
12.5–
38.25
22.5-3.75
1.0 - 5.66
25.0045.00
18.75
6.25
3.00
25.00
100
<17.5
26.25
0
>17.5
8.75
∞
<1.0
8.75
35.00
Morphogenesis influenced by total available
nitrogen. Others have found that the total nitrogen
content of culture media influences morphogenesis
more than the relative ammonium concentration.
Results of Margara and Leydecker (1978) indicated
that adventitious shoot formation from rapeseed
callus was optimal in media containing 30-45mM
total nitrogen. The percentage of explants forming
shoots was reduced on media containing smaller or
greater amounts (e.g. on MS medium). Increasing the
ratio of NH4+ to total N in media, from 0.20 to 0.33
was also detrimental. Similarly, Gertsson (1988)
found that a small number of adventitious shoots was
obtained on petiole segments of Senecio hybridus
when the total nitrogen in MS medium was increased
to 75 mM, but that an increased number of shoots
was produced when the total nitrogen was reduced to
Reference
Biederman
(1987)
Grimes and
Hodges (1990)
35.00
30 mM (while keeping the same ratio of NO3⎯ to
NH4+). Shoot production was more than doubled if,
at the same time as the total N was reduced, the
potassium ion concentration was fixed at 15 mM,
instead of 20 mM.
The total amount of nitrogen in a medium was
shown by Roest and Bokelmann (1975) to affect the
number of adventitious shoots formed directly on
Chrysanthemum pedicels. The combined amount of
KNO3 + NH4NO3 in MS medium (60 mM), was
adjusted as is shown in Fig. 3.3 while the ratio of
NO3⎯ to NH4+ (66:34) was unchanged. From 30-120
mM total nitrogen was optimal. However there was
clearly a strong effect of genotype, because the
cultivar `Bravo’ was much more sensitive to
increased nitrogen than ‘Super Yellow’.
Chapter 3
Nitrogen x sugar interaction. The enhancement
of morphogenesis caused by high nitrogen levels may
not be apparent unless there is an adequate sucrose
concentration in the medium (Margara and Rancillac,
1966; Gamborg et al., 1974). In Dendrobium, the
uptake of NO3⎯ is slower than that of NH4+. Uptake is
dependent on the nature and concentration of the
sugar in the medium, being slower in the presence of
77
fructose than when sucrose or glucose are supplied
(Hew et al., 1988). The rate of growth of Rosa
‘Paul’s Scarlet’ suspensions was influenced by the
ratio of NO3⎯ to sucrose in the medium. A high ratio
favoured the accumulation of reduced nitrogen, but
not the most rapid rate of cell growth (Fletcher,
1980).
Fig. 3.3 The number of adventitious shoots formed directly on Chrysanthemum explants with increasing total nitrogen concentration in an
otherwise normal MS medium [from data of Roest and Bokelmann, 1975]
Embryogenesis and embryo growth. It is
accepted that the presence of some reduced nitrogen
is necessary for somatic embryogenesis in cell and
callus cultures (Halperin and Wetherell, 1965;
Reinert et al., 1967); but although reduced nitrogen
compounds are beneficial to somatic embryo
induction, apparently they are not essential until the
stage of embryo development (Kamada and Harada,
1979). A relatively high level of both nitrate and
ammonium ions then seems to be required. Some
workers have also noted enhanced embryogenesis
and/or improved embryo growth when media have
been supplemented with amino acids in addition to
NO3⎯ and NH4+. Street (1979) thought that an
optimum level of NH4+ for embryogenesis was about
10 mM (from NH4Cl) in the presence of 12-40 mM
NO3 (from KNO3): that is:
[NO3⎯ to NH4+ ratio, from 55:45 to 80:20; Total N 22-50 mM]
Walker and Sato (1981) obtained no
embryogenesis in alfalfa callus in the absence of
either ammonium or nitrate ions. Miller’s medium
[Miller (1961; 1963)] (12.5 mM NH4+) supported a
high rate of embryogenesis:
[NO3⎯ to NH4+ ratio, 68:32; Total N 39.1 mM], but only a
small number of embryos were produced on Schenk
and Hildebrandt (1972) medium (SH):
[NO3⎯ to NH4+ ratio, 90:10, Total N 27.32 mM], unless it was
supplemented with NH4+ from either ammonium
carbamate, ammonium chloride or ammonium
sulphate. An optimal level of ammonium in SH
medium was 12.5 mM:
78
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
[NO3⎯ to NH4+ ratio, then 66:34, total N, 37.22],
although
embryogenesis was still at a high level with 100 mM
ammonium ion:
[NO3⎯ to NH4+ ratio, then, 20:80; total N, 124.72 mM].
By contrast, Coffea arabica leaf callus (Sondahl
and Sharp, 1977), which was formed on a medium
containing MS salts, was induced to become
embryogenic by first being cultured on a medium
with MS salts (and high auxin):
[NO3⎯ to NH4+ ratio, 66:34; total N, 30.0 mM],
and then moved to another medium containing MS
salts with an extra 2850 mg/l KNO3:
[NO3⎯ to NH4+ ratio, 82:18; total N, 58.2 mM] (and low
auxin)
Zygotic embryos. The presence of some reduced
nitrogen in the growth medium is also required for
the continued growth of zygotic embryos in culture.
Nitrate alone is insufficient (Mauney et al., 1967;
Norstog, 1967, 1973). An optimum concentration of
NH4+ for the development of barley embryos in
culture was 6.4 mM (Umbeck and Norstog, 1979). A
similar provision seems to be necessary in most
plants for the in vitro growth of somatic embryos.
Flower bud formation and growth. Nitrate was
essential for the formation of adventitious buds on
leaf segments of Begonia franconis. The greatest
proportion of flower buds was obtained with 5 mM
NO3⎯ and 1.5 mM NH4+. Above this level, NH4+
promoted vegetative sprouts (Berghoef and
Bruinsma, 1979b). The best in vitro growth of
Begonia franconis flower buds detached from young
inflorescences, occurred on a medium with 10-15
mM total nitrogen (NO3⎯ to NH4+ ratio, 50:50 to
67:33) (Berghoef and Bruinsma, 1979a). Detached
flower buds of Cleome iberidella were found to grow
best in vitro with 25 mM total nitrogen (NO3⎯ to NH4+
ratio, 80:20) (De Jong and Bruinsma, 1974; De Jong
et al., 1974), but the complete omission of NH4NO3
from MS medium, where the salts had been diluted to
their original concentration, promoted the
development (but not the initiation) of adventitious
floral buds of Torenia fournieri (Tanimoto and
Harada, 1979, 1981, 1982).
Effect on the action of growth regulants. The
ratio of NO3⎯ to NH4+ present in the culture medium
has been found to affect the activity of plant growth
substances and plant growth regulators.
The
mechanisms by which this occurs are not fully
elucidated. It has been noted, for example, that cells
will divide with less added cytokinin when the
proportion of reduced nitrogen is reasonably high.
To induce tobacco protoplasts to divide, it was
necessary to add 0.5-2 mg/l BAP to a medium
containing only NO3⎯ nitrogen. The presence of
glutamine or NH4+ in the medium together with NO3⎯,
reduced the cytokinin requirement, and division
proceeded without any added cytokinin when urea,
NH4+, or glutamine were the sole N-sources of the
medium (Meyer and Abel, 1975b). Sargent and King
(1974) found that soybean cells were dependent on
cytokinin when cultured in a medium containing
NO3⎯ nitrogen, but independent of cytokinin when
NH4+ was present as well.
The relative proportion of nitrate and ammonium
ions also affects the response of cells to auxin growth
regulators in terms of both cell division and
morphogenesis. It is possible that this is through the
control of intracellular pH (see below). Carrot
cultures that produce somatic embryos when
transferred from a high- to a low-auxin medium, can
also be induced into embryogenesis in a high-auxin
medium, if it contains adequate reduced nitrogen.
Only root initials are formed in high-auxin media
which do not contain reduced nitrogen (Halperin,
1967). The number of plants regenerated from rice
callus grown on Chu et al., (1975) medium
containing 0.5 mg/l 2,4-D, depended on the ratio of
NO3⎯ to NH4+. It was high in the unaltered medium
(ratio 4:1), but considerably less if, with the same
total N, the ratio of the two ions was changed to 1:1
(Grimes and Hodges, 1990).
Cells of Antirrhinum majus regenerated from
isolated protoplasts were stimulated to divide with a
reduced quantity of auxin in a medium containing
39.77 mM total nitrogen
[NO3⎯ to NH4+ ratio, 39:77 (2.98)]
by adding further ammonium ion to give a total
nitrogen content of 54.72 mM
[NO3⎯ to NH4+ ratio, 54:46 (1.19)].
or, alternatively, 400 mg/l of casein hydrolysate
(Poirier-Hamon et al., 1974).
In experiments of Koetje et al., (1989) and
Grimes and Hodges (1990) (Table 3.5), when the
NO3⎯ to NH4+ ratio in N6 medium was 80:20, there
was a strong dose response curve to the auxin 2,4-D
with 0.5 mg/l being the best concentration to induce
embryogenesis in Oryza sativa callus; if the medium
was modified, so that the NO3- to NH4+ ratio was
66:34 or 50:50, 2,4-D was less effective, and there
was little difference in the number of plants
regenerated between 0.5 and 3 mg/l 2,4-D. The ratio
of NO3⎯ to NH4+ therefore seemed either to to alter
the sensitivity of cells to the auxin, or to affect its
uptake or rate of metabolism.
79
Chapter 3
Walker and Sato (1981) also found that the
proportion of ammonium ion in the medium can
influence the way in which growth regulants control
morphogenesis. Having been placed for 3 days on a
medium which would normally induce root formation
[Schenk and Hildebrandt (1972) medium containing
5 mM 2,4-D and 50 mM kinetin], suspension cultured
cells were subsequently plated on a modification of
the same medium (which contains 24.8 mM NO3⎯)
without regulants, in which the concentration of NH4+
had been adjusted to various levels. Table 3.6 shows
that the morphogenesis experienced, depended on the
concentration of ammonia in the regeneration
medium. Media containing high levels of ammonium
ion would have tended to become acid, especially as
the extra ammonium was added as ammonium
sulphate. Possibly this affected the uptake or action of
the regulants?
2.1.10. Addition of amino acids
Amino acids can be added to plant media to
satisfy the requirement of cultures for reduced
nitrogen, but as they are expensive to purchase, they
will only be used in media for mass propagation
where this results in improved results. For most
tissue culture purposes, the addition of amino acids
may be unnecessary, providing media contain
adequate amounts, and correct proportions, of nitrate
and ammonium ions. For example, Murashige and
Skoog (1962) found that when cultures were grown
on media such as Heller (1953; 1955), Nitsch and
Nitsch (1956) N1, and Hildebrandt et al., (1946)
Tobacco, containing sub-optimal amounts of
inorganic chemicals, a casein hydrolysate (consisting
mainly of a mixture of amino acids, see later)
substantially increased the yield of tobacco callus,
whereas it gave only marginal increases in yield
when added to their revised MS medium. Arginine
(0.287 mM) increased the growth of sugar cane callus
and suspension cultures grown on Nickell and
Maretzski (1969) medium (Nickell and Maretzski,
1969) but was without effect on cultures of this plant
grown on a medium based on Scowcroft and
Adamson (1976) CS5 macronutrients (Larkin, 1982).
It is noticeable from the literature that organic
supplements (particularly amino acids) have been
especially beneficial for growth or morphogenesis
when cells or tissues were cultured on media such as
White (1943a), which do not contain ammonium
ions. White (1937) and Bonner and Addicott (1937),
for example, used known amino acids to replace the
variable mixture provided by yeast extract. For the
culture of Picea glauca callus, Reinert and White
(1956) supplemented Risser and White (1964)
medium with 17 supplementary amino acids, and
similar, or greater numbers, were used by Torrey and
Reinert (1961) and Filner (1965) in White (1943)
medium for the culture of carrot, Convolvulus
arvensis, Haplopappus gracilis and tobacco tissues.
Dependence on the nitrate to ammonium ratio.
Grimes and Hodges (1990) found that when both
NO3⎯ and NH4+ are present in the medium, the
response to organic nitrogen depends on the ratio of
these two ions.
Twice as many plants were
regenerated from embryogenic rice callus when 1g/l
CH was added to Chu et al., (1975) N6 medium,
providing the proportion of NO3⎯ to NH4+ was also
changed to 1 (50:50). There was little response to
CH with the same amount of total N in the medium,
if the NO3⎯/NH4+ ratio was 4 (i.e. 80%:20%, as in the
original medium), or more.
2.1.11. Amino acids as the sole N source
As most of the inorganic nitrogen supplied in
culture media is converted by plant tissues to amino
acids, which are then assimilated into proteins, it
should be possible to culture plants on media in
which amino acids are the only nitrogen source. This
has been demonstrated: for example, Nicotiana
tabacum callus can be cultured on MS salts lacking
NO3⎯ and NH4+ (but with an extra 20.6 mM K+), if 0.1
mM glycine, 1mM arginine, 2 mM aspartic acid and
6 mM glutamine are added (Muller and Grafe, 1978);
wild carrot suspensions can be grown on a medium
Table 3.6 The effect of total nitrogen, and the ratio of NO3⎯ to NH4+ on the type of organ produced by alfalfa cells which have
been subcultured on a root-inducing medium (Walker and Sato, 1981)
Type of organ
produced
Roots
Roots and somatic
embryos
Somatic embryos
NH4+
(mM)
<2.5
12.5-37.5
NO3⎯
(mM)
27.2
37.3-62.3
Ratio of NO3⎯ to
NH4+
100:0 to 91:9
66:34 to 40:60
50-100
74.7-124.7
33:67 to 20:80
80
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
containing glutamine or casein hydrolysate as the sole
nitrogen source (Anderson, 1976). Amino acids
provide plant cells with an immediately available
source of nitrogen, and uptake can be much more
rapid than that of inorganic nitrogen in the same
medium (Thom et al., 1981). Only the L- form of
amino acids is biologically active.
Amino acids can also provide reduced nitrogen in
culture media in place of NH4+ and as a supplement
to NO3⎯. However they are usually employed as
minor additions to media containing both NH4+ and
NO3⎯. Uptake of amino acids into cultured tissues
causes a decrease in the pH of the medium, similar to
that which occurs when NH4+ ions are absorbed.
Sugar-based amines such as glucosamine and
galactosamine can also serve as a source of reduced
nitrogen in morphogenesis (Margara, 1969a; Margara
and Leydecker, 1978).
2.1.12. Biologically-active amino acids
Amino acids are classified according to their
stereoisomers and according to the relative positions
of the amino group and the acidic radical. Only the
L- isomers of the α-amino acids are important for
plant tissue culture media. They have the general
structure:
NH2
|
R-CH- COOH
β-Amino acids are present in plants but tend to
result from secondary metabolism. They have the
general structure:
NH2
|
R-CHCH2- COOH
(where R = functional groups)
Unfortunately the particular amino acid, or
mixture of amino acids, which promotes growth or
morphogenesis in one species, may not do so in
another.
For instance, L-α-alanine, glutamine,
asparagine, aspartic acid, glutamic acid, arginine and
proline could serve as a source of reduced nitrogen in
a medium containing 20 mM NO3⎯, and were
effective in promoting embryogenesis in Daucus
carota callus and suspensions, but lysine, valine,
histidine, leucine and methionine were ineffective
(Kamada and Harada, 1982).
Competitive inhibition. Some amino acids are
growth inhibitory at fairly low concentrations and this
is particularly observed when mixtures of two or
more amino acids are added to media. Inhibition is
thought to be due to the competitive interaction of
one compound with another. In oat embryo cultures,
phenylalanine and L-tyrosine antagonise each other,
as do L-leucine and DL-valine; DL-isoleucine and
DL-valine, and L-arginine and L-lysine (Harris,
1956). Lysine and threonine often exert a cooperative inhibition when present together, but do not
inhibit growth when added to a medium singly
(Cattoir-Reynaerts et al., 1981).
Glycine. Glycine is an ingredient of many media.
It has usually been added in small amounts, and has
been included by some workers amongst the vitamin
ingredients. Despite frequent use, it is difficult to
find hard evidence that glycine is really essential for
so many tissue cultures, but possibly it helps to
protect cell membranes from osmotic and
temperature stress (Orczyk and Malepszy, 1985).
White (1939) showed that isolated tomato roots
grew better when his medium was supplemented with
glycine rather than yeast extract and that glycine
could replace the mixture of nine amino acids that
had been used earlier. It was employed as an organic
component by Skoog (1944) and continued to be used
in his laboratory until the experiments of Murashige
and Skoog (1962). They adopted the kinds and
amounts of organic growth factors specified by White
(1943a) and so retained 2 mg/l glycine in their
medium without further testing.
Linsmaier and Skoog (1965), furthering the study
of medium components to organic ingredients,
omitted glycine from MS medium and discovered
that low concentrations of it had no visible effect on
the growth of tobacco callus, while at 20 mg/l it
depressed growth. No doubt the success of MS
medium has caused the 2 mg/l glycine of Skoog
(1944) to be copied in many subsequent experiments.
Many workers overlook the later Linsmaier and
Skoog paper.
Casein and other protein hydrolysates.
Proteins, which have been hydrolysed by acid, or
enzymes, and so broken down into smaller molecules,
are less costly than identified amino acids. The
degree of degradation varies: some protein
hydrolysates consist of mixtures of amino acids
together with other nitrogenous compounds such as
peptide fragments, vitamins, and elements which
might (if they can form inorganic ions, or are
associated with organic compounds that can be taken
Chapter 3
up by plant tissues) be able to serve as macro- or
micro-elements. Peptones are prepared from one or
several proteins in a similar fashion but generally
consist of low molecular weight proteins. Although
protein hydrolysates are a convenient source of
substances which may promote plant growth, they are
by nature relatively undefined supplements. The
proportion of individual amino acids in different
hydrolysates depends on the nature of the source
protein and the method by which the product has
been prepared.
The hydrolysate most often used in culture media
is that of the milk protein, casein, although
lactalbumin hydrolysate has been employed (La
Motte and Lersten, 1971). Peptones and tryptone
have been used less frequently, but there are reports
of their having been added to media with advantage
(e.g. Muralidhar and Mehta, 1982; Pierik et al.,
1988). Casein hydrolysates can be a source of
calcium, phosphate, several microelements, vitamins
and, most importantly, a mixture of up to 18 amino
acids. Several casein hydrolysates (CH) are available
commercially but their value for plant tissue culture
can vary considerably. Acid hydrolysis can denature
some amino acids and so products prepared by
enzymatic hydolysis are to be preferred. The best can
be excellent sources of reduced nitrogen, as they can
contain a relatively large amount of glutamine.
Casein hydrolysate produces an improvement in
the growth of Cardamine pratensis and Silene alba
suspensions, only if the medium is deficient in
phosphorus. Glutamine has the same effect; it is the
most common amino acid in CH, and its synthesis
requires ATP. For these reasons, Bister-Miel et al.,
(1985) concluded that CH overcomes the shortage of
glutamine when there is insufficient phosphorus for
adequate biosynthesis.
However several investigators have concluded
that casein hydrolysate itself is more effective for
plant culture than the addition of the major amino
acids which it provides. This has led to speculation
that CH might contain some unknown growthpromoting factor (Inoue and Maeda, 1982). In
prepared mixtures of amino acids resembling those in
CH, competitive inhibition between some of the
constituents is often observed. For instance, the
induction of embryogenesis in carrot cell suspensions
on a medium containing glutamine as the only
nitrogen source, was partly inhibited by the further
addition of L-amino acids similar in composition to
those in CH. This suppression was mainly caused by
the L-tyrosine in the mixture (Anderson, 1976).
81
There may be a limit to the amount of CH, which
can be safely added to a medium. Anstis and
Northcote (1973) reported that the brand of CH
known as ‘N-Z-amine’, can produce toxic substances
if concentrated solutions are heated, or if solutions
are frozen and thawed several times. Possibly these
are reasons why mixtures of amino acids occasionally
provide more valuable supplements than CH.
Nicotiana tabacum callus grew better on a nitrogenfree MS medium when a mixture of the amino acids
L-glutamine (6 mM), L-aspartic acid (2 mM), Larginine (1 mM) and glycine (0.1 mM) was added,
rather than 2 g/l casein hydrolysate (which would
have provided about 2 mM glutamine, 0.6 mM
aspartic acid, 0.2 mM arginine and 0.3 mM glycine)
(Muller and Grafe, 1978).
2.1.13. Beneficial effects of amino acid additions
Improved growth. The growth rate of cell
suspensions is frequently increased by the addition of
casein hydrolysate or one or more amino acids
(particularly glutamine) to media containing both
nitrate and ammonium ions. Some workers have
included a mixture of several amino acids in their
medium without commenting on how they improved
growth. In other cases the benefit resulting from a
specific compound has been clearly shown. The lag
phase of growth in suspensions of Pseudotsuga
menziesii cultured on Cheng (1977; 1978) medium
was eliminated by the addition of 50 mM glutamine,
and the final dry weight of cells was 4 times that
produced on the unammended medium (Kirby, 1982).
Similarly the rate of growth of Actinidia chinensis
suspensions was improved by the addition of 5mM
glutamine (Suezawa et al., 1988) and those of Prunus
amygdalus cv. ‘Ferragnes’ could not be maintained
unless 0.2% casamino acids was added to the medium
(Rugini and Verma, 1982). Molnar (1988b) found
that the growth of Brassica nigra cell suspensions
was improved by adding 1-4 g/l CH or a mixture of 4
mM alanine, 4 mM glutamine and 1 mM glutamic
acid. In this case the medium contained MS salts (but
less iron) and B5 vitamins.
Amino acid supplements have also been used to
boost the rate of growth of callus cultures. For
instance, Short and Torrey (1972) added 5 amino
acids and urea to a medium containing MS salts for
the culture of pea root callus, and Sandstedt and
Skoog (1960) found that aspartic and glutamic acids
promoted the growth of tobacco callus as much as a
mixture of several amino acids (such as found in
yeast extract). Glutamic acid seemed to be primarily
responsible for the growth promotion of sweet clover
82
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
callus caused by casein hydrolysate on a medium
containing 26.6 mM NO3⎯, 12.5 mM NH4+ and 2.0
mM PO43- (Taira et al., 1977).
Amino acids are often added to media for
protoplast culture. It was essential to add 2 mM
glutamine and 2 mM asparagine to a medium
containing MS salts, to obtain cell division, colony
growth and plantlet differentiation from Trigonella
protoplasts (Shekhawat and Galston, 1983).
Shoot cultures. Many shoot cultures are grown
on MS medium containing glycine, although in most
cases the amino acid is probably not an essential
ingredient. Usually it is unnecessary to add amino
acids to media supporting shoot cultures, but
methionine may represent a special case. Druart
(1988) found that adding 50-100 mg/l L-methionine
to the medium seemed to stimulate cytokinin activity
and caused cultures of Prunus glandulosa var.
sinensis to have high propagation rates through
several subcultures. This promotive effect of Lmethionine was thought to be due to it acting as a
precursor of ethylene (see Chapter 7). Glutamine
inhibited the growth of apical domes excised from
Coleus blumei shoots (Smith, 1981) and 50-100 mg/l
glutamic acid inhibited shoot growth, the formation
of axillary buds and shoot proliferation in cultures of
woody plants (Druart, loc. cit.).
L-Citrulline is an important intermediate in
nitrogen metabolism in the genus Alnus. The
addition of 1.66 mM (4.99 mM NH2) to WPM
medium improved the growth of A. cordata and
A. subcordata shoot cultures (Cremiere et al., 1987).
Contaminants grow more rapidly on media
containing amino acids.
Casein hydrolysate is
therefore sometimes added to the media for Stage I
shoot cultures so that infected explants can be
rejected quickly (Schulze, 1988). The health of
shoots grown from seedling shoot tips of Feijoa
(Acca) sellowiana was improved when 500 mg/l CH
was added to Boxus (1974) medium (which does not
contain ammonium ions) (Bhojwani et al., 1987).
Organogenesis. The presence of amino acids can
enhance morphogenesis, either when they provide the
only source of reduced nitrogen, or when they are
used as a supplement to a medium containing both
NO3⎯ and NH4+. In a medium containing 25 mM
nitrate, but no NH4+, direct adventitious shoot
formation on cauliflower peduncle explants was
induced by the addition of a mixture of the amino
acids asparagine, proline, tyrosine and phenylalanine,
each at a concentration of only 0.1 mM. (Margara,
1969b).
A high rate of adventitious shoot
regeneration and embryogenesis, from Beta vulgaris
petioles or petiole callus, was achieved on a medium
comprised of several amino acids and a complex
vitamin mixture with MS salts (Freytag et al., 1988).
The addition of CH to MS medium was found to be
essential for shoot formation from callus (Chand and
Roy, 1981).
Adding only 1-10 mg/l of either L-leucine or Lisoleucine to Gamborg et al., (1968) B5 medium,
decreased callus growth of Brassica oleracea var
capitata, but increased adventitious shoot formation.
Basu et al., (1989) thought that this might be due to
these amino acids being negative effectors of
threonine deaminase (TD) enzyme, the activity of
which was diminished in their presence. Threonine,
methionine and pyruvic acid, which increased callus
growth in this species, enhanced TD activity.
There are several examples of the amino acid Lasparagine being able to stimulate morphogenesis.
This may be because it too can be a precursor of
ethylene (Durzan, 1982), the biosynthesis of which
may be increased by greater substrate availability.
Kamada and Harada (1977) found that the addition of
5 mM L-asparagine stimulated both callus and bud
formation in stem segments of Torenia fournieri,
while alanine (and, to a lesser extent, glutamic acid)
increased flower bud formation from Torenia
internode segments when both an auxin and a
cytokinin were present. An increase in the number of
adventitious buds formed on the cotyledons and
hypocotyl of Chamaecyparis obtusa seedlings
occurred when 1.37 mM glutamine and 1.51 mM
asparagine were added together to Campbell and
Durzan (1975) medium, but not when they were
supplied on their own (Ishii, 1986). L-asparagine was
also added to MS medium by Green and Phillips
(1975), to obtain plant regeneration from tissue
cultures of maize; adding it to Finer and Nagasawa
(1988) 10A4ON medium caused there to be more
embryogenic clumps in Glycine max suspension
cultures (Finer and Nagasawa, 1988).
Amino acid additions do not invariably enhance
morphogenesis.
Supplementing Linsmaier and
Skoog (1965) medium with 0.5-5 mM glutamine,
caused callus of Zamia latifolia to show greatly
decreased organogenesis (Webb and Rivera, 1981)
and ammending Linsmaier and Skoog (1965)
medium with 100 mg/l CH, prevented adventitious
shoot formation from stem internode callus of apple
and cherry rootstocks (James et al., 1984).
Embryogenesis. The presence the ammonium
ion is usually sufficient for the induction of
Chapter 3
embryogenesis in callus or suspension cultures
containing NO3⎯, but on media where NH4+ is lacking
[e.g. White (1954)], casein hydrolysate, or an amino
acid such as alanine, or glutamine, is often promotory
(Ranga Swamy, 1958; Ammirato and Steward, 1971;
Street, 1979). For embryogenesis in carrot cultures,
Wetherell and Dougall (1976) have shown that in a
medium containing potassium nitrate, reduced
nitrogen in the form of ammonium chloride matched
the effectiveness of an equivalent concentration of
nitrogen from casein hydrolysate. Casein hydrolysate
could be replaced by glutamine, glutamic acid, urea
or alanine. Suspensions of wild carrot cells grew and
produced somatic embryos on a medium containing
either glutamine or CH as the sole nitrogen source
(Anderson, 1976).
There have also been many reports of
embryogenesis being promoted by the addition of
casein hydrolysate, or one or more specific amino
acids, when both NO3⎯ and NH4+ were available in the
medium. Some examples are given in Table 3.7. In
many cases, embryogenic callus and/or embryo
formation did not occur without the presence of the
amino acid source, suggesting that without amino
acid, the medium was deficient in NH4+ or total
nitrogen. Armstrong and Green (1985) found that the
frequency of friable callus and somatic embryo
formation from immature embryos of Zea mays
increased almost linearly with the addition of up to 25
mM proline to Chu et al., (1975) N6 medium
[total N, 34.99 mM; NO3/NH4 ratio, 3.99],
but there was no benefit from adding proline to MS
medium (containing 150 mg/l asparagine hydrate).
[total N, 60.01 mM; NO3/NH4 ratio, 1.91]
The growth of somatic embryos can also be
affected by the availability of reduced nitrogen. That
of Coronilla varia embryos was poor on Gamborg et
al., (1968) B5 medium (Total N 26.74 mM; NH4+
2.02 mM), unless 10 mM asparagine or 20 mM
NH4Cl was added to the medium, or unless the
embryos were moved to Saunders and Bingham
(1972) BOi2Y medium, which has 37.81 mM total
inorganic N, 12.49 mM NH4+ and 2000 mg/l casein
hydrolysate (approx. 9.9 mM NH4+ equivalence)
(Moyer and Gustine, 1984).
However, the
germination of Triticum aestivum somatic embryos
was completely prevented by adding 800 mg/l CH to
MS medium (Ozias Akins and Vasil, 1982; Carman
et al., 1988).
Culture of immature cotyledons.
Young
storage cotyledons isolated from immature zygotic
embryos accumulate protein efficiently when cultured
with amino acids in a medium without nitrate and
83
ammonium ions. A medium such as that of Millerd
et al. (1975), or of Thompson et al., (1977) is
normally used, but where the effect of different
amino acids on protein assimilation is being studied,
the amino acid content of the medium is varied.
Glutamine is often found to be the most efficient
nitrogen source for this purpose (Thompson et al.,
1977; Haga and Sodek, 1987), but protein increase
from culture with asparagine and glutamate (glutamic
acid) is usually also significant (Lea et al., 1979).
2.1.14.
acids
Causes of the stimulatory effect of amino
We may conclude therefore, that for many
cultural purposes, amino acids are not essential media
components; but their addition as identified pure
compounds, or more cheaply through casein
hydrolysates, can be an easy way of ensuring against
medium deficiency, or of providing a source of
nitrogen that is immediately available to cultured
cells or tissues. An observation by Murashige and
Skoog (1962) that the presence of casein hydrolysate
allowed vigorous organ development over a broader
range of IAA and kinetin levels, may be of
significance.
In gram moles per litre, amino acids can be a
much more efficient source of reduced nitrogen than
ammonium compounds. For instance, the mixture of
amino acids provided by 400 mg/l of casein
hydrolysate (containing at most as much reduced
nitrogen as 3.3 mM NH4+) was as effective as 14.95
mM NH4Cl in stimulating the division of protoplastderived cells of Antirrhinum (Poirier-Hamon et al.,
1974).
Why should this be, and why can additions of
amino acids (sometimes in comparatively small
amounts) stimulate growth or morphogenesis when
added to media, which already contain large amounts
of NH4+? Some hypotheses, which have been
advanced are:
• Conservation of ATP - alleviating phosphate
deficiency. Durzan (1982) pointed out that when
plant tissues take up the ammonium ion, they
consume adenosine tri-phosphate (ATP) in
converting it to amino acids. If suitable amino acids
are available from the medium, some of ATP may be
conserved. Bister-Miel et al., (1985) noted that CH
promoted growth in cultures where phosphate
became growth-limiting. They suggested that amino
acids compensated for phosphate deficiency. With
the plant well supplied with amino acids, some of the
phosphate, which is normally used for ATP
production can be diverted to other uses. Several
84
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
Table 3.7 Some examples of the promotion of embryogenesis by amino acids in media containing NO3- and NH4+
Type of culture
Aesculus
hippocastrum
Dactylis glomerata
Daucus carota
Zygotic embryo
callus
Suspension-derived
callus
Hypocotyl callus
Dioscorea
rotundata
Glycine max
Zygotic embryo
callus
Suspension
Gossypium
klotzschianum
Larix decidua
Basal medium used
MS
Schenk and
Hildebrandt (1972)
Gamborg et al.
(1968) B5
MS
Amino acid
supplements
CH (250 mg/l) +
Proline (250 mg/l)
CH (1.5 g/l)
Reference
Radojevic (1988)
Gray et al. (1984)
Proline (100 mM) + Nuti Ronchi et al.
Serine (100 mM)
(1984)
CH (1 g/l)
Osifo (1988)
Kartha et al.
(1974a)
Suspension
Gamborg et al.
(1968) B5
Gametophyte callus Litvay et al. (1981)
LM
Nigella sativa
Roots or leaf callus
Trigonella foenumgraecum
Leaf callus
Triticum aestivum
Anther
Vitis vinifera
Zea mays
Anther
Zygotic embryo
callus
L-asparagine
Finer and Nagasawa
(5 mM)
(1988)
Glutamine (10 mM) Price and Smith
(1979a,b)
CH (1 g/l) +
Nagmani and
Glutamine
Bonga (1985)
(500 mg/l)
MS
CH
Bannerjee and
(100 – 500 mg/l)
Gupta (1976)
MS
CH (50 mg/l)
Gupta et al. (1987)
(500 mg/l was
inhibitory)
Chu and Hill (1988) Serine, proline,
Chu and Hill (1988)
MN6
arinine, aspartic
acid and alanine
(each at 40 mg/l) +
glutamine
(400 mg/l)
½ MS
CH (250 mg/l)
Mauro et al. (1986);
Chu et al. (1975)
Proline (20-25 mM) Kamo et al. (1985)
N6
Armstrong and
Green (1985)
authors have pointed out that CH itself is also a
source of phosphate. For example, Bridson (1978)
and some chemical catalogues, show that some casein
hydrolysates normally contain about 1.3g P2O5 per
100g. The addition of 2 g/l of CH will therefore
increase the phosphate content of MS medium by
11% and that of White (1954) medium by 44%
(assuming complete phosphate availability).
• A capacity to act as chelating agents. Some
amino acids can act as chelating agents (see later in
section on chelates)
• Enhanced nitrogen assimilation. Glutamine and
glutamic acid are directly involved in the assimilation
of NH4+. A direct supply of these amino acids should
therefore enhance the utilization of both nitrate and
ammonium nitrogen and its conversion into amino
acids.
• A replacement for toxic ammonium ions.
Certain plant tissues are particularly sensitive to
NH4+. Ochatt and Caso (1986) and Ochatt and Power
(1988a, b) found that protoplasts of Pyrus spp. would
not tolerate the ion, and that to obtain sustained cell
division it was necessary to eliminate it from MS
medium, and use 50 mg/l casein hydrolysate as a
source of reduced nitrogen. CH can however be
extremely toxic to freshly isolated protoplasts of
some species and varieties of plants (Ranch and
Widholm, 1980; Russell and McCown, 1988).
Conifer tissues too are unable to cope with high
Chapter 3
concentrations of NH4+, but cultures can be supplied
with equivalent levels of reduced nitrogen in the form
of amino acids without the occurrence of toxicity
(Durzan, 1982). In soybean suspension cultures, the
high level of ammonium in MS medium has been
shown to inhibit isocitrate dehydrogenase (a Krebs’
cycle enzyme) and glutamine synthetase, which
contribute to the conversion of NH4+ to glutamine
(Gamborg and Shyluk, 1970).
• Adjustment of intracellular pH.
As
intracellular pH is important for the activation of sea
urchin eggs, and amino acids can promote
embryogenesis, Nuti Ronchi et al., (1984) speculated
that the uptake and assimilation of amino acids might
help to regulate cellular pH in plants.
As mentioned before, there is commonly a
minimum inoculation density below which growth
cannot be initiated in vitro. This minimum varies
according to both the source of the cells and the
nature of the medium. It can usually be lowered by
employing a ‘conditioned’ medium (i.e. a fresh
medium into which the products of another medium
in which cells are actively growing, have been
added). Alternatively, initial growth at low densities
can be supported by the close presence of other
actively growing plant cells (‘nurse cultures’).
Compounds responsible for this effect must be freely
diffusible from living cells and could include growth
substances, reducing sugars, vitamins and amino
acids. Addition of such supplements has been found
to overcome the inhibited growth of some cells at low
densities (Kao and Michayluk, 1975).
2.2. PHOSPHATE
Phosphorus is a vital element in plant
biochemistry. It occurs in numerous macromolecules
such as nucleic acids, phospholipids and co-enzymes.
It functions in energy transfer via the pyrophophate
bond in ATP. Phosphate groups attached to different
sugars provide energy in respiration and
photosynthesis and phosphate bound to proteins
regulates their activity. Phosphorus is absorbed into
plants in the form of the primary or secondary
orthophosphate anions H2PO4- and HPO42- by an
active process, which requires the expenditure of
respiratory energy. Phosphate, in contrast to nitrate
and sulphate, is not reduced in plants, but remains in
the highly oxidized form. It is used in plants as the
fully oxidized orthophosphate (PO43-) form.
In culture media the element is provided as
soluble potassium mono- and di-hydrogen
phosphates. The di- and mono-valent phosphate
85
anions respectively provided by these chemicals are
interconvertible in solution depending on pH.
Monovalent H2PO4- predominates at pH values below
7, characteristic of most tissue culture media, and it is
this ion, which is most readily absorbed into plants
(Devlin, 1975). Conversion of H2PO4- into divalent
HPO42- begins to occur as solutions become more
alkaline. The divalent ion is said to be only sparingly
available to plants but Hagen and Hopkins (1955) and
Jacobsen et al., (1958) thought that its absorption
could be significant, because even though the ion is
normally at a relatively low concentration in nutrient
solutions, its affinity with the site of absorption is
greater than that of the mono-valent form. Trivalent
PO43-, which appears in alkaline solutions, is not
generally absorbed by plants.
In some early tissue culture media, all (e.g.
Bouharmont, 1961), or part (e.g. Vacin and Went,
1949) of the phosphorus was supplied as sparinglysoluble phosphates. A slow rate of phosphorus
availability seems to be possible from such
compounds.
The optimum rate of uptake of
phosphate (HPO42-) into cultured Petunia cells
occurred at pH 4 (Chin and Miller, 1982) but Zink
and Veliky (1979) did not observe any decline in the
absorption of phosphate by Ipomoea suspension
cultures at pH 6.5, when HPO42- and H2PO4- were
present in approximately equal concentrations. Plant
tissue cultures secrete phosphatase enzymes into the
medium (Ciarrocchi et al., 1981), which could release
phosphate ions from organic phosphates.
In the cytoplasm, phosphate is maintained at a
constant concentration of 5-10 mM, more or less
independent of the external concentration. Phosphate
in the vacuole fluctuates according to the external
concentration but does not increase above 25 mM
(Schachtman et al., 1998). When there is a high
supply of phosphate and it is taken up at rates that
exceed the demand, a number of processes act to
prevent toxic phosphate concentrations, among others
storage into inorganic compounds such as phytic
acid. High concentrations of dissolved phosphate can
depress growth, possibly because calcium and some
microelements are precipitated from solution and/or
their uptake reduced. In Arabidopsis thaliana, four
different phophate transporter genes have been
isolated (APT1-4).
In vivo, the genes are
predominantly expressed in the roots and their
expression is constitutive or induced by phosphate
starvation. Overexpression of APT1 gene in tobacco
cell cultures increased the rate of phosphate uptake
(Mitsukawa et al., 1997).
86
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
Although the concentration of phosphate
introduced into plant culture media has been as high
as 19.8 mM, the average level is 1.7 mM and most
media contain about 1.3 mM. However many reports
indicate that such typical levels may be too low for
some purposes. When phosphate is depleted from
MS medium, there is an increase in free amino acids
in Catharanthus roseus cells, because protein
synthesis has ceased and degradation of proteins is
occurring (Ukaji and Ashihara, 1987). Phosphate
(starting concentration 2.64 mM) and sucrose were
the only nutrients completely depleted in
Catharanthus roseus batch suspension cultures, and
the period of growth could be prolonged by
increasing the levels of both (MacCarthy et al.,
1980).
MS medium contains only 1.25 mM
phosphate which may be insufficient for suspension
cultures of some plants. The phosphate in MS
medium is insufficient for Cardamine pratensis
suspension cultures, all having been absorbed in 5
days: it is however adequate for Silene alba
suspensions (Bister-Miel et al., 1985).
The phosphate in MS medium is also inadequate
for static cultures of some plants, or where a large
amount of tissue or organs are supported on a small
amount of medium (for example where many
separate shoots are explanted together in a static
shoot culture). The concentration of the ion is then
likely to be reduced almost to zero over several
weeks (Barroso et al., 1985; Singha et al., 1987;
Lumsden et al.,1990). Insufficient levels of phosphate
were present from MS during culture of
Hemerocallis, Iris and Delphinium (Leiffert et al.,
1995). Although growth can continue for a short
while after the medium is depleted of phosphate, for
some purposes it has been found to be beneficial to
increase the phosphate concentration of MS to 1.86
mM (Jones and Murashige, 1974), 2.48 mM
(Murashige et al., 1972; Murashige, 1974; Jakobek
et al., 1986), 3.1 mM (Miller and Murashige, 1976)
or 3.71 mM (Thorpe and Murashige, 1968, 1970), for
example, to induce adventitious shoot formation from
callus, or to increase the rate of shoot multiplication
in shoot cultures. It should be noted that there is in
vivo a significant retranslocation of phosphate from
older leaves to the growing shoot (Schachtman et al.,
1998). Retranslocation also occurs in tissue culture.
In Dahlia culture in liquid medium, phosphate is
almost completely taken up after 2 weeks (Fig. 3.4a).
In spite of this, the concentration in tissues formed
after the exhaustion is ‘normal’ (Fig. 3.4b). The
depletion of phosphate early during culture has also a
major effect on the pH of tissue culture media in
which added phosphate is the major buffering
component. When phosphate levels are increased to
obtain a more rapid rate of growth of a culture, it can
be advisable to investigate the simultaneous
enhancement of the level of myo-inositol in the
medium
2.3. POTASSIUM
Potassium is the major cation (positive ion) within
plants reaching in the cytoplasm and chloroplasts
concentrations of 100 – 200 mM. The biphasic
uptake kinetics suggest two uptake systems: a highaffinity and a low-affinity one. K+ is not metabolised.
It contributes significantly to the osmotic potential of
cells. K+ counterbalances the negative charge of
inorganic and organic anions. It functions in cell
extension through the regulation of turgor, it has a
major role in stomatal movements and functions in
long-distance nutrient flow. Potassium ions are
transported quickly across cell membranes and two of
their major roles are regulating the pH and osmotic
environment within cells.
Potassium, calcium,
sodium and chloride ions conserve their electrical
charges within the plant, unlike the cation NH4+ and
the anions NO3-, SO42-, and H2PO4-, which are rapidly
incorporated into organic molecules. In intact plants,
potassium ions are thought to cycle. They move,
associated with cations (particularly NO3⎯), upwards
from the roots in the xylem. As nitrate is reduced to
ammonia and assimilated, carboxylic acid ions
(RCO3-, malate) are produced.
These become
associated with the released K+ ions and are
transported in the phloem to the roots, where they are
decarboxylated, releasing K+ for further anion
transport (Ben-Zioni et al., 1971). Carboxylate
transported to the roots gives rise to OH ⎯ ion, which
is excreted into the soil (or medium) to
counterbalance NO3⎯ uptake (Touraine et al., 1988).
Potassium ions will clearly have a similar role in
cultured tissues, but obvious transport mechanisms
will usually be absent.
Many proteins show a high specificity for
potassium which, acting as a cofactor, alters their
configuration so that they become active enzymes.
Potassium ions also neutralise organic anions
produced in the cytoplasm, and so stabilise the pH
and osmotic potential of the cell. In whole plants,
deficiency of potassium results in loss of cell turgor,
flaccid tissues and an increased susceptibility to
drought, salinity, frost damage and fungal attack. A
high potassium to calcium ratio is said to be
Chapter 3
characteristic of the juvenile stage in woody plants
(Boulay, 1987). Potassium deficiency in plant culture
media is said to lead to hyperhydricity (Pasqualetto
et al., 1988), and a decrease in the rate of absorption
of phosphate (Chin and Miller, 1982). However quite
wide variations in the potassium content of MS
medium had little effect on the growth or
proliferation of cultured peach shoots (Loreti et al.,
1988).
Lavee and Hoffman (1971) reported that the
optimum rate of callus growth of two apple clones
was achieved in a medium containing 3.5 mM K+:
when the concentration was much higher than this, or
when it was less than 1.4 mM, the callus grew less
vigorously. However, the growth rate of wild carrot
suspensions was said by Brown et al., (1976) to be at,
or near, the maximum when K+ concentration was 1
mM: for embryogenesis 10-50 mM K+ was required.
Uptake of potassium into plants is reduced in the
absence of calcium (Devlin, 1975).
Within a large sample of different macronutrient
compositions, it is found that authors have tended to
relate the concentration of potassium to the level of
nitrate. This is correlated with a coefficient of 0.78,
P<0.001 (George et al., 1988).
The average
concentration of potassium in these media was 13.6
mM and the most common value (median), 10.5 mM.
Murashige and Skoog (1962) medium contains 20.04
mM K+.
87
Sodium only appears to be essential to those salttolerant plants, which have a C4 (crassulacean acid)
metabolism. Examples are Bryophyllum tubiflorum
(Crassulaceae) and Mesembryanthemum crystallimum
(Aizoaceae). In these plants the element is necessary
for CO2 fixation in photosynthesis.
2.4. SODIUM
Sodium ions (Na+) are taken up into plants, but in
most cases they are not required for growth and
development and many plants actively secrete them
from their roots to maintain a low internal
concentration. The element can function as an
osmotic stabilizer in halophytic plants; these have
become adapted so that, in saline soils with low water
potential, they can accumulate abnormally high
concentrations of Na+ ion in vacuoles, and thereby
maintain sufficient turgor for growth.
Sodium does appear to have a beneficial
nutritional effect on some plants and is therefore
considered as a functional element (Subbarao et al.,
2003). Small amounts of sodium chloride (e.g. 230
mg/l) can stimulate the growth of plants in the
families Chenopodiaceae and Compositae even when
there is no limitation on the availability of K+
(Brownell, 1979). In other plants such as wheat, oats,
cotton and cauliflower (Sharma and Singh, 1990),
sodium can partially replace potassium, but is not
essential.
Fig. 3.4 Top: Depletion of P in the medium compared to the
growth of Dahlia cultures.
Bottom (Right) P content in 1-week old Dahlia shoots taken from
the culture after 1 week when P had not yet been exhausted.
(Left) 6-week old shoots in which the upper part had been formed
after all P had been taken up from the medium. The high content
in the newly formed upper part of the shoots indicates massive
retranslocation of P after uptake from older to newer tissue
(G. de Klerk, unpub. data).
Most macronutrient formulations do not contain
any sodium at all, and the average concentration in
615 different preparations was 1.9 mM (George et al.,
1988). Even if the element is not deliberately added
as a macronutrient, small amounts are incorporated in
most media from the salts added to provide
micronutrients. Plant macronutrient preparations
88
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
containing high concentrations of both sodium and
chloride ions are not well formulated.
2.5. MAGNESIUM
Magnesium is an essential component of the
chlorophyll molecule and is also required nonspecifically for the activity of many enzymes,
especially those involved in the transfer of phosphate.
ATP synthesis has an absolute requirement for
magnesium and it is a bridging element in the
aggregation of ribosome subunits. Magnesium is the
central atom in the porphyrin structure of the
chlorophyll molecule. Within plants, the magnesium
ion is mobile and diffuses freely and thus, like
potassium, serves as a cation balancing and
neutralising anions and organic acids. Macklon and
Sim (1976) estimated there to be 2.1 mM Mg2+ in the
cytoplasm of Allium cepa roots while McClendon
(1976) put the general cytoplasmic requirement of
plants as high as 16 mM. Plant culture media
invariably contain relatively low concentrations of
magnesium (average 6.8 mM, median 5.3 mM).
Very often MgSO4 is used as the unique source of
both magnesium and sulphate ions.
Walker and Sato (1981) found there to be a large
reduction in the number of somatic embryos formed
from Medicago sativa callus when Mg2+ was omitted
from the medium. In sympathy with this finding,
Kintzios et al., (2004) observed in tissue culture of
melon that the highest level of magnesium occurred
in direct somatic embryogenic cultures and the lowest
level in callus cultures.
2.6. SULPHUR
The sulphur utilised by plants is mainly absorbed
as SO42-, which is the usual source of the element in
plant culture media. Uptake is coupled to nitrogen
assimilation (Reuveny et al., 1980), and is said to be
independent of pH. It results in the excretion of OHions by the plant, making the medium more alkaline.
However, according to Mengel and Kirkby (1982),
plants are relatively insensitive to high sulphate levels
and only when the concentration is in the region of 50
mM, is growth adversely affected. Although sulphur
is mainly absorbed by plants in the oxidized form,
that which is incorporated into chemical compounds
is mainly as reduced -SH, -S- or -S-S- groups. The
sulphur-containing amino acids cysteine and
methionine become incorporated into proteins.
Sulphur is used by plants in lipid synthesis and in
regulating the structure of proteins through the
formation of S-S bridges. The element also acts as a
ligand joining ions of iron, zinc and copper to
metalloproteins and enzymes. The reactive sites of
some enzymes are -SH groups. Sulphur is therefore
an essential element and deficiency results in a lack
of protein synthesis. Sulphur-deficient plants are
rigid, brittle and thin-stemmed. Important sulphur
compounds are glutathione, which acts in
detoxification of oxygen radicals, and the proteins
thioredoxin and ferrodoxin that are involved in redox
chemistry.
Growth and protein synthesis in tobacco cell
suspensions were reduced on a medium containing
only 0.6 mM SO42- instead of 1.73 mM (Klapheck
et al., 1982) and when the supply of S in the medium
was used up, large amounts of soluble nitrogen
accumulated in the cells. Most media contain from 25 meq/l SO42- (1 – 2.5 mM).
2.7. CALCIUM
As a major cation, calcium helps to balance
anions within the plant, but unlike potassium and
magnesium, it is not readily mobile. Because of its
capacity to link biological molecules together with
coordinate bonds, the element is involved in the
structure and physiological properties of cell
membranes and the middle lamella of cell walls. The
enzyme β-(1→3)-glucan synthase depends on
calcium ions, and cellulose synthesis by cultured cells
does not occur unless there are at least micro-molar
quantities of Ca2+ in the medium. Many other plant
enzymes are also calcium-dependent and calcium is a
cofactor in the enzymes responsible for the hydrolysis
of ATP.
Although calcium can be present in millimolar
concentrations within the plant as a whole, calcium
ions are pumped out of the cytoplasm of cells. Ca2+
is sequestered in the vacuole, complexes with
calcium-binding proteins and may precipitate into
calcium oxalate crystals to maintain the concentration
at around only 0.1 mM. The active removal of Ca2+
is necessary to prevent the precipitation of phosphate
(and the consequent disruption of phosphatedependent metabolism) and interference with the
function of Mg2+. The uniquely low intra-cellular
concentration of Ca2+ allows plants to use calcium as
a chemical ‘second messenger’ (Hepler and Wayne,
1985; Sanders et al., 1999). Regulatory mechanisms
are initiated when Ca2+ binds with the protein
calmodulin, which is thus enabled to modify enzyme
activities. A temporary increase in Ca2+concentration
to 1 or 10 mM does not significantly alter the ionic
environment within the cell, but is yet sufficient to
Chapter 3
trigger fundamental cell processes such as polarized
growth (for example that of embryos - Shelton et al.,
1981), response to gravity and plant growth
substances, cytoplasmic streaming, and mitosis
(Ferguson and Drbak, 1988; Poovaiah, 1988).
Physiological and developmental processes, which
are initiated through the action of phytochrome are
also dependent on the presence of Ca2+ (Shacklock
et al., 1992). A short-term increase in cytosolic free
Ca2+ has been observed for osmoadaptation (Taylor
et al., 1996), phytoalexin synthesis (Knight et al.,
1991), thermotolerance (Gong et al., 1998) and
induction of free-radical scavengers (Price et al.,
1994).
Large quantities of calcium can be deposited
outside the protoplast, in cell vacuoles and in cell
walls. Calcification strengthens plant cell walls and
is thought to increase the resistance of a plant to
infection. By forming insoluble salts with organic
acids, calcium immobilises some potentially
damaging by-products. The element gives protection
against the effects of heavy metals and conveys some
resistance to excessively saline conditions and low
pH.
The Ca2+ ion is involved in in vitro
morphogenesis and is required for many of the
responses induced by plant growth substances,
particularly auxins and cytokinins. In the moss
Funaria, cytokinin causes an increase in membraneassociated Ca2+ specifically in those areas which are
undergoing differentiation to become a bud (Saunders
and Hepler, 1981). Protocorm formation from callus
of Dendrobium fibriatum was poor on Mitra et al.,
(1976) A medium when calcium was omitted (Mitra
et al., 1976) and in Torenia stem segments,
adventitious bud formation induced by cytokinin
seems to be mediated, at least in part, by an increase
in the level of Ca2+ within cells (Tanimoto and
Harada, 1986).
Exogenous Ca2+ enhanced the
formation of meristemoids and the first phases of
outgrowth into organs in tobacco pith explants
(Capitani and Altamura, 2004). In carrot, somatic
embryogenesis coincides with a rise of free cytosolic
Ca2+ (Timmers et al., 1996) and applied Ca2+
increases the number of somatic embryos (Jansen
et al., 1990).
2.7.1. Shoot tip necrosis
Calcium deficiency in plants results in poor root
growth and in the blackening and curling of the
margins of apical leaves, often followed by a
cessation of growth and death of the shoot tip. The
latter symptoms are similar to aluminium toxicity
89
(Wyn Jones and Hunt, 1967). Tip necrosis has been
especially observed in shoot cultures, sometimes
associated with hyperhydricity. It often occurs after
several subcultures have been accomplished (e.g. in
Cercis canadensis - Yusnita et al., 1990). After
death of the tip, shoots often produce lateral
branches, and in extreme cases the tips of these will
also die and branch again. The cause of tip necrosis
has not always been determined [e.g. in Pistacia
shoot cultures (Barghchi, 1986), where shoots
showing symptoms may die after planting out
(Martinelli, 1988)]. The occurrence of necrosis was
reduced in Pistacia (Barghchi loc. cit.) and Prunus
tenella (Alderson et al., 1987) by more frequent
subculturing, but this is a costly and time-consuming
practice. In Pictacia, calcium reduced necrosis
(Barghchi and Alderson, 1996).
Tip necrosis was found in Psidium guajava shoot
cultures after prolonged subculturing, if shoots were
allowed to grow longer than 3 cm, and was common
in rapidly growing cultures (Amin and Jaiswal,
1988); it occurred on Sequoiadendron giganteum
shoots only when they were grown on relatively
dilute media (Monteuuis et al., 1987). Necrosis of
Rosa hybrida ‘White Dream’, was cured by adding
0.1 mg/l GA3 to the medium (Valle and Boxus,
1987).
Analysis of necrotic apices has shown them to be
deficient in calcium (Debergh, 1988), and a shortage
of this element has been associated with tip necrosis
in Amelanchier, Betula, Populus, Sequoia, Ulmus,
Cydonia and other woody plants, although the extent
of damage is variable even between genotypes within
a species (Sha et al., 1985; Singha et al., 1990). As
calcium is not remobilised within plant tissues,
actively growing shoots need a constant fresh supply
of ions in the transpiration stream. An inadequate
supply of calcium can result from limited uptake of
the ion, and inadequate transport, the latter being
caused by the absence of transpiration due to the high
humidity in the culture vessel. A remedy can
sometimes be obtained by reducing the culture
temperature so that the rate of shoot growth matches
calcium supply, using vessels which promote better
gas exchange (thereby increasing the transpiration
and xylem transport), or by increasing the
concentration of calcium in the medium (McCown
and Sellmar, 1987). The last two remedies can have
drawbacks: the medium will dry out if there is too
free gas exchange; adding extra calcium ions to the
medium is not always effective (e.g. in cultures of
Castanea sativa - Mullins, 1987); and can introduce
90
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
undesirable anions. Chloride toxicity can result if too
much calcium chloride is added to the medium (see
below). To solve this difficulty, McCown et al.,
(Zeldin and McCown, 1986; Russell and McCown,
1988) added 6 mM calcium gluconate to Lloyd and
McCown (1981) WPM medium to correct Ca2+
deficiency, without altering the concentrations of the
customary anions.
There is a limit to the
concentration of calcium, which can be employed in
tissue culture media because several of its salts have
limited solubility.
2.8. CHLORIDE
The chloride ion (Cl-) has been found to be
essential for plant growth (Broyer et al., 1954;
Johnson et al., 1957; Ozanne et al., 1957; Ozanne,
1958), but seems to be involved in few biological
reactions and only very small quantities are really
necessary.
Rains (1976) listed chlorine as a
micronutrient. Chloride is required for the watersplitting protein complex of Photosystem II, and it
can function in osmoregulation in particular in
stomatal guard cells. The chloride ion is freely
transported and many plants can tolerate the presence
of high concentrations without showing toxicity. The
chief role of chloride seems to be in the maintenance
of turgor and in balancing rapid changes in the level
of free cations such as K+, Mg2+ and Na+. Plants
deprived of Cl- are liable to wilting (Johnson et al.,
1957).
In isolated chloroplasts, chloride (together with
Mn2+) ions are required for oxygen evolution in
photosystem II of photosynthesis (Bov et al., 1963;
Mengel and Kirkby, 1982; Shkolnik, 1984), although
there has been some doubt whether this requirement
exists in vivo (Terry, 1977). Chloride ions are best
taken into plants at slightly acid pH (Jacobson et al.,
1971).
The most common concentration of chloride in
culture media is 3 mM, the average 6 mM. MS
medium contains 6 mM Cl-; Quoirin and Lepoivre
(1977) medium, only 0.123 μM. Some species are
sensitive to chloride ions. McCown and Sellmer
(1987) reported that too high a concentration, seemed
to cause woody species to have yellow leaves and
weak stems: sometimes tissues collapsed and died.
An excess of Cl- has been thought to be one cause of
the induction of hyperhydricity, and omission of the
ion does seem to prevent the development of these
symptoms in Prunus (Volume 2). Pevalek-Kozlina
and Jelaska (1987) deliberately omitted chloride ions
from WPM medium for the shoot culture of Prunus
avium and obtained infrequent hyperhydricity in only
one genotype. The presence of 7 mM Cl- can be toxic
to pine suspension cultures (Teasdale, 1987).
As chlorine has only a relatively small nutritional
significance, steps are sometimes taken to reduce the
concentration of chloride ion in culture media, but in
order to adjust the concentration of other ions, it is
then often necessary to make a marked increase in
SO42-. For example, using ammonium sulphate
instead of ammonium chloride to supply NH4+ in
Eeuwens (1976) Y3 medium, would increase the
sulphate level from 2 to 12 meq/l (from 1 to 6 mM).
3. MICRONUTRIENTS
Plant requirements for microelements have only
been elucidated over the past 50-60 years. Before the
end of the last century, it had been realised that too
little iron caused chlorophyll deficiency in plants, but
the importance of other elements took many years to
prove conclusively.
Mazé, for example, used
hydroponic techniques during the years 1914-1919 to
show that zinc, manganese and boron improved the
growth of maize plants. Sommer and Lipman (1926)
also showed the essentiality of boron, and Sommer
(1931) of copper, but uncertainty over which
elements were really indispensible to growth still
existed in 1933 when Hoagland and Snyder proposed
two supplementary nutrient solutions for water
culture which in total contained 26 elements. It took
several further years to prove that molybdenum
(Arnon and Stout, 1939) and cobalt in very small
amounts, were most important for healthy plant
growth. Early plant tissue culture work was to both
profit from, and contribute to the findings of previous
hydroponic studies. Our understanding has been
enhanced by investigations into the biochemical role
of minor elements.
3.1. EARLY USE IN PLANT TISSUE CULTURE MEDIA
At the time of the early plant tissue culture
experiments, uncertainty still existed over the nature
of the essential microelements. Many tissues were
undoubtedly grown successfully because they were
cultured on media prepared from impure chemicals
(see below) or solidified with agar, which acted as a
micronutrient source.
Chapter 3
In the first instance, the advantage of adding
various micronutrients to culture media was mainly
evaluated by the capability of individual elements to
improve the growth of undifferentiated callus or
isolated root cultures. Knudson (1922) incorporated
Fe and Mn in his very successful media for the nonsymbiotic germination of orchid seeds, and,
following a recommendation by Berthelot (1934),
Gautheret (1939) and Nobécourt (1937) included in
their media (in addition to iron) copper, cobalt,
nickel, titanium and beryllium. Zinc was found to be
necessary for the normal development of tomato root
systems (Eltinge and Reed, 1940), and without Cu,
roots ceased to grow (Glasstone, 1947). Hannay and
Street (1954) showed that Mo and Mn were also
essential for root growth.
An advantage adding five micronutrients to tissue
culture media was perhaps first well demonstrated by
Heller in 1953 who found that carrot callus could be
maintained for an increased number of passages when
Fe, B, Mn, Zn and Cu were present.
3.2. MICRONUTRIENTS FROM TRACE IMPURITIES
Micronutrients tend to be added to modern media
by the addition of fairly standard chemicals. Street
(1977) rightly emphasised that even analytical grade
chemicals contain traces of impurities that will
provide a hidden supply of micronutrients to a
medium. An illustration of this, comes from the work
of Dalton et al., (1983) who found traces of silicon
(Si) in a precipitate from MS medium which had been
made up with analytical grade laboratory chemicals.
Gelling agents contain inorganic elements but
whether cultures can utilize them is unclear.
Amounts of contaminating substances in chemicals
would have been greater in times past, so that an
early medium such as Knudson (1922; 1943) B,
prepared today with highly purified chemicals, will
not have quite the same composition as when it was
first used by Knudson in 1922; the addition of some
micronutrients might improve the results obtained
from a present-day formulation of such early media.
3.3. OPTIMUM MICRO-ELEMENT CONCENTRATIONS
Most modern culture media use the
microelements of Gamborg et al., (1968) B5 medium,
or the more concentrated mixtures in MS or Bourgin
and Nitsch (1967) H media. Several research
workers have continued to use Heller (1953) micronutrient formulation, even though higher levels are
now normally recommended. Quoirin and Lepoivre
91
(1977) showed clearly that in conjunction with MS or
their Quoirin and Lepoivre (1977) B macro-elements,
the concentration of Mn in Heller’s salts should be
increased by 100-fold to obtain the most effective
growth of Prunus meristems.
Cell growth and morphogenesis of some species
may even be promoted by increasing the level of
micronutrients above that recommended by
Murashige and Skoog (1962). The induction and
maintenance of callus and growth of cell suspensions
of juvenile and mature organs of both Douglas fir and
loblolly pine, was said to be improved on Litvay et
al., (1981) LM medium in which Mg, B, Zn, Mo, Co
and I were at 5 times the concentration of MS microelements, and Mn and Cu at 1.25 and 20 times
respectively (Litvay et al., 1981; Verma et al., 1982).
Other authors to have employed high micronutrient
levels are Barwale et al., (1986) who found that the
induction of adventitious shoots from callus of 54
genotypes of Glycine max was assisted by adding
four times the normal concentration of minor salts to
MS medium.
A further example of where more concentrated
micro-elements seemed to promote morphogenesis is
provided by the work of Wang, et al., (1980, 1981).
Embryogenesis could be induced most effectively in
callus derived from Hevea brasiliensis anthers, by
doubling the concentration of microelements in MS
medium, while at the same time reducing the level of
macronutrients to 60-80% of the original.
Despite these reports, few research workers seem
to have accepted the need for such high micronutrient
levels. To diminish the occurrence of hyperhydricity
in shoot culture of carnation, Dencso (1987) reduced
the level of micronutrients (except iron, which was as
recommended by Dalton et al., 1983) to those in MS
medium, but this mixture was inadequate for Gerbera
shoot cultures and the rate of propagation was less
than that with the normal MS formulation.
The need for macronutrient concentrations to be
optimised as the first step in media development
seems to be emphasised by results of Eeuwens
(1976). In an experiment with factorial combinations
of the macro- and micro-nutrient components of four
media, his Eeuwens (1976) Y3 micronutrients gave a
considerable improvement in the growth of coconut
callus, compared with other micro-element mixtures,
when they were used with Y3 and MS
macronutrients, but not when used with those of
White (1942) or Heller (1953; 1955).
92
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
3.4. CELLULAR DIFFERENTIATION
AND MORPHOGENESIS
Welander (1977) obtained evidence, which
suggested that plant cells are more demanding for
minor elements when undergoing morphogenesis.
Petiole explants of Begonia hiemalis produced callus
on media without micronutrients, but would only
produce adventitious shoot buds directly when
micronutrients were added to the macronutrient
formulation. The presence of iron is particularly
important for adventitious shoot and root formation
(Legrand, 1975).
That mineral nutrition can influence cellular
differentiation in combination with growth hormones,
was shown by Beasley et al., (1974). Cotton ovules
cultured on a basic medium containing 5.0 mM IAA
and 0.5 mM GA3, required 2 mM calcium (normally
present in the medium) for the ovules to develop
fibres. Magnesium and boron were essential for fibre
elongation.
3.5. THE ROLES OF MICRONUTRIENTS
3.5.1. Manganese
The essential micronutrient metals Fe, Mn, Zn, B,
Cu, Co and Mo are components of plant cell proteins
of metabolic and physiological importance. At least
five of these elements are, for instance, necessary for
chlorophyll synthesis and chloroplast function
(Sundqvist et al., 1980). Micronutrients have roles in
the functioning of the genetic apparatus and several
are involved with the activity of growth substances.
Manganese (Mn) is one of the most important
microelements and has been included in the majority
of plant tissue culture media. It is generally added in
similar concentrations to those of iron and boron, i.e.
between 25-150 mM.
Manganese has similar
chemical properties to Mg2+ and is apparently able to
replace magnesium in some enzyme systems (Hewitt,
1948). However there is normally 50- to 100-fold
more Mg2+ than Mn2+ within plant tissues, and so it is
unlikely that there is frequent substitution between
the two elements.
The most probable role for Mn is in definition of
the structure of metalloproteins involved in
respiration and photosynthesis (Clarkson and Hanson,
1980). It is known to be required for the activity of
several enzymes, which include decarboxylases,
dehydrogenases, kinases and oxidases and superoxide
dismutase enzymes. Manganese is necessary for the
maintenance of chloroplast ultra-structure. Because
Mn(II) can be oxidized to Mn(IV), manganese plays
an important role in redox reactions. The evolution
of oxygen during photosystem II of the
photosynthetic process, is dependent on a Mncontaining enzyme and is proportional to Mn content
(Mengel and Kirkby, 1982; Shkolnik, 1984). Mn is
toxic at high concentration (Sarkar et al., 2004).
In tissue cultures, omission of Mn ions from
Doerschug and Miller (1967) medium reduced the
number of buds initiated on lettuce cotyledons. A
high level of manganese could compensate for the
lack of molybdenum in the growth of excised tomato
roots (and vice versa) (Hannay and Street, 1954).
Natural auxin levels are thought to be reduced in the
presence of Mn2+ because the activity of IAA-oxidase
is increased. This is possibly due to Mn2+ or Mncontaining enzymes inactivating oxidase inhibitors,
or because manganous ions are one of the cofactors
for IAA oxidases in plant cells (Galston and Hillman,
1961). Manganese complexed with EDTA increased
the oxidation of naturally-occurring IAA, but not the
synthetic auxins NAA or 2,4-D (MacLachlan and
Waygood, 1956).
However, Chée (1986) has
suggested that, at least in blue light, Mn2+ tends to
cause the maintenance of, or increase in, IAA levels
within tissues by inactivating a co-factor of IAA
oxidase. When the Mn2+ level in MS medium was
reduced from 100 mM to 5 mM, the production in
blue light, of axillary shoots by Vitis shoot cultures
was increased.
3.5.2. Zinc
Zinc is a component of stable metallo-enzymes
with many diverse functions, making it difficult to
predict the unifying chemical property of the element,
which is responsible for its essentiality (Clarkson and
Hanson, 1980). Zinc is required in more than 300
enzymes including alcohol dehydrogenase, carbonic
anhydrase, superoxide dismutase and RNApolymerase. Zinc forms tetrahedral complexes with
N-, O-, and S-ligands. In bacteria, Zn is present in
RNA and DNA polymerase enzymes, deficiency
resulting in a sharp decrease in RNA levels. DNA
polymerase is concerned with the repair of incorrectly
formed pieces of DNA in DNA replication, and RNA
polymerase locates the point on the DNA genome at
which initiation of RNA synthesis is to take place.
Divalent Mg2+, Mn2+ or Co2+ are also required for
activation of these enzymes (Eichhorn, 1980).
Zinc deficient plants suffer from reduced enzyme
activities and a consequent diminution in protein,
nucleic acid and chlorphyll synthesis. Molybdenumand zinc-deficient plants have a decreased
chlorophyll content and poorly developed
Chapter 3
chloroplasts. Plants deprived of zinc often have short
internodes and small leaves.
2+
The concentration of Zn in MS medium is 30
μM but amounts added to culture media have often
varied widely between 0.1-70 μM and experimental
results to demonstrate the most appropriate level are
limited. When Eriksson (1965) added 15 mg/l
Na2ZnEDTA.2H2O (40μM Zn2+)to Haplopappus
gracilis cell cultures, he obtained a 15% increase in
cell dry weight which was thought to be due to the
presence of zinc rather than the chelating agent. Zinc
was also shown to increase growth of a rice
suspension. The highest concentration tested, 520
µM, resulted in the fastest rate of growth and it was
suggested that zinc had increased auxin activity (see
below) (Hossain et al., 1997). Zinc is required for
adventitious root formation in Eucalyptus
(Schwambach et al., 2005). In cassava, additional
zinc promotes somatic embryogenesis and rooting
(C.J.J.M Raemakers, pers. commun.). However, very
high concentrations of zinc are found to be inhibitory,
and the microelement has been noted to prevent root
growth at a concentration higher than 50 μM.
There is a close relationship between the zinc
nutrition of plants and their auxin content (Skoog,
1940). It has been suggested that zinc is a component
of an enzyme concerned with the synthesis of the
IAA precursor, tryptophan (Tsui, 1948).
The
importance of Zn for tryptophan synthesis is
especially noticeable in crown gall callus which
normally produces sufficient endogenous auxin to
maintain growth on a medium without synthetic
auxins, but which becomes auxin-deficient and ceases
to grow in the absence of Zn (Klein et al., 1962).
3.5.3. Boron
Boron is involved in plasma membrane integrity
and functioning, probably by influencing membrane
proteins, and cell wall intactness. Reviews have been
provided by Lewis (1980) and by Blevins and
Lukaszewski (1998). The element is required for the
metabolism of phenolic acids, and for lignin
biosynthesis: it is probably a component, or co-factor
of the enzyme which converts p-coumaric acid to
caffeate and 5-hydroxyferulate. Boron is necessary
for the maintenance of meristematic activity, most
likely because it is involved in the synthesis of Nbases (uracil in particular); these are required for
RNA synthesis (Mengel and Kirkby, 1982). It is also
thought to be involved in the maintenance of
membrane structure and function, possibly by
stabilizing natural metal chelates which are important
93
in wall and membrane structure and function (Pollard
et al., 1977; Clarkson and Hanson, 1980). Boron is
concerned with regulating the activities of phenolase
enzymes; these bring about the biosynthesis of
phenylpropane compounds, which are polymerized to
form lignin. Lignin biosynthesis does not take place
in the absence of boron. Boron also mediates the
action of phytochrome and the response of plants to
gravity (Tanada, 1978).
Use in culture media. In the soil, boron occurs
in the form of boric acid and it is this compound,
which is generally employed as the source of the
element in tissue cultures. Uptake of boric acid
occurs most readily at acid pH, possibly in the
undissociated form (Oertli and Grgurevic, 1974) or as
H2BO3- (Devlin, 1975). A wide range of boron
concentrations has been used in media, the most usual
being between from 50 and 100 μM: MS medium
contains 100 μM. Bowen (1979) found boron to be
toxic to sugarcane suspensions above 2 mg/l (185
μM), but there are a few reports of higher
concentrations being employed (Table 3.8). High
concentrations of boron may have a regulatory
function; for example, 1.6-6.5 mM have been used in
simple media to stimulate pollen germination
(Brewbaker and Kwack, 1963; Taylor, 1972).
Boric acid reacts with some organic compounds
having
two
adjacent
cis-hydroxyl
groups
(Greenwood, 1973). This includes o-diphenols,
hexahydric alcohols such as mannitol and sorbitol
(commonly used in plant tissue culture as osmotic
agents), and several other sugars, but excludes
sucrose which forms only a weak association. Once
the element is complexed it appears to be unavailable
to plants. This led Lewis (1980) to suggest that
because boric acid was required for lignin
biosynthesis, vascular plants were led, during
evolution, to use sucrose exclusively for the transport
of carbohydrate reserves.
Although the addition of sugar alcohols and
alternative sugars to sucrose can be beneficial during
plant tissue culture (see Chapter 4), it is clearly
necessary to return to a sucrose-based medium for
long-term culture, or boron deficiency may result.
Deficiency symptoms. Boron is thought to
promote the destruction of natural auxin and increase
its translocation. Endogenous IAA levels increase in
the absence of boron and translocation is reduced, the
compound probably being retained at the site of
synthesis (Goldbach and Amberger, 1986). Plants
suffering from boron deficiency have restricted root
systems (Odhnoff, 1957; Whittington, 1959) and a
94
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
reduced capacity to absorb H2PO4- and some other
ions. High levels of auxins can have the same effect
on growth and ion uptake (Pollard et al., 1977).
Neales (1959, 1964) showed that isolated roots
stopped growing unless a minimum concentration of
boron was present (although the necessity for the
element was not so apparent when cultures were
grown in borosilicate glass vessels). Inhibition of
root elongation in the absence of boron has been
shown to be due to the cessation of mitosis and the
inhibition of DNA synthesis (Moore and Hirsch,
1981). Boron deficiency also results in depressed
cytokinin synthesis. Cell division is inhibited in the
absence of boron, apparently because there is a
decrease in nuclear RNA synthesis (Ali and Jarvis,
1988). However, deficiency often leads to increased
cambial growth in intact dicotyledonous plants.
Table 3.8 Examples of cultures grown with unusually high concentrations of boron
Plant
Antirrhinum majus
Brassica napus
Capsella bursa-pastoris
Citrus medica
Hevea brasiliensis
Hordeum crosses
Larix deciduas
Lycopersicon esculentum
Nicotiana tabacum
Petunia hybrida
Prunus amygdalus
Concentration of
boron (μM)
323
Type of Culture
Reference
200
Embryos from protoplast
colonies
Embryo
Monnier (1976)
646
320
242
250
250
242
323
566
200
Anther callus
Anther: plant regeneration
Embryo rescue
Direct morphogenesis
Callus and embryogenesis
Isolated root
Protoplant culture
Callus and root formation
Shoot
Drira and Benbadis (1975)
Chen (1984)
Jensen (1974)
Bonga (1984))
Nagmani and Bonga (1985)
Street and McGregor (1952)
Ohyama and Nitsch (1972)
Sangwan and Norreel (1975)
Hisjima (1982a)
One of the changes seen in some plants grown
under boron deficiency is the outgrowth of lateral
buds resulting in plants with a bushy or rosette
appearance. In pea, this was associated with a sharp
decrease in IAA-export from the apex (Li et al.,
2001). It is generally accepted that the outgrowth of
lateral buds is inhibited by polar auxin transport in
the stem and that disruption of this transport by
decapitation or auxin transport inhibitors results in
the outgrowth of lateral buds (Tamas, 1987).
Cotton ovules which otherwise develop fibres
when cultured, commence extensive callus formation
when placed on a medium deficient in boron. On the
other hand, the growth rate of callus cultures of
Helianthus annuus and Daucus carota (Krosing,
1978), and cell cultures of sugar cane (Bowen, 1979),
was much reduced when boron was not present in the
growth medium. Boron influences the development
of the suspensor of somatic embryos in Larix
deciduas (Behrendt and Zoglauer, 1996). Boron had
no influence on the induction of embryogenesis in
Daucus carota but altered the development of
Poirier-Hamon et al., (1974)
embryos: root development was promoted at low
concentrations and shoot development at high. This
coincided with a high and low auxin-cytokinin ratio,
respectively (Mashayekhi and Neumann, 2006).
Adventitious root formation. Boron is thought
to promote the destruction of auxin. Although auxin
is required for the formation of adventitious root
initials, boron is necessary in light grown-plants for
the growth of these primordia (Middleton et al.,
1978); possibly boron enhances the destruction of
auxin in these circumstances, which in high
concentrations is inhibitory to root growth (Jarvis,
1986). An interaction between boron and auxins in
the rooting of cuttings has been noticed in several
species (Hemberg, 1951; Weiser, 1959; Weiser and
Blaney, 1960; Bowen et al., 1975; Josten and
Kutschera, 1999) and a supply of exogenous borate
has been shown to be essential (Ali and Jarvis, 1988).
However, excessive boron concentrations lead to a
reduction in the number of roots formed (Jarvis,
1986). Boron deficiency had no observed effect
Chapter 3
on the rooting of Eucalyptus
(Schwambach et al., 2005).
microcuttings
3.6. COPPER AND MOLYBDENUM
Copper is an essential micronutrient, even though
plants normally contain only a few parts per million
of the element. Two kinds of copper ions exist; they
are the monovalent cuprous [Cu(I)] ion, and the
divalent cupric [Cu(II)] ion: the former is easily
oxidized to the latter; the latter is easily reduced. The
element becomes attached to enzymes, many of
which bind to, and react with oxygen. They include
the cytochrome oxidase enzyme system, responsible
for oxidative respiration, and superoxide dismutase
(an enzyme which contains both copper and zinc
atoms). Detrimental superoxide radicals, which are
formed from molecular oxygen during electron
transfer reactions, are reacted by superoxide dismutase and thereby converted to water. Copper atoms
occur in plastocyanin, a pigment participating in
electron transfer.
Several copper-dependent enzymes are involved
in the oxidation and hydroxylation of phenolic
compounds, such as ABA and dopamine (Lerch,
1981).
The hydroxylation of monophenols by
copper-containing enzymes leads to the construction
of important polymeric constituents of plants, such as
lignin.
These same enzymes can lead to the
blackening of freshly isolated explants. Copper is a
constituent of ascorbic acid oxidase and the
characteristic growth regulatory effects of ethylene
are thought to depend on its metabolism by an
enzyme, which contains copper atoms.
High concentrations of copper can be toxic. Most
culture media include ca. 0.1-1.0 µM Cu2+. Ions are
usually added through copper sulphate, although
occasionally cupric chloride or cupric nitrate have
been employed. In hydroponic culture of Trifolium
pratense, uptake of copper into the plant depended on
the amount of nitrate in solution. Uptake was
considerably reduced when NO3⎯ was depleted
(Jarvis, 1984). The concentration of Cu in tissue
culture media is very small relative to the level in
plants (Table 3.1). It is therefore not surprising that
various authors report strong increases of growth
when Cu is added at 1- 5 µM (Dahleen, 1995; Nirwan
and Kothari, 2003; Kintzios et al., 2001; Nas and
Read, 2004; Bouman and Tiekstra, 2005)
Plants utilise hexavalent molybdenum and absorb
the element as the molybdate ion (MoO42-). This is
normally added to culture media as sodium molybdate
at concentrations up to 1 mM. Considerably higher
95
levels have occasionally been introduced [e.g. in the
media of Abou-Mandour (1977) and Asahira and
Kano (1977)] apparently without adverse effect,
although Teasdale (1987) found pine suspension
cultures were injured by 50 mM. Molybdenum is a
component of several plant enzymes, two being
nitrate reductase and nitrogenase, in which it is a
cofactor together with iron: it is therefore essential
for nitrogen utilisation. Tissues and organs presented
with NO3⎯ in a molybdenum-deficient medium can
show symptoms of nitrate toxicity because the ion is
not reduced to ammonia.
3.7. COBALT
Cobalt is not regarded as an essential element.
Nevertheless, it was found to have been included in
approximately half of a large sample of published
plant culture media (George, et al., 1987). Murashige
and Skoog (1962) included Co in their medium
because it had been shown to be required by lower
plants (Holm-Hansen et al., 1954) and that it might
have a role in regulating morphogenesis in higher
plants (Miller, 1954; Salisbury, 1959). However, no
stimulatory effect on the growth of tobacco callus
was observed by adding cobalt chloride to the
medium at several concentrations from 0.1 µM and
above, and at 80.0 and 160 µM the compound was
toxic. Similarly Schenk and Hildebrandt (1972)
obtained no clear evidence for a Co requirement in
tests on a wide variety of plants, but retained the
element in their medium because they occasionally
observed an apparent stimulation to the callus growth
of some monocotyledons. Pinus suspension cultures
do not require cobalt (Teasdale, 1987).
The
concentration most commonly added to a medium is
ca. 0.1 µM, although ten times this amount has
sometimes been used. Cobalt is the metal component
of Vitamin B12 which is concerned with nucleic acid
synthesis (Fries, 1962), but evidence that the element
has any marked stimulatory effect on growth or
morphogenesis in plant tissue cultures is hard to find.
Cobalt may replace nickel in urease and thereby
render it inactive, e.g., in potato (Witte et al., 2002).
Advantage from adding cobalt to plant culture
media might be derived from the fact that the element
can have a protective action against metal chelate
toxicity and it is able to inhibit oxidative reactions
catalysed by ions of copper and iron (Albert, 1958).
The Co2+ ion can inhibit ethylene synthesis.
96
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
3.8. ALUMINIUM AND NICKEL
Several workers, following Heller (1953), have
included aluminium and nickel in their micronutrient
formulations. However, the general benefit of adding
the former metal does not seem to have been
adequately demonstrated.
It was believed that in most plants Ni2+ is not
absolutely required for normal growth and
development (Mishra and Kar, 1975). However,
more recently, it has been found by careful
experimentation that nickel is essential (Gerendás
et al., 1999). The ion is a component of urease
enzymes (Dixon et al., 1975; Polacco, 1977a), which
convert urea to ammonia. It has been shown to be an
essential micronutrient for some legumes and to
actvate urease in potato microshoot cultures (Witte
et al., 2002). In tissue cultures the presence of
0.1 mM Ni2+ strongly stimulates the growth of
soybean cells in a medium containing only urea as a
nitrogen source. Slow growth occurs on urea without
the deliberate addition of nickel, possibly supported
by trace amounts of the element remaining in the
cells (Polacco, 1977b). Cells and tissues are not
normally grown with urea as a nitrogen source, and
as urease is the only enzyme, which has been shown
to have a nickel component, it could be argued that
nickel is not essential. However, without it soybean
plants grown hydroponically, accumulate toxic
concentrations of urea (2.5%) in necrotic lesions on
their leaf tips, whether supplied with inorganic
nitrogen, or with nitrogen compounds obtained from
bacterial symbiotic nitrogen fixation.
These
symptoms can be alleviated in plants growing in
hydroponic culture by adding 1 mg/l Ni to the
nutrient solution. Absence of nickel in a hydroponic
solution also results in reduced early growth and
delayed nodulation (Eskew et al., 1983).
Despite these findings nickel has not been added
deliberately to tissue culture media. However, it
should be noted that agar contains relatively high
levels of nickel (Table 3.2) and the possibility of urea
toxicity may have been avoided because, in tissue
cultures, urea diffuses into the medium (Teasdale,
1987). Quoirin and Lepoivre (1977) showed that at
the concentrations recommended by Heller, Al3+ and
Ni2+ were without effect on the growth of Prunus
meristems and were inhibitory at higher levels. If it
is thought that Ni should be added to a culture
medium, 0.1 mM is probably sufficient.
Aluminium has been said to be necessary for the
growth of some ferns (Taubck, 1942), but is not
generally added to tissue culture media for fern
propagation.
3.9. IODINE
Iodine is not recognised as an essential element
for the nutrition of plants (Rains, 1976), although it
may be necessary for the growth of some algae, and
small amounts do accumulate in higher plants (ca.12
and 3 mol/kg dry weight in terrestrial and aquatic
plants respectively – Raven, 1986). However, the
iodide ion has been added to many tissue culture
media (e.g. to 65% of micronutrient formulations).
The practice of including iodide in plant culture
media began with the report by White (1938) that it
improved the growth of tomato roots cultured in
vitro. Hannay (1956) obtained similar results and
found that root growth declined in the absence of
iodine which could be supplied not only from
potassium iodide, but also from iodoacetate or
methylene iodide, compounds which would only
provide iodide ions very slowly in solution by
hydrolysis. Street (1966) thought that these results
indicated that iodine could be an essential nutrient
element, but an alternative hypothesis is that any
beneficial effect may result from the ability of iodide
ions to act as a reducing agent (George et al., 1988).
Oxidants convert iodide ions to free iodine. Eeuwens
(1976) introduced potassium iodide into his Y3
medium at 0.05 mM (ten times the level used by
Murashige and Skoog), as it prevented the browning
of coconut palm tissue cultures. The presence of 0.06
µM potassium iodide slightly improved the survival
and growth of cultured Prunus meristems (Quoirin
and Lepoivre, 1977).
Although Gautheret (1942) and White (1943) had
recommended the addition of iodine to media for
callus culture, Hildebrandt et al., (1946) obtained no
statistically significant benefit from adding potassium
iodide to tumour callus cultures of tobacco and
sunflower. However, as the average weight of
tobacco callus was 11% less without it, the compound
was included (at different levels) in both of the media
they devised. Once again iodine also had no
appreciable effect on tobacco callus yield in the
experiments of Murashige and Skoog (1962), but was
nevertheless included in their final medium. Other
workers have omitted iodine from MS medium (e.g.
Roest and Bokelmann, 1975; Périnet et al., 1988;
Gamborg, 1991) or from new media formulations
without any apparent ill effects. However, Teasdale
et al., (1986); Teasdale, (1987) reported a definite
requirement of Pinus taeda suspensions for 25 mM
Chapter 3
KI when they were grown on Litvay et al., (1981)
LM medium.
There seems, at least in some plants, to be an
interaction between iodine and light. Eriksson (1965)
left KI out of his modification of MS medium,
finding that it was toxic to Haplopappus gracilis cells
cultured in darkness: shoot production in Vitis shoot
cultures kept in blue light was reduced when iodine
was present in the medium (Chée, 1986), but the
growth of roots on rooted shoots was increased.
Chée thought that these results supported the
hypothesis that iodine enhanced the destruction
and/or the lateral transport of IAA auxin. This seems
to be inconsistent with the suggestion that I- acts
mainly as a reducing agent.
3.10. SILICON
Silicon (Si) is the second most abundant element
on the surface of the earth. Si has been demonstrated
to be beneficial for the growth of plants and to
alleviate biotic and abiotic stress (Epstein, 1971).
The silicate ion is not normally added to tissue
culture media, although it is likely to be present in
low concentrations.
Deliberate addition to the
medium might, however, improve the growth of some
plants. Adatia and Besford (1986) found that
cucumber plants depleted silicate from a hydroponic
solution and in consequence their leaves were more
rigid, had a higher fresh weight per unit area and a
higher chlorophyll content than the controls. The
resistance of the plants to powdery mildew was also
much increased.
3.11. IRON
Chelating agents. Some organic compounds are
capable of forming complexes with metal cations, in
which the metal is held with fairly tight chemical
bonds. The complexes formed may be linear or ringshaped, in which case the complex is called a chelate
(from the Greek word meaning a crab’s claw).
Metals can be bound (or 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. For
example, Cu2+ complexed with amino acids is more
active biologically than the free ion (Cruickshank
et al., 1987).
Chelating agents vary in their
sequestering capacity (or avidity) according to
chemical structure and their degree of ionisation,
which changes with the pH of the solution. Copper is
chelated by amino acids at relatively high pH, but in
97
conditions of greater acidity, it is more liable to be
complexed with organic acid ligands (White et al.,
1981). The higher the stability of a complex, the
higher the avidity of the complexing agent. One, and
in many cases, two or three molecules of a
complexing agent may associate with one metal ion,
depending on its valency.
Despite tight bonding, there is always an
equilibrium between different chelate complexes and
between ions in solution. Complexing agents also
associate with some metal ions more readily than
with others. In general Fe3+ (for agents able to
complex with trivalent ions) complexes have a higher
stability than those of Cu2+, then (in descending
order), Ni2+, Al3+ (where possible), Zn2+, Co2+, Fe2+,
Mn2+ and Ca2+ (Albert, 1958; Reilley and Schmid,
1958). For a chelated metal ion to be utilised by a
plant there must be some mechanism whereby the
complex can be broken. This could occur if it is
absorbed directly and the ion displaced by another
more avid binding agent, or if the complex is
biochemically denatured. Metals in very stable
complexes can be unavailable to plants; copper in
EDTA chelates may be an example (Coombes et al.,
1977). High concentrations of avid chelating agents
are phytotoxic, probably because they competitively
withdraw essential elements from enzymes.
Naturally-occurring compounds act as chelating
agents. Within the plant very many constituents such
as proteins, peptides, porphyrins, carboxylic acids
and amino acids have this property (Albert, 1958;
Martin, 1979): some of those with high avidity are
metal-containing enzymes. Amino acids are able to
complex with divalent metals (Fig. 3.5). Grasses are
thought to secrete a chelating agent from their roots
to assist the uptake of iron (Römheld and Marschner,
1986). There are also synthetic chelating agents with
high avidities (stability constants) for divalent and
trivalent ions. Some are listed in Table 3.9, and the
structure of those most commonly used in plant
culture media is illustrated in Fig 3.6.
The
application of synthetic chelating agents and chelated
micronutrients to the roots of some plants growing in
alkaline soils can improve growth by supplying
essential metals such as iron and zinc which are
otherwise unavailable.
The addition of such
compounds to tissue culture media can help to make
macro- and micro-nutrients more accessible to plant
cells.
98
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
Fig. 3.5 Copper chelated with amino acid, glycine.
Iron chelates. A key property 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. In plants, iron is primarily used in the
chloroplasts, mitochondria and peroxisomes of plants
for effecting oxidation/reduction (redox) reactions.
The element is required for the formation of amino
laevulinic acid and protoporphyrinogen (which are
respectively early and late precursors of chlorophyll)
and deficiency leads to marked leaf chlorosis. Iron is
also a component of ferredoxin proteins, which
function as electron carriers in photosynthesis.
Iron is therefore an essential micronutrient for
plant tissue culture media and can be provided from
either ferrous or ferric salts. In early experiments,
ferrous sulphate or ferric citrate or tartrate were used
in media as a source of the element. Citric and and
tartaric acids can act as chelating agents for some
divalent metals (Bobtelsky and Jordan, 1945), but are
not very efficient at keeping iron in solution (Fig 3.6).
If Fe2+ and Fe3+ ions escape from the chelating agent,
they are liable to be precipitated as iron phosphate.
The iron may then not be available to plant cells,
unless the pH of the medium falls sufficiently to
bring free ions back into solution. The problem of
precipitation is more severe in aerated media and
where the pH of the medium drifts towards alkalinity.
Under these conditions Fe2+ (ferrous) ions are
oxidized to Fe3+ (ferric) ions and unchelated ferric
ions may then also be converted to insoluble
Fe(OH)3.
For plant hydroponic culture, the
advantages of adding iron to nutrient solutions in the
form of a chelate with EDTA was first recognised in
the 1950’s (Jacobson, 1951; Weinstein et al., 1951).
Street et al., (1952) soon found that iron in this form
was less toxic and could be utilised by in vitro
cultures of isolated tomato roots over a wider pH
range than ferric citrate. Klein and Manos (1960)
showed that callus cultures of several species grew
more rapidly on White (1954) medium if Fe3+ ions
from Fe2(SO4)3 were chelated with EDTA, rather than
added to the medium from the pure compound, and
Doerschug and Miller (1967), that 0.036 mM Fe from
NaFeEDTA was as effective as 0.067 mM Fe as
ferric citrate, in promoting shoot bud initiation on
lettuce cotyledons. Iron presented as ferric sulphate
(0.025 mM Fe) was much less effective than either
chelated form.
Skoog and co-workers began to use EDTA in
media for tobacco callus cultures in 1956 and
discussed their findings in the same paper that
describes MS medium (Murashige and Skoog, 1962).
The addition of an iron (Fe)-EDTA chelate once
again greatly improved the availability of the
element. Following this publication, (Fe)-EDTA
complexes were rapidly recognised to give generally
improved growth of all types of plant cultures
(Nitsch, 1969). EDTA has now become almost a
standard medium component and is generally
preferred to other alternative chelating agents
(Table 3.8).
Preparation and use. (Fe)-EDTA chelates for
tissue cultures are prepared in either of two ways.
• A ferric or ferrous salt is dissolved in water with
EDTA and the solution is heated;
• A ready-prepared salt of iron salt of EDTA is
dissolved and heated.
Heating can take place during the preparation of
chelate stock solutions, or during the autoclaving of a
medium.
The form of iron complexed is invariably Fe(III).
If iron has been provided from ferrous salts, it is
oxidised during heating in aerated solutions. The rate
of oxidation of the ferrous ion is enhanced in some
complexes and retarded in others (Albert, 1958).
That of Fe2+-EDTA is extremely rapid (Kolthoff and
Auerbach (1952). Only a small proportion of Fe2+ is
likely to remain: its chelate with EDTA is much less
stable than the Fe(III) complex. Iron is however
thought to be absorbed into plants in the ferrous form.
Uptake of iron from EDTA probably occurs when
molecules of Fe(III)-chelate bind to the outer plasma
membrane (the plasmalemma) of the cytoplasm,
where Fe(III) is reduced to Fe(II) and freed from the
chelate (Chaney et al., 1972; Römheld and
Marschner, 1983).
Chapter 3
In most recent plant tissue culture work, EDTA
has been added to media at an equimolar
concentration with iron, where it will theoretically
form a chelate with all the iron in solution. However,
it has been found in practice that the Fe(III)-EDTA
chelate, although stable at pH 2-3, is liable to lose
some of its bound iron in culture media at higher pH
levels; the displaced iron may form insoluble ferric
hydroxides and iron phosphate (Dalton et al., 1983).
If this occurs, free EDTA will tend to form chelates
with other metal ions in solution.
Some
micronutrients complexed with EDTA may then not
be available to the plant tissues. Re-complexing may
also happen if the EDTA to Fe ratio is increased by
decreasing the amount of iron added to the medium
(as has been proposed to solve the precipitation
problem, see Chapter 4). It is not possible to add
very much more than 0.1 mM EDTA to culture media
because the chelating agent can become toxic to some
plants (see below).
Hill-Cottingham and Lloyd-Jones (1961) showed
that tomato plants absorbed iron from FeEDTA more
rapidly than they absorbed EDTA itself, but
concluded that both Fe and Fe-chelate were probably
taken up. They postulated that EDTA liberated by
the absorption of Fe, would chelate other metals in
the nutrient solution in the order given at the
beginning of Section 3.6.. Teasdale (1987) calculated
that in many media, nearly all the copper and zinc,
and some manganese ions might be secondarily
chelated, but it is unclear whether micronutrients in
this form are freely available to plant tissues. One
presumes they are, for deficiency symptoms are not
reported from in vitro cultures.
Ambiguous descriptions. In many early papers
on plant tissue culture, the authors of scientific papers
have failed to describe which form of EDTA was
used in experiments, or have ascribed weights to
EDTA, which should refer to its hydrated sodium
salts. Singh and Krikorian (1980) drew attention to
this lack of precision. They assumed that in papers
where Na2EDTA is described as a medium
constituent, it indicates the use of the anhydrous salt
(which would give 11 mol/l excess of EDTA to iron,
with unknown consequences).
However, the
disodium salt of EDTA is generally made as the
dihydrate (Beilstein’s Handbuch der Organischen
Chemie) and this is the form which will almost
invariably have been used, Na2EDTA merely being a
99
shorthand way of indicating the hydrated salt without
being intended as a precise chemical formula.
Further confusion has arisen through workers
using ready-prepared iron-EDTA salts in media
without specifying the weight or molar concentration
of actual Fe used. Mono-, di-, tri-, and tetra-sodium
salts of EDTA are possible, each with different (and
sometimes alternative) hydrates, so that when a
research report states only that a certain weight of
‘FeEDTA’ was used, it is impossible to calculate the
concentration of iron that was employed with any
certainty.
The compound ‘monosodium ferric EDTA’ with
the formula NaFeEDTA (no water of hydration)
exists, and is nowadays commonly selected as a
source of chelated iron. However in some papers
‘NaEDTA’ has been used as an abbreviation for some
other form of iron-EDTA salt. For example the paper
of Eeuwens (1976) describing Y3 medium, says that
to incorporate 0.05 mM iron, 32.5 mg/l ‘sodium ferric
EDTA’ was used. The weight required using a
compound with the strict molecular formula
NaFeEDTA would be 18.35 mg/l. Hackett (1970)
employed ‘Na4FeEDTA’. Gamborg and Shyluk
(1981) and Gamborg (1982) said that to prepare B5
or MS medium with 0.1 mM Fe, 43 mg/l of ‘ferric
EDTA’ or ‘Fe-versenate’ (EDTA) should be
weighed. The compound recommended in these
papers was probably the Na2FeEDTA.2H2O chelate
(theoretical mol. Wt. 428.2) as was the ‘FeEDTA’
(13% iron) employed by Davis et al., (1977). It
should be noted that NaFeEDTA is the only source of
Na in MS medium apart from the contamination in
the gelling agents.
Alternatives to EDTA. A few other chelating
agents have been used in culture media in place of
EDTA. The B5 medium of Gamborg et al., (1968)
was originally formulated with 28 mg/l of the iron
chelate ‘Sequestrene 330 Fe’. According to HeberleBors (1980), ‘Sequestrene 330 Fe’ is FeDTPA (Table
3.9), containing 10% iron (Anon, 1978). This means
that the concentration of Fe in B5 medium was
originally 0.05 mM. Gamborg and Shyluk (1981)
have proposed more recently that the level of Fe
should be increased to 0.1 mM. B5 medium is now
often used with 0.1 mM FeEDTA, but some
researchers still prefer FeDTPA, for example (Garton
and Moses, 1986) used it in place of FeEDTA in
Lloyd and McCown (1981) WPM medium for shoot
culture of several woody plants.
.
100
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
Fig. 3.6 Chemical structures of some chelating agents and iron chelates.
Growth regulatory effects of chelating agents.
Although most iron, previously complexed to
chelating agents such as EDTA, EDDHA and DTPA
(Table 3.9) is absorbed as uncomplexed ions by plant
roots, there is evidence that the chelating agents
themselves can be taken up into plant tissues
(Weinstein et al., 1951; Tiffin et al., 1960; Tiffin and
Brown, 1961). Chelating compounds such as EDTA,
in low concentrations, exert growth effects on plants,
which are similar to those produced by auxins. The
effects include elongation of oat coleoptiles (Heath
and Clark, 1956a.b), and etiolated lupin hypocotyls
(Weinstein et al., 1956), the promotion of leaf
epinasty (Weinstein et al., 1956) and the inhibition of
root growth (Burstrom, 1961, 1963). Hypotheses put
Chapter 3
forward to explain these observations have included
that:
• chelating agents act as auxin synergists by
sequestering Ca from the cell wall (Thimann and
Takahashi, 1958);
• the biological properties of the natural auxin IAA
may be related to an ability to chelate ions; other
chelating agents therefore mimic its action (Heath
and Clark, 1960).
Burstrom (1960) noted that EDTA inhibited root
growth in darkness (not in light) but that the growth
inhibition could be overcome by addition of Fe3+ or
several other metal ions (Burstrom, 1961). He
recognised that reversal of EDTA action by a metal
does not mean that the metal is physiologically active
but that it might only release another cation, which
had previously been made unavailable to the tissue by
chelation.
Effects in tissue culture. Growth and morphogenesis in tissue cultures have been noted on several
occasions to be influenced by chelating agents other
than EDTA. It has not always been clear whether the
observed effects were caused by the chelation of
metal ions, or by the chelating agent per se.
The growth rate of potato shoot tips was increased
by 0.01-0.3 mg/l 8-hydroxyquinoline (8-HQ) when
cultured on a medium which also contained EDTA
(Goodwin, 1966), and more callus cultures of a
haploid tobacco variety formed shoots in the absence
of growth regulators when DHPTA was added to
Kasperbauer and Reinert (1967) medium which
normally contains 22.4 mg/l EDTA. The DHPTA
appears to have been used in addition to the EDTA,
not as a replacement, and was not effective on callus
of a diploid tobacco (Kochhar et al., 1970). In the
same experimental system, Fe-DHPTA and FeEDDHA were more effective in promoting shoot
formation from the haploid-derived tissue than Fe
with CDTA, citric acid or tartaric acid (Kochhar
et al., 1971).
The inclusion of EDTA into a liquid nutrient
medium caused the small aquatic plant Lemna
perpusilla to flower only in short day conditions
whereas normally the plants were day-neutral
(Hillman, 1959, 1961). In the related species Wolffia
microscopica, plants did not flower unless EDTA
was present in the medium, and then did so in
response to short days (Maheshwari and Chauhan,
1963). When, however, Maheshwari and Seth (1966)
substituted Fe-EDDHA for EDTA and ferric citrate,
they found that plants not only flowered more freely
under short days, but also did so under long days.
The physiological effect of EDTA and EDDHA as
chelating agents was thus clearly different. This was
again shown by Chopra and Rashid (1969) who
found that the moss Anoectangium thomsonii did not
form buds as other mosses do, when grown on a
simple medium containing ferric citrate or Fe-EDTA,
but did so when 5-20 mg/l Fe-EDDHA was added to
the medium instead. An optimum concentration was
between 5 and 8 mg/l. Rashid also discovered that
haploid embryoids developed more freely from in
vitro cultures of Atropa belladonna pollen
microspores when Fe-EDDHA was incorporated into
the medium, rather than Fe-EDTA (Rashid and
Street, 1973). Heberle-Bors (1980) did not obtain the
same result, and found that FeEDTA was superior to
FeEDDHA for the production of pollen plants from
anthers of this species and of two Nicotianas. In
tobacco, the production of haploid plants was greatest
with FeEDTA, next best with FeDTPA, FeEGTA,
FeEDDHA, and poorest with Fe citrate. Each
complex was tested at or about the same iron
concentration. Heberle-Bors also showed that chelating agents are differentially absorbed by activated
charcoal (see Chapter 7). In tissue culture of rose
(Van Der Salm, 1994), Prunus (Mallosiotis et al.,
2003), citrus (Dimassi et al., 2003) and red raspberry
(Zawadzka and Orlikowska, 2006), it is advantageous
to use FeEDDHA rather than FeEDTA.
Toxicity caused by chelating agents. Although
low concentrations of EDTA markedly stimulate the
growth of whole plants in hydroponic cultures by
making iron more readily available, the compound
begins to be toxic at higher levels. By comparisons
Table 3.9 Some common chelating agents.
EDTA
EGTA
EDDHA
DTPA
DHPT
101
Etyhylenediaminetetraaceticacid
Ethyleneglycol-bis(2-aminoethylether)tetraaceticacid
Etyhylenediamine-di(o-hydroxyphenyl)acetic acid
Dietyhylenetriaminepentaacetic acid
1,3-diamino-2-hydroxypropane-tetraaceticacid
102
The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients
with observations on animal tissues, Weinstein et al.,
(1951) suggested that toxicity arose through
competition between EDTA and enzymes (and other
physiologically-active complexes) in the plant, for
metals essential to their activity. This will occur if
the avidity of the chelating agent is greater than the
metal binding capacity of proteins on the surface of
cells (Albert, 1958).
Toxicity can also occur in in vitro cultures.
Legrand (1975) found that an optimum rate of
adventitious shoot initiation occurred in endive leaf
segments when only 7.5 mg/l EDTA (one fifth the
concentration used in MS medium) was employed. In
these circumstances, higher levels of EDTA were
clearly inhibitory and more than 55 mg/l prevented
shoot formation. Dalton et al., (1983) found that 0.3
mM EDTA (compared to the 0.1 mM in MS medium)
reduced the growth rate of Ocimum cell suspensions.
Flower buds of Begonia franconis died within a
few days if cultured with a high level of FeEDTA (11.5 mM, i.e. 10-15 times the normal level) together
with 0.4-1.6 mM H2PO4-. Berghoef and Bruinsma
(1979a) thought that Fe3+ released from the FeEDTA
complex, had precipitated the phosphate. Necrosis
was avoided by increasing H2PO4- concentration to
6.4 mM.
Tissues may be damaged by culture in media
containing synthetic chelating agents where the pH
approaches neutrality, because at these pH levels,
EDTA and EGTA have been shown to remove
calcium ions from the membranes of mitochondria
and this inhibits NAD(P)H oxidation and respiration
(Moller and Palmer, 1981). Chelating agents have
been found to inhibit the action of the growth
substance ethylene (see Chapter 7) and are thought to
do so by sequestering Cu ions within plant tissues,
thereby interfering with the synthesis or action of a
Cu-containing enzyme responsible for ethylene
metabolism. EDTA can also inhibit the activity of
plant polyphenol oxidase enzymes in vitro (Weinstein
et al., 1951) and Smith (1968) thought that this might
occur because EDTA made Cu ions less available for
enzyme incorporation, when he found the chelating
agent was able to prevent the blackening of freshlyisolated Carex flacca shoot tips. Several oxidative
reactions are also biochemically catalysed by ions
such as Cu2+, Co2+ and Zn2+, and where this is the
case [e.g. the oxidation of glutathione – Martin
(1979); catechol amine oxidation – Kiss and Gergely
(1979)], chelating agents such as EDTA and CDTA
are inhibitory.
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