Plant
Physiol.
(1970)
45,
598-600
The
Culture
of
Plant
Cells
with
Ammonium
Salts
as
the
Sole
Nitrogen
Source
Received
for
publication
December
9,
1969
OLuF
L.
GAMBORG
AND
J.
P.
SHYLUK
Prairie
Regional
Laboratory,
National
Research
Council
of Canada,
Saskatoon,
Saskatchewan,
Canada
ABSTRACT
Soybean
cell
suspension
cultures
grew
on
defined
media
with
ammonium
as
the
sole
nitrogen
source
if
Krebs
cycle
acids
were
added.
Satisfactory
growth
was
obtained
with
ammonium
salts
of
citrate,
malate,
fumarate,
or
succinate,
when
compared
with
the
regular
medium
containing
nitrate
and
ammonium.
Little
or
no
growth
occurred
when
am-
monium
salts
of
shikimate,
tartrate,
acetate,
carbonate,
or
sulfate
were
used.
The
cells
also
grew
well
with
L-glutamine
as
nitrogen
source.
The
specific
activities
of
glutamine
syn-
thetase
and
isocitrate
dehydrogenase
(nicotinamide
ade-
nine
dinucleotide
phosphate)
were
lower
than
in
cells
grown
on
a
nitrate
medium,
but
ammonium
enhanced
the
activity
of
glutamate
dehydrogenase.
Cells
of
soybean,
wheat,
and
flax
have
been
cultured
for
an
extended
period
on
the
am-
monium
citrate
medium.
The
most
suitable
inorganic
nitrogen
source
for
growing
plant
cells
is
a
mixture
of
ammonium
and
nitrate
(2,
3,
7).
Plant
cells
in
culture
do
not
grow
readily
on
ammonium
salts
alone
although
ammonium
is
a
more
direct
nitrogen
source
than
nitrate
for
protein
production.
Filner
(3)
could
not
grow
to-
bacco
cells
on
ammonium
salts.
Rice
callus
did
not
grow
on
agar
media
containing
ammonium
sulfate
but
grew
on
am-
monium
citrate
(13).
We
observed
that
soybean
cells,
grown
in
a
liquid,
defined
medium,
needed
ammonium
in
order
to
utilize
nitrate
(7).
Ammonium
was
most
effective
at
concentrations
of
2
to
4
mm.
At
higher
or
lower
concentrations
the
cell
yields
decreased.
Glutamine,
but
not
other
amino
acids
or
asparagine,
could
replace
ammonium
(5).
The
cell
cultures
provided
a
convenient
system
to
determine
the
effect
of
ammonium
on
enzyme
activity
in
the
cells.
The
concentrations
of
glutamine
synthetase
(EC
6.3.1.2),
gluta-
mate
dehydrogenase
(EC
1.
4.
1.
2),
and
isocitrate
dehydrogenase
(EC
1.1.1.42)
were
determined.
This
paper
reports
that
soy-
bean
cells
will
grow
with
ammonium
salts
as
the
sole
nitrogen
source
if
certain
Krebs
cycle
acids
are
added.
Ammonium
en-
hanced
glutamate
dehydrogenase
activity
and
reduced
the
levels
of
glutamine
synthetase
and
isocitrate
dehydrogenase.
MATERIAL
AND
METHODS
Culture
Conditions.
The
soybean
cells
were
cultured
in
De
Long
flasks
in
liquid
B5
medium
as
described
earlier
(4,
6,
7).
This
medium
contains
mineral
salts,
sucrose,
the
B-vitamins,
and
2,4-dichlorophenoxyacetic
acid
(1
mg/liter).
Nitrogen
was
supplied
as
25
mm
potassium
nitrate
and
1
mM
ammonium
sul-
fate.
The
pH
was
5.5
which
is
optimal
for
growth
(7).
The
cell
inoculum
was
washed
in
fresh
medium
which
contained
no
nitrate
or
ammonium
before
being
added
to
the
culture
flasks.
The
cells
used
for
inoculum
were
grown
in
the
B5
medium
but
grew
immediately
when
transferred
to
a
suitable
ammonium
medium
without
requiring
a
period
of
adaptation.
The
car-
boxylic
acids
were
filter-sterilized
and
neutralized
with
am-
monium
hydroxide
and
potassium
hydroxide.
The
cells
were
usually
harvested
after
5
or
6
days
by
filtration
and
then
washed
with
water.
Dry
weights
were
determined
after
drying
aliquots
of
cells
in
a
vacuum
oven
at
60
C
for
18
hr.
Total
protein
was
estimated
by
the
micro-Kjeldahl
method.
Enzyme
Extraction.
The
cells
were
collected
by
filtration
on
Miracloth
and
washed.
A
weighed
amount
of
cells
was
mixed
with
1-mm
glass
beads
and
50
mm
Tricine
(N-tris(hydroxy-
methyl)methylglycine)
buffer,
pH
7.5,
containing
1
mM
2-mer-
captoethanol,
in
the
proportions
1:1-5:1
(w/w/v)
in
a
75-ml
homogenizer
flask
and
homogenized
for
90
sec
at
2
to
4
C
in
a
Braun
homogenizer,
model
MSK.
The
homogenate
was
sepa-
rated
from
the
beads
by
decantation.
A
measured
aliquot
of
the
homogenate
was
added
to
2
volumes
of
ethanol
at
-20
C,
and
the
precipitate
was
used
for
total
protein
analysis.
The
remainder
of
the
homogenate
was
centrifuged,
and
the
super-
natant
was
lyophilized
and
stored.
The
freeze-dried
preparations
were
suspended
in
water,
centrifuged,
and
passed
through
a
column
of
Sephadex
G-50,
and
the
protein
fraction
was
im-
mediately
assayed
for
enzyme
activity.
Enzyme
Assays.
Isocitrate
dehydrogenase,
shikimate
dehy-
drogenase,
and
glutamate
dehydrogenase
were
determined
by
recording
the
change
in
absorption
of
the
pyridine
nucleotides
at
340
nm
on
a
Gitford
automatic
spectrophotometer
in
the
presence
of
the
appropriate
substrate.
The
formation
of
'y-glutamylhydroxamate
was
used
to
measure
glutamine
syn-
thetase
(12).
One
unit
of
enzyme
is
the
amount
required
to
produce
1
,umole/min
at
30 C.
Assays
Conditions
Isocitrate
Dehydrogenase.
The
reaction
mixture
consisted
of
5
,umoles
of
MnCl2;
5
,umoles
of
DL-alloisocitrate;
0.2
,umole
of
NADP
or
NAD;
50
;umoles
of
Tricine
buffer,
pH
7.5;
and
en-
zyme
preparation
in
a
total
volume
of
1.0
ml.
Glutamate
Dehydrogenase.
The
reaction
mixture
had
the
following
composition:
20
,moles
of
ammonium
sulfate;
10
,umoles
of
a-ketoglutarate;
0.1
,mole
of
NADH;
50
,umoles
of
Tricine
buffer,
pH
8.0;
and
enzyme
in
a
total
volume
of
1.0
ml.
Shikimate
Dehydrogenase.
The
reaction
mixture
consisted
of
598
