Essential Nutrients for Plant Tissue Cultures: A Comprehensive Guide
Nutrient
Media
for
Plant
T
issue
Cultures
Functions
of
medium
Provide
Provide
Provide
Provi
d
e
wat
e
r
mi
n
er
a
l
n
utr
i
ti
o
n
a
l
vita
m
ins
gr
o
wth
re
g
u
l
a
t
ors
n
e
e
ds
Ac
c
ess
to at
m
osp
h
ere
for g
a
s
excha
n
ge
Re
m
oval
of
p
l
a
nt
m
e
t
a
b
o
l
ite waste
S
o
urces
o
f
en
e
r
g
y
Carb
o
n
S
o
urce
Nitr
o
g
e
n
S
o
urce
-
-
-
-
-
Sucrose
2
-5%
F
r
uc
t
ose
Lactose
Mal
t
o
se
Starch
D
ef
i
ned
Unde
f
ined
-
Mi
l
k
o
f
cocunut
-
Extrac
t
s of
malt
y
e
ast
& corn
M
a
jor
I
n
o
r
g
anic
-
NH
4
+
-
NO
3
-
Min
o
r
ions
- Am
i
no
ac
id
s
- Gl
y
cine
- Glu
t
amine
83
Com
p
osition
of C
u
lture
media
•
Cu
l
tre
Me
d
ia
is
co
m
po
sed
of
•
In
o
rg
a
nic
n
utrie
n
ts
which
i
n
clu
d
es
m
a
c
r
o
n
u
trie
n
ts
li
k
e
n
i
tr
o
g
e
n,
p
h
o
s
p
h
o
r
ous,
po
tassiu
m
,
cal
c
i
u
m
e
t
c.
and
m
ic
r
o
n
u
t
rien
t
s
l
i
ke
b
o
ro
n
,
cop
p
e
r
,
ir
on
,
m
anganese,
zinc et
c
.
O
r
g
a
nic
nu
t
rients
i
n
clu
d
es
V
it
a
m
i
n
s
l
i
k
e
V
it
a
m
in
B
1
,
B
6
,
B
3
,
B
5
•
et
c
.
A
m
i
n
o
a
cids
li
k
e
L-
a
r
g
i
n
i
ne,
L-asparagi
n
e,
L
-c
ystei
n
e
HCL,
L-g
l
u
ta
m
i
n
e
etc,
Carb
o
n
s
o
u
rce
li
k
e
glucose
o
r
m
a
l
t
o
se,
Growth
h
o
r
m
o
n
es/
r
eg
u
lato
r
s
li
k
e
au
x
i
n
,
c
y
t
o
k
i
n
i
n
s
and
g
i
b
b
erellin
s
,
eth
y
lene,
abscisic
acid.
Oth
e
rs
me
d
ia
subst
a
nces
li
k
e
p
r
o
t
ein
h
y
d
r
olysate
s
,
y
ea
s
t
•
extaracts,
f
r
u
it
(
e
.g
.
ba
n
ana)
extracts,
coco
n
ut
m
i
l
k,
sol
i
d
i
f
y
ing
agen
t
s
li
k
e
aga
r
,
algi
n
ate,
gelatin
et
c
.
,
Ir
o
n
so
u
rce
e
.g.
E
D
T
A
,
Anti
b
iot
i
c
s
.
p
H
o
f
t
h
e
m
e
d
i
u
m
sh
o
u
l
d
b
e
in
a
ran
g
e
o
f
5
.6
-
6.
0
bef
o
re
aut
o
clavi
n
g
t
h
e
cult
u
re
m
edi
u
m
•
I
n
o
r
g
a
n
i
c
Ma
c
ro
n
u
tr
i
e
n
ts
f
o
r
p
l
a
nt
ti
s
s
u
e
c
u
lt
u
r
e
s
85
Con
s
tit
u
e
n
t
s
He
l
l
e
r
N
i
ts
c
h &
N
i
ts
c
h
Wh
i
te
H
i
l
d
e
brand
t Ric
ke
r
&
D
u
g
g
e
r
M
usa
s
hige
&
S
k
o
o
g
Gauther
e
t
KCl
750
1500
65
65
N
a
NO
3
600
MgS
O
4
7H
2
0
250
250
720
180
370
125
N
a
H
2
P
O
4
H
2
0
125
250
16.5
33
C
a
Cl
2
2H
2
0
75
440
KN
O
3
2000
80
80
1900
125
C
a
Cl
2
25
N
a
2
SO
4
200
800
C
a
(N
O
3
)
2
NH
4
NO
3
1650
KH
2
P
O
4
170
125
MgSO
4
C
a
(N
o
3
)
2
4H
2
0
300
400
500
In
o
r
g
a
n
ic
Mi
c
ro
n
u
tr
i
e
n
ts
for
p
l
a
n
t
tis
s
ue
c
u
lt
u
r
e
s
6
Con
s
tit
u
e
n
t
s
He
l
l
e
r
N
i
ts
c
h &
N
i
ts
c
h
Wh
i
te
H
i
l
d
e
bran
d
t
Ric
ke
r &
D
u
g
g
e
r
M
usa
s
hige
&
S
k
o
o
g
Gauther
e
t
NiSO
4
0.05
F
e
SO
4
7H
2
0
2
7
.
8
0.05
MnS
O
4
4H
2
0
0.01
3
7
4
.
5
22.3
3
KI
0.01
0
.
75
3.0
0.83
0.5
NiCl
2
6H
2
0
0.03
CoCl
2
0.025
Ti(SO
4
)
3
0.2
Z
n
S
O
4
7H
2
0
0.01
0.5
3
6
8.6
0.18
C
u
SO
4
5H
2
0
0.03
0.025
0.025
0.05
B
e
S
O
4
0.1
H
3
B
O
3
1.0
0.5
1.5
0.38
6.2
0.05
H
2
SO
4
1.0
F
e
Cl
3
6H
2
0
1.0
M
g
2
M
O
4
0.025
0.25
AlCl
3
0.03
F
e
2
(S
O
4
)
3
2.5
F
e
rrict
a
rt
a
r
a
te
40
8
I
n
or
g
a
n
ic n
u
tri
e
n
t
s:
•
Min
e
r
al
e
lem
e
nts
play
v
ery
im
p
or
t
ant
r
ole
i
n
t
h
e
gr
ow
t
h
of
pl
a
nt
F
u
nct
io
n
o
f
nu
t
ri
e
nts
i
n
p
lant
g
r
o
wt
h
.
E
s
s
e
nt
i
a
l
ly
abo
ut
1
5
el
e
m
en
t
s
fou
n
d
i
mp
or
t
ant
f
or
who
l
e
pl
a
nt
gr
o
w
t
h
h
a
v
e
al
s
o
b
ee
n
p
r
o
ve
d
n
e
c
es
s
ary
f
or
the
g
ro
wth
of
tis
s
u
e(
s)
in
cu
l
tur
e
.
M
a
c
ro
nu
t
ri
e
nt:
Elem
e
n
t
s
r
eq
u
i
re
d
•
i
n
t
h
e
l
i
f
e
o
f
a
plant
g
re
at
e
r
than
0
.
5
mmo
l
/lit
are
referred
a
s macr
o
nu
tri
en
ts.
The
macron
u
ti
r
e
n
ts
inc
l
ude
six major
el
e
me
n
ts
as follo
w
s:
•
N
i
trogen
(N),
Potassi
u
m
(K),
Phos
p
hor
o
us
(P), C
a
lc
i
um
(C
a
),
Mag
n
e
sium
(Mg),
Sulfur
(S).
N
i
t
r
o
g
e
n
2
-
2
0
m
m
o
l
/
l
i
t
–
I
n
f
l
u
e
n
c
e
s
p
l
a
n
t
g
r
o
w
t
h
r
a
t
e
,
e
s
s
e
n
t
i
a
l
i
n
p
l
a
n
t
n
u
c
l
e
i
c
a
c
i
d
s
(
D
N
A
)
,
p
r
o
t
e
i
n
s
,
c
h
l
o
r
o
p
h
y
l
l
,
a
m
i
n
o
a
c
i
d
s
,
a
n
d
h
o
r
m
o
n
e
s
.
P
h
o
s
p
h
o
r
u
s
1
-
3
m
m
o
l
/
l
i
t
–
A
b
u
n
d
a
n
t
i
n
m
e
r
i
s
t
i
m
a
t
i
c
a
n
d
•
fast
growing
tissue,
essent
i
a
l
in
ph
o
tosynthe
s
is,
respiratio
n
.
P
o
t
a
s
s
i
u
m
2
0
me
r
istematic
-
3
0
m
mol/li
t
–
N
e
ces
s
ary
for
cell
d
i
v
isi
o
n,
•
tissue,
helps
in
the
path
w
ays
for
carbohydrate,
protein
and chl
o
rophy
l
l
synthesi
s
.
C
a
l
c
i
u
m
1
-
3
m
m
o
l
/
l
i
t
-
I
n
v
o
l
v
e
d
i
n
f
o
r
m
a
t
i
o
n
o
f
c
e
l
l
w
a
l
l
s
•
and
r
o
ot
and
l
e
a
f
dev
e
lo
p
men
t
.
P
a
rt
i
cipa
t
es
in
t
r
ans
l
ocat
i
on
of
suga
r
s,
amino
acids,
and
ties
up
o
x
al
ic
acid
(
t
o
x
i
n
)
M
a
g
n
e
s
i
u
m
1
-
3
mm
o
l/lit
-
Involv
e
d
in
photosynth
e
t
i
c
•
and
re
s
p
i
r
ation
syst
e
m
.
A
c
tive
i
n
uptake
o
f
ph
o
sp
h
ate
and
t
r
ans
l
ocation
of ph
o
sphate
and sta
r
ches.
S
u
l
f
u
r
1
-
3
m
m
o
l
/
l
i
t
-
I
n
v
o
l
v
e
d
i
n
f
o
r
m
a
t
i
o
n
o
f
n
o
d
u
l
e
s
an
d
chlo
r
o
p
hy
l
l
syn
t
h
e
si
s
,
s
t
r
uctural
co
m
p
o
nent
of
•
I
n
or
g
a
n
ic n
u
tri
e
n
t
s:
con
t
d…
•
Micr
o
n
utr
ie
nt:
Elem
e
n
t
s
r
e
qui
r
e
d
i
n
t
h
e
l
i
fe
o
f
a
p
l
a
nt
l
e
s
s
than
0
.
5
mmo
l
/lit
are
referred
a
s micr
o
n
ut
r
ients.
•
O
v
erall
t
h
e
p
l
ant
th
r
i
v
es
on
se
ve
nt
ee
n
el
e
m
e
nts
o
ut
of
w
h
ich
f
o
ur
l
ike
c
ar
b
o
n,
h
y
d
ro
g
e
n,
o
x
y
g
en
an
d
ni
t
r
oge
n
a
r
e
d
er
i
ve
d
f
r
om
t
h
e
a
tmo
s
ph
e
r
e
and
the
rest
thir
t
ee
n
micr
o
el
e
m
e
nts
l
i
k
e
B
oro
n,
c
op
p
e
r
,
iron,
ma
ng
a
nese,
z
in
c
,
c
o
balt,
m
oly
b
de
n
u
m,
n
i
c
k
e
l
,
a
lum
i
nium,
i
o
di
n
e,
f
e
r
ro
us,
sodi
u
m,
chlor
i
n
e
.
A
media
l
a
cking
i
n
t
h
e
s
e
micr
o
n
utr
ie
nt
d
oes
not
•
s
upp
ort
e
xh
ibits
h
ealthy
and
w
h
ole
s
o
me
gr
o
w
t
h
and
t
h
e
pl
a
nt
d
efici
e
ncy
v
esse
l
s,
sy
m
pt
o
ms
p
re
se
n
c
e
su
c
h
as
pig
m
e
n
t
ation,
a
b
s
ence
of
of
nar
r
ow
c
a
m
b
ial
z
one,
c
ell
u
l
ar
h
y
per
tro
phy
and
sy
m
p
t
o
m
s
o
f
c
h
loro
s
is
du
e
t
o
a
b
se
nce
of
ferrous
an
d
su
l
phu
r
.
◦
◦
M
i
c
r
o
n
u
t
r
i
e
n
t
s
c
o
n
c
e
n
t
r
a
t
i
o
n
s
a
n
d
t
h
e
r
e
r
o
l
e
(Fe
)-
1
m
M
/l
-
I
r
on
I
n
volved
in
Ce
l
l
d
iv
i
s
io
n,
respirat
i
on,
Fe
N
aE
D
T
A
chlo
r
o
p
h
y
ll
synthesis
and
p
h
otosynthes
i
s
.
E
g. sod
i
um
salt of
E
D
T
A
.
Man
g
a
n
e
s
e
(Mn)
20
-
9
0
m
M/l
-
I
nvolved
i
n
Ce
l
l
elongation
re
g
u
l
a
t
i
on
of
enzy
m
es
and
gr
o
wth
horm
o
nes.
Ass
i
sts
in
ph
o
tosynthesis
and respiratio
n
◦
.
◦
B
o
ron
(B)
2
-
5100
m
M/l
respon
s
ib
l
e
e
l
o
n
g
a
tion
C
o
pp
e
r
(Cu)
0
.
1
m
M/l
Molybd
e
num
(
M
o) 5
m
M/l
C
o
b
a
lt
(Co)
0
.
1
m
M/l
Z
i
nc (Zn) 1
.
5
-
30
m
M/l
Iodine
(I
)
N
i
ck
e
l
(
N
i),
Alumi
n
um
(Al),
Ferrous,
chl
o
ri
n
e and
sod
i
um
(Na) are
a
l
so
requ
i
red
for
cell
di
vision
a
n
d
cell
◦
◦
◦
◦
◦
•
O
r
ganic
n
u
tr
i
ents
It
in
c
ludes
Ni
t
rogen
s
u
b
s
t
a
n
c
es,
V
i
t
a
m
ins,
A
m
ino
a
c
ids,
Carbon
s
o
urc
e
,
G
r
o
w
th
hor
m
ones/r
e
gul
a
tors
Nit
r
ogen so
u
r
ce
Mo
s
t
cu
l
tur
e
d
p
l
ant
cells
a
r
e
c
a
p
ab
l
e
of
s
y
nt
h
esising
essent
i
a
l
v
i
ta
m
ins
but
not
i
n
s
u
f
fi
c
i
e
nt
amount.
T
o
a
c
h
i
e
ve
best
gro
w
th
i
t
i
s
es
s
e
nti
a
l
t
o
s
u
pp
l
e
m
ent
the
t
is
s
ue
c
u
l
t
ure
m
ed
i
um
w
i
th
one
or
m
ore
vi
t
amins
and amino
a
c
i
d
.
V
itam
i
ns
Th
i
amine
(
V
it
a
m
in
B
1
)
-
essent
i
al
a
s
a
co
e
n
z
y
m
e
i
n
the
c
i
t
r
ic
a
c
i
d
c
y
c
l
e
.
I
t
i
s
re
q
u
ir
e
d
m
ost
l
y
i
n
t
i
s
s
ue
cu
l
ture
and
i
s
c
o
nsider
e
d
t
o
be
e
s
s
en
t
i
a
l
.
T
h
i
a
mi
ne
h
y
dro
c
h
o
loride
i
n
0
.
1-
1
m
g/l
i
t conc
e
ntra
t
ion
i
s requir
ed
.
Nico
t
inic
a
c
i
d
(
ni
a
c
i
n-
V
i
t
a
m
in
B
3
)
0
.
5
m
g/li
t
,
P
y
rido
x
ine
(
V
i
ta
m
in
B
6
)
0
.
5
m
g
/
l
i
t
,
C
a
l
c
ium
pen
t
oth
e
n
a
t
e
(
V
i
t
a
m
in
B
5
)
0
.
1
m
g/
l
it,
are
known
t
o
improve
gr
o
wth
of
the t
i
s
s
ue
cu
l
tu
r
e
m
a
t
er
i
a
l
.
M
y
o
-inosi
t
o
l
-
pa
r
t
of
t
h
e
B
c
o
m
pl
e
x,
i
n
ph
o
sphate
form
i
s pa
r
t
of
c
e
l
l
m
embran
e
s,
o
r
gane
l
l
e
s and
i
s
not
es
s
en
t
i
al
t
o
gro
w
th
but
b
e
nefi
c
i
al
and
ha
v
e
i
m
portant
role
i
n
m
a
n
y
bios
y
nth
e
t
i
c
pa
t
hwa
y
s.
C
y
noco
b
a
l
amin
(
V
i
t
amin
B
12
),
R
ibof
l
av
i
n
(
V
ita
m
in
B
1,
),
Folic
ac
i
d
(
V
i
t
amin
M
)
0
.
5
m
g/
l
i
t,
Bio
t
in
(
V
i
t
a
m
in
H),
p-amino ben
z
oic
acid
(
P
ABA),
A
s
co
r
b
ic
a
c
i
d
(
V
i
t
a
mi
n
C),
α
-
to
c
ophe
r
ol
(vit
am
in
E
)
are
ad
d
ed
i
n
spe
c
i
a
l
c
as
e
s
but
their
e
xa
c
t
r
o
le
i
s
n
o
t
y
et
we
l
l
est
a
bl
i
she
d
.
•
•
A
m
ino
Acids
So
m
e
c
u
l
t
u
r
ed
p
l
a
n
t
-ce
l
ls
can
s
y
n
t
h
e
s
i
z
e
a
l
l
a
m
i
n
o
ac
id
s,
none
a
re
c
o
n
s
i
d
ered
e
s
s
e
n
t
i
a
l
.
The
m
o
s
t
c
o
m
m
on
s
o
u
r
ces
o
f
o
r
g
a
n
i
c
n
i
t
r
ogen
u
s
e
d
i
n
c
u
l
t
u
re
m
e
d
ia
are
•
a
m
i
n
o
ac
i
d
m
i
x
t
u
re
s
,
(e
.g.
,
ca
s
ein
h
y
dr
o
l
y
sat
e
),
L
-g
l
u
t
a
m
i
n
e,
L
-
a
s
p
a
ra
g
i
ne
,
o
r
g
i
n
i
n
e
,
m
e
t
h
i
o
n
i
n
e
a
n
d a
d
e
n
i
ne
.
W
h
e
n
a
m
i
n
o
ac
i
ds are
a
d
d
e
d
a
l
o
n
e,
t
h
ey can
b
e
inhibito
r
y
t
o ce
l
l g
r
owt
h
.
T
y
r
o
s
i
ne
h
a
s
b
e
en
used
t
o
s
t
i
m
u
l
a
t
e
m
o
r
p
ho
g
e
n
e
s
i
s
i
n
ce
l
l
c
u
ltur
e
s
but
sho
u
l
d
on
l
y
b
e
u
s
ed
i
n
a
n
a
g
ar
m
e
d
i
u
m
.
L
-
t
y
rosi
n
e
-
s
t
imu
l
a
t
es
sh
o
ot
f
o
r
m
a
t
i
o
n
.
Supple
m
en
t
at
i
on
o
f
th
e
cu
l
ture
m
ed
i
um
w
i
t
h
ad
e
n
i
ne
s
u
l
f
ate
can
s
t
i
m
u
l
ate
ce
l
l
g
r
owth
a
n
d
g
r
ea
t
ly
e
n
h
a
n
c
e
shoo
t
f
o
r
m
a
t
i
on
.
Carb
o
n
s
o
u
r
ce
•
•
•
•
•
Car
b
o
h
y
drates
are
used
i
n
t
i
s
s
u
e
cult
u
re
m
e
d
ia
as
an
e
n
e
r
gy
s
ource
of
car
b
on.
Mo
s
t
p
l
a
n
t
tissue
c
u
l
t
u
r
e
are
nonau
t
o
t
r
o
p
ic
a
n
d
are
t
h
er
ef
o
r
e
e
n
t
i
re
l
y
d
e
p
e
n
d
e
n
t
o
n
an e
x
t
e
r
n
al
s
o
u
r
ce
o
f
car
b
o
n
.
The
m
o
s
t
c
o
mm
o
n
ly
u
s
ed car
b
on
s
o
u
r
ce
i
s
Suc
r
o
s
e
(
2
-
5
%
o
r
20
-
3
0
g
/
l
i
t)
Gluc
o
se
a
n
d
Fructo
s
e
are u
s
ed
f
or
goo
d
g
r
owt
h
.
•
•
•
Ma
l
tose
a
n
d
r
a
f
f
in
o
se
are
us
e
d
i
n so
m
e
c
a
se
s
.
•
I
n
g
e
n
e
ral
e
xcised
d
i
c
o
tyledono
u
s
ro
o
t
s
gr
o
w
b
e
t
t
er
wi
t
h
s
u
c
rose
w
here
a
s
m
o
n
o
c
o
t
s
d
o
b
e
st with
d
e
x
t
rose
(g
l
u
c
os
e)
.
•
Oth
e
r
c
a
rbo
h
ydrat
e
s
l
i
ke
m
a
n
n
o
se,
s
or
b
i
t
o
l
,
pen
t
o
s
e
s,
s
u
g
ar
a
l
coh
o
l,
g
l
yco
l
s, h
e
xoses,
uron
i
c
a
c
i
d,
l
a
c
t
ose, g
a
l
a
c
t
os
e
, p
o
t
a
t
o
st
a
rc
h
, gr
a
in
starch
and
even
g
l
y
co
s
i
d
es
ca
n
b
e
u
sed d
e
p
e
n
d
i
n
g
o
n
t
h
e
e
x
p
er
i
m
e
n
t
al
c
o
n
d
i
t
i
o
n
s
.
•
G
r
owth hor
m
ones
/
r
e
g
ul
a
to
r
s/
Modul
a
to
r
s
T
h
e
su
c
c
e
ss
o
f
p
l
a
n
t
t
i
ssu
e
,
cel
l
a
n
d
o
r
g
a
n
c
u
l
t
ure
will
d
e
p
e
nds
o
n
t
he
a
m
o
u
nt
o
f
p
l
a
n
t
h
o
r
m
o
n
es
a
n
d
gr
o
w
t
h
substa
n
ce
a
d
ded
i
n
to
n
u
trient
m
e
d
i
u
m
.
Au
x
i
ns,
e
t
hyl
e
n
e
,
abscisic
a
c
i
d
,
cytoki
n
i
ns
a
nd
g
ib
b
ere
l
l
in
s
are
c
o
m
m
o
n
ly
r
e
c
o
g
n
i
z
e
d
a
s
t
he
fi
v
e
m
a
i
n
c
l
asses
o
f
n
a
t
ur
a
l
l
y
o
c
c
u
r
r
ing
p
l
a
n
t h
o
r
m
o
n
e
s
.
The
req
u
ir
e
m
e
n
t
o
f
t
h
ese
h
o
r
m
o
n
es
v
a
r
ies
c
o
n
s
i
d
erab
l
e
with
t
h
e
i
r
e
n
d
o
g
e
n
o
us
l
e
v
e
l
s
.
Oth
e
r
p
l
a
n
t
ho
r
m
o
n
es
l
i
ke
p
o
ly
a
m
i
n
es,
j
a
s
m
o
n
a
t
es,
sa
l
i
c
ylat
e
s
are
a
l
s
o
us
e
d
d
e
pen
d
i
ng
on
t
h
e
e
x
p
er
i
m
e
n
t
a
l
c
o
n
d
itions
a
nd
p
l
a
n
t
s
to
be
c
u
l
t
ur
e
d
.
P
lant
Gr
o
w
th Re
g
ula
t
o
rs (Horm
o
nes)
Gibberel
l
ins
Auxins
-
St
i
mula
t
e
C
y
tok
i
nins
-
P
rom
o
te cell
d
i
vis
i
o
n
-
E
l
o
nga
t
e
in
t
ernodes
ce
l
l
elonga
t
ion
N
a
tu
r
al
Aden
i
ne
Z
eat
i
n
Syn
t
he
t
ic
K
i
ne
t
in
Benzy
l
aden
i
ne
S
y
n
t
he
t
ic
N
A
A
2
,
4
-
D
N
a
tu
r
al
I
AA
94
Auxi
n
s
Auxins
sh
o
w
a
st
r
ong
influe
n
ce
ov
e
r
proce
s
ses
such
as
cell
growth
ex
pa
nsio
n
,
ce
ll
wa
l
l a
c
i
d
ifi
c
a
tion, i
n
it
i
ati
o
n
of c
e
ll
d
i
v
ision
and
o
r
ga
n
iza
t
ion
of
m
eris
t
e
m
s
giving
r
ise
to
eith
e
r
c
allus
or
defined
o
r
gan
s
.
I
n
o
r
ga
n
ized
tissue,
au
x
i
n
s
cause
root
for
m
at
i
o
n
,
dela
y
ing
leaf
senescenc
e
,
f
ruit r
i
pening
and
used
i
n
e
m
bry
o
gen
e
si
s
.
C
o
m
m
only
used
n
a
tur
a
l
au
x
in
i
s
in
d
ol
e
-
3
-
a
c
e
tic
a
c
id
(I
AA
-
1
-
50
m
g/
l
it),
b
u
t
depen
d
ing
o
n
the
sp
e
c
ies,
other
n
atur
a
l
au
x
ins
a
re
4-
chlo
r
oindo
l
e
-
3
-
a
c
e
tic
ac
i
d,
indo
le-
3
-
bu
t
yric
ac
i
d
(
I
B
A
)
.
C
o
m
m
only
used
syn
t
h
e
tic
au
x
ins
are
1
-
nap
h
tha
l
eneac
e
tic
ac
id
(NA
A-
0.1
-
10
m
g/
l
it)
a
n
d
2,
4
dich
l
o
roph
e
noxy
a
c
e
tic
a
cid
(
2
,
4
-
D
-
0.05
-
0.5
m
g/
l
i
t
)
.
95
Cytokinins
C
y
tok
i
nins
are
usef
u
l
i
n
cu
l
ture
for
s
t
i
m
u
l
a
t
ion
of
ce
l
l
div
i
sion
(c
y
tok
i
nesis
)
,
rel
e
ase
o
f
l
a
t
e
ral
bud
dor
m
ancy
and
i
n
d
u
ce
adven
t
i
t
ious bud for
m
a
t
io
n
.
Ce
l
l
div
i
sion
i
s
regul
a
t
e
d
b
y
the
j
oint
ac
t
ions
of
aux
i
ns
and
cytoki
n
in
s
.
Auxins
a
f
fect
DN
A
repl
i
ca
t
ion
where
a
s
c
y
tok
i
nins
se
e
m
s
t
o
exert
so
m
e
con
t
rol over
the even
t
s
l
e
ad
i
ng
t
o
m
i
t
osi
s
.
In
in
t
act
pl
a
nts,
c
y
tok
i
n
i
ns
pro
m
ote
l
a
t
e
ral
bud
growth
and l
e
af
exp
a
nsion,
p
ro
m
ote
ch
l
orop
h
y
ll
s
y
nt
h
esis
and
enh
a
nce
ch
l
orop
l
ast
deve
l
op
m
en
t
.
The
m
ost
co
m
m
only
us
e
d
c
y
t
okin
i
ns
are
the
su
b
sti
t
ut
e
d
pur
i
nes
such
as
synthe
t
ic
derived
kin
e
t
i
n
(0.1
-
10
m
g/
l
i
t
),
BA
(
6-
benzyladen
i
ne)
.
Z
e
a
t
in
and
2-
i
P
(6
-γ-
γ-di
m
e
t
h
y
l
a
m
ino
pur
i
ne)
are
na
t
ura
l
ly
occurr
i
ng
c
y
tok
i
nin
s
.
Other
c
y
tok
i
nins
are
adenosine
and adenylic ac
i
d.
Kine
t
in
i
s 30,00
0
t
i
m
es
m
ore pot
e
nt th
a
n aden
i
ne.
96
G
i
bbe
r
e
l
lins
Gibbe
r
e
l
l
ins
wi
l
l
pro
m
ote
flo
w
er
i
ng,
seed
g
e
r
m
ination
a
nd
st
e
m
or shoot elong
a
t
i
o
n
.
There
are
o
ver
20
known
Gibbe
r
e
llin
s
.
Gibb
e
rel
l
in
(G
A
3
)
is
usua
l
ly
used
t
o
i
n
c
r
e
a
se
the shoot elong
a
t
i
o
n
.
Gibberel
l
i
ns
are
used ra
r
ely
co
m
pared
t
o
auxin
and cytokin
i
n.
Cultu
r
ed
ca
l
lus
cell
s
syn
t
hes
i
ze
t
h
e
i
r
own
Gibbe
r
el
l
i
n
s
.
A
b
sc
i
sic acid
(AB
A
)
A
b
sci
s
ic
a
c
i
d
(A
B
A)
i
s
nat
u
ra
l
ly
p
roduced
i
n
pl
a
n
t
t
issue
s
.
ABA
and
other
s
t
r
u
c
t
u
rally
re
la
ted
n
a
tu
r
a
l
c
o
m
pounds
are
m
ost
l
i
kely
produ
c
ed
by
the cl
e
avage
of xanthophy
ll
.
ABA
i
s
often
r
e
ga
r
d
ed
as
being
an
inh
i
b
ito
r
,
a
s
i
t
m
aint
a
i
n
s
bud
and
seed
dor
m
an
c
y
,
inhi
bi
ts
auxi
n
-
pro
m
oted
c
e
l
l
wa
l
l
ac
i
dification
loos
e
ning
and slows
ce
l
l
elong
a
t
i
o
n
.
ABA
plays
a
key
role
i
n
c
los
i
n
g
of
sto
m
atal
a
p
ertu
r
e
s
(
r
educ
i
ng
t
r
ansp
i
rat
i
on)
and
absc
i
ss
i
on
of le
a
ve
s
.
AB
A
al
s
o
cont
r
ol
of water and
ion
uptake
by roo
t
s
.
97
Eth
y
lene
I
t
i
s
a
gaseous
hor
m
o
n
e
syn
t
he
s
ied
i
n
c
u
l
t
u
red
cell,
fungi
and
bac
t
er
i
a
.
Eth
y
le
n
e
g
as
pro
m
ot
e
s
f
ruit
r
ip
e
ning,
sene
s
cen
c
e
,
and
leaf
absc
i
ss
i
o
n
.
A
t
higher
c
once
n
tra
t
io
n
s
the
gas
decreas
e
d
c
ell
elonga
t
ion
but
inc
r
ea
s
ed
ce
l
l
expans
i
o
n
.
The
r
o
le
of
eth
y
l
ene
ca
n
be
di
f
f
ic
ul
t
t
o
un
d
ers
t
and
be
c
ause
i
ts
e
f
fects
vary
with
can
d
ev
e
lop
m
ent
st
a
g
e
a
n
d
because
low
concen
t
ra
t
i
o
ns
whereas higher
pro
m
ote
(or
s
o
m
et
i
m
es
i
n
hi
b
it)
a
p
ro
c
ess,
lev
e
ls
have
the oppos
i
te
e
f
fect.
Eth
y
le
n
e
i
s
synthesi
z
ed
f
r
o
m
m
et
hi
onin
e
.
A
u
x
i
n
sti
m
ulate
the
production
of
ethyle
n
e
but
the
p
hy
s
i
o
l
o
g
ic
a
l
signi
f
ican
c
e
of
ethy
l
ene
i
n
t
i
ssue
cul
t
ure
i
s
qui
t
e
obscu
re
.
98
G
r
o
w
t
h
r
e
g
u
l
a
t
o
r
s
/
H
o
r
m
o
n
e
s
a
n
d
t
h
e
i
r
f
u
n
c
t
i
o
n
s
•
•
-
auxin
– promote
roots
gro
w
th
and
Ce
l
l divis
i
o
n
.
-
cyto
k
inin
–
pr
o
m
ote
sho
o
ts
gro
w
th
and
Cell
di
v
i
s
ion
-
gib
b
e
r
e
l
l
i
n –
promote
c
e
ll enl
a
r
ge
m
ent
and
sho
o
t
e
l
o
n
gation
-
absc
i
s
i
c
a
c
i
d – plant
s
t
re
s
s
h
ormone
and in
h
ibits
au
x
in
-
e
t
h
y
l
e
ne
– low
concent
r
a
t
ions
c
a
n
promote
(or
so
m
e
t
i
m
es
in
h
ibit)
a
pr
o
c
e
s
s
,
w
h
er
e
as
hi
g
her
l
e
vels
ha
v
e
the op
p
osite
e
f
fe
c
t
•
•
•
•
Ot
h
e
r
s
media
s
u
b
stanc
e
s
which
pro
m
otes
growth
of
the
tissue
c
u
l
tu
r
e
like
protein
hydrol
y
sa
t
es
(
e
.
g
.,
soy
-
protein
hydro
l
y
z
a
tes),
yeast
e
x
tra
c
ts,
fru
i
t
(
e.g.
b
a
n
ana)
e
x
tra
c
ts,
coconut
m
i
l
k fresh
/
pas
t
eu
r
ize
d
.
•
Phenol
i
c
c
ompoun
d
s
l
i
ke
P
h
lo
r
o
g
l
u
cinol
-
S
t
i
m
ula
t
es
rooting
of shoot sec
t
ion
s
.
Activ
a
ted
c
ha
r
coal
i
s
u
sed
as
a
de
t
o
xifying
a
g
en
t
.
D
e
to
x
i
f
ies
wast
e
s
from plant
t
i
ssues
and i
m
pur
i
t
i
e
s
.
Adsorp
t
ion
qual
i
ty
va
r
y
,
concent
r
at
i
on
nor
m
al
l
y
us
e
d
i
s
0.3
%
•
•
or
lowe
r
.
It
adso
r
bs
t
h
e
s
e
conda
r
y
products
s
ecr
t
ed
by
t
h
e
cul
t
ure
t
i
ssu
e
.
Charcoal
for
tissue
c
u
l
t
ure
a
c
id
w
a
shed
and
n
e
utra
l
ized
n
e
ver
re
u
se
d
.
Contro
l
s
the
supp
l
y of
endogenous
growth
hor
m
one
s
.
D
e
-
m
ine
r
al
i
ses
wa
t
e
r
.
•
•
•
Soli
d
ifying
a
g
ent
Sol
i
d
m
edia
are
often
p
referred
b
ecau
s
e
its
i
m
prov
e
d
oxy
ge
n
supply
and support cul
t
ure
growth.
Ther
e
fo
r
e,
subs
t
a
n
ce
w
i
th
st
r
ong
ge
l
ling
c
apac
i
ty
i
s
added
i
n
to
the l
i
quid
m
edi
a
.
The
m
ost
com
m
only
u
sed
subs
t
a
n
ce
i
s
ag
a
r
and
ot
h
ers
are
algi
n
ate,
c
a
rrag
e
enan,
g
el
a
t
i
n,
s
tar
c
h,
silica
gel,
hydroxy
ethyl
ce
l
lu
l
ose
and polya
c
ry
l
a
m
id
e
.
A
g
ar
i
s
e
x
t
rao
r
d
in
a
ry
r
esista
n
t
t
o
enzy
m
atic
hydroly
s
is
at
incub
a
t
i
on
te
m
pe
r
ature
and
o
n
ly
fe
w
bacte
r
ia
exist
wh
i
ch
are
capab
l
e
of produ
c
ing
deg
r
ading
enzy
m
e
.
101
I
r
on sou
r
ce
E
D
T
A
can
be used
a
s
a i
r
on
sourc
e
.
pH
of
the
m
edium
sh
o
uld
be
i
n
a
ra
n
ge
of
5.
6-
6.0
befo
r
e
auto
c
laving
the cu
l
t
u
re
m
edium
A
n
t
i
bio
t
ics
are
us
e
d
for
p
r
eve
n
tion
of
exce
s
sive
conta
m
inat
i
on
i
n
the
c
u
l
tu
r
e
m
ediu
m
.
Genera
l
ly
fungi
c
i
d
es
and
ba
c
te
r
icides
a
re
us
e
d
ve
r
y
useful
because
they
i
n
c
u
l
t
u
re
m
edium
but
ar
e
not
b
e
e
n
can
be
to
x
ic
t
o
the
e
x
plant
and
t
h
e
conta
m
inant
so
m
et
i
m
es
re
a
p
p
e
a
rs
as
soon
are
as
t
h
ey
are
re
m
oved.
C
o
m
m
only
used
ant
i
bi
o
tics
Carbon
i
c
i
l
lin
(
500
m
g/
l
i
t
)
and
Aug
m
ent
i
n
(
250
m
g/
l
i
t
)
The composition of culture media for plant tissue cultures includes inorganic and organic nutrients, sources of energy like sucrose and amino acids, and essential macro and micronutrients. Providing gas exchange, waste removal, and growth regulators, the medium supports plant growth by offering access to vital elements and maintaining optimal conditions. Inorganic macro and micro nutrients, along with organic components like vitamins and growth hormones, play crucial roles in the growth and development of plant tissues in culture.
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Nutrient Media for Plant Tissue Cultures
Functions of medium Provide Provide Provide Provide Access to atmosphere for gas exchange Removal of plant metabolite waste water mineral nutritional vitamins growth regulators needs
Sources of energy Carbon Source Nitrogen Source - - - - - Sucrose 2-5% Fructose Lactose Maltose Starch Defined Undefined - Milk of cocunut -Extracts of malt yeast & corn Major Inorganic - NH4+ - NO3- Minor ions - Amino acids - Glycine - Glutamine 83
Composition of Culture media Cultre Media is composed of Inorganic nutrients which includes macronutrients like nitrogen, phosphorous, potassium, calcium etc. and micronutrients like boron, copper, iron, manganese, zinc etc. Organic nutrients includes Vitamins like Vitamin B1, B6, B3, B5 etc. Amino acids like L-arginine, L-asparagine, L-cysteine HCL, L-glutamine etc, Carbon source like glucose or maltose, Growth hormones/regulators like auxin, cytokinins and gibberellins, ethylene,abscisic acid. Others media substances like protein hydrolysates, yeast extaracts, fruit (e.g. banana) extracts, coconut milk, solidifying agents like agar, alginate, gelatin etc., Iron source e.g.EDTA, Antibiotics. pH of the medium should be in a range of 5.6-6.0 before autoclaving the culture medium
Inorganic Macro nutrients for plant tissue cultures Constituents Heller Nitsch & Nitsch White Hildebrand t Ricker & Dugger Musashige & Skoog Gautheret KCl 750 1500 65 65 NaNO3 600 250 250 720 180 370 125 MgSO47H20 125 250 16.5 33 NaH2PO4H20 75 440 CaCl22H20 KNO3 2000 80 80 1900 125 CaCl2 25 Na2SO4 200 800 Ca(NO3)2 NH4NO3 1650 KH2PO4 170 125 MgSO4 300 400 500 Ca(No3)24H20 85
Inorganic Micro nutrients for plant tissue cultures Constituents Heller Nitsch & Nitsch White Hildebrandt Ricker & Dugger Musashige & Skoog Gautheret NiSO4 0.05 27.8 0.05 FeSO47H20 MnSO44H20 KI 0.01 3 7 4.5 22.3 3 0.01 0.75 3.0 0.83 0.5 0.03 NiCl26H20 CoCl2 Ti(SO4)3 ZnSO47H20 CuSO45H20 BeSO4 H3BO3 H2SO4 FeCl36H20 Mg2MO4 AlCl3 Fe2(SO4)3 Ferrictartarate 0.025 0.2 0.01 0.5 3 6 8.6 0.18 0.03 0.025 0.025 0.05 0.1 1.0 0.5 1.5 0.38 6.2 0.05 1.0 1.0 0.025 0.25 0.03 2.5 40 6 8
Inorganic nutrients: Mineral elements play very important role in the growth of plant Function of nutrients in plant growth. Essentially about 15 elements found important for whole also been proved necessary for the growth of tissue(s) in culture. Macronutrient: Elements required than 0.5 mmol/lit are referred as macronutrients. The macronutirents include six major elements as follows: Nitrogen (N), Potassium (K), Phosphorous (P), Calcium (Ca), Magnesium (Mg), Sulfur (S). Nitrogen 2-20mmol/lit Influences plant growth rate, essential in plant nucleic acids (DNA), proteins, chlorophyll, amino acids, and hormones. plant growth have in the life of a plant greater
Phosphorus 1-3 mmol/lit Abundant in meristimatic and fast growing tissue, essential respiration. in photosynthesis, Potassium 20 meristematic carbohydrate, protein and chlorophyll synthesis. -30 mmol/lit Necessary for cell division, tissue, helps in the pathways for Calcium 1-3 mmol/lit - Involved in formation of cell walls and root and leaf development. translocation of sugars, amino acids, and ties up oxalic acid (toxin) Participates in Magnesium 1-3 mmol/lit - Involved in photosynthetic and respiration system. Active in uptake of phosphate and translocation of phosphate and starches. Sulfur 1-3 mmol/lit - Involved in formation of nodules and chlorophyll synthesis, structural component of
Inorganic nutrients: contd Micronutrient: Elements required in the life of a plant less than 0.5 mmol/lit are referred as micronutrients. Overall the plant thrives on seventeen elements out of which four like carbon, hydrogen, oxygen and nitrogen are derived from the atmosphere microelements like Boron, copper, iron, manganese, zinc, cobalt, molybdenum, nickel, aluminium, iodine, ferrous, sodium, chlorine. A media lacking in these micronutrient does not healthy and wholesome growth and the plant deficiency vessels, presence of narrow hypertrophy and symptoms of chlorosis due to absence of ferrous and sulphur. and the rest thirteen support exhibits symptoms such as pigmentation, absence of cambial zone, cellular
Micronutrients concentrations and there role (Fe)-1 mM/l - chlorophyll synthesis and photosynthesis. Eg. sodium salt of EDTA. Manganese (Mn) 20-90 mM/l - Involved in Cell elongation regulation of enzymes and growth hormones. Assists in photosynthesis and respiration Involved in Cell division,respiration, Iron FeNaEDTA . Boron (B) 2-5100 mM/l responsible elongation Copper (Cu) 0.1 mM/l Molybdenum (Mo) 5mM/l Cobalt (Co) 0.1 mM/l Zinc (Zn) 1.5-30 mM/l Iodine (I) Nickel (Ni), Aluminum (Al), Ferrous, chlorine and sodium (Na) are also required for cell division and cell
Organic nutrients It includes Nitrogen substances, Vitamins, Amino acids, Carbon source, Growth hormones/regulators Nitrogen source Most cultured plant cells are capable of synthesising essential vitamins but not in sufficient amount. To achieve best growth it is essential to supplement the tissue culture medium with one or more vitamins and amino acid. Vitamins Thiamine (Vitamin B1) - essential as a coenzyme in the citric acid cycle. It is required mostly in tissue culture and is considered to be essential. Thiamine hydrocholoride in 0.1- 1mg/lit concentration is required. Nicotinic acid (niacin-Vitamin B3) 0.5 mg/lit, Pyridoxine (Vitamin B6) 0.5 mg/lit, Calcium pentothenate (Vitamin B5) 0.1 mg/lit, are known to improve growth of the tissue culture material. Myo-inositol - part of the B complex, in phosphate form is part of cell membranes, organelles and is not essential to growth but beneficial and have important role in many biosynthetic pathways. Cynocobalamin (Vitamin B12), Riboflavin (Vitamin B1,), Folic acid (Vitamin M) 0.5 mg/lit, Biotin (Vitamin H), p-amino benzoic acid (PABA), Ascorbic acid (Vitamin C), - tocopherol (vitamin E) are added in special cases but their exact role is not yet well established.
AminoAcids Some cultured plant-cells can synthesize all amino acids, none are considered essential. The most common sources of organic nitrogen used in culture media are amino acid mixtures, (e.g., casein hydrolysate), L-glutamine, L- asparagine, orginine, methionine and adenine. When amino acids are added alone, they can be inhibitory to cell growth. Tyrosine has been used to stimulate morphogenesis in cell cultures but should only be used in an agar medium. L-tyrosine - stimulates shoot formation. Supplementation of the culture medium with adenine sulfate can stimulate cell growth and greatly enhance shoot formation. Carbon source Carbohydrates are used in tissue culture media as an energy source of carbon. Most plant tissue culture are nonautotropic and are therefore entirely dependent on an external source of carbon. The most commonly used carbon source is Sucrose (2-5% or 20-30 g/lit) Glucose and Fructose are used for good growth.
Maltose and raffinose are used in some cases. In general excised dicotyledonous roots grow better with sucrose where as monocots do best with dextrose (glucose). Other carbohydrates like mannose, sorbitol, pentoses, sugar alcohol, glycols, hexoses, uronic acid, lactose, galactose, potato starch, grain starch and even glycosides can be used depending on the experimental conditions. Growth hormones/regulators/ Modulators The success of plant tissue, cell and organ culture will depends on the amount of plant hormones and growth substance added into nutrient medium. Auxins, ethylene, abscisic acid, cytokinins and gibberellins are commonly recognized as the five main classes of naturally occurring plant hormones. The requirement of these hormones varies considerable with their endogenous levels. Other plant hormones like polyamines, jasmonates, salicylates are also used depending on the experimental conditions and plants to be cultured.
Plant Growth Regulators (Hormones) Gibberellins - Elongate internodes Auxins - Stimulate cell elongation Cytokinins - Promote cell division Natural Adenine Zeatin Synthetic Kinetin Benzyladenine Synthetic NAA 2,4-D Natural IAA 94
Auxins Auxins show a strong influence over processes such as cell growth expansion, cell wall acidification, initiation of cell division and organization of meristems giving rise to either callus or defined organs. In organized tissue, auxins cause root formation, delaying leaf senescence, fruit ripening and used in embryogenesis. Commonly used natural auxin is indole-3-acetic acid (IAA-1-50 mg/lit), but depending on the species, other natural auxins are 4- chloroindole-3-acetic acid, indole-3-butyric acid (IBA). Commonly used synthetic auxins are 1-naphthaleneacetic acid (NAA-0.1-10 mg/lit) and 2, 4 dichlorophenoxyacetic acid (2, 4-D- 0.05-0.5 mg/lit). 95
Cytokinins Cytokinins are useful in culture for stimulation of cell division (cytokinesis), release of lateral bud dormancy and induce adventitious bud formation. Cell division is regulated by the joint actions of auxins and cytokinins. Auxins affect DNA replication where as cytokinins seems to exert some control over the events leading to mitosis. In intact plants, cytokinins promote lateral bud growth and leaf expansion, promote chlorophyll synthesis and enhance chloroplast development. The most commonly used cytokinins are the substituted purines such as synthetic derived kinetin (0.1-10 mg/lit), BA (6- benzyladenine). Zeatin and 2-iP (6- - -dimethylamino purine) are naturally occurring cytokinins. Other cytokinins are adenosine and adenylic acid. Kinetin is 30,000times more potent than adenine. 96
Gibberellins Gibberellins will promote flowering, seed germination and stem or shoot elongation. There are over 20 known Gibberellins. Gibberellin (GA3) is usually used to increase the shoot elongation. Gibberellins are used rarely compared to auxin and cytokinin. Cultured callus cells synthesize their own Gibberellins. Abscisic acid (ABA) Abscisic acid (ABA) is naturally produced in plant tissues. ABA and other structurally related natural compounds are most likely produced by the cleavage of xanthophyll. ABA is often regarded as being an inhibitor, as it maintains bud and seed dormancy, inhibits auxin-promoted cell wall acidification loosening and slows cell elongation. ABA plays a key role in closing of stomatal apertures (reducing transpiration) and abscission of leaves. ABAalso control of water and ion uptake by roots. 97
Ethylene It is a gaseous hormone synthesied in cultured cell, fungi and bacteria. Ethylene gas promotes fruit ripening, senescence, and leaf abscission. At higher concentrations the gas decreased cell elongation but increased cell expansion. The role of ethylene can be difficult to understand because its effects vary with can concentrations whereas higher levels have the opposite effect. Ethylene is synthesized from methionine. Auxin stimulate the production of ethylene but the physiological significance of ethylene in tissue culture is quite obscure. development promote (or sometimes inhibit) a process, stage and because low 98
Growth regulators/Hormones and their functions -auxin promote roots growth and Cell division. -cytokinin promote shoots growth and Cell division -gibberellin promote cell enlargement and shoot elongation -abscisic acid plant stress hormone and inhibits auxin -ethylene low concentrations can promote (or sometimes inhibit) a process, whereas higher levels have the opposite effect
Others media substances which promotes growth of the tissue culture like protein hydrolysates (e.g., soy-protein hydrolyzates), yeast extracts, fruit (e.g. banana) extracts, coconut milk fresh/pasteurized. Phenolic compounds like Phloroglucinol - Stimulates rooting of shoot sections. Activated charcoal is used as a detoxifying agent. Detoxifies wastes from plant tissues and impurities. Adsorption quality vary, concentration normally used is 0.3 % or lower. It adsorbs the secondary products secrted by the culture tissue. Charcoal for tissue culture acid washed and neutralized never reused. Controls the supply of endogenous growth hormones. De-mineralises water.
Solidifying agent Solid media are often preferred because its improved oxygen supply and support culture growth. Therefore, substance with strong gelling capacity is added into the liquid media. The most commonly used substance is agar and others are alginate, carrageenan, gelatin, starch, silica gel, hydroxy ethyl cellulose and polyacrylamide. Agar is extraordinary resistant to enzymatic hydrolysis at incubation temperature and only few bacteria exist which are capable of producing degrading enzyme. 101
Iron source EDTAcan be used as a iron source. pH of the medium should be in a range of 5.6-6.0 before autoclaving the culture medium Antibiotics contamination in the culture medium. Generally fungicides and bactericides are used very useful because they can be toxic to the explant and the contaminant sometimes reappears as soon removed. Commonly used antibiotics (500mg/lit) andAugmentin (250 mg/lit) are used for prevention of excessive in culture medium but are not been as they are Carbonicillin are
