Callus induction and thallus regeneration of the red alga Meristotheca papulosa (Rhodophyta, Gigartinales)


Huang, W.; Fujita, Y.

Botanica Marina 40(1): 55-61

1997


Suitable medium and culture conditions for callus induction from thallus explants and thallus regeneration in the red alga Meristotheca papulosa (Montagne) J. Agardh were examined. Axenic explants were obtained by treatment of thallus segments in iodine solution followed by an antibiotic mixture. The highest level of callus induction from axenic explants was obtained on ASP12NTA medium with 1.5% agar incubated at 20 degree C temperature and a photon flux density of 10 mu-mol photons m-2s-1. The callus was induced mainly from the cortical and medullary tissues at the cut surface and the callus cells were red pigmented. The callus cells were ellipsoidal or spherical and in the initial stages the cells were uniseriate, with time the callus grew irregularly. The callus induction rate was influenced by thallus collection time. The explants from thalli collected during December-May showed a higher rate of callus induction than the June-July collections. Addition of 0.1 mg L -1 indole-3-acetic acid (IAA) in combination with 0.05 mg L-1 6-benzylaminopurine (BAP) was effective in enhancing the callus size, but not the induction rate. Sucrose was most effective carbon compound tested, enhancing callus induction rate and size. Glycerol, mannitol and sorbitol showed an inhibitory effect. The excised callus from the explant showed sustained growth on subculture. The callus mass formed shoots when transferred to aerated PES liquid medium in 2 - 3 weeks. At 20 or 25 degree C, 40 mu-mol photons m-2s-1 were favourable for shoot formation. In 3 months these shoots regenerated 4.5 cm long thallus.

Botanica
Marina
Vol.
40,
1997,
pp.
55-61
©
1997
by
Walter
de
Gruyter
Berlin
New
York
Callus
Induction
and
Thallus
Regeneration
of
the
Red
Alga
Meristotheca
papulosa
(Rhodophyta,
Gigartinales)
W.
Huang'
and
Y.
Fujitab*
aGraduate
School
of
Marine
Science
and
Engineering,
Nagasaki
University,
Bunkyo
Machi
1-14,
Nagasaki
852,
Japan
b
Faculty
of
Fisheries,
Nagasaki
University,
Bunkyo
Machi
1-14,
Nagasaki
852,
Japan
*
Corresponding
author
Suitable
medium
and
culture
conditions
for
callus
induction
from
thallus
explants
and
thallus
regeneration
in
the
red
alga
Meristotheca
papulosa
(Montagne)
J.
Agardh
were
examined.
Axenic
explants
were
obtained
by
treatment
of
thallus
segments
in
iodine
solution
followed
by
an
antibiotic
mixture.
The
highest
level
of
callus
induction
from
axenic
explants
was
obtained
on
ASP12NTA
medium
with
1.5%
agar
incubated
at
20
°C
temper-
ature
and
a
photon
flux
density
of
10
µmol
photons
m
-2
s
-1
.
The
callus
was
induced
mainly
from
the
cortical
and
medullary
tissues
at
the
cut
surface
and
the
callus
cells
were
red
pigmented.
The
callus
cells
were
ellipsoidal
or
spherical
and
in
the
initial
stages
the
cells
were
uniseriate,
with
time
the
callus
grew
irregularly.
The
callus
induction
rate
was
influenced
by
thallus
collection
time.
The
explants
from
thalli
collected
during
December-
May
showed
a
higher
rate
of
callus
induction
than
the
June-July
collections.
Addition
of
0.1
mg
L
-1
indole-3-
acetic
acid
(IAA)
in
combination
with
0.05
mg
L
-1
6-benzylaminopurine
(BAP)
was
effective
in
enhancing
the
callus
size,
but
not
the
induction
rate.
Sucrose
was
most
effective
carbon
compound
tested,
enhancing
callus
induction
rate
and
size.
Glycerol,
mannitol
and
sorbitol
showed
an
inhibitory
effect.
The
excised
callus
from
the
explant
showed
sustained
growth
on
subculture.
The
callus
mass
formed
shoots
when
transferred
to
aerated
PES
liquid
medium
in
2-3
weeks.
At
20
or
25
°C,
40
pimol
photons
m
-2
s
-1
were
favourable
for
shoot
formation.
In
3
months
these
shoots
regenerated
4.5
cm
long
thallus.
Introduction
Tissue
culture
of
flowering
plants
is
a
well
established
technique
applied
for
crop
improvement,
micropro-
pagation
and
breeding
because
of
the
greater
under-
standing
of
the
physiological
and
biochemical
basis
of
cultured
cells.
Tissue
culture
technique
are
now
being
applied
to
seaweeds.
Thus
far
48
species
of
brown
and
red
algae
have
been
studied
for
callus
in-
duction
and
regeneration
(Aguirre-Lipperheide
et
al.
1995).
Apart
from
this
the
different
parameters
ef-
fecting
callus
induction,
growth
and
differentiation
were
also
reported
(Saga
et
al.
1982,
Gusev
et
al.
1987,
Lawlor
et
al.
1989,
Bradley
and
Cheney
1990,
Liu
and
Kloareg
1991,
Kaczyna
and
Megnet
1993).
However,
the
field
of
cell
and
tissue
culture
of
sea-
weeds
is
not
as
advanced
as
that
of
higher
plants
probably
because
of
lack
of
knowledge
of
factors
controlling
callus
induction
and
differentiation.
Meristotheca
papulosa
(Montagne)
J.
Agardh
a
sol-
ieriacean
red
alga,
has
been
commercially
used
for
seaweed
salad
or
as
garnishing
for
sliced
raw
fish
(=
sashimi).
In
this
report
we
studied
the
callus
in-
duction
and
regeneration
from
the
thallus
explants
of
Meristotheca
papulosa.
Materials
and
Methods
Meristotheca
papulosa
was
obtained
from
Kurose
Kaisan
Corp.
,at
Amakusa,
Kumamoto
Prefecture,
Japan.
Fronds
of
15-40
cm
length
were
collected.
They
were
cleaned
of
epiphytes
after
arrival
in
the
laboratory
with
a
soft
brush.
Pieces
of
3-4
cm
were
cut
after
cleaning
and
were
immersed
in
autoclaved
seawater
containing
0.1%
detergent
(`Charmy
Green',
Lion
Co.
Ltd.,
Japan)
solution
for
5
min
and
were
further
cleaned
with
a
brush.
Then
they
were
im-
mersed
in
0.02%
of
0.5
M
iodine
solution
(Wako
Pure
chemicals
Ltd.)
for
5
min,
in
autoclaved
seawater.
Sterilization
was
continued
by
further
incubation
in
autoclaved
seawater
for
48
h
at
20
°C
supplemented
with
2%
filter
sterilized
antibiotic
mixture
of
Polne-
Fuller
and
Gibor
(1984)
modified
by
the
addition
of
fosfomycin
(5
g
L
-1
),
erythromycin
(400
mg
L
-1
)
(Wako
Pure
chemicals
Ltd.)
and
polymixin
B
(400
mg
L
-1
)
(Pfizer
Co.
Ltd.).
After
each
treatment
the
thallus
segments
were
rinsed
2-3
times
with
auto-
claved
seawater.
The
segments
were
cut
into
about
3
x
3
mm
explants
for
callus
induction.
The
axenicity
of
the
explants
was
tested
on
a
marine
bacterial
cul-
ture
medium
(Oppenheimer
and
ZoBell
1952).
The
explants
from
thalli
collected
during
March
1992
were
inoculated
on
solid
(0.5
and
1.5%
agar)
or
liquid
ASP12NTA
(Provasoli
1963)
and
PES
(Prova-
soli
1968).
The
cultures
were
incubated
at
different
combinations
of
temperature
(15,
20
and
25
°C)
and
photon
flux
density
(0,
10,
40
and
80
Amol
photons
m
-2
s
-1
).
The
culture
media
were
sterilized
by
auto-
claving
for
20
min
at
120
°C.
After
sterilization
the
56
W.
Huang
and
Y.
Fujita
medium
was
supplemented
with
filter
sterilized
vit-
amins,
and
20
mL
of
the
medium
was
distributed
into
each
petri
dish
(9
cm
dia.).
Ten-twelve
explants
were
placed
in
each
petri
dish.
To
study
the
effect
of
thallus
collection
time
on
callus
induction
rate
and
size,
thalli
were
collected
at
monthly
intervals
between
1992
to
1994.
The
addition
of
plant
growth
regulators
indole-3-
acetic
acid
(IAA)
(0.01-0.5
mg
L'),
6-benzylamino-
purine
(BPA)
(0.01-0.5
mg
L
-1
)
individually
or
in
combination
and
carbon
compounds
(glycerol,
man-
nitol,
sorbitol,
glucose,
galactose,
mannose
and
su-
crose)
(Wako
Pure
chemicals
Ltd.)
at
100
mm
concen-
tration
were
tested
on
callus
induction
rate
and
size
(thalli
collected
in
January
1993).
A
filter
sterilized
stock
solution
of
each
plant
growth
regulator
and
autoclaved
stock
solution
of
carbon
compounds
were
added
separately
to
the
autoclaved
medium.
The
explants
were
observed
under
a
stereoscopic
microscope
(Olympus,
SZH10).
Callus
size
was
meas-
ured
as
the
height
from
the
cut
surface
of
the
explant.
The
callus
induction
rate
was
calculated
as
the
per-
centage
callus
induction
on
explants
to
the
total
ex-
plants
inoculated.
About
1
mm
diameter
of
callus
mass
was
excised
from
the
explant
and
was
maintained
by
subculturing
every
2-3
months.
For
callus
induction
and
subcul-
ture,
ASP12NTA
medium
with
1.5%
agar
was
used.
To
study
the
shoot
formation,
5-10
pieces
of
cal-
lus
of
1-2
mm
diameter
were
transferred
to
aerated
culture
flasks
with
200
mL
liquid
ASP12NTA
or
PES
medium.
They
were
cultivated
at
different
combi-
nations
of
temperature
(15,
20
and
25
°C)
and
pho-
ton
flux
density
(10,
40
and
80
gmol
photons
111
-2
s").
At
weekly
intervals
the
shoot
formation
was
observed.
Thallus
regeneration
was
obtained
from
the
culture
of
the
shoots
formed
in
aerated
cul-
tures.
All
the
cultures
were
usually
maintained
under
day
light
fluorescent
lamps
at
10
gmol
photons
m
-2
s',
12
:
12,
L
:
D
photoperiod,
20
°C
unless
otherwise
mentioned.
Results
Treatment
of
the
explants
with
iodine
solution
fol-
lowed
by
the
antibiotic
mixture
resulted
in
about
85-
93%
viable,
axenic
explants
on
bacterial
culture
me-
dium.
Sterility
was
maintained
during
the
culture.
Callus
was
induced
after
3-7
days
in
cultures
from
cortical
and
medullary
tissue
at
the
cut
surface.
The
red
pigmented
callus
gradually
increased
to
the
size
of
200
gm
in
2
weeks
on
ASP12NTA
(1.5%
agar)
medium
incubated
at
20
°C
and
10
grnol
photons
m
-2
s
-1
(Fig.
1).
Rarely
callus
was
induced
on
the
surface
of
the
explant..After
3
months
in
culture
the
explants
were
completely
covered
by
the
callus
tissue
and
grew
to
1-2
mm
size
(Fig.
2).
The
callus
cells
were
ellipsoidal
or
spherical.
In
the
initial
stages
of
callus
induction
the
callus
showed
a
chain
of
cells
of
15-25
gm
in
diameter,
with
time
the
callus
grew
irregularly
and
a
cell
diameter
of
20-80
gm
was
ob-
served
(Fig.
3).
In
the
temperature
range
of
15-25
°C
cultures
grown
at
20
°C
showed
the
highest
induction
rate.
A
temperature
of
20
°C
and
a
low
photon
flux
density
of
10
gmol
photons
m
-2
s'
were
most
favourable
for
callus
induction.
Increase
of
photon
flux
density
has
shown
a
tendency
of
decreased
induction
rate.
At
25
°C
and
a
high
photon
flux
density
of
80
gmol
photons
M
-2
s'
no
callus
was
induced
and
the
ex-
plants
bleached
and
died
after
2
weeks
in
culture.
The
explants
incubated
in
the
dark
also
could
not
induce
callus,
they
eventually
died
after
1
week
in
culture.
Higher
callus
induction
rate
and
size
were
observed
on
ASP12NTA
medium
than
on
PES
medium.
Callus
induction
was
not
observed
on
a
medium
with
lower
concentration
of
agar
(0.5%)
or
in
liquid
medium.
The
ASP12NTA
(1.5%
agar)
medium
incubated
at
20
°C
and
10
gmol
photons
111
-2
s'
was
found
most
favourable
for
callus
induction
(Table
I).
Thalli
collected
during
December
to
May
showed
the
highest
induction
rates.
The
highest
induction
rate
of
89%
was
observed
from
the
December
1992
collection.
Conversely
during
June
and
July
a
tend-
ency
for
a
decreased
callus
induction
rate
was
ob-
served.
No
callus
was
induced
from
thalli
collected
during
July
1992
(Table
II).
Additions
of
IAA
and
BAP
at
different
concen-
trations
and
combinations
showed
an
enhancing
ef-
fect
on
callus
size,
but
no
obvious
effect
on
callus
induction
rate.
Addition
of
IAA
at
0.1
mg
L'
in
combination
with
BAP
.0.05
mg
L
-1
showed
an
in-
creased
effect
on
size
(462
pin)
of
the
callus.
While
IAA
at
0.5
mg
L
-1
showed
a
lower
callus
size
and
induction
rate
than
the
control
explants
(Fig.
5).
The
addition
of
carbon
compounds
showed
a
marked
effect
on
both
callus
size
and
induction
rate.
Sucrose,
glucose,
galactose
and
mannose
enhanced
callus
size
and
induction
rate.
Sucrose
was
most
ef-
fective
with
the
greatest
callus
size
(516
gm)
and
in-
duction
rate
(86%).
Glycerol,
mannitol
and
sorbitol
showed
an
inhibitory
effect
(Fig.
6).
The
induced
callus
mass
was
excised
from
the
ex-
plant
and
continued
irregular
growth
(Fig.
4)
and
was
maintained
by
subculturing
for
over
two
years
on
ASP12NTA
medium
supplemented
with
IAA
(0.1
mg
L
-1
),
BAP
(0.05
mg
L
-1
)
and
sucrose
(100
mm).
The
callus
growth
stopped
when
the
callus
mass
was
transferred
to
the
aerated
liquid
medium.
The
callus
cells
dedifferentiated
into
the
outer
cortical
cells
and
the
central
medullary
cells
similar
to
the
thallus
organization.
Shoot
formation
was
observed
in
2-3
weeks
by
the
division
of
cells
from
the
surface
of
the
callus
mass
(Fig.
7).
After
3
months
a
thallus
of
4.5
cm
length
was
regenerated
by
the
shoot
growth
(Fig.
8).
The
effect
of
temperature,
light
and
medium
on
shoot
formation
were
studied.
At
15
°C
no
shoot
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Figs
1-4.
Fig.
1.
Callus
induction
from
cut
surface
on
explant
of
Meristotheca
papulosa
after
2
weeks
on
solid
(1.5%
agar)
ASP12NTA
medium,
E
=
explant;
C
=
callus
(bar
=
200
gm).
Fig.
2.
3
months
old
callus
mass
on
explant,
E
=
explant;
C
=
callus
(bar
=
1
mm).
Fig.
3.
3
months
old
callus
cells
showing
irregular
growth
(bar
=
100
pm).
Fig.
4.
1
mm
diameter
excised
callus
mass
cultured
for
4
months
(bar
=
1
mm).
Table
I.
Effect
of
various
culture
conditions
and
media
on
callus
induction
in
Meristotheca
papulosa
cultured
for
4
weeks
(n
=
20-24).
Temp.
Photon
flux
density
Culture
media
Agar
conc.
(°C)
(Imo]
photon
r11
-2
S
-1
)
(%)
Callus
induction
Rate
(%)'
Size
(Iim)
2
15
10
ASP12NTA
1.5
33
235
±
32
15
40
ASP12NTA
1.5
39
254
±
24
15
80
ASP12NTA
1.5
13
156
±
15
20
0
ASP12NTA
1.5
0
0
20
10
PES
0
0
0
20
10
PES
0.5
0
0
20
10
PES
1.5
13
150
±
36
20
10
ASP12NTA
0
0
0
20
10
ASP12NTA
0.5
0
0
20
10
ASP12NTA
1.5
68
350
±
33
20
40
ASP12NTA
1.5
50
286
±
41
20
80
ASP12NTA
1.5
23
108
±
29
25
10
ASP12NTA
1.5
54
295
±
32
25
40
Aspl2NTA
1.5
5
75
±
18
25
80
ASP12NTA
1.5
0
0
I
Percentage
callus
induction
of
explants
to
total
Number
of
explants
inoculated.
2
Average
of
callus
height
from
the
cut
surface
of
the
explant,
expressed
as
mean
±
SD.
3
11111131=11.1111.1m.11
4
4
4;
ti
)-1;
1=1111•111119=1.1...11
0:
yr
if
-
80
-
60
r.
-
40
H
ti
-
20
0
0.010.05
0.1
0.05
0.5
0
0.01
0.05
0.1
p.5
0.1
0
0.01
0.05
0.1
0.5
0.5
0
Ca
llu
s
in
du
c
t
io
n
ra
te
(
%)
500
-
Callus
size
0
Callus
induction
rate
400
-
'X<
Fl
200-
100-
0
0.01
0.05
0.1
0.5
0.01
r r
r
i
0
IAA
0
0.01
0.05
0.1
0.5
BP
0
58
W.
Huang
and
Y.
Fujita
Table
II.
Favourable
thallus
collection
time
for
callus
in-
duction
from
Meristotheca
papulosa
cultured
for
4
weeks.
Date
of
No.
of
Callus
induction
Collection
explants
Rate
(%)
Size
(pm)
1992
March
622
66
May
370
54
June
271
27
July
208
0
December
270
89
1993
January
280
63
May
520
42
June
403
10
1994
January
349
74
June
230
3
formation
was
observed
and
at
increasing
photon
flux
density
(40
and
80
Imo]
photons
m
-2
s
-1
)
the
callus
mass
bleached
and
died
after
4
weeks
in
cul-
ture.
With
increasing
temperature
(20-25
°C)
and
photon
flux
density
(40-80
i.tmol
photons
m
-2
s
-1
)
a
tendency
for
early
shoot
formation
was
also
ob-
served.
At
a
high
photon
flux
density
of
80
Amol
pho-
tons
111
-2
s
-1
(at
20
°C
and
25
°C)
the
shoots
died
in
3-5
weeks
in
cultures.
The
shoot
formation
was
very
slow
in
ASP12NTA
medium
in
comparison
to
PES
medium.
For
shoot
formation
PES
liquid
medium
in
aerated
cultures
maintained
at
20-25
°C
and
40
µmol
photons
m
-2
s
-1
was
found
to
be
the
most
favourable
(Table
III).
Discussion
Meristotheca
papulosa
callus
developed
primarily
from
cortical
and
medullary
tissue
and
showed
no
difference
in
cell
size
and
pigmentation.
Callus
was
developed
from
filamentous
cells
that
grow
out
from
cortical
cells
of
Eucheuma
uncinatum
Sitchell
et
Gardner
and
E.
alvarezii
var.
tamblang
Doty
(Polne-
Fuller
and
Gibor
1987),
while
Dawes
and
Koch
(1991)
reported
that
callus
from
Eucheuma
denticula-
turn
(Burman)
Collins
et
Hervey
and
Kappaphycus
al-
varezii
(Doty)
Doty
developed
from
the
medullary
tissue.
Our
results
show
no
functional
difference
of
cortical
and
medullary
tissue
in
callus
formation.
In
our
study,
callus
mostly
originated
from
the
cut
surface
of
the
explant.
From
microscopic
obser-
vation,
it
was
evident
that
2-3
rows
of
cells
from
the
cut
surface
will
form
new
cells
after
injury.
There
is
also
a
requirement
for
a
solid
substratum.
For
ex-
ample
in
a
liquid
medium
supplemented
with
0.5%
agar,
no
callus
induction
was
recorded.
This
phenom-
enon
supports
the
need
of
air-medium
interface
for
callus
formation
(Polne-Fuller
and
Gibor
1987).
The
condition
of
the
thallus
itself,
is
a
controlling
factor
for
successful
induction
of
callus.
The
differ-
ence
in
induction
rate
was
mainly
related
to
thallus
collection
time,
with
thalli
collected
during
winter
and
spring
showing
maximum
induction
rate.
This
correlates
with
the
life
of
the
annual
species
Meristo-
theca
papulosa
that
grows
from
autumn
to
spring
with
thalli
maturing
during
early
summer,
and
disap-
pearing
in
late
summer
(Shinmura
1974).
Maybe
the
decreased
or
absence
of
callus
induction
from
the
355
±
34
310
±
46
135
±
29
0
322
±
37
305
±
52
298
±
43
250
±
48
211
±
35
108
±
32
Concentration
(mg
L
1
)
Fig.
5.
Effect
of
indole-3-acetic
acid
(IAA)
and
6-benzylaminopurine
(BAP)
on
callus
induction
of
Meristotheca
papulosa
cultured
for
4
weeks
(n
=
20-24).
Error
bars
=
SD
f•
O.
C
r,
-N.
1
,7e'k;i2144,„,
'".,
/
,
(
Z-i
.
*
t
4
:
1
t'
'
L
'
'
ii
e
,
.
.
..
*
'''
1
';
•;r
,,I.:,,
•''C#:--,,.,
..
.-
,
.,.
7.‘
:7
Z
.
4:s.1'4
,1
:.
,,1s,...
-1
.'
-
:10,
:1
,5.
-
4
,
s
pia'
';'''
.
,
t
,;,..
,.:4
V
4
"4
-
1.4
,e
,
)
,
;
.'
y,..:
-
,
.
c:
.
1"-•
.-:-
;-
..,
'
-
J
.
,!:'
'
.--
,
"
Callus
induction
in
Meristotheca
59
Callus
size
Callus
induction
rate
500-
400-
0
Con.A
B
C
D
E
F
G
Carbon
compounds
Fig.
6.
Effect
of
carbon
compounds
(100
mm)
on
callus
in-
duction
of
Meristotheca
papulosa
cultured
for
4
weeks
(n
=
20-24).
Con.,
Control
(no
carbon);
A,
glycerol;
B,
mannitol;
C,
sorbitol;
D,
glucose,
E,
galactose;
F,
mannose;
G,
sucrose.
Error
bars
=
SD
thalli
collected
during
June
and
July
corresponds
to
the
functional
status
of
the
mature
thallus
cells.
The
effect
of
thallus
collection
time
on
callus
formation
were
also
reported
in
Laminaria
digitata
(Hudson)
Lamour.
and
L.
hyperborea
(Gunner)
Foslie
(Fries
1980)
and
Ecklonia
cava
Kjellman
(Kawashima
and
Tokuda
1990).
In
most
flowering
plants,
induction
of
callus,
growth
and
differentiation
are
controlled
by
plant
growth
regulators
(Flick
et
al.
1983).
Even
in
some
seaweeds
the
induction
of
callus
or
cell
division
may
be
controlled
by
auxin
or
cytokinin
individually
and
in
some
species
at
different
combinations
and
con-
centrations
(Bradley
and
Cheney
1990,
Dawes
and
Koch
1991,
Liu
and
Kloareg
1991,
Kaczyna
and
Megnet
1993).
The
effect
of
IAA
(auxin)
and
BAP
(cytokinin)
in-
dividually
or
in
combination
showed
no
obvious
ef-
fect
on
callus
induction
rate
while
IAA
in
combi-
nation
with
BAP
showed
an
enhancing
effect
on
cal-
lus
size.
We
have
also
used
plant
growth
regulators
to
try
to
induce
callus
from
thalli
with
a
low
induc-
tion
rate,
but
no
enhancing
effect
was
found
(data
not
shown).
These
results
suggest
that
plant
growth
regulators
cannot
influence
the
callus
inducing
ability
of
an
explant
but
can
enhance
the
growth
of
the
in-
duced
callus.
Reports
on
seaweed
callus
cultures
using
carbon
compounds
in
the
medium,
give
varying
results.
Saga
et
al.
(1982)
reported
an
enhancing
effect
of
callus
formation
and
growth
of
the
marine
brown
alga
Dictyosiphon
foeniculaceus
(Hudson)
Greville
on
the
addition
of
165
mm
mannitol
to
the medium.
Gusev
et
al.
(1987)
also
reported
the
exchange
of
58
mm
su-
crose
with
110
mm
mannitol
stimulated
callus
forma-
tion
in
explants
from
the
leaf
blades
of
Phyllophora
nervosa
Greville.
On
other
hand,
in
callus
formation
from
explants
of
Ecklonia
radiata
(C.
Agardh)
J.
Agardh
the
addition
of
mannitol
and
several
other
carbon
compounds
at
1
and
10
mm
concentrations
showed
no
obvious
enhancing
effect
(Lawlor
et
al.
1989).
Kaczyna
and
Megnet
(1993)
emphasized
the
growth
promoting
effect
of
glycerol
in
combination
with
phytohormones,
while
Robaina
et
al.
(1990)
ob-
served
glycerol
alone
can
enhance
callus
on
explants
from
Grateloupia
doryphora
(Montage)
Howe.
In
this
report
we
found
callus
induction
rate
and
size
were
greatly
affected
by
the
kinds
of
carbon
com-
300-
a
•—
u)
(1)
200-
=
Tts
"
0
100-
r
.
-80
0
-
-60
a)
0
-
4.0
g
_
20
Tti
C.)
,
c(*ts•-
Figs
7-8.
Fig.
7.
Shoot
formation
from
callus
mass
of
Meristotheca
papulosa
in
PES
liquid
medium
for
3
weeks.
Section
showing
differentiation
into
outer
cortical
and
inner
medullary
tissue,
S
=
shoot
(bar
=
100
pm).
Fig.
8.
Thallus
regenerated
by
shoot
growth
in
liquid
PES
medium
for
3
months
(bar
=
1
cm).
r-)
7
.
60
W.
Huang
and
Y.
Fujita
Table
III.
Effect
of
various
culture
conditions
and
media
on
shoot
formation
from
excised
callus
of
Meristotheca
papulosa.
Temp.
(°C)
Photon
flux
density
(µmol
photon
m
-2
s
-1
)
Liquid
media
Culture
Time
(weeks)
1
2
3
4
5
15
15
15
20
20
20
20
20
25
25
25
10
40
80
10
10
40
40
80
10
40
80
PES
PES
PES
PES
A
SP12NTA
PES
ASP12NTA
PES
PES
PES
PES
x
x
x
x
+
+
+
+
+
+
+
+
+
+
(+)
+
+
+
+
+
+
+
x
x
x
+:
Shoot
formation,
-:
No
shoot
formation,
x:
Callus
death,
(+):
Shoot
death.
pounds.
Sucrose
enhanced
callus
induction
rate
and
size.
While
glycerol,
sorbitol
and
mannitol
had
an
in-
hibitory
effect
on
callus
induction
rate
and
size.
Fries
(1984)
also
reported
inhibitory
effect
of
mannitol
and
glycerol
during
plantlet
induction
from
Fucus
spiralis
Linnaeus
rhizoids.
The
choice
of
sucrose
by
Meristo-
theca
papulosa
is
similar
to
some
flowering
plant
cal-
lus
(Street
1977),
but
so
far
there
is
no
clear
evidence
of
carbon
metabolism
in
seaweed
cell
cultures.
More
research
information
is
needed
on
the
carbon
utilis-
ation
in
cultures,
to
formulate
a
medium
for
tissue
culture
of
seaweeds.
The
callus
exposed
to
an
air-medium
interface
grows
irregularly,
but
in
liquid
medium
it
showed
a
regular
organogenesis
and
experienced
dedifferen-
tiation
into
shoot
and
regenerated
the
whole
thallus.
Dawes
and
Koch
(1991)
also
made
similar
obser-
vations.
That
callus
induction
and
growth
were
fav-
orable
at
low
photon
flux
density,
correlates
with
the
natural
habitat
of
Meristotheca
papulosa.
High
tern-
perature
and
light
favoured
shoot
formation.
Similar
results
were
obtained
in
some
species
of
the
Lami-
nariales
(Notoya
et
al.
1994).
On
the
contrary,
long
incubation
at
80
prmol
photons
m
-2
s
-1
showed
an
inhibitory
effect
on
shoot
growth.
Thus,
during
callus
dedifferentiation
a
short
time
exposure
to
high
light
and
temperature
has
a
stimulatory
effect
on
shoot
formation,
but
is
not
favourable
for
shoot
growth.
Acknowledgements
We
wish
to
thank
Prof.
C.
J.
Dawes
(Department
of
Biology,
University
of
South
Florida,
U.
S.
A.)
for
helpful
suggestions
with
the
manuscript
and
Mr
U.
S.
Rao
for
help
in
preparation
of
the
manuscript.
This
work
is
supported
in
part
by
a
grant-in-aid
for
Sci-
ence
Research
from
Ministry
of
Education,
Science
and
Culture
of
Japan.
Accepted
3
November
1996
References
Aguirre-Lipperheide,
M.,
F.
J.
Estrada-Rodriguez
and
L.
V.
Evans.
1995.
Facts,
problems,
and
needs
in
seaweed
tis-
sue
culture:
An
appraisal.
J.
Phycol.
31:
677-688.
Bradley,
P.
M.
and
D.
P.
Cheney.
1990.
Some
effects
of
plant
growth
regulators
on
tissue
culture
of
the
marine
red
alga
Agardhiella
subulata
(Gigartinales,
Rhodo-
phyta).
Hydrobiologia
2041205:
353-360.
Dawes,
C.
J.
and
E.
W.
Koch.
1991.
Branch,
micropropag-
ule
and
tissue
culture
of
the
red
algae
Eucheuma
denticul-
atuni
and
Kappaphycus
alvarezii
farmed
in
the
Philip-
pines.
J.
Appl.
Phycol.
3:
247-257.
Flick,
C.
E.,
D.
A.
Evans
and
W.
R.
Sharp.
1983.
Organo-
genesis.
In:
(D.
A.
Evans,
W.
R.
Sharp,
P.
V.
Ammirato
and
Y.
Yamada,
eds)
Handbook
of
Plant
Cell
Culture.
Macmillan
Publications,
NY.
pp.
13-82.
Fries,
L.
1980.
Axenic
cultures
from
sporophytes
of
Lanzi-
naria
digitata
and
Laminaria
hyperborea
(Phaeophyta).
J.
Phycol.
16:
475-477.
Fries,
L.
1984.
Induction
of
plantlets
in
axenically
culti-
vated
rhizoids
of
Fucus
spiralis.
Can.
J.
Bot.
62:
1616-
1620.
Gusev,
M.
V.,
A.
H.
Tambiev,
N.
N.
Kirikova,
N.
N.
Shely-
astina
and
R.
R.
Aslanyan.
1987.
Callus
formation
in
seven
species
of
agarophyte
marine
algae.
Mar.
Biol.
95:
593-597.
Kaczyna,
F.
and
R.
Megnet.
1993.
The
effects
of
glycerol
and
plant
growth
regulators
on
Gracilaria
verrucosa
(Gi-
gartinales,
Rhodophyceae).
Hydrobiologia
268:
57-64.
Kawashima,
Y.
and
H.
Tokuda.
1990.
Callus
formation
in
Ecklonia
cava
Kjellnian
(Laminariales,
Phaeophyta).
Hydrobiologia
2041205:
375-380.
Lawlor,
H.
J.,
J.
A.
McComb
and
M.
A.
Borowitzka.
1989.
Tissue
culture
of
Ecklonia
radiata
(Phaeophyceae,
Lami-
nariales):
effects
on
growth
of
light,
organic
carbon
source
and
vitamins.
J.
Appl.
Phycol.
1:
105-112.
Callus
induction
in
Meristotheca
61
Liu,
X-W.
and
B.
Kloareg.
1991.
Tissue
culture
of
Porphyra
umbilicalis
(Bangiales,
Rhodophyta).
I.
The
effects
of
plant
hormones
on
callus
induction
from
tissue
explants.
C.
R.
Acad.
Sci.
Paris.
312:
517-522.
Notoya,
M.,
M.
Nagashima
and
Y.
Aruga.
1994.
Influence
of
light
intensity
and
temperature
on
callus
development
in
young
sporophytes
of
three
species
of
Laminariales
(Phaeophyta).
J.
Mar
Biotech.
2:
15-18.
Oppenheimer,
C.
H.
and
C.
E.
ZoBell.
1952.
The
growth
and
viability
of
sixty-three
species
of
marine
bacteria
as
influenced
by
hydrostatic
pressure.
J.
Mar.
Res.
11:
10-18.
Polne-Fuller,
M.
and
A.
Gibor.
1984.*
Developmental
stud-
ies
in
Porphyra.
I.
Blade
differentiation
in
Porphyra
per-
forata
as
expressed
by
morphology,
enzymatic
digestion,
and
protoplast
regeneration.
J.
PhycoL
20:
609-619.
Polne-Fuller,
M.
and
A.
Gibor.
1987.
Calluses
and
callus-
like
growth
in
seaweeds:
Induction
and
culture.
Hydro-
biologia
1511152:
131-138.
Provasoli,
L.
1963.
Growing
marine
seaweeds.
Proc.
Int.
Seaweed
Synip.
4:
9-17.
Provasoli,
1968.
Media
and
prospects
for
the
cultivation
of
marine
algae.
In:
(A.
Watanabe
and
A.
Hattori,
eds)
Cultures
and
Collections
of
Algae.
Jpn.
Soc.
Plant
Phy-
siol.
Hakone.
pp.
63-75.
Robaina,
R.
R.,
P.
Garcia,
G.
Garcia-Reina
and
A.
Luque.
1990.
Morphogenetic
effect
of
glycerol
on
tissue
cultures
of
the
red
seaweed
Grateloupia
doryphora.
J.
Appl.
Phy-
col.
2:
137-143.
Saga,
N.,
T.
Motomura
and
Y.
Sakai.
1982.
Induction
of
callus
from
the
marine
brown
alga
Dictyosiphon
foenicu-
laceus.
Plant
Cell
Physiol.
23:
727-730.
Shinmura,
I.
1974.
The
seasonal
variation
of
growth
and
the
period
of
spore-liberation
in
Meristotheca
papulosa.
Bull.
Jpn.
Soc.
Phycol.
22:
124-129
(in
Japanese).
Street,
H.
E.
1977.
Plant
Tissue
and
Cell
Culture.
Blackwell
Science
Publications,
Oxford.
614
pp.