Plant regeneration from leaf explants of Adromischus nussbaumerianus Poelln. and Echeveria laui Moran et Meyran


Wojciechowicz, M., K.; Kazimierczak-Grygiel, E.; Olejnik, S.; Zenkteler, E.

Biological Bulletin of Poznan 38(1): 71-78

2001


Leaf fragments of Adromischus nussbaumerianus and Echeveria laui were placed on the Murashige and Skoog (MS) medium with various combinations of growth regulators. The best growth of callus was observed on the MS medium supplemented with 4 mg l-1 6-benzylaminopurine and 2 mg l-1 2,4-dichlorophenoxyacetic acid. The most effective leaf and root induction was achieved for of A. nussbaumerianus callus on the MS medium with macronutrient content reduced four times, with the addition of 1 mg l-1 zeatin, 1 mg l-1 beta-indoleacetic acid and 1% sucrose. On the other hand, the most effective for E. laui callus was the half-strength MS medium supplemented with 6 mg l-1 zeatin or 2 mg l-1 zeatin and 1 mg l-1 beta-indoleacetic acid for leaf and root development, respectively. Histological analysis of explants cultured in vitro showed that cell divisions leading to callus formation were initiated in the subepidermal layer of the leaf. Subsequent cell divisions were observed in deeper layers of the mesophyll. In A. nussbaumerianus cell divisions were also noticed in some epidermal cells. Additionally, in E. laui multinucleate cells were noticed in meristematic regions of callus. Transverse sections of callus showed that the first leaf primordia were initiated in callus after 2 and 8 weeks of culture in A. nussbaumerianus and E. laui, respectively. This experiment resulted in full regeneration of both species. After acclimation, young plants were planted in the soil.

BIOLOGICAL
BULLETIN
OF
POZNAN
38(I):
71-78,2001
(PRINTED
IN
POLAND)
SPECIAL
COMMEMORATIVE
VOLUME
DEDICATED
TO
PROFESSOR
DR.
MACIEJ
ZENKTELER
ON
THE
OCCASION
OF
HIS
SEVENTIETH
BIRTHDAY
PLANT
REGENERATION
FROM
LEAF
EXPLANTS
OF
ADROMISCHUS
NUSSBAUMERIANUS
POELLN.
AND
ECHEVERIA
LAUI
MORAN
ET
MEYRAN
MARIA
K.
WOJC1ECHOWICZ',
EWA
KAZIMIERCZAK-GRYGIEL
2
,
SYLWIA
OLEJNIK'
and
ELZBIETA
ZENKTELER'
'
Department
of
General
Botany,
Institute
of
Experimental
Biology,
Adam
Mickiewicz
University,
al.
NiepodlegloSci
14,
61-713
Poznan,
Poland
2
Botanical
Garden,
Adam
Mickiewicz
University,
Dabrowskiego
165,
60-594
Poznan,
Poland
(Received
on
15th
December
2000.•
Accepted
on
4th
March
2001)
A
b
s
t
r
a
c
t:
Leaf
fragments
of
Adromischus
nussbaumerianus
and
Echeveria
/aui
were
placed
on
the
Murashige
&
Skoog
(MS)
medium
with
various
combinations
of
growth
regulators.
The
best
growth
of
callus
was
observed
on
the
MS
medium
supplemented
with
4
mg
1
-
'
6-benzylaminopurine
and
2
mg
1
-
'
2,4-dichlorophenoxyacetic
acid.
The
most
effective
leaf
and
root
induction
was
achieved
for
of
A.
nussbaumerianus
callus
on
the
MS
medium with
macronutrient
content
reduced
four
times,
with
the
addition
of
1
mg
1
-
'
zeatin,
1
mg
1
-
'
0-indoleacetic
acid
and
I%
sucrose.
On
the
other
hand,
the
most
effective
for
E.
laui
callus
was
the
half-strength
MS
medium
supplemented
with
6
mg
zeatin
or
2
mg
1
-1
zeatin
and
I
mg
1
-
'13-indoleacetic
acid
for
leaf
and
root
development,
respectively.
Histological
analysis
of
explants
cultured
in
vitro
showed
that
cell
divisions
leading
to
callus
formation
were
initiated
in
the
subepidermal
layer
of
the
leaf.
Subsequent
cell
divisions
were
observed
in
deeper
layers
of
the
mesophyll.
In
A.
nussbaumerianus
cell
divisions
were
also
noticed
in
some
epidermal
cells.
Additionally,
in
E.
laui
multinucleate
cells
were
noticed
in
meristematic
regions
of
callus.
Transverse
sections
of
callus
showed
that
the
first
leaf
primordia
were
initiated
in
callus
after
2
and
8
weeks
of
culture
in
A.
nussbaumerianus
and
E.
laui,
respectively.
This
experiment
resulted
in
full
regeneration
of
both
species.
After
acclimation,
young
plants'were
planted
in
the
soil.
Key
words:
Adrotnischus
nussbaumerianus,
Echeveria
laui,
in
vitro,
leaves,
propagation,
histology
INTRODUCTION
Succulents
are
usually
propagated
from
seed
or
cuttings
(KRut.ix
1980).
These
conventional
methods
of
succulent
propagation
are
not
always
satisfactory
(STUPPv
&
NAGI
1992).
Growing
from
seed
is
the
best
if
the
plant
produces
lots
of
seed
and
72
M
K.
WojciechoMez,
E.
Ka...iinierczak-Grygiel,
S.
Olejnik
and
E.
Zenkteler
seedlings
grow
quickly.
Propagation
from
cuttings
(of
stems,
roots,
leaves
or
branches)
is
a
slower
method
(KRUI
IK
1980).
Outside
professional
collections
of
succulent
plants,
the
rooting
and
establishment
of
cuttings
can
be
difficult,
since
many
species
are
very
susceptible
to
rots
caused
by
bacteria
and
fungi
(GRAT
-
roN
&
FAY
1990),
Hitherto,
it
has
been
shown
that
propagation
of
succulents
by
tissue
culture
is
feasible
(MAU-
SETH
1977,
JOHNSONS
&
EMINIO
1979.
KRut.ix
1980,
STARLING
&
DODDS
1983).
The
plants
of
A.
nussbaunterianus
do
not
produce
seed
in
greenhouse
culti-
vation
so
they
are
propagated
from
leaf
and
stern
cuttings
(PILBEAM
&
et.
al
1998).
The
propagation
of
E.
laui
species
in
collections
is
difficult:
the
plants
rarely
pro-
duce
seed,
do
not
produce
offsets,
only
a
small
percentage
of
leaf
cuttings
take
root,
and
seedlings
are
very
sensitive
to
fungal
infection
(RINA
&
SUBIK
1992).
In
this
study
we
investigated
the
possibility
of
regeneration
in
Adromischus
nussbaumeranus
and
Echeveria
law;
two
interesting
species
of
crassulaceaean
suc-
culents,
from
leaf
fragments
using
in
vitro
techniques.
MATERIALS
AND
METHODS
Plant
material
Leaves
of
two
species
Adromischus
nussbaumerianus
von
Poellnitz,
1936
and
Echeveria
laui
Moran
et
Meyran,
1976
used
in
this
study
came
from
the
collection
of
the
Botanical
Garden
of
the
Adam
Mickiewicz
University
Poznan,
Poland.
A.
nussbaumerianus
grows
in
semi-arid
areas
of
South
Africa.
Like
other
spe-
cies
of
the
genus
Adromischus,
it
is
attractive
because
of
the
beautiful
shape
and
markings
of
its
leaves.
The
leaves
are
4-4.5
cm
long,
broadly
triangular,
wider
at
the
apex,
frequently
with
an
undulating
apical
margin.
In
cultivation,
the
species
need
warm
and
sunny
places
and
slightly
acidic
soil
(PILBEAM
et
al.
1998).
E.
laui,
an
endemic
and
endangered
species,
grows
in
the
Tomellin
Canyon
in
Oaxaca
(central
Mexico)
at
an
altitude
of
500-600
m.
The
plant
forms
a
beautiful
compact
rosette
of
succulent,
silvery-blue,
glaucous
leaves,
about
20
cm
in
diameter.
In
cultivation,
it
requires
high
temperatures
throughout
the
year,
with
winter
tem-
peratures
above
14°C,
slightly
acidic
soil,
and
sparse
watering
(RmA
&
SUBIK
1992).
In
vitro
culture
Leaves
of
different
ages
isolated
from
plants
were
surface-sterilized
in
70%
ethanol
for
1
min.,
then
in
0.5%
(v/v)
sodium
hypochloride
for
5
min.
After
three
times
rinsing
in
sterile
distilled
water,
leaves
were
cut
into
pieces
(0.25
cm'
in
area).
The
initial
explants
divided
into
distal
and
middle
parts
of
leaves
were
cultured
on
the
MS
medium
(MGRAsniGE
&
SKOOG
1962),
supplemented
with
auxins
and
cyto-
kinins'
at
various
concentrations
and
combinations
(Table
1).
The
cultures
were
incubated
in
a
growth
chamber
under
continuous
fluorescent
light
(40
µmot
m
s
-
')
at
24°C
and
70-80%
relative
humidity.
'
Abbreviations:
IAA
-
0-indole
acetic
acid;
2,4-D
-
2,4-dichlorophenoxyacetic
acid;
NAA
-
a-naph-
thalene
acetic
acid;
BAP
-
6-benzylaminopurine;
TDZ
-
I-Phenyl-3-(1,2,3-thiadiazol-5-yOurea
=
thidiazuran;
ZEA
(6[441ydroxy-3methylbut-2-enylamino]purine)
=
zeatin.
IN
VITRO
REGENERATION
OF
ADROMISCHUS
AND
ECHEVERIA
73
Table
1.
Modifications
of
the
MS
medium
used
in
this
study
MS
medium
variant
Callus
induction
C-1
BAP
2
mg
r'
+
NAA
0.5
mg
r'
C-2
BAP
4
mg
I'
l
+
NAA
I
mg
rl
C-3
BAP
4
mg
1'
1
+
NAA
2
mg
I
.
'
C-4
BAP
4
mg
1
-
'
+
2.4-D
0.5
mg
C-5
BAP
4
mg
r'
+
2,4-D
2
mg
I'
l
C-6
TDZ
0.5
mg
1"'
+
IAA
I
mg
1
-
'
C-7
TDZ
1
mg
1'
1
+
2,4-D
0.5
mg
I"'
MS
medium
variant
Shoot
induction
K-1
ZEA
1
mg
I
-I
K-2
ZEA
2
mg
r'
K-3
ZEA
4
mg1
-1
K-4
1
/
2
macro
+
ZEA
1
mg
r'
K-5
+
ZEA
4
mg
r'
K-6
+ZEA
6
mg
1"'
K-7
+
ZEA
I
mg
1
4
+
sucrose
1%
K-8
1
/
4
macro
+
ZEA
I
mg
r'
K-9
+
ZEA
I
mg
1
-1
+
sucrose
2%
K-10
+
ZEA
1
mg
r'+
sucrose
1%
K-1
I
+
ZEA
2
mg
r'
+
sucrose
2%
K-12
+
ZEA
6
mg
1'
1
K-13
'/
3
macro
+
BAP
0.2
mg
I
-1
+
IAA
lmg
r'
MS
medium
variant
Root
induction
R-1
/2
macro
+
ZEA
2
mg
1'
1
+
IAA
1
mg
r'
R-2
1
/
4
macro
+
ZEA
1
mg
rl
+
IAA
I
mg
r'
R-3
+
ZEA
l
mg
t'
+
sucrose
1%
+
IAA
1
mg
I
-I
R-4
+
ZEA
2
mg
r'+
IAA
I
trig
r'
R-5
1
/
2
macro
+
IAA
I
mg
r'
R-6
1
/
4
macro
+
IAA
I
mg
r'
R-7
1
/
2
macro
R-8
1
/
4
macro
After
6
weeks,
the
formed
callus
was
subcultured
on
several
variants
of
MS
medium
for
shoot
induction
(K
1-13
in
Table
1).
Explants
that
formed
leaf
primor-
dia
were
transplanted
onto
rooting
media
(R
1-8
in
Table
1).
In
order
to
stimulate
root
elongation,
the
plantlets
were
put
to
Erlenmeyer
flasks
with
the
half-strength
liquid
MS
medium
and
perlite,
or
perlite
and
filter
paper,
or
filter
paper
alone.
The
rooting
cultures
in
each
species
were
divided
into
two
groups:
50%
were
kept
under
continuous
fluorescent light
(40
µmot
m
-
'
s
-
'),
and
the
other
50%
were
kept
under
a
16h/8h
photoperiod.
For
acclimatisation,
all
plants
were
transferred
to
sterile
soil
mix
of
leaf
mould,
sand
and
clay
(4
parts
:
2
parts
:
1
part)
and
kept
in
glass
jars.
Three
months
later
the
plants
were
transplanted
to
pots
containing
non-sterile
soil.
74
M.K.
Wojciechowicz,
E.
Kazimierczak-Grygiel,
S.
Olejnik
and
E.
Zenkteler
Histology
Material
for
light
microscopic
examination
was
fixed
in
mixture
of
acetic
acid-
alcohol.
Paraffin
microtome
sections
(12
mm
thick)
were
stained
with
safranin
and
counterstained
with
fast
green
(JENsEN
1962).
Photographs
of
these
sections
were
taken
with
an
Amplival
Optiphot
Microscope
(Carl
Zeiss,
Jena,
Germany).
For
transmis-
sion
electron
microscopy,
the
samples
were
fixed
with
4%
glutaraldehyde
and
4%
paraformaldehyde
(1:1)
for
2
h
and
post-fixed
with
2%
0s0
4
.
The
fixed
material
was
counterstained
for
12
h
with
2%
uranyl
acetate.
Samples
wcrc
dehydrated
using
an
acetone
series
and
embedded
in
Spurr
resin
(Smut
1969).
Ultra-thin
sections
were
cut
using
an
LKB
ultramicrotome
fitted
with
a
glass
knife,
and
examined
with
a
JEM
1200
EX
II,
JE
DL.
a
RESULTS
In
vitro
culture
The
effectiveness
of
the
individual
MS
medium
variants
on
callus
tissue
for-
mation,
shoot
induction
and
rooting
is
presented
in
Figures
1,
2
and
3,
respectively.
Callus
fonnation
on
leaf
fragments
of
A.
nussbaumerianus
was
observed
after
2
weeks.
However,
in
E.
laui
callus
appeared
after
3
weeks.
In
both
cases
the
earli-
est
callus
was
formed
on
the
C-5
medium,
on
the
youngest
leaves.
The
callus
tissue
of
A.
nussbaumerianus
was
compact,
hard
and
green.
First
the
callus
of
E.
laui
was
friable,
glass-like,
and
light
yellow,
but
turned
green
after
next
two
weeks
in
culture.
In
both
species
the
strongest
callus
proliferation
was
observed
on
the
C-5
medium.
The
strongest
formation
of
callus
tissue
was
noted
on
the
middle
parts
of
the
leaf.
After
3
weeks
of
culture,
leaf
primordia
developed
on
the
callus
of
E.
laui
on
the
media:
K-5,
K-6,
K-11
and
K-12.
Four
weeks
later
small
leaves
were
observed
(Fig.
4).
The
first
leaf
primordia
of
A.
nussbaumerianus
were
produced
2
weeks
after
90
50
ro
1
00
ZD'
50
C
0)
40
30
20
10
0
1
C-7
C1
C•3
C-4
C-6
Variant
of
MS
medium
ClAdromischus
nussbaumerianus
m
Echeveria
laui
Fig.
1.
Callus
tissue
formation
on
explants
after
6
weeks
of
cultivation;
an
average
value
of
16
explants
per
each
medium
was
calculated.
EN
VITRO
REGENERATION
OF
ADROMISCHUS
AND
ECHEVERIA
75
210
70
0,
so
g
g
40
2
30
0.
X
'1
20
10
1I
K-1
K
2
K-3
K-4
K-5
K-6
K
7
K-8
K-9
K-10
K-11
K-12
K-13
Variant
of
MS
medium
0
Adromischus
nussbaumerianus
•Echeveria
laui
Fig.
2.
Effect
of
medium
composition
on
shoot
initiation;
an
average
value
of
II
explants
per
each
medium
was
calculated.
!-:
R-1
R-2
R
3
R-4
R-5
R-6
R-7
R-8
Variant
of
MS
medium
OAdromischus
nussbaumerianus
l
Echeveria
laui
Fig.
3.
Effect
of
medium
composition
on
rooting
explants;
an
average
value
of
11
explants
per
each
medium
was
calculated,
th6
transfer
onto
the
regeneration
medium
K-12
(Fig.
5).
The
highest
percentage
of
regenerated
shoots
for
both
species
was
observed
on
the
K-12
medium,
where
73%
and
70%
of
A.
nussbaumerianus
and
E.
laui
explants
formed
buds,
respectively.
The
effectiveness
of
medium
variants
used
for
primordium
induction
in
both
species
is
shown
in
the
Figure
2.
Root
formation
on
A.
nussbaumerianus
explants
was
observed
after
3
months
on
the
R-1
medium
(Fig.
7).
In
E.
laui
first
roots
were
formed
on
the
R-7
medium
4-
5
months
later
(Fig.
6).
These
cultures
were
kept
under
a
16/8
h
photoperiod.
Under
76
M.K.
Wojciechowicz.
E.
Kazimicrczak-Grygiel,
S.
Olejnik
and
E.
Zenktcler
continuous
illumination,
rooting
was
delayed
in
both
species
as
in
these
light
condi-
tions
the
explants
formed
roots
4
weeks
or
4
months
later
than
in
photoperiod
re-
gime
for
E.
/aui
and
A.
nussbaumerianus,
respectively.
The
highest
percentage
of
rooting
explants
of
E.
laui
was
observed
on
the
R-1
medium
(Fig.
3).
In
contrast,
the
most
intensive
rooting
of
explants
of
A.
miss-
baumerianus
took
place
on
the
R-3
medium.
After
18
weeks,
30%
of
explants
formed
roots
(5-10
cm;
Fig.
6).
The
best
subsequent
growth
of
roots
in
both
species
was
observed
in
Erlenmeyer
flasks
with
perlite
and
pieces
of
filter
paper
submerged
in
the
half-strength
MS
medium.
Thirty-six
percent
of
plantlets
of
E.
laui
and
41%
of
A.
nussbaumerianus
were
successfully
acclimated
and
are
now
grown
in
the
greenhouse
(Figs
8,
9).
Anatomical
analysis
In
control,
transverse
sections
of
A.
nussbaumerianus
and
B
laui
leaves
showed
a
regular
arrangement
of
epidermal
cells.
The
tightly
packed
epidermal
cells
were
surrounded
by
cellulose
walls
with
a
clearly
visible
cuticle.
Stomata
were
present
both
on
adaxial
and
on
abaxial
surfaces.
Thin-walled
mesophyll
cells,
approximate-
ly
oval
in
shape,
were
found
right
under
the
single
epidermal
layer
(Fig.
10).
The
cells
were
considerably
larger
than
epidermal
cells,
and
small
intercellular
spaces
occurred
(Fig.
10).
No
differentiation
into
palisade
or
spongy
mesophyll
could
be
observed
(Fig.
10).
Additionally,
cells
containing
mucous
material
were
noticed
in
A.
nussbaumerianus
under
the
epidermis
(Fig.
11).
Collateral
vascular
bundles
were
found
in
the
mesophyll
(Fig.
12).
As
revealed
by
transmission
electron
microscopy,
mesophyll
cells
contained
mucous
and
lipid
bodies
(Figs
13,
14).
The
peripheral
band
of
cytoplasm
between
the
plasmalemma
and
the
tonoplast
contained
plastids
with
numerous
plastoglobuli
and
large
starch
grains
(Fig.
15).
In
A.
nussbaumerianus,
after
one
week
of
culture,
mesophyll
cells
underlying
the
epidermis
shrank
and
assumed
an
oblong
shape
(Fig.
19).
En
E.
/aui
similar
changes
of
shape
of
subepidermal
parenchyma
cells
were
observed
after
three
weeks
of
cul-
ture.
In
contrast,
after
three
weeks
of
culture
of
A.
nussbaumerianus
leaf
fragments,
numerous
meristematic
centres
were
already
observed
in
the
mesophyll.
Initially
a
meristematic
centre
was
formed
in
the
subepidermal
layer
of
leaves
and
later
in
deeper
layers
of
mesophyll
cells.
The
meristematic
centres
in
the
callus
of
E.
laui
were
formed
after
four
weeks
of
subculture.
The
cells
of
the
meristematic
centres
contained
nu-
merous
amyloplasts
(Fig.
16)
and
a
dense
cytoplasm
(Fig.
17),
and
formed
a
unise-
Hate
layer.
Under
a
transmission
electron
microscope,
the
cells
in
the
meristematic
centre
had
a
large
nucleus
with
one
nucleolus
(Fig.
18).
The
anatomical
sections
of
in
vitro
cultivated
leaf
fragments
revealed
that
in
A.
nussbaumerianus
the
first
leaf
primordia
were
present
after
2
weeks,
and
in
E.
laui,
after
8
weeks
(Fig.
20).
-->
Fig.
4.
Regenerated
leaves
of
E.
laui
formed
on
the
K-8
medium
after
18
weeks
Fig.
5.
Leaf
primordia
of
A.
nussbaumerianus
on
the
K-2
medium
after
3
weeks
Fig.
6.
Plantlets
of
A.
nussbaumerianus
on
the
R-7
medium
after
16
weeks
Fig.
7.
Plantlets
of
E.
laui
on
the
R-10
medium
after
16
weeks
Figs
8,
9.
Pot
culture
of
E.
/aui
(8)
and
A.
nussbaumerianus
(9)
regenerants
growing
in
the
greenhouse
6
7
-
Jl
fr.
11
8..
12
Fig.
10.
Transverse
section
of
the
leaf
explant.
Epidermis
(arrow)
and
mesophyll
tissue
Fig.
11.
Single
layer
of
cells
with
mucous
idiob-
lasts
beneath
the
epidermis
Fig.
12.
A
collateral
bundle
with
cambium
in
a
leaf;
scale
bars
=
100
gm
Figs
10-12
A.
nussbaun2ennnus
Fig.
13.
Numerous
lipid
bodies
in
a
mesophyll
cell
of
leaf
explant
Fig.
14.
Mucous
material
in
a
mesophyll
cell
of
leaf
explant
Fig.
15.
Plastids
with
large
starch
grains
and
numerous
plastoglobuli
in
a
mesophyll
cell
of
an
explant
leaf
Fig.
16.
Amyloplasts
with
large
starch
grains
in
a
mesophyll
cell
of
explant
leaves
Fig.
17.
A
meristematic
cell
in
a
leaf
after
4
weeks
of
culture
Fig.
18.
A
cell
ot'meristematic
region
from
a
leaf
after
4
weeks
of
culture
CH
chloroplast,
CHr
chromatin,
CW
cell
wall,
ER
endoplasmic
reticulum,
LB
lipid
body,
M
mitochondrion,
Mu
mucus
material,
N
nucleus,
NU
nucleolus,
PL
plastid,
S
starch,
V
vacuole;
scale
bars
=
4µm;
Figs
13-18
E.
laui
4
1111
r
4.
,
4
k..„
5
.
41
4
M
i
r
.„,41•
1,
V
.
7.7
C)
\k
-
\
fi
t
19
-
.
21
/'
4
!..
••-
.
..
.
eY
1
4
;
'
/
1
";
.2;
,
4
c.
22
Fig.
19.
Oblong
cells
under
the
epidermis
of
E
law
after
one
week
of
culture
Fig.
20.
Leaf
primordium
formed
after
4
weeks
of
A.
nussbaumerianus
culture
on
the
K-12
medium
Fig.
21.
Meristematic
centres
in
epidermis
of
A.
nussbaumerianus•
after
3
weeks
of
culture
on
the
C-5
medium
Fig.
22.
Multinucleated
cell
in
the
meristematic
centres
of
E.
laui
after
5
weeks
on
the
C-3
medium;
scale
bars
=
100
j..im
IN
VITRO
REGENERATION
OF
ADROMISCHUS
A
ND
ECHEVERIA
77
Additionally,
in
A.
nussbaumerianus
meristematic
centres
were
located
also
among
epidermal
cells
(Fig.
21).
In
ineristematic
centres
of
E.
laui
multinucleated
cells
were
frequently
observed
(Fig.
22).
DISCUSSION
Members
of
the
family
Crassulaceae
have
been
widely
used
in
the
develop-
ment
of
tissue
culture
techniques
and
in
biochemical
and
physiological
experiments
(FAY
&
GRATTON
1992).
In
the
available
literature,
references
to
only
the
following
few
species
of
the
genus
Echeveria
and
Adromischus
propagated
by
in
vitro
tech-
niques
have
been
found:
E.
elegans(Rmu
&
MANN
1970,
1971)
E.
ciliata,
E.
sanchez-
rnejoradae
(FAY
&
GRArroN
1992)
and
A.
marianea
(FAv
&
GRATroN
1992).
In
the
case
of
A.
marianea,
rooting
of
shoots
has
not
been
achieved.
In
our
study,
we
ob-
tained
fully
developed
plants
of
E.
laui
and
A.
nussbaumerianus
by
tissue
culture.
Nearly
40%
of
these
plants
adapted
to
greenhouse
conditions.
The
major
factor
influencing
the
outcome
of
an
in
vitro
propagation
experi-
ment
is
the
choice
of
explants.
The
regeneration
ability
of
each
organ
varies
with
age;
also
different
tissues
of
the
same
organ
have
various
degrees
of
developmental
potential
(MLODZIANOWSKI
1984).
The
earliest
study
of
regeneration
of
E.
elegans
(R,uu
&
HINES
1973)
showed
that
explant
age
affected
markedly
the
regeneration
ability.
Young
leaves
growing
near
the
apex
had
higher
regeneration
ability
than
the
older
ones.
The
same
paper
(RAJU
&
HINES
1973)
reported
differences
between
distal
and
proximal
parts
of
leave,
as
the
leaf
base
had
the
highest
regeneration
ability.
Results
of
our
study
confirmed
that
young
leaves
of
E.
laui
and
A.
nussbaumerianus
regen-
erated
better
than
did
the
older
ones.
The
fastest
and
the
most
intensive
callus
regen-
eration
was
observed
in
the
middle
part
of
the
leaf.
Callus
proliferation
on
the
distal
part
of
the
leaf
was
slightly
slower.
In
our
study
the
leaf
bases
were
not
put
on
the
medium
because
of
damage
during
excision
and
sterilization.
The
most
frequently
used
medium
for
succulent
propagation
by
tissue
culture
had
been
the
MS
medium
(KRULIK
1980,
STARLING
&
DODDS
1983,
GRATTON
&
FAY
1990,
FAY
&
GRATTON
1992).
On
the
basis
of
this
trend,
in
our
experiment
we
used
the
MS
medium
supplemented
with
cytokinins
and
auxins.
In
studies
of
succulent
plant
propagation,
BAP
has
been
the
most
frequently
applied
cytokinin,
but
zeatin
and
kinetin
have
also
been
used
with
supplements
of
the
following
auxins:
IAA,
NAA,
and
2,4-D
(STARLING
&
DODDS
1983).
In
our
study,
the
optimal
medium
for
callus
induction
was
MS
with
4
mg
1
-
'
BAP
and
0.5
mg
2,4-D
in
A.
nussbaumerianus.
In
E.
laui,
the
same
induction
was
achieved
with
4
mg
l
-
'
BAP
and
2
mg
2,4-D.
For
callus
induction,
2,4-D
is
commonly
used,
and
in
some
cases
it
can
induce
somatic
embryogenesis
in
the
resulting
cultures
(FAY
&
GRArroN
1992).
In
this
work,
a
histological
analysis
of
the
cultured
leaf
fragments
of
E.
laui
and
A.
nussbaume-
rianus
indicated
that
plant
regeneration
occurred
by
indirect
organogenesis.
Somatic
embryos
were
not
observed.
To
stimulate
rooting
in
previous
experiments,
the
half-strength
MS
was
used,
or
the
half-strength
MS
with
auxins
IAA
or
NAA
added
at
concentrations
ranging
from
0.01
mg
I
-
'
to
1
mg
1
-
'
(FAY
&
GRArroN
1992).
In
this
study,
the
highest
percent-
age
of
rooting
in
A.
nussbaumerianus
was
achieved
on
the
MS
medium
with
macronutrients
and
no
growth
regulators.
78
M.K.
Wojciechowicz,
E.
Kazimierezak-Grygiel,
S.
(=Melnik
and
E.
Zenkteler
Our
study
enabled
the
development
of
efficient
methods
of
in
vitro
regener-
ation
of
whole
plants
from
leaf
fragments
of
A.
nussbaumerianus
and
E
lard.
The
results
can
be
used
as
guidelines
for
application
of
tissue
culture
techniques
for
propa-
gation
of
succulents.
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M.
F.,
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J.,
1992.
Tissue
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Bradleya
10:
33-48.
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J.,
FAY
M.
F.,
1990.
Vegetative
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of
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J.
W.,
WALKER
J.
M.,
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W.
A.,
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J.
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R.,
1979.
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G.,
1980.
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F.,
1984.
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J.
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1977.
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E.,
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H.
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