Micropropagation of self-heading Philodendron via direct shoot regeneration

Abstract

The present study describes a direct shoot regeneration-based micropropagation procedure for the self-heading cultivars of Philodendron. Three types of explant (i.e. leaf lamina, petiole, stem nodal segment) were screened for their shoot induction potential following a three months treatment with 0.5 mg l−1 of either 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ) or both. Results indicated that the leaf laminas were poor candidates for shoot induction whereas the petioles showed potential for adventitious shoot production at frequencies of 2.8–11.1% in two of the cultivars tested. Stem nodal segments were the most responsive among the three as shoots formed directly following the TDZ treatment at frequencies of 16.7–41.7% depending on the cultivar. When comparing the effectiveness of different cytokinins to induce shoot proliferation on stem nodal segments, it was found that the 0.5 and 1 mg l−1 of kinetin (Kn) and 6-benzyladenine (BA) treatments resulted in higher shoot formation percentages compared to the 0.5 and 1 mg l−1 of TDZ treatments in two of the three cultivars. Furthermore, more shoots were produced on BA than on Kn-supplemented media in all the three cultivars. Shoots derived from the 0.5 mg l−1 of BA treatment can be induced to root following one month incubation with 0.1–1 mg l−1 of indole-3-butyric-acid (IBA). The rooted shoots showed 100% survival after acclimatization in the greenhouse. The procedure reported in the present study can assist in the large-scale multiplication of elite self-heading cultivars of Philodendron in the future.

Full text

Scientia
Horticulturae
141
(2012)
23–29
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Horticulturae
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l
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Micropropagation
of
self-heading
Philodendron
via
direct
shoot
regeneration
F.C.
Chena, C.Y.
Wangb,
J.Y.
Fangc,∗
aDepartment
of
Plant
Industry,
National
Pingtung
University
of
Science
and
Technology,
No.1
Shueh
Fu
Road,
Neipu,
Pingtung
91201,
Taiwan
bSouthern
Taiwan
Service
Center,
Food
Industry
Research
and
Development
Institute,
No.31
Gongye
2nd
Road,
Annan
District,
Tainan
70955,
Taiwan
cDepartment
of
Tropical
Agriculture
and
International
Cooperation,
National
Pingtung
University
of
Science
and
Technology,
No.1
Shueh
Fu
Road,
Neipu,
Pingtung
91201,
Taiwan
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
16
February
2012
Received
in
revised
form
6
April
2012
Accepted
10
April
2012
Keywords:
Philodendron
Stem
nodal
segment
Leaf
lamina
Petiole
Direct
shoot
regeneration
Plant
growth
regulator
a
b
s
t
r
a
c
t
The
present
study
describes
a
direct
shoot
regeneration-based
micropropagation
procedure
for
the
self-
heading
cultivars
of
Philodendron.
Three
types
of
explant
(i.e.
leaf
lamina,
petiole,
stem
nodal
segment)
were
screened
for
their
shoot
induction
potential
following
a
three
months
treatment
with
0.5
mg
l−1of
either
2,4-dichlorophenoxyacetic
acid
(2,4-D),
thidiazuron
(TDZ)
or
both.
Results
indicated
that
the
leaf
laminas
were
poor
candidates
for
shoot
induction
whereas
the
petioles
showed
potential
for
adventitious
shoot
production
at
frequencies
of
2.8–11.1%
in
two
of
the
cultivars
tested.
Stem
nodal
segments
were
the
most
responsive
among
the
three
as
shoots
formed
directly
following
the
TDZ
treatment
at
frequencies
of
16.7–41.7%
depending
on
the
cultivar.
When
comparing
the
effectiveness
of
different
cytokinins
to
induce
shoot
proliferation
on
stem
nodal
segments,
it
was
found
that
the
0.5
and
1
mg
l−1of
kinetin
(Kn)
and
6-benzyladenine
(BA)
treatments
resulted
in
higher
shoot
formation
percentages
compared
to
the
0.5
and
1
mg
l−1of
TDZ
treatments
in
two
of
the
three
cultivars.
Furthermore,
more
shoots
were
produced
on
BA
than
on
Kn-supplemented
media
in
all
the
three
cultivars.
Shoots
derived
from
the
0.5
mg
l−1of
BA
treatment
can
be
induced
to
root
following
one
month
incubation
with
0.1–1
mg
l−1of
indole-3-
butyric-acid
(IBA).
The
rooted
shoots
showed
100%
survival
after
acclimatization
in
the
greenhouse.
The
procedure
reported
in
the
present
study
can
assist
in
the
large-scale
multiplication
of
elite
self-heading
cultivars
of
Philodendron
in
the
future.
©
2012
Elsevier
B.V.
All
rights
reserved.
1.
Introduction
The
genus
Philodendron
is
the
second
largest
member
of
the
Araceae
family
and
is
composed
of
more
than
500
species
native
to
tropical
and
subtropical
America
and
the
West
Indies
(Mayo
et
al.,
1997).
Philodendrons
are
highly
appreciated
for
their
attractive
foliage
and
tolerance
of
interior
environments
and
have
been
pro-
duced
for
use
extensively
in
interiorscaping.
Two
distinct
growth
types
can
be
found
in
Philodendron:
the
vining
type
which
dom-
inated
foliage
plant
sales
from
the
1950s
to
the
early
1970s,
and
the
self-heading
type
which
has
become
popular
in
the
last
40
years
due
to
an
increasing
number
of
new
hybrids
with
red,
yel-
low
or
orange
foliage
that
were
released
to
the
market
(Chen
et
al.,
2002).
In
Taiwan,
Philodendron
constitutes
an
important
share
in
the
foliage
plant
market
and
the
raising
popularities
of
self-heading
cultivars
have
made
them
to
rank
among
the
top
ten
most
popular
plants
in
the
floricultural
trade.
To
fulfill
grower’s
demands
for
potted
plants
of
Philodendron,
procedures
for
rapid
propagation
of
elite
cultivars
are
essential.
Conventional
propagation
of
Philodendron
by
stem
cuttings
is
slow
∗Corresponding
author.
Tel.:
+886
87703202x6615;
fax:
+886
87740446.
E-mail
address:
jyfang@mail.npust.edu.tw
(J.Y.
Fang).
due
to
the
low
number
of
cuttings
that
can
be
made
from
each
plant.
It
is
also
technically
inconvenient
as
the
sap
of
the
plant
can
cause
contact
dermatitis
(Reffstrup
and
Boll,
1985).
Micropropagation
is
a
comparatively
more
attractive
means
for
Philodendron
propaga-
tion
as
a
higher
number
of
plants
can
be
generated
in
a
shorter
time
period
compared
to
the
conventional
procedure.
In
addition,
tissue
culture
of
tropical
foliage
plants
has
been
proposed
as
a
means
to
eliminate
various
systemic
viral,
fungal
and
bacterial
diseases
that
are
often
prevalent
in
stock
plants
(Hartman,
1974;
Henny,
1988).
Tissue
culture
has
also
led
to
improved
plant
forms
compared
to
those
propagated
by
traditional
methods.
It
was
found
that
tis-
sue
culture-derived
Dieffenbachia,
Spathiphyllum
and
Syngonium,
also
belonging
to
the
Araceae
family,
produced
a
fuller
and
more
compact
plant
when
compared
to
those
propagated
by
traditional
cuttings
(Conover,
1985).
Although
tissue
culture
has
been
performed
by
private
firms
for
the
production
and
supply
of
Philodendron
liners,
their
micro-
propagation
protocols
are
generally
undisclosed
to
the
public.
Few
procedures
have
been
published
to
date
for
the
micropropagation
of
Philodendron
species
and
mostly
used
lateral
buds
(Jámbor-
Benczúr
and
Márta-Riffer,
1990;
Gangopadhyay
et
al.,
2004)
and
stem
nodal
segments
(Sreekumar
et
al.,
2001)
as
starting
explants.
These
procedures
were
however
constrained
by
the
low
quality
of
the
regenerated
shoots
(i.e.
poor
elongation
and
rooting,
poor
0304-4238/$
–
see
front
matter
©
2012
Elsevier
B.V.
All
rights
reserved.
http://dx.doi.org/10.1016/j.scienta.2012.04.011

24
F.C.
Chen
et
al.
/
Scientia
Horticulturae
141
(2012)
23–29
conversion
success
into
plant)
as
a
result
of
long-term
exposure
to
high
concentrations
of
plant
growth
regulators
(PGRs)
used
for
shoot
proliferation
(Jámbor-Benczúr
and
Márta-Riffer,
1990;
Vardja
and
Vardja,
2001;
Sreekumar
et
al.,
2001;
Gangopadhyay
et
al.,
2004),
species
and
cultivars-dependent
requirement
of
PGR
treatments
for
optimal
shoot
proliferation
(Sreekumar
et
al.,
2001),
and
the
low
number
of
species
and
cultivars
studied
(notably
the
self-heading
types).
The
present
study
therefore
aims
at
developing
an
efficient
micropropagation
procedure
for
the
self-heading
cul-
tivars
of
Philodendron.
The
procedure
is
expected
to
achieve
a
high
shoot
multiplication
rate,
obtain
healthy
plantlets
and
be
readily
applicable
to
different
cultivars.
To
achieve
this,
the
influence
of
explant
type,
as
well
as
PGR
type
and
concentration
on
shoot
induc-
tion
and
proliferation
was
studied.
The
quality
of
the
induced
shoots
was
evaluated
based
on
their
ability
to
root
and
to
adapt
to
the
ex
vitro
environment.
2.
Materials
and
methods
2.1.
Plant
material
and
aseptic
culture
establishment
Three
commercial
self-heading
cultivars
of
Philodendron
(i.e.
‘Imperial
Green’,
‘Imperial
Red’
and
‘Imperial
Rainbow’)
were
used
in
the
present
study.
The
plants
were
purchased
from
a
local
nurs-
ery
and
sprayed
with
fungicides
Ridomil
MZ
and
Mancozeb
(1000×
dilution)
each
for
one
week
prior
to
explant
excision
and
establish-
ment
in
vitro.
After
defoliation,
shoot
cuttings
measuring
5–10
cm
long
were
washed
under
running
tap
water
for
15
min
and
divided
into
single
nodal
segments
each
containing
a
lateral
bud.
The
nodal
segments
were
then
surface-sterilized
with
70%
EtOH
for
1
min
fol-
lowed
by
1%
(v/v)
sodium
hypochlorite
(NaOCl)
containing
several
drops
of
Tween-20
for
20
min
on
a
rotary
shaker.
After
three
rinses
with
sterile
water
and
removal
of
the
damaged
ends,
the
explants
were
cultured
on
MS
(Murashige
and
Skoog,
1962)
medium
supple-
mented
with
0.1
mg
l−1of
1-naphthaleneacetic
acid
(NAA),
1
mg
l−1
of
6-benzyladenine
(BA),
3%
(w/v)
of
sucrose
and
0.7%
(w/v)
of
agar.
The
pH
of
the
medium
was
adjusted
to
5.7
prior
to
autoclaving.
Cultures
were
checked
regularly
for
contaminations
and
those
pre-
sented
apparent
infection
symptoms
were
immediately
discarded.
The
outgrowth
of
the
lateral
bud
(i.e.
approximately
2
cm
long)
was
excised
from
the
nodal
segments
and
subcultured
on
the
same
medium
for
further
shoot
multiplication.
Regenerated
shoot
clus-
ters
were
divided
and
subcultured
every
eight
weeks
to
build
up
a
stock
shoot
culture.
Shoots
from
the
fifth
subculture
and
mea-
suring
approximately
3
cm
in
height
were
used
for
the
subsequent
experiments.
2.2.
Effect
of
explant
and
plant
growth
regulator
types
on
the
growth
and
shoot
induction
percentages
of
three
Philodedron
cultivars
The
explants
tested
included
leaf
petiole
(i.e.
∼5
mm
in
length),
leaf
lamina
(∼8
mm
×
3
mm
section
with
two
cuts
made
across
the
midvein),
and
stem
nodal
segment
(containing
2
nodes
each
and
measuring
∼4
mm
in
length).
The
explants
were
cultured
on
MS
medium
supplemented
with
either
0.5
mg
l−1of
2,4-
dichlorophenoxyacetic
acid
(2,4-D),
0.5
mg
l−1of
thidiazuron
(TDZ)
or
both.
All
the
explants
were
placed
horizontally
on
the
medium
and
the
lamina
explants
were
placed
with
their
adaxial
side
touching
the
medium.
The
growth
as
well
as
the
shoot
induction
percentages
of
the
different
explants
was
evaluated
three
months
following
the
treatments.
Growth
was
recorded
when
any
type
of
new
structures
were
produced
from
the
original
explant.
Shoot
induction
was
recorded
when
at
least
one
visible
shoot
formed
on
the
explant.
Three
replicate
Petri
dishes
were
used
for
each
treatment
with
six
explants
in
each
dish.
2.3.
Effect
of
cytokinin
type
and
concentration
on
shoot
proliferation
Three
cytokinins,
i.e.
kinetin
(Kn),
BA
and
TDZ,
at
0.5
and
1
mg
l−1
were
tested
for
their
effectiveness
in
inducing
shoot
proliferation
in
previously
selected
explants.
Three
replicate
Petri
dishes
were
used
for
each
treatment
with
six
explants
in
each
dish.
The
percentage
of
explants
exhibiting
growth
and
shoot
formation,
as
well
as
the
number
of
shoots
produced
per
responding
explant
was
recorded
after
three
months.
The
regenerated
shoots
were
divided
into
small
shoot
clusters
and
were
transferred
to
PGR-free
MS
medium
for
shoot
elongation
during
a
period
of
two
months.
2.4.
Effect
of
IBA
concentration
on
root
induction
Shoots
from
the
best
cytokinin
treatment
and
obtained
at
the
end
of
the
elongation
stage
with
at
least
four
leaves
were
individu-
ally
placed
inside
glass
test
tubes
(i.e.
2.5
cm
in
diameter
and
15
cm
in
height)
containing
10
ml
of
MS
medium
supplemented
with
0,
0.1,
0.5
and
1
mg
l−1of
IBA.
Ten
replicated
test
tubes
were
used
in
each
medium
treatment.
The
percentage
of
shoots
producing
roots,
as
well
as
the
number
and
length
of
the
induced
roots
were
recorded
after
30
days.
2.5.
Greenhouse
acclimatization
After
two
months
on
the
rooting
medium,
well
developed
shoots
(2–2.5
cm
long;
4–6
leaves)
with
at
least
five
roots
were
collected
from
the
best
IBA
treatment
for
acclimatization.
Rooted
shoots
were
carefully
retrieved
from
the
test
tubes
and
washed
under
tap
water
to
remove
sticking
medium
from
the
roots.
The
plantlets
were
then
transplanted
into
10.1
cm
plastic
pots
filled
with
a
potting
mixture
(4:1)
of
peat
moss
and
perlite.
Potted
plants
were
directly
grown
in
a
partially
shaded
greenhouse
with
temperatures
ranging
from
25
to
30 ◦C,
relative
humidity
of
between
70
and
100%,
and
light
inten-
sity
of
35
␮mol
m−2s−1under
a
10
h
photoperiod.
Plantlets
were
considered
successfully
acclimatized
when
these
have
produced
at
least
one
new
leaf
after
two
months.
2.6.
Culture
conditions,
experimental
design
and
data
analysis
The
in
vitro
experiments
were
performed
with
an
incuba-
tion
temperature
of
25
±
2◦C
and
a
16/8
h
light/dark
photoperiod
provided
by
cool
fluorescent
lamps
at
40
␮mol
m−2s−1.
All
the
experiments
were
set
up
in
a
completely
randomized
design
and
were
conducted
twice.
Data
were
subjected
to
the
analysis
of
vari-
ance
using
SAS
(SAS
Institute
Inc.,
1999)
software,
and
means
were
separated
by
Duncan’s
multiple
range
test
at
the
95%
level
(Duncan,
1955).
3.
Results
3.1.
Effect
of
explant
and
plant
growth
regulator
types
on
the
growth
and
shoot
induction
percentages
of
three
Philodendron
cultivars
Three
months
following
the
treatments,
none
of
the
explants
grown
on
the
PGR-free
control
medium
showed
any
growth
(Table
1).
In
contrary,
explants
grown
on
PGR-containing
media
presented
different
growth
patterns
depending
on
the
type
of
explants
cultured.
With
the
lamina
explants,
growth
was
initiated
by
the
enlargement
of
the
explants
followed
by
the
production
of
small
and
round
globules
along
the
cuts
across
the
mid-vein

F.C.
Chen
et
al.
/
Scientia
Horticulturae
141
(2012)
23–29
25
Table
1
Effect
of
explant
and
plant
growth
regulator
types
on
the
growth
(A)
and
shoot
induction
(B)
percentages
of
three
Philodendron
cultivars.
(A)
Cultivar
Explant
Control
0.5
mg
l−12,4-D
0.5
mg
l−1TDZ
0.5
mg
l−12,4-D+
0.5
mg
l−1TDZ
‘Imperial
Green’
Lamina
0a*
0b*
0b*
0c*
Petiole
0a***
50.0
±
11.1a*
0b***
33.3
±
7.9b**
Stem
0a***
37.5
±
18.2a**
26.7
±
8.6a**
88.9
±
11.7a*
‘Imperial
Red’ Lamina 0a**
2.8
±
3.9b**
0b**
36.1
±
7.3b*
Petiole 0a***
2.8
±
3.9b**
8.3
±
5.3b**
97.2
±
3.9a*
Stem
0a***
88.9
±
7.9a*
55.6
±
11.7a**
88.9
±
7.9a*
‘Imperial
Rainbow’
Lamina
0a**
0c**
0c**
13.9
±
11.3c*
Petiole
0a*
33.3
±
11.1b*
20.0
±
4.3b*
41.7
±
13.3b*
Stem 0a**** 77.8
±
5.0a** 58.3
±
8.1a*** 97.2
±
3.9a*
(B)
Cultivar Explant
Control
0.5
mg
l−12,4-D
0.5
mg
l−1TDZ
0.5
mg
l−12,4-D+
0.5
mg
l−1TDZ
‘Imperial
Green’
Lamina
0a*
0b*
0b*
0b*
Petiole
0a*
0b*
0b*
0b*
Stem 0a** 5.6
±
5.0a** 16.7
±
6.1a* 2.8
±
3.9a**
‘Imperial
Red’ Lamina 0a*
0b*
0b*
0a*
Petiole
0a*
2.8
±
3.9b*
2.8
±
3.9b*
0a*
Stem
0a**
2.8
±
3.9a**
38.9
±
11.7a*
0a**
‘Imperial
Rainbow’
Lamina
0a*
0b*
0c*
0a*
Petiole 0a*0
b* 11.1
±
10.0b*0
a*
Stem
0a**
8.3
±
8.1a**
41.7
±
5.3a*
0a**
Data
(±SE)
are
the
mean
values
of
three
replicates
of
six
explants
each,
with
the
experiment
conducted
twice.
For
each
cultivar,
different
lowercase
letters
and
star
numbers
indicate
significant
differences
among
treatments
within
each
column
and
row
respectively
(Duncan’s
multiple
range
test,
P

0.05).
(Fig.
1a).
These
globular
structures,
however,
were
not
able
to
develop
further
and
convert
into
shoots.
With
the
petiole
explants,
growth
also
manifested
as
small
globular
structures
and
occurred
at
the
proximal
ends
of
the
petioles
(Fig.
1b).
Some
of
these
protruding
globules
later
developed
into
shoots,
appearing
either
individually
or
in
clusters.
With
the
stem
nodal
explants,
swelling
of
the
nodes
was
followed
by
the
emergence
of
dormant
axillary
buds.
In
some
cases,
adventitious
buds
were
formed
at
the
base
of
the
developing
axillary
shoot
to
form
a
shoot
cluster
(Fig.
1c).
Occasionally,
roots
were
observed
to
develop
from
the
node
region.
The
type
of
explants
tested
had
an
influence
on
the
growth
responses
of
the
three
Philodendron
cultivars
as
shown
in
Table
1A.
Among
the
three
explants
tested,
lamina
explants
were
the
least
responsive
as
their
growth
was
only
observed
following
the
2,4-D
treatment
(i.e.
2.8%)
in
‘Imperial
Red’
and
following
the
combined
2,4-D
and
TDZ
treatment
in
‘Imperial
Red’
(i.e.
36.1%)
and
‘Impe-
rial
Rainbow’
(i.e.
13.9%).
The
petiole
explants
were
comparatively
more
responsive
as
growth
manifested
in
all
the
three
cultivars
and
under
different
PGR
treatment
conditions
except
for
‘Imperial
Green’
with
the
TDZ
treatment.
In
addition,
the
growth
percent-
ages
of
petiole
explants
were
significantly
higher
than
the
lamina
explants
in
most
of
the
cases.
The
stem
nodal
segments
were
the
most
responsive
of
the
three
explant
types.
Growth
was
apparent
in
all
the
PGR
treatments
and
cultivars
tested.
The
percentages
of
growth
of
the
stem
nodal
explants
were
significantly
higher
then
the
petiole
explants,
in
exception
of
the
2,4-D
treatment
for
‘Impe-
rial
Green’
and
the
combined
2,4-D
and
TDZ
treatment
for
‘Imperial
Red’
where
growth
percentages
were
comparable
between
the
two
explant
types.
The
growth
responses
of
the
different
Philodendron
cultivars
were
also
affected
by
the
PGR
treatments
which
they
were
sub-
jected
to
(Table
1A).
Growth
was
observed
in
petiole
and
stem
nodal
explants
in
all
the
three
cultivars
tested
and
following
all
the
PGR
treatments,
except
for
‘Imperial
Green’
with
the
TDZ
treat-
ment.
It
was
found
that
the
growth
percentages
of
the
different
explants
under
the
combined
2,4-D
and
TDZ
treatment
were
sig-
nificantly
higher
than
under
single
PGR
treatments,
except
for
the
petiole
explants
of
‘Imperial
Green’
and
‘Imperial
Rainbow’,
and
the
Fig.
1.
Morphogenic
response
of
leaf
lamina
(a),
petiole
(b)
and
stem
nodal
segments
(c)
following
different
PGR
treatments.
(a)
Production
of
small
and
round
globules
along
the
cuts
across
the
mid-vein
in
an
‘Imperial
Red’
lamina
explant
following
0.5
mg
l−1of
2,4-D
treatment.
(b)
Small
globular
structures
occurred
at
the
proximal
ends
of
an
‘Imperial
Rainbow’
petiole
explant
following
0.5
mg
l−1of
TDZ
treatment.
(c)
Direct
emergence
of
the
dormant
axillary
buds
in
an
‘Imperial
Red’
stem
nodal
segment
following
0.5
mg
l−1of
TDZ
treatment.
(Bar
=
0.1
cm.)

26
F.C.
Chen
et
al.
/
Scientia
Horticulturae
141
(2012)
23–29
stem
nodal
explants
of
‘Imperial
Red’.
When
comparing
the
two
sin-
gle
PGR
treatments,
it
can
be
seen
that
the
growth
percentage
of
the
different
explants
was
comparable
in
most
cases
except
for
the
stem
nodal
explants
of
‘Imperial
Red’
and
‘Imperial
Rainbow’,
and
the
petiole
explants
of
‘Imperial
Green’
where
the
2,4-D
treatment
produced
a
higher
growth
response
than
the
TDZ
treatment.
As
for
the
growth
response,
both
the
explant
and
PGR
types
tested
influenced
the
shoot
induction
response
of
the
three
Philo-
dendron
cultivars.
It
was
observed
that
the
stem
nodal
explants
exhibited
a
higher
shoot
induction
percentage
compared
to
the
petiole
explants
in
all
the
three
cultivars
tested,
and
no
shoot
was
formed
on
the
lamina
explants
(Table
1B).
For
the
stem
nodal
explants,
the
TDZ
treatment
produced
a
significantly
higher
shoot
induction
response
than
the
2,4-D
and
combined
2,4-D
and
TDZ
treatments
in
all
the
three
cultivars.
3.2.
Effect
of
cytokinin
type
and
concentration
on
shoot
proliferation
Stem
nodal
explants
were
selected
because
of
their
high
poten-
tial
for
inducing
shoots
as
observed
in
the
previous
experiment.
Three
cytokinins
(i.e.
kinetin,
BA
and
TDZ)
at
two
different
con-
centrations
(i.e.
0.5
and
1
mg
l−1)
were
tested
for
their
influence
on
growth
and
shoot
formation
from
stem
nodal
explants.
Explant
growth
was
observed
in
all
cytokinin
treatments
for
all
three
cul-
tivars
(Table
2).
No
significant
difference
in
growth
between
the
different
cytokinin
treatments
was
observed
for
‘Imperial
Red’
and
‘Imperial
Rainbow’,
but
Kn
and
BA
treatments
gave
significantly
higher
growth
response
than
the
control
treatment
in
the
case
of
‘Imperial
Green’.
Shoot
formation
was
observed
following
all
the
cytokinin
treat-
ments
except
the
TDZ
treatments
in
‘Imperial
Green’.
For
all
cultivars,
production
of
shoot
clusters
at
the
nodes
was
observed
in
the
presence
of
Kn
or
BA.
The
shoots
produced
in
the
presence
of
Kn
were
comparatively
larger
(Fig.
2a)
than
those
in
the
BA
treatment
(Fig.
2b),
and
the
shoots
developed
roots
on
the
Kn-supplemented
medium
(Fig.
2a).
When
TDZ
was
used,
shoot
formation
was
initi-
ated
with
the
appearance
of
green
buds,
which
then
later
grew
into
leaf-like
structures
in
the
bud
tips
and
developed
into
leafy
shoots
for
‘Imperial
Red’
and
‘Imperial
Rainbow’,
or
which
failed
to
convert
into
shoots
in
the
case
of
‘Imperial
Green’
(Fig.
2c).
For
‘Imperial
Green’,
a
significantly
higher
percentage
of
shoot
formation
was
observed
with
the
0.5
mg
l−1Kn
treatment
(i.e.
88.8%)
compared
to
the
1
mg
l−1BA
treatment
(i.e.
58.3%).
But
there
was
no
significant
difference
between
the
0.5
and
1
mg
l−1Kn
treatments
and
the
0.5
mg
l−1BA
treatment.
The
number
of
shoots
produced
per
explant
was
significantly
higher
following
the
BA
treatments
(i.e.
48.7–49.4)
compared
to
Kn
treatments
(i.e.
6.5–7.8).
For
‘Imperial
Red’,
the
shoot
formation
percentage
was
significantly
higher
following
the
Kn
and
BA
treatments
compared
to
the
TDZ
treatments.
Although
no
significant
difference
in
shoot
formation
frequency
was
noted
between
the
Kn
and
BA
treatments,
the
pres-
ence
of
BA
induced
a
significantly
higher
number
of
shoots
(i.e.
46.3–47.4)
than
Kn
(i.e.
2.5–2.9).
For
‘Imperial
Rainbow’,
no
signifi-
cant
difference
in
shoot
formation
frequency
was
recorded
among
the
different
cytokinin
treatments.
However,
the
highest
number
of
shoots
produced
per
explant
was
found
with
the
0.5
mg
l−1BA
treatment
(i.e.
50.4),
followed
by
the
1
mg
l−1BA
treatment
(i.e.
40.8)
and
the
Kn
and
TDZ
treatments
(i.e.
5.3–6.9).
Clumps
of
small
shoots
produced
on
the
0.5
mg
l−1BA-
supplemented
medium
were
transferred
onto
PGR-free
MS
medium
for
shoot
elongation.
Two
months
after
transfer,
the
small
shoots
increased
in
size
and
were
100%
normal
looking
in
all
the
cultivars
(Fig.
3a).
3.3.
Effect
of
IBA
concentration
on
root
induction
Since
root
formation
was
spontaneous
in
Kn-derived
shoots,
this
experiment
used
only
rootless
shoots
derived
from
the
0.5
mg
l−1
BA
treatment.
IBA
at
0
(control),
0.1,
0.5
and
1
mg
l−1were
tested
for
their
influence
on
the
frequency
of
root
induction,
the
number
of
roots
produced
per
shoot,
as
well
as
the
root
length
in
three
Philo-
dendron
cultivars.
It
was
observed
that
100%
of
the
shoots
tested
produced
roots
following
the
different
IBA
treatments
(including
the
control)
so
only
the
number
of
roots
produced
per
shoot
as
well
as
the
length
of
the
roots
produced
are
presented
in
Table
3.
A
sig-
nificantly
higher
number
of
roots
were
generated
on
shoots
in
the
presence
of
0.1–1
mg
l−1IBA
(i.e.
6.2–7.0)
compared
to
the
control
(i.e.
3.0)
for
‘Imperial
Green’.
For
‘Imperial
Red’,
shoots
subjected
to
IBA
treatments
at
0.5
and
1
mg
l−1produced
a
significantly
higher
number
of
roots
(i.e.
9.3–10.8)
than
those
subjected
to
0.1
mg
l−1
of
IBA
(i.e.
4.8)(Fig.
3b)
and
the
control
(i.e.
3.6).
A
similar
trend
was
observed
for
‘Imperial
Rainbow’
which
yielded
7.1–7.9
roots
per
shoot
in
the
presence
of
0.5
and
1
mg
l−1IBA
compared
to
only
4.8
and
3.5
roots
with
the
0.1
mg
l−1IBA
treatment
and
the
control
respectively.
Shoots
in
the
control
produced
significantly
longer
roots
compared
to
those
in
the
IBA
treatments
for
both
‘Imperial
Green’
and
‘Imperial
Red’.
However,
no
significant
difference
in
root
length
was
found
between
the
different
IBA
treatments
and
the
control
in
‘Imperial
Rainbow’.
3.4.
Greenhouse
acclimatization
A
total
of
120
rooted
shoots
of
the
three
cultivars
following
the
0.5
and
1
mg
l−1of
IBA
treatments
were
used
for
acclimatization
in
the
greenhouse.
Results
showed
that
all
the
acclimatized
plantlets
grew
vigorously
in
the
greenhouse
with
100%
survival.
New
leaves
were
produced
and
leaf
variegation
in
‘Imperial
Red’
became
more
apparent
with
time
(Fig.
3c).
4.
Discussion
Contamination
is
frequently
observed
during
the
tissue
culture
of
ornamental
aroids
such
as
Aglaonema
(Chen
and
Yeh,
2007),
Anthurium
(Kunisaki,
1980),
Dieffenbachia
(Brunner
et
al.,
1995),
Spathyphyllum
and
Syngonium
(Kneifel
and
Leonhardt,
1992),
Zan-
tesdeschia
(Kritzinger
et
al.,
1998),
as
well
as
Philodendron
(Fisse
et
al.,
1987).
Therefore,
the
present
study
was
initiated
by
estab-
lishing
an
aseptic
shoot
stock
culture
from
nodal
axillary
buds.
The
regenerated
shoots
were
then
divided
to
provide
the
leaf
lamina,
petiole
and
stem
nodal
explants.
The
use
of
leaves
and
stem
nodal
segments
as
explants
for
micropropagation
has
been
reported
in
many
ornamental
Aroid
species
(Martin
et
al.,
2003;
Qu
et
al.,
2002;
Thao
et
al.,
2003;
Azza
and
Khalafalla,
2010;
Sreekumar
et
al.,
2001),
and
different
explants
were
found
with
different
shoot
regeneration
potentials.
Results
from
this
study
showed
that
the
growth
and
shoot
induc-
tion
responses
of
Philodendron
were
greatly
influenced
by
the
type
of
explant
used.
Lamina
explants
were
the
least
responsive
of
the
three
explant
types
tested
as
growth
was
only
observed
for
two
of
the
three
cultivars
when
2,4-D
or
both
the
2,4-D
and
TDZ
were
supplemented
to
the
medium.
In
any
case,
no
shoots
were
formed.
In
contrast,
Martin
et
al.
(2003)
reported
that
direct
shoot
regeneration
can
be
achieved
from
young
lamina
explants
in
two
Anthurium
andreanum
cultivars.
Similarly,
callus
formation
and
subsequent
shoot
regeneration
was
observed
with
lamina
explants
of
Dieffenbachia
cv.
Camouflage
(Shen
et
al.,
2007).
In
Anthurium
andraeanum
hybrids,
embryogenic
calli
were
formed
on
whole
of
the
leaf
blade
explants
(Kuehnle
et
al.,
1992).
It
is
possible
that
different
plant
species
vary
in
the
morphogenetic
competence
of

F.C.
Chen
et
al.
/
Scientia
Horticulturae
141
(2012)
23–29
27
Table
2
Effect
of
cytokinin
type
and
concentration
on
shoot
proliferation
of
stem
nodal
explants
in
three
Philodendron
cultivars.
Cytokinin
(mg
l−1)
‘Imperial
Green’
‘Imperial
Red’
‘Imperial
Rainbow’
Growth
(%)
Shoot
(%)
No.
shoot
Growth
(%)
Shoot
(%)
No.
shoot
Growth
(%)
Shoot
(%)
No.
shoot
0.5
TDZ
(control)
33.3
±
15.2d0c0b47.2
±
9.5b36.1
±
9.5b3.9
±
0.8b66.7
±
9.6a53.3
±
8.1a6.9
±
2.0c
1
TDZ
36.1
±
7.3cd 0c0b69.4
±
9.5ab 41.7
±
13.3b4.7
±
1.5b75.0
±
22.6a44.4
±
19.9a6.6
±
3.7c
0.5
Kn
88.8
±
7.9a88.8
±
7.9a7.8
±
2.4b73.3
±
10.9ab 73.3
±
10.9a2.9
±
1.1b62.2
±
12.3a62.2
±
12.3a5.3
±
0.7c
1
Kn 77.8 ±
9.9ab 77.8
±
9.9ab 6.5
±
1.5b72.2
±
11.7ab 72.2
±
11.7a2.5
±
0.4b63.9
±
7.3a63.9
±
7.3a6.2
±
1.3c
0.5
BA 66.7
±
12.7ab 66.7
±
12.7ab 48.7
±
7.4a80.6
±
11.3a80.6
±
11.3a47.4
±
5.9a55.6
±
15.7a55.6
±
15.7a50.4
±
3.0a
1
BA
58.3
±
19.9bc 58.3
±
19.9b49.4
±
3.2a73.3
±
14.6ab 73.3
±
14.6a46.3
±
1.0a61.1
±
7.9a61.1
±
7.9a40.8
±
1.0b
Data
(±SE)
are
the
mean
values
of
three
replicates
of
six
explants
each,
with
the
experiment
conducted
twice.
Different
lowercase
letters
indicate
significant
differences
among
treatments
within
each
column
(Duncan’s
multiple
range
test,
P

0.05).
Fig.
2.
Shoot
proliferation
of
stem
nodal
explants
on
medium
enriched
with
Kn
(a),
BAP
(b)
and
TDZ
(c).
(Bar
=
0.5
cm.)
their
lamina
explants.
Petiole
explants
were
more
responsive
than
lamina
explants
as
direct
shoot
formation
was
achieved
with
TDZ
in
‘Imperial
Red’
and
‘Imperial
Rainbow’,
and
with
2,4-D
in
‘Impe-
rial
Red’,
treatments
for
which
no
shoot
was
formed
on
the
lamina
explants.
The
morphogenic
superiority
of
petiole
explants
over
lamina
explants
was
also
demonstrated
in
Epipremnum
aureum
where
shoot
induction
from
petiole
explants
was
greater
than
leaf
lamina
sections
(Qu
et
al.,
2002).
In
Syngonium
podophyllum
Fig.
3.
Shoot
elongation,
rooting
and
acclimatization
ex
vitro.
(a)
Two
months
Elongation
of
shoots
on
PGR-free
MS
medium.
(b)
Rooting
of
‘Imperial
Red’
shoots
on
medium
supplemented
with
0.1
mg
l−1of
IBA.
(c)
Greenhouse
grown
‘Imperial
Red’
plantlets
after
one
month
acclimatization.

28
F.C.
Chen
et
al.
/
Scientia
Horticulturae
141
(2012)
23–29
Table
3
Effect
of
IBA
concentration
on
root
induction
in
three
Philodendron
cultivars.
IBA
(mg
l−1)
‘Imperial
Green’
‘Imperial
Red’
‘Imperial
Rainbow’
Root
number
Root
length
(cm)
Root
number
Root
length
(cm)
Root
number
Root
length
(cm)
0
3.0
±
0.6b1.1
±
0.1a3.6
±
0.5b1.0
±
0.1a3.5
±
0.5b1.0
±
0.2a
0.1
6.2
±
0.8a0.4
±
0.1c4.8
±
0.6b0.6
±
0.1b4.8
±
0.8b1.3
±
0.2a
0.5
7.0 ±
0.8a0.8 ±
0.1b9.3 ±
1.2a0.7 ±
0.1b7.9 ±
1.4a1.2
±
0.1a
1 6.3 ±
0.9a0.7
±
0.2b10.8
±
1.4a0.6
±
0.1b7.1
±
1.3a1.0
±
0.2a
Data
(±SE)
are
the
mean
values
of
ten
replicates,
with
the
experiment
conducted
twice.
Different
lowercase
letters
indicate
significant
differences
among
treatments
within
each
column
(Duncan’s
multiple
range
test,
P

0.05).
‘Variegatum’,
somatic
embryos
formed
directly
on
the
petiole
explants,
whereas
no
embryo
formation
was
observed
from
leaf
explants
(Zhang
et
al.,
2006).
Stem
nodal
segments
proved
to
be
the
most
responsive
explants
in
the
present
study.
Growth
was
observed
following
all
the
PGR
treatments
in
all
the
three
cultivars,
and
shoot
formation
was
recorded
in
seven
out
of
the
nine
PGR
treatments.
The
choice
of
an
appropriate
PGR
to
induce
shooting
in
Philo-
dendron
was
essential
as
shown
with
the
stem
nodal
explants
in
this
study.
The
highest
shoot
induction
percentage
was
achieved
using
the
TDZ
treatment,
with
an
average
of
16.7–41.7%
depend-
ing
on
the
cultivar.
Shoots
were
also
formed
following
the
2,4-D
treatment
in
all
the
three
cultivars,
but
at
lower
fre-
quencies
(i.e.
2.8–8.3%)
compared
to
the
TDZ
treatment.
Shoot
formation
on
cytokinin-free
medium
has
also
been
reported
in
other
Aroid
species.
In
Aglaonema
for
instance,
shoots
were
observed
to
form
from
the
inflorescence
explants
on
media
devoid
of
cytokinins
(Yeh
et
al.,
2007).
Similar
observation
was
made
with
Dieffenbachia
where
shoot
buds
differentiated
into
shoots
on
media
without
cytokinin
(Azza
and
Khalafalla,
2010).
Despite
the
high
growth
response
(i.e.
88.9–97.2%)
of
the
stem
nodal
explants
under
the
combined
2,4-D
and
TDZ
treatment,
their
shoot
induction
percentage
was
only
0-2.8%
for
the
three
cultivars.
This
was
not
the
case
for
E.
aureum
where
a
com-
bination
of
0.18
mg
l−1NAA
and
2.20
mg
l−1TDZ
allowed
shoot
regeneration
(Qu
et
al.,
2002).
Shoot
regeneration
was
also
obtained
for
A.
andreanum
lamina
explants
following
a
com-
bination
of
0.25
mg
l−1BA,
0.19
mg
l−1IAA
and
0.09
mg
l−1Kn
treatments
(Martin
et
al.,
2003).
It
appears
that
the
PGR
require-
ment
for
shoot
regeneration
varies
from
one
plant
species
to
another.
Since
the
application
of
TDZ
alone
has
proved
successful
to
induce
shoots
from
the
stem
nodal
explants
of
Philodendron,
the
subsequent
experiment
was
conducted
to
compare
the
efficiency
of
different
cytokinins
on
shoot
proliferation.
Cytokinins
such
as
BA,
Kn
and
TDZ
have
frequently
been
used
in
the
shoot
proliferation
of
different
members
of
the
Araceae
family
(Sreekumar
et
al.,
2001;
Dewir
et
al.,
2006;
Jo
et
al.,
2008;
Kozak
and
Stelmaszczuk,
2009;
Azza
and
Khalafalla,
2010;
Mariani
et
al.,
2011).
This
study
showed
that
both
the
cytokinin
type
and
concentration
had
a
significant
impact
on
shoot
regeneration
in
Philodendron.
For
‘Imperial
Green’
and
‘Imperial
Red’,
the
BA
and
Kn
treatments
showed
a
higher
shoot
formation
percentage
than
the
TDZ
treatment,
whereas
no
sig-
nificant
difference
was
observed
between
the
different
cytokinin
treatments
for
‘Imperial
Rainbow’.
In
terms
of
the
number
of
shoot
produced
per
explant,
BA
proved
superior
to
Kn
and
TDZ
in
all
three
cultivars.
No
difference
was
noticed
between
the
0.5
and
1
mg
l−1
BA
treatments
in
two
of
the
three
cultivars.
The
superiority
of
BA
over
other
cytokinins
has
already
been
reported
in
Spathiphyllum
cannifolium
(Dewir
et
al.,
2006),
Zantedeschia
aethiopica
(Kozak
and
Stelmaszczuk,
2009),
Caladiums
bicolor
(Ali
et
al.,
2007),
Dieffen-
bachia
compacta
(Azza
and
Khalafalla,
2010),
and
six
Philodendron
cultivars
(Sreekumar
et
al.,
2001).
In
this
study,
the
efficiency
in
shoot
proliferation
of
TDZ
was
inferior
to
BA
and
Kn.
This
is
in
opposition
to
many
studies.
For
example,
TDZ
was
the
best
cytokinin
for
pothos
regeneration
(supe-
rior
to
zeatin
and
2iP)
(Qu
et
al.,
2002).
TDZ
was
more
effective
in
multiple
shoot
proliferation
of
Alocasia
amazonica
compared
to
BA
and
Kn
(Jo
et
al.,
2008).
It
was
found
in
the
present
study
that
TDZ-treated
explants
tend
to
induce
a
large
amount
of
globules-like
structures
at
the
level
of
the
nodes
which
seemed
to
have
difficulty
to
convert
into
shoots
at
a
later
stage.
Reasons
for
the
low
shoot
conversion
success
of
TDZ
might
include
first
the
dual
auxin-
and
cytokinin-like
activities
of
the
TDZ
(Singh
et
al.,
2003)
which
might
lead
to
an
increase
in
endogenous
levels
of
auxins
(Hutchinson
et
al.,
1996),
and
second
higher
concentrations
of
TDZ
have
some-
times
been
associated
with
morphological
abnormalities
in
several
species
(Huetteman
and
Preece,
1993).
In
terms
of
the
length
of
shoots
produced,
it
was
found
that
the
shoots
obtained
following
the
Kn
treatment
were
comparatively
larger
than
those
from
the
BA
treatment.
The
promotive
effect
of
Kn
on
shoot
length
was
also
observed
with
D.
compacta
and
Dif-
fenbachia
picta
‘Tropica’
(El-sawy
and
Bakheet,
1999).
The
small
sized-shoots
found
in
the
BA
treatment
can
be
attributed
to
the
concentration
of
BA
used
which
might
restrain
the
shoots
from
outgrowing.
Similar
observation
was
made
in
another
Philodendron
study
where
the
use
of
BA
(up
to
2.5
mg
l−1)
had
an
inhibitory
effect
on
root
formation
of
the
shoots
at
later
stage
of
culture
(Sreekumar
et
al.,
2001).
Similarly,
although
BA
(at
4.95
mg
l−1)
allowed
rapid
multiplication
of
Philodendron
‘Xanadu’,
a
gradual
decline
of
BA
from
4.95
down
to
0.45
mg
l−1was
essential
for
obtaining
healthy
plantlets
otherwise
BA
would
arrest
growth
and
development
of
the
multiplied
shoots
(Gangopadhyay
et
al.,
2004).
It
was
observed
that
the
BA-derived
shoots
could
successfully
elongate
to
produce
normal
shoots
in
eight
weeks,
suggesting
that
the
low
BA
concen-
trations
used
in
the
present
study
not
only
allowed
a
high
shoot
multiplication
rate
but
also
allowed
healthy
shoots
to
be
produced.
The
success
of
micropropagation
on
a
commercial
level
relies
on
adequate
rooting
of
the
shoots
as
well
as
high
survival
rate
of
the
acclimatized
plantlets.
IBA
has
commonly
been
employed
for
the
in
vitro
rooting
of
many
Araceae
species,
such
as
0.5
mg
l−1
IBA
for
nine
Dieffenbachia
cultivars
(Zhu
et
al.,
1999),
1.5
mg
l−1of
IBA
for
D.
compacta
(Azza
and
Khalafalla,
2010),
and
3
mg
l−1of
IBA
for
Aglaonema
‘Cochin’
(Mariani
et
al.,
2011).
The
rooting
of
BA-derived
shoots
was
evaluated
in
the
present
study
using
IBA
at
0.1–1
mg
l−1.
Results
showed
100%
of
rooting
on
all
the
media
(including
the
control)
four
weeks
after
the
treatment.
In
general,
the
frequency
of
rooting
was
higher
in
0.5–1
mg
l−1IBA-treated
shoots
compared
to
0–0.1
mg
l−1IBA-treated
shoots.
However,
the
control
treatment
produced
longer
roots
than
with
the
different
IBA
treatments,
except
for
‘Imperial
Rainbow’
where
no
difference
in
root
length
was
observed
between
the
different
treatments.
Longer
roots
were
also
produced
in
the
IBA-free
treatment
in
Diffenbachia
compacta
(Azza
and
Khalafalla,
2010).
In
the
present
study,
all
the
shoots
were
successfully
acclimatized
with
a
survival
rate
of
100%
in
all
the
three
cultivars.

F.C.
Chen
et
al.
/
Scientia
Horticulturae
141
(2012)
23–29
29
5.
Conclusion
The
present
study
constitutes
the
first
report
on
the
microprop-
agation
of
self-heading
cultivars
of
Philodendron.
Results
in
the
present
study
demonstrated
that
the
stem
nodal
segments
showed
a
higher
potential
to
produce
shoot
than
the
leaf
lamina
and
peti-
ole
explants
under
the
treatment
conditions
tested.
Shoots
can
be
induced
to
form
following
the
0.5
and
1
mg
l−1of
BA
treatments
at
frequencies
of
55.6–80.6%
and
with
an
average
shoot
number
of
40.8–50.4
per
explant
depending
on
the
cultivar.
The
induced
shoots
could
elongate
on
PGR-free
MS
medium
to
produce
normal
looking
shoots,
and
rooted
successfully
on
IBA-contained
media.
Following
transfer
to
the
greenhouse
conditions,
100%
of
the
rooted
shoots
survived.
The
procedure
developed
in
the
present
study
can
be
easily
implemented
for
the
mass
production
of
Philodendron
on
a
commercial
level.
Acknowledgement
This
research
was
financially
supported
by
a
grant
from
the
Council
of
Agriculture,
Taiwan
(Contract
no.
96AS-1.1.1-FD-Z2).
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