Hort. Environ. Biotechnol. 55(1):56-62. 2014.

ISSN (p rint) : 2211-3452

DOI 10.1007/s13580-014-0022-9 ISSN (online) : 2211-3460

Research Report

Adventitious Shoot Induction from Leaf Segments in Anthurium andreanum is

Affected by Age of Explant, Leaf Orientation and Plant Growth Regulator
1* 2
Jean Carlos Cardoso and Gustavo Habermann
1
Departamento de Desenvolvimento Rural, Centro de Cincias Agrrias, UFSCar, Km 174, ArarasSP, 13600-970, Brazil
2
Departamento de Botanica, IB, Univ Estadual Paulista (UNESP), Rio Claro-SP, 13506-900, Brazil

*Corresponding author: jeancardosoctv@gmail.com

Received February 16, 2013 / Revised December 17, 2013 / Accepted January 28, 2014
GKorean Society for Horticultural Science and Springer 2014

Abstract. Anthurium is one of the main potted plants on the flower market, and micropropagation is the most used
technique for clonal and large scale propagation of this species. Calli, adventitious shoots and regenerated plantlets
were obtained from leaf segments of Anthurium andraeanum. The influence of genotype, explant orientation, plant
growth regulator, and age of donor plants were tested. We obtained regenerated plants in three (Red One, Red
Dark, and Snow White) out of four different cultivars. The White Beauty, however, was considered to be recalcitrant
under these conditions. Therefore, the second study was conducted only with this recalcitrant cultivar, and the use of
juvenile rather than adult leaf explants enhanced plantlet regeneration at the frequency of 0.3-2.8 plantlets per leaf
segment. The use of 1.0 mgL-1 6-benzylaminopurine (BAP) and no addition of 2,4-D in the culture medium resulted
in direct plantlet regeneration of Anthurium. Plantlets obtained from Red One and White Beauty were successfully
micropropagated and acclimatized. In ex vitro phases, leaf-derived plants of these two genotypes maintained their
most important horticultural and ornamental characteristics.
Additional key words: direct and indirect organogenesis, ex vitro cultivation, micropropagation, recalcitrant cultivars,
true-to-type plants

Introduction elik, 2009). However, the use of leaf segments shows

The Anthurium genus is comprised of 1,500 species from
tropical areas, and Anthurium andreanum is of great ornamental
value (Dufour and Guerin, 2003).
The propagation of Anthurium andreanum using seeds is
limited because seeds are available in small number and
they develop slowly (Dufour and Guerin, 2003). In addition,
seed propagation causes high heterozygosity, resulting in
genetic segregation and uneven production (Bejoy et al., 2008).
Vegetative propagation by traditional methods is challenging
and time-consuming due to the slow growth behavior of this
species (Martin et al., 2003). Therefore, micropropagation is
an useful alternative for propagating Anthurium as well as a
rapid method for providing pathogen-free plants (DOnghia
et al., 2001; Martin et al., 2003).
Indirect organogenesis from leaf tissues is the most fre-
quently used technique for in vitro regeneration of this species
(Gantait and Mandal, 2010). Explants from vegetative and
reproductive organs have been successfully used (Atak and
wider applications in indirect organogenesis of Anthurium,
because it enhances the regeneration potential and maintains
the genetic fidelity of regenerated plantlets (Gantait and
Sinniah, 2011). Therefore, callus induction and in vitro regen-
eration from leaf explants is highly dependent on genotype
of Anthurium (Gantait and Mandal, 2010; Silva et al., 2005).
Notwithstanding, direct regeneration of Anthurium plantlets
from adult tissues is still a challenge.
Unsuccessful results of regeneration in ornamental species
have been overcome when the explants are inserted at different
orientations into the medium. Horizontal explant orientation,
with adaxial side-up, shows higher inductions of direct somatic
embryogenesis in Phalaenopsis amabilis, P. Nebula (Gow
et al., 2009) and Oncidium Gower Ramsey orchids (Chen
and Chang, 2002) as compared to the abaxial side-up orien-
tation.
We tested different genotypes and explant orientations on
leaf-segmented organogenesis and plantlet regeneration in
A. andraeanum. Additionally, we attempted to directly and
 Hort. Environ. Biotechnol. 55(1):56-62. 2014.
indirectly induce regeneration of plantlets using the same
medium, i.e. without transferring leaf explants or calli to
another medium. We tested whether the age of donor plants
is a factor controlling leaf organogenesis in recalcitrant
cultivars of A. andraeanum. We aimed to obtain an efficient
protocol as well as to discover the main factors controlling
shoot organogenesis in recalcitrant cultivars of this species.
Materials and Methods
3ODQW 0DWHULDO
The leaves used for in vitro experiments were obtained
from Anthurium andreanum Red One and Red Dark with buds, and shoots per leaf segment were measured after 90
red spathes, and White Beauty with white spathes. These
cultivars have been developed for the potted plant market.
Leaves from the Snow White with white spathes (originally
developed for the cut flower market) were also used in this
study.
Donor plants were obtained from micropropagation, and
were grown in plastic pots containing coconut fiber as the
substrate (Ecogrow crush type, Biogrow, Conde-BA, Brazil).
These plants were maintained under greenhouse conditions,
-2 -1
with an average irradiance of 300 molm s PPFD (11.5 h
photoperiod), 25 5C, and 60% as the minimum air
relative humidity. A drip irrigation and fertigation system
was used as recommended by horticultural standard procedures
(IMAC and Anthura, 2007). The pH of the substrate was
maintained between 5.0-6.0 and the EC (electroic conduc-
-1
tivity) between 1.6-1.8 dSm for fertigation.
6WXG\ RI ([SODQW 2ULHQWDWLRQ DQG *HQRW\SHV RQ
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The four above-mentioned cultivars and three leaf-segmented
explant orientations were tested. Explant orientations were:
horizontal, with the abaxial leaf surface in contact with the
medium (ABA), horizontal, with the adaxial leaf surface in
contact with the medium (ADA), and vertical, with 50% of
the leaf segment inserted into the medium (VER).
The medium used for callus and shoot induction consisted
of a MS medium (Murashige and Skoog, 1962) with a
-1
reduced concentration of NH4NO3 to 250 mgL (Atak and
-1 -1
elik, 2009; Nhut et al. 2006), 30 gL sucrose, 100 mgL
-1 -1
myo-inositol, 1.0 mgL thiamine, 1.0 mgL 6-benzylamino-
-1
purine (BAP), and 0.1 mgL 2,4-diclorophenoxiacetic acid
(2,4-D). The medium pH was adjusted to 5.8 before solid-
-1
ification, which was achieved by adding 6.0 gL Agar (Algagel,
Type 900, Campinas-SP, Brazil). Test tubes (13 cm 2.1 cm)
containing 10 mL of medium was used as culture vessels,
-2
which were autoclaved at 121C under 1.0546 kgcm (= 15
-2
psi, 15 lbin ) for 20 min.
Leaves of donor plants were first washed with detergent
57
under tap water. Subsequently, these leaves were immersed
in 75% (v/v) alcohol for one min, followed by immersion in
a 1% (v/v) active chlorine solution. Next, the leaves were
washed three times in autoclaved distilled water.
Leaf midribs were cut and discarded, and squares of 1
2
cm were cut from the leaf lamina using only distal and mid
parts of young brown-green leaves (Martin et al., 2003).
Test tubes with leaf segments were maintained in dark
conditions for 60 days, before being transferred to 16 h pho-
-2 -1
toperiod (35 molm s PPFD provided by white fluorescent
lamps) for 30 days. The room temperature of both dark and
light conditions was 26 1C. The number of calli, adventitious
days of incubation.
A two-way ANOVA was run to test the effects of genotypes
(4), explant positions (3), and their interactions on the ad-
ventitious shoot induction from leaf segments. For each
treatment, ten test tubes, containing one explant each, were
used. The experiments were repeated twice.
$JH RI WKH 'RQRU 3ODQWV RQ $GYHQWLWLRXV /HDI
6KRRW ,QGXFWLRQ
The second experiment was conducted to study the
influence of the donor plant age and plant growth regulators
(PGRs) used in the culture medium on adventitious shoot
induction from leaf explants. The ages of donor plants were:
(D1) six-month-old plants in the vegetative phase (after
acclimatization), showing six fully expanded leaves, and no
flowers; and (D2) 12-month-old plants in the reproductive
phase (after acclimatization), displaying flowers and 12-15
fully expanded leaves.
Using the same culture medium described previously, we
also tested the following PGRs and combinations: (An1)
-1 -1
2,4-D at 0.1 mgL and BAP at 1.0 mgL ; (An2) BAP at
-1 -1 -1
1.0 mgL ; (An3) 2,4-D at 0.1 mgL and kinetin at 1.0 mgL ;
-1
and (An4) kinetin at 1.0 mgL .
For this study, only the White Beauty was used because
it showed a strong recalcitrant behavior regarding the plantlet
regeneration from in vitro adventitious shoot induction from
leaf segments in the first study.
A two-way ANOVA was run to test effects of the donor
plant age (2), PGRs used in the medium (4), and their inter-
actions on the adventitious shoot induction from leaf segments.
For each treatment, ten test tubes, containing one leaf segment
each, were used as replications.
The growth period and conditions as well as evaluated
parameters were the same as those previously described in
the first experiment.
In both experiments, mean values were compared by the
Tukey test ( = 0.05). The Assistat 7.6 (Silva, 2011) statistical
software was used. The experiment was repeated twice.
58 Jean Carlos Cardoso and Gustavo Habermann
0XOWLSOLFDWLRQ LQ 9LWUR DQG H[ 9LWUR *URZWK RI
5HJHQHUDWHG 3ODQWV
After shoot organogenesis was obtained in both studies,
shoots were multiplied in the MS medium in which 25 gL
-1
-1 -1
sucrose, 1.5 gL activated charcoal, 0.1 mgL myo-inositol,
-1
and 0.5 mgL BAP were added. The medium used for
rooting and stem elongation was the same as the medium
used in the multiplication phase. However, BAP was replaced
-1
by indol-3-butyric acid at 0.1 mgL . In both media, pH was
adjusted to 5.8 0.05 before achieving solidification with
-1
2.2 gL of gelling agent Gelrite (Duchefa, The Netherlands).
Growth conditions used in this phase were also the same
as those used in the first and the second experiments de-
scribed previously.
Two hundred plantlets of Red One and White Beauty
were obtained. Rooted plantlets were acclimatized and grown
in plastic pots containing coconut fiber as the substrate
(Ecogrow fiber, Biogrow, Conde-BA, Brazil). These plants
were maintained in a greenhouse, where temperature was
maintained between 20 (minimum) and 30C (maximum)
-2 -1
and an average irradiance of 150 molm s PPFD for the
-2 -1
first three months and 300 molm s PPFD in the fol-
lowing period were used.
Table 1. Effects of genotype and explant orientation on adventitious shoot induction from the leaf segments of Anthurium andraeanum.
The ex vitro plant development was observed for 18
months. During this phase, morphological alterations in
plants obtained from shoot induction were monitored. The
time for vegetative growth and flowering, as well as the
color of leaves and flowers were closely observed.
Results and Discussion
In the first experiment, three out of four cultivars showed
shoot induction and regeneration. Only the White Beauty
showed a strong recalcitrant behavior (callus was formed,
but no shoot developed). For this cultivar, leaf segments,
which were originally green, turned yellow after 60 days
under dark conditions, becoming darker and no shoot regen-
eration was observed at the end of 90 days of in vitro
incubation (Table 1). Raaj et al. (2012) also observed that
callus obtained from petioles of A. andreanum Casino
turned brown and regenerated no plantlets.
The Red Dark showed the highest rate of adventitious
bud induction and shoot regeneration (8.13 shoots/leaf
segment). Although the callus induction rate was higher in
the Red One and Snow White, these genotypes showed
lower adventitious shoot induction as compared to the Red

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 Hort. Environ. Biotechnol. 55(1):56-62. 2014.
Dark (Table 1). We also observed two types of callus
formed from leaf segments: cream-colored callus, which
was usually small, and green callus with vigorous growth.
Both types gave shoot induction and plantlet regeneration in
all cultivars, except for the White Beauty (Table 1).
We observed that shoot induction of Anthurium differed
among genotypes. Particularly for the White Beauty, we
found a conspicuous rate of unsuccessful regeneration. Nhut
et al. (2006) also observed two Anthurium cultivars did not
produce callus and that no plantlet regeneration from calli
was obtained in other eight cultivars. A similar recalcitrant
behavior was observed in the Octopus of Dieffenbachia
genus, which is related to Anthurium genus. In that cultivar,
Shen et al. (2008) reported that no shoots or plantlet regenera-
tion was obtained from the leaf explants. Therefore, observations
made by Nhut et al. (2006), Raaj et al. (2012) and our
observations on White Beauty in Anthurium, and also by
Shen et al. (2008) on Dieffenbachia largely exemplify im-
portant factors involved in the recalcitrant behavior of
Anthurium when trying to obtain shoot induction and
plantlet regeneration.
Results from the first experiment showed that leaf segments
with adaxial (ADA-type) surface in direct contact with the
culture medium was the best leaf explant orientation for
obtaining Anthurium shoot induction. Similar results were
obtained for Prunus armeniaca, when young expanded leaves
with the adaxial side touching the culture medium produced
Table 2. Effect of explant age on adventitious shoot induction from the leaf explants of Anthurium andraeanum White Beauty cultivated
on the culture medium composed of different PGRs (An1, An2, An3, and An4).
59
the best results (Prez-Tornero et al., 2000). On the other
hand, the abaxial position (ABA-type) enhanced direct somatic
embryogenesis in Phalaenopsis amabilis and P. Nebula (Gow
et al., 2009) and in Oncidium Gower Ramsey (Chen and
Chang, 2002) orchids. Therefore, in the present study, the
adaxial leaf surface must be in direct contact with the
culture medium during the callus induction and plantlet
regeneration. Differences in shoot induction from the ADA-
or ABA-type leaf orientation might be related to genetic,
physiological, and morphological factors such as leaf polarity,
which is ontogenetically established in the shoot meristem
(Sessions and Yanofsky, 1999; Xu et al., 2003). Leaf surfaces
also show distinct capacities for water and ion uptake
(Schnherr, 2006).
The age of donor plants seems to play a critical role in the
control of shoot organogenesis in Anthurium, as observed in
the White Beauty (Table 2 and Fig. 1). The PGR treatment
(An2) that induced 2.8 shoots per leaf segment in juvenile
tissues, induced only 0.3 shoots per leaf segments from
adult donor plants (Table 2 and Fig. 1). This developmental
switch is frequently observed in woody species. In Prunus
serotyna, 91.4% shoot regeneration was obtained using
juvenile explants, while only 40% when using mature
explants (Liu and Pijut, 2008). Optimization of regeneration
protocols for adult-derived explants has been attempted in
trees, which have long juvenile cycles. For this, genetic
transformation has been used to enhance cell competence
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60 Jean Carlos Cardoso and Gustavo Habermann

Fig. 1. Micropropagation and in vitro development of plantlets of Anthurium andraeanum obtained by adventitious shoot induction from
-1
the leaf segments: A, leaf segments from juvenile donor plant; B, leaf segments from adult donor plant; [1, BA (1.0 mgL ) + 2,4-D
-1 -1 -1 -1 -1
(0.1 mgL ); 2, BA (1.0 mgL ); 3, kinetin (1.0 mgL ) + 2,4-D (0.1 mgL ); 4, kinetin (1.0 mgL )]; C, details of plantlet regeneration
from the leaf segment of juvenile (D1) and adult (D2) explants; D, multiplication phase; and E, rooting phase.

for regeneration (Cervera et al., 2008; Pea-Ramrez et al.,
2010). The reason why adult Anthurium plants lose its in
vitro regeneration capacity is not well understood. High
secretion of phenols and shifts in the IAA:ABA ratio in
mature tissues are among the main causes of low
regeneration capacity in adult explants of woody species
(Beck et al. 1998; Chauvin and Salesses, 1988). For some
species showing episodic recalcitrance, the manipulating
phase may be a solution (McCown, 2000), but in many
recalcitrant plants, adequate procedures have not been
identified. However, the recalcitrance of adult tissues does
not only involve shifts in plant development, but also
includes the silencing of many genes that can be reverted
with osmotic or thermo stress and/or PGRs application
(Park et al. 2010; von Aderkas and Bonga, 2000).
Leaf segments from the adult White Beauty plants in-
oculated onto the culture medium containing 2,4-D and BAP
(An1) resulted in brown callus and tissues, and no shoots
were observed after 90 days of incubation. Replacing BAP
with kinetin, with (An3) or without 2,4-D (An4), did not
result in shoot induction from the leaf explants obtained
from adult plants (Table 2 and Fig. 1).
Our results combined with those from previous studies
(Bejoy et al., 2008; Jahan et al., 2009) support the hypothesis
that 2,4-D can serve as an inhibitor of shoot induction in
shoots of most adult Anthurium cultivars. In addition, BAP
is absolutely necessary in the medium for shoot induction
(Table 2).
Our results propose a protocol for direct shoot induction
from leaf segments. The direct shoot induction and plantlet
regeneration obtained is a fast clonal micropropagation
method and results in true-to-type regenerated plants. In
fact, plantlet regeneration from callus shows high somaclonal
variations, as observed in Dieffenbachia (Shen et al., 2007),
potato (Bordallo et al., 2004) and strawberry (Nehra et al.,
1992).
Furthermore, most protocols for induction and regeneration
of Anthurium shoots obtained from leaf segments (Atak and
elik, 2009; Jahan et al., 2009; Joseph et al. 2003) proposed
two phases: (1) production of callus and (2) induction and
regeneration of shoots. As a novelty, we induced callus and
regenerated shoots using the same medium, with no transfer
of callus to another medium for shoot regeneration.
Shoots of Red Dark and White Beauty were multiplied
in vitro and rooted (Figs. 1D and 1E), acclimatized, and
grown under greenhouse conditions. The multiplication rate
observed for the Red Dark was 5:1 (five shoots were
obtained from each explant), requiring approximately 160
days for the production of 200 plants. Approximately 93% of
these plants were successfully rooted and 89% of them were
 Hort. Environ. Biotechnol. 55(1):56-62. 2014.
Fig. 2. Eighteen-month-old plants, grown in the greenhouse, obtained from adventitious shoot induction in leaf segment culture.
successfully acclimatized under greenhouse conditions. For the
White Beauty, the multiplication rate was 3.5:1, which required
approximately 200 days for the production of 200 plants.
Approximately 98% of these plants were successfully rooted
and 96% of them were successfully acclimatized under
greenhouse conditions.
The multiplication, rooting, and acclimatization patterns
observed for Anthurium plantlets in the present study were
similar to those observed in other Anthurium cultivars (Atak
and elik, 2009; Martin et al., 2003). Every acclimatized plant
that was subsequently cultivated in pots grew vigorously and
showed no apparent morphological variations, maintaining
the same characteristics of the cultivars used as donor plants
(Fig. 2). For ornamental plants, monitoring horticultural traits
in the field is important to detect any somaclonal variations
which have occurred in the in vitro phase. This is important
because molecular markers, when available, are likely to fail
to detect somaclonal variations. In fact, RAPD markers failed
to separate mutants of Doritaenopsis orchid obtained from
tissue culture (Park et al., 2009). In Anthurium, SSR markers
have also failed to detect genetic fidelity in micropropagated
plants (Gantait and Sinniah, 2011).
We showed that leaf segments with the adaxial surface in
direct contact with the culture medium is the best explant
orientation for obtaining regenerated Anthurium plantlets.
We also demonstrated that obtaining calli and regenerated
plantlets from the leaf segments was genotype-dependent,
and was best achieved by using juvenile donor plants, espe-
cially for the recalcitrant White Beauty. Finally, BAP, and
not 2,4-D, in the culture medium was demonstrated to be the
best PGR to achieve the highest number of shoots and
plantlets per leaf segment. In conclusion, we showed that
direct regeneration of shoots from adult tissues is possible,
but few shoots and plantlets were obtained when only BAP
was added to the medium.
Acknowledgements: J. C. Cardoso acknowledges Vliet Flora
Co. for the financial support and experimental conditions
61
provided for this study. G. Habermann acknowledges the
Brazilian National Council for Scientific and Technological
Development (CNPq) for a research productivity fellowship
(CNPq Proc. 306119/2011-0).
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