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Micropropagation of self-heading Philodendron via direct shoot regeneration

Article in Scientia Horticulturae · June 2012

DOI: 10.1016/j.scienta.2012.04.011

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 Scientia Horticulturae 141 (2012) 23–29

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Micropropagation of self-heading Philodendron via direct shoot regeneration

F.C. Chen a , C.Y. Wang b , J.Y. Fang c,∗
a
Department of Plant Industry, National Pingtung University of Science and Technology, No.1 Shueh Fu Road, Neipu, Pingtung 91201, Taiwan
b
Southern Taiwan Service Center, Food Industry Research and Development Institute, No.31 Gongye 2nd Road, Annan District, Tainan 70955, Taiwan
c
Department 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 a b s t r a c t

Article history: The present study describes a direct shoot regeneration-based micropropagation procedure for the self-
Received 16 February 2012 heading cultivars of Philodendron. Three types of explant (i.e. leaf lamina, petiole, stem nodal segment)
Received in revised form 6 April 2012 were screened for their shoot induction potential following a three months treatment with 0.5 mg l−1 of
Accepted 10 April 2012
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
Keywords:
shoot production at frequencies of 2.8–11.1% in two of the cultivars tested. Stem nodal segments were the
Philodendron
most responsive among the three as shoots formed directly following the TDZ treatment at frequencies
Stem nodal segment
Leaf lamina
of 16.7–41.7% depending on the cultivar. When comparing the effectiveness of different cytokinins to
Petiole induce shoot proliferation on stem nodal segments, it was found that the 0.5 and 1 mg l−1 of kinetin (Kn)
Direct shoot regeneration and 6-benzyladenine (BA) treatments resulted in higher shoot formation percentages compared to the
Plant growth regulator 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.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction 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
The genus Philodendron is the second largest member of the contact dermatitis (Reffstrup and Boll, 1985). Micropropagation is
Araceae family and is composed of more than 500 species native to a comparatively more attractive means for Philodendron propaga-
tropical and subtropical America and the West Indies (Mayo et al., tion as a higher number of plants can be generated in a shorter time
1997). Philodendrons are highly appreciated for their attractive period compared to the conventional procedure. In addition, tissue
foliage and tolerance of interior environments and have been pro- culture of tropical foliage plants has been proposed as a means to
duced for use extensively in interiorscaping. Two distinct growth eliminate various systemic viral, fungal and bacterial diseases that
types can be found in Philodendron: the vining type which dom- are often prevalent in stock plants (Hartman, 1974; Henny, 1988).
inated foliage plant sales from the 1950s to the early 1970s, and Tissue culture has also led to improved plant forms compared to
the self-heading type which has become popular in the last 40 those propagated by traditional methods. It was found that tis-
years due to an increasing number of new hybrids with red, yel- sue culture-derived Dieffenbachia, Spathiphyllum and Syngonium,
low or orange foliage that were released to the market (Chen et al., also belonging to the Araceae family, produced a fuller and more
2002). In Taiwan, Philodendron constitutes an important share in compact plant when compared to those propagated by traditional
the foliage plant market and the raising popularities of self-heading cuttings (Conover, 1985).
cultivars have made them to rank among the top ten most popular Although tissue culture has been performed by private firms
plants in the floricultural trade. for the production and supply of Philodendron liners, their micro-
To fulfill grower’s demands for potted plants of Philodendron, propagation protocols are generally undisclosed to the public. Few
procedures for rapid propagation of elite cultivars are essential. procedures have been published to date for the micropropagation
Conventional propagation of Philodendron by stem cuttings is slow 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.
∗ Corresponding author. Tel.: +886 87703202x6615; fax: +886 87740446. These procedures were however constrained by the low quality
E-mail address: [email protected] (J.Y. Fang). 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 on the explant. Three replicate Petri dishes were used for each
to high concentrations of plant growth regulators (PGRs) used treatment with six explants in each dish.
for shoot proliferation (Jámbor-Benczúr and Márta-Riffer, 1990;
Vardja and Vardja, 2001; Sreekumar et al., 2001; Gangopadhyay 2.3. Effect of cytokinin type and concentration on shoot
et al., 2004), species and cultivars-dependent requirement of PGR proliferation
treatments for optimal shoot proliferation (Sreekumar et al., 2001),
and the low number of species and cultivars studied (notably the Three cytokinins, i.e. kinetin (Kn), BA and TDZ, at 0.5 and 1 mg l−1
self-heading types). The present study therefore aims at developing were tested for their effectiveness in inducing shoot proliferation in
an efficient micropropagation procedure for the self-heading cul- previously selected explants. Three replicate Petri dishes were used
tivars of Philodendron. The procedure is expected to achieve a high for each treatment with six explants in each dish. The percentage
shoot multiplication rate, obtain healthy plantlets and be readily of explants exhibiting growth and shoot formation, as well as the
applicable to different cultivars. To achieve this, the influence of number of shoots produced per responding explant was recorded
explant type, as well as PGR type and concentration on shoot induc- after three months. The regenerated shoots were divided into small
tion and proliferation was studied. The quality of the induced shoots shoot clusters and were transferred to PGR-free MS medium for
was evaluated based on their ability to root and to adapt to the ex shoot elongation during a period of two months.
vitro environment.
2.4. Effect of IBA concentration on root induction

2. Materials and methods Shoots from the best cytokinin treatment and obtained at the
end of the elongation stage with at least four leaves were individu-
2.1. Plant material and aseptic culture establishment 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,
Three commercial self-heading cultivars of Philodendron (i.e. 0.1, 0.5 and 1 mg l−1 of IBA. Ten replicated test tubes were used
‘Imperial Green’, ‘Imperial Red’ and ‘Imperial Rainbow’) were used in each medium treatment. The percentage of shoots producing
in the present study. The plants were purchased from a local nurs- roots, as well as the number and length of the induced roots were
ery and sprayed with fungicides Ridomil MZ and Mancozeb (1000× recorded after 30 days.
dilution) each for one week prior to explant excision and establish-
ment in vitro. After defoliation, shoot cuttings measuring 5–10 cm 2.5. Greenhouse acclimatization
long were washed under running tap water for 15 min and divided
into single nodal segments each containing a lateral bud. The nodal After two months on the rooting medium, well developed shoots
segments were then surface-sterilized with 70% EtOH for 1 min fol- (2–2.5 cm long; 4–6 leaves) with at least five roots were collected
lowed by 1% (v/v) sodium hypochlorite (NaOCl) containing several from the best IBA treatment for acclimatization. Rooted shoots were
drops of Tween-20 for 20 min on a rotary shaker. After three rinses carefully retrieved from the test tubes and washed under tap water
with sterile water and removal of the damaged ends, the explants to remove sticking medium from the roots. The plantlets were then
were cultured on MS (Murashige and Skoog, 1962) medium supple- transplanted into 10.1 cm plastic pots filled with a potting mixture
mented with 0.1 mg l−1 of 1-naphthaleneacetic acid (NAA), 1 mg l−1 (4:1) of peat moss and perlite. Potted plants were directly grown in
of 6-benzyladenine (BA), 3% (w/v) of sucrose and 0.7% (w/v) of agar. a partially shaded greenhouse with temperatures ranging from 25
The pH of the medium was adjusted to 5.7 prior to autoclaving. to 30 ◦ C, relative humidity of between 70 and 100%, and light inten-
Cultures were checked regularly for contaminations and those pre- sity of 35 ␮mol m−2 s−1 under a 10 h photoperiod. Plantlets were
sented apparent infection symptoms were immediately discarded. considered successfully acclimatized when these have produced at
The outgrowth of the lateral bud (i.e. approximately 2 cm long) least one new leaf after two months.
was excised from the nodal segments and subcultured on the same
medium for further shoot multiplication. Regenerated shoot clus- 2.6. Culture conditions, experimental design and data analysis
ters were divided and subcultured every eight weeks to build up
a stock shoot culture. Shoots from the fifth subculture and mea- The in vitro experiments were performed with an incuba-
suring approximately 3 cm in height were used for the subsequent tion temperature of 25 ± 2 ◦ C and a 16/8 h light/dark photoperiod
experiments. provided by cool fluorescent lamps at 40 ␮mol m−2 s−1 . All the
experiments were set up in a completely randomized design and
were conducted twice. Data were subjected to the analysis of vari-
2.2. Effect of explant and plant growth regulator types on the ance using SAS (SAS Institute Inc., 1999) software, and means were
growth and shoot induction percentages of three Philodedron separated by Duncan’s multiple range test at the 95% level (Duncan,
cultivars 1955).

The explants tested included leaf petiole (i.e. ∼5 mm in length), 3. Results

leaf lamina (∼8 mm × 3 mm section with two cuts made across
the midvein), and stem nodal segment (containing 2 nodes each 3.1. Effect of explant and plant growth regulator types on the
and measuring ∼4 mm in length). The explants were cultured growth and shoot induction percentages of three Philodendron
on MS medium supplemented with either 0.5 mg l−1 of 2,4- cultivars
dichlorophenoxyacetic acid (2,4-D), 0.5 mg l−1 of thidiazuron (TDZ)
or both. All the explants were placed horizontally on the medium Three months following the treatments, none of the explants
and the lamina explants were placed with their adaxial side grown on the PGR-free control medium showed any growth
touching the medium. The growth as well as the shoot induction (Table 1). In contrary, explants grown on PGR-containing media
percentages of the different explants was evaluated three months presented different growth patterns depending on the type of
following the treatments. Growth was recorded when any type of explants cultured. With the lamina explants, growth was initiated
new structures were produced from the original explant. Shoot by the enlargement of the explants followed by the production
induction was recorded when at least one visible shoot formed 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−1 2,4-D 0.5 mg l−1 TDZ 0.5 mg l−1 2,4-D+ 0.5 mg l−1 TDZ

‘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−1 2,4-D 0.5 mg l−1 TDZ 0.5 mg l−1 2,4-D+ 0.5 mg l−1 TDZ

‘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 * 0b * 11.1 ± 10.0b * 0a *
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 Green’ with the TDZ treatment. In addition, the growth percent-
develop further and convert into shoots. With the petiole explants, ages of petiole explants were significantly higher than the lamina
growth also manifested as small globular structures and occurred at explants in most of the cases. The stem nodal segments were the
the proximal ends of the petioles (Fig. 1b). Some of these protruding most responsive of the three explant types. Growth was apparent
globules later developed into shoots, appearing either individually in all the PGR treatments and cultivars tested. The percentages of
or in clusters. With the stem nodal explants, swelling of the nodes growth of the stem nodal explants were significantly higher then
was followed by the emergence of dormant axillary buds. In some the petiole explants, in exception of the 2,4-D treatment for ‘Impe-
cases, adventitious buds were formed at the base of the developing rial Green’ and the combined 2,4-D and TDZ treatment for ‘Imperial
axillary shoot to form a shoot cluster (Fig. 1c). Occasionally, roots Red’ where growth percentages were comparable between the two
were observed to develop from the node region. explant types.
The type of explants tested had an influence on the growth The growth responses of the different Philodendron cultivars
responses of the three Philodendron cultivars as shown in Table 1A. were also affected by the PGR treatments which they were sub-
Among the three explants tested, lamina explants were the least jected to (Table 1A). Growth was observed in petiole and stem
responsive as their growth was only observed following the 2,4-D nodal explants in all the three cultivars tested and following all
treatment (i.e. 2.8%) in ‘Imperial Red’ and following the combined the PGR treatments, except for ‘Imperial Green’ with the TDZ treat-
2,4-D and TDZ treatment in ‘Imperial Red’ (i.e. 36.1%) and ‘Impe- ment. It was found that the growth percentages of the different
rial Rainbow’ (i.e. 13.9%). The petiole explants were comparatively explants under the combined 2,4-D and TDZ treatment were sig-
more responsive as growth manifested in all the three cultivars nificantly higher than under single PGR treatments, except for the
and under different PGR treatment conditions except for ‘Imperial 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−1 of 2,4-D treatment. (b) Small globular structures occurred at the proximal ends
of an ‘Imperial Rainbow’ petiole explant following 0.5 mg l−1 of TDZ treatment. (c) Direct emergence of the dormant axillary buds in an ‘Imperial Red’ stem nodal segment
following 0.5 mg l−1 of 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- 3.3. Effect of IBA concentration on root induction
gle PGR treatments, it can be seen that the growth percentage of
the different explants was comparable in most cases except for the Since root formation was spontaneous in Kn-derived shoots, this
stem nodal explants of ‘Imperial Red’ and ‘Imperial Rainbow’, and experiment used only rootless shoots derived from the 0.5 mg l−1
the petiole explants of ‘Imperial Green’ where the 2,4-D treatment BA treatment. IBA at 0 (control), 0.1, 0.5 and 1 mg l−1 were tested for
produced a higher growth response than the TDZ treatment. their influence on the frequency of root induction, the number of
As for the growth response, both the explant and PGR types roots produced per shoot, as well as the root length in three Philo-
tested influenced the shoot induction response of the three Philo- dendron cultivars. It was observed that 100% of the shoots tested
dendron cultivars. It was observed that the stem nodal explants produced roots following the different IBA treatments (including
exhibited a higher shoot induction percentage compared to the the control) so only the number of roots produced per shoot as well
petiole explants in all the three cultivars tested, and no shoot as the length of the roots produced are presented in Table 3. A sig-
was formed on the lamina explants (Table 1B). For the stem nodal nificantly higher number of roots were generated on shoots in the
explants, the TDZ treatment produced a significantly higher shoot presence of 0.1–1 mg l−1 IBA (i.e. 6.2–7.0) compared to the control
induction response than the 2,4-D and combined 2,4-D and TDZ (i.e. 3.0) for ‘Imperial Green’. For ‘Imperial Red’, shoots subjected to
treatments in all the three cultivars. IBA treatments at 0.5 and 1 mg l−1 produced 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
3.2. Effect of cytokinin type and concentration on shoot per shoot in the presence of 0.5 and 1 mg l−1 IBA compared to only
proliferation 4.8 and 3.5 roots with the 0.1 mg l−1 IBA treatment and the control
respectively. Shoots in the control produced significantly longer
Stem nodal explants were selected because of their high poten- roots compared to those in the IBA treatments for both ‘Imperial
tial for inducing shoots as observed in the previous experiment. Green’ and ‘Imperial Red’. However, no significant difference in
Three cytokinins (i.e. kinetin, BA and TDZ) at two different con- root length was found between the different IBA treatments and
centrations (i.e. 0.5 and 1 mg l−1 ) were tested for their influence the control in ‘Imperial Rainbow’.
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 3.4. Greenhouse acclimatization
different cytokinin treatments was observed for ‘Imperial Red’ and
‘Imperial Rainbow’, but Kn and BA treatments gave significantly A total of 120 rooted shoots of the three cultivars following the
higher growth response than the control treatment in the case of 0.5 and 1 mg l−1 of IBA treatments were used for acclimatization in
‘Imperial Green’. the greenhouse. Results showed that all the acclimatized plantlets
Shoot formation was observed following all the cytokinin treat- grew vigorously in the greenhouse with 100% survival. New leaves
ments except the TDZ treatments in ‘Imperial Green’. For all were produced and leaf variegation in ‘Imperial Red’ became more
cultivars, production of shoot clusters at the nodes was observed in apparent with time (Fig. 3c).
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 4. Discussion
(Fig. 2b), and the shoots developed roots on the Kn-supplemented
medium (Fig. 2a). When TDZ was used, shoot formation was initi- Contamination is frequently observed during the tissue culture
ated with the appearance of green buds, which then later grew into of ornamental aroids such as Aglaonema (Chen and Yeh, 2007),
leaf-like structures in the bud tips and developed into leafy shoots Anthurium (Kunisaki, 1980), Dieffenbachia (Brunner et al., 1995),
for ‘Imperial Red’ and ‘Imperial Rainbow’, or which failed to convert Spathyphyllum and Syngonium (Kneifel and Leonhardt, 1992), Zan-
into shoots in the case of ‘Imperial Green’ (Fig. 2c). tesdeschia (Kritzinger et al., 1998), as well as Philodendron (Fisse
For ‘Imperial Green’, a significantly higher percentage of shoot et al., 1987). Therefore, the present study was initiated by estab-
formation was observed with the 0.5 mg l−1 Kn treatment (i.e. lishing an aseptic shoot stock culture from nodal axillary buds. The
88.8%) compared to the 1 mg l−1 BA treatment (i.e. 58.3%). But regenerated shoots were then divided to provide the leaf lamina,
there was no significant difference between the 0.5 and 1 mg l−1 Kn petiole and stem nodal explants.
treatments and the 0.5 mg l−1 BA treatment. The number of shoots The use of leaves and stem nodal segments as explants for
produced per explant was significantly higher following the BA micropropagation has been reported in many ornamental Aroid
treatments (i.e. 48.7–49.4) compared to Kn treatments (i.e. 6.5–7.8). species (Martin et al., 2003; Qu et al., 2002; Thao et al., 2003;
For ‘Imperial Red’, the shoot formation percentage was significantly Azza and Khalafalla, 2010; Sreekumar et al., 2001), and different
higher following the Kn and BA treatments compared to the TDZ explants were found with different shoot regeneration potentials.
treatments. Although no significant difference in shoot formation Results from this study showed that the growth and shoot induc-
frequency was noted between the Kn and BA treatments, the pres- tion responses of Philodendron were greatly influenced by the type
ence of BA induced a significantly higher number of shoots (i.e. of explant used. Lamina explants were the least responsive of
46.3–47.4) than Kn (i.e. 2.5–2.9). For ‘Imperial Rainbow’, no signifi- the three explant types tested as growth was only observed for
cant difference in shoot formation frequency was recorded among two of the three cultivars when 2,4-D or both the 2,4-D and TDZ
the different cytokinin treatments. However, the highest number were supplemented to the medium. In any case, no shoots were
of shoots produced per explant was found with the 0.5 mg l−1 BA formed. In contrast, Martin et al. (2003) reported that direct shoot
treatment (i.e. 50.4), followed by the 1 mg l−1 BA treatment (i.e. regeneration can be achieved from young lamina explants in two
40.8) and the Kn and TDZ treatments (i.e. 5.3–6.9). Anthurium andreanum cultivars. Similarly, callus formation and
Clumps of small shoots produced on the 0.5 mg l−1 BA- subsequent shoot regeneration was observed with lamina explants
supplemented medium were transferred onto PGR-free MS of Dieffenbachia cv. Camouflage (Shen et al., 2007). In Anthurium
medium for shoot elongation. Two months after transfer, the small andraeanum hybrids, embryogenic calli were formed on whole of
shoots increased in size and were 100% normal looking in all the the leaf blade explants (Kuehnle et al., 1992). It is possible that
cultivars (Fig. 3a). 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 ± 15.2d 0c 0b 47.2 ± 9.5b 36.1 ± 9.5b 3.9 ± 0.8b 66.7 ± 9.6a 53.3 ± 8.1a 6.9 ± 2.0c
1 TDZ 36.1 ± 7.3cd 0c 0b 69.4 ± 9.5ab 41.7 ± 13.3b 4.7 ± 1.5b 75.0 ± 22.6a 44.4 ± 19.9a 6.6 ± 3.7c
0.5 Kn 88.8 ± 7.9a 88.8 ± 7.9a 7.8 ± 2.4b 73.3 ± 10.9ab 73.3 ± 10.9a 2.9 ± 1.1b 62.2 ± 12.3a 62.2 ± 12.3a 5.3 ± 0.7c
1 Kn 77.8 ± 9.9ab 77.8 ± 9.9ab 6.5 ± 1.5b 72.2 ± 11.7ab 72.2 ± 11.7a 2.5 ± 0.4b 63.9 ± 7.3a 63.9 ± 7.3a 6.2 ± 1.3c
0.5 BA 66.7 ± 12.7ab 66.7 ± 12.7ab 48.7 ± 7.4a 80.6 ± 11.3a 80.6 ± 11.3a 47.4 ± 5.9a 55.6 ± 15.7a 55.6 ± 15.7a 50.4 ± 3.0a
1 BA 58.3 ± 19.9bc 58.3 ± 19.9b 49.4 ± 3.2a 73.3 ± 14.6ab 73.3 ± 14.6a 46.3 ± 1.0a 61.1 ± 7.9a 61.1 ± 7.9a 40.8 ± 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 explants. The morphogenic superiority of petiole explants over
lamina explants as direct shoot formation was achieved with TDZ lamina explants was also demonstrated in Epipremnum aureum
in ‘Imperial Red’ and ‘Imperial Rainbow’, and with 2,4-D in ‘Impe- where shoot induction from petiole explants was greater than
rial Red’, treatments for which no shoot was formed on the lamina 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−1 of 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 ± 0.6b 1.1 ± 0.1a 3.6 ± 0.5b 1.0 ± 0.1a 3.5 ± 0.5b 1.0 ± 0.2a
0.1 6.2 ± 0.8a 0.4 ± 0.1c 4.8 ± 0.6b 0.6 ± 0.1b 4.8 ± 0.8b 1.3 ± 0.2a
0.5 7.0 ± 0.8a 0.8 ± 0.1b 9.3 ± 1.2a 0.7 ± 0.1b 7.9 ± 1.4a 1.2 ± 0.1a
1 6.3 ± 0.9a 0.7 ± 0.2b 10.8 ± 1.4a 0.6 ± 0.1b 7.1 ± 1.3a 1.0 ± 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 In this study, the efficiency in shoot proliferation of TDZ was
explants, whereas no embryo formation was observed from leaf inferior to BA and Kn. This is in opposition to many studies. For
explants (Zhang et al., 2006). Stem nodal segments proved to be example, TDZ was the best cytokinin for pothos regeneration (supe-
the most responsive explants in the present study. Growth was rior to zeatin and 2iP) (Qu et al., 2002). TDZ was more effective
observed following all the PGR treatments in all the three cultivars, in multiple shoot proliferation of Alocasia amazonica compared to
and shoot formation was recorded in seven out of the nine PGR BA and Kn (Jo et al., 2008). It was found in the present study that
treatments. TDZ-treated explants tend to induce a large amount of globules-like
The choice of an appropriate PGR to induce shooting in Philo- structures at the level of the nodes which seemed to have difficulty
dendron was essential as shown with the stem nodal explants in to convert into shoots at a later stage. Reasons for the low shoot
this study. The highest shoot induction percentage was achieved conversion success of TDZ might include first the dual auxin- and
using the TDZ treatment, with an average of 16.7–41.7% depend- cytokinin-like activities of the TDZ (Singh et al., 2003) which might
ing on the cultivar. Shoots were also formed following the lead to an increase in endogenous levels of auxins (Hutchinson
2,4-D treatment in all the three cultivars, but at lower fre- et al., 1996), and second higher concentrations of TDZ have some-
quencies (i.e. 2.8–8.3%) compared to the TDZ treatment. Shoot times been associated with morphological abnormalities in several
formation on cytokinin-free medium has also been reported species (Huetteman and Preece, 1993).
in other Aroid species. In Aglaonema for instance, shoots were In terms of the length of shoots produced, it was found that the
observed to form from the inflorescence explants on media shoots obtained following the Kn treatment were comparatively
devoid of cytokinins (Yeh et al., 2007). Similar observation was larger than those from the BA treatment. The promotive effect of
made with Dieffenbachia where shoot buds differentiated into Kn on shoot length was also observed with D. compacta and Dif-
shoots on media without cytokinin (Azza and Khalafalla, 2010). fenbachia picta ‘Tropica’ (El-sawy and Bakheet, 1999). The small
Despite the high growth response (i.e. 88.9–97.2%) of the stem sized-shoots found in the BA treatment can be attributed to the
nodal explants under the combined 2,4-D and TDZ treatment, concentration of BA used which might restrain the shoots from
their shoot induction percentage was only 0-2.8% for the three outgrowing. Similar observation was made in another Philodendron
cultivars. This was not the case for E. aureum where a com- study where the use of BA (up to 2.5 mg l−1 ) had an inhibitory effect
bination of 0.18 mg l−1 NAA and 2.20 mg l−1 TDZ allowed shoot on root formation of the shoots at later stage of culture (Sreekumar
regeneration (Qu et al., 2002). Shoot regeneration was also et al., 2001). Similarly, although BA (at 4.95 mg l−1 ) allowed rapid
obtained for A. andreanum lamina explants following a com- multiplication of Philodendron ‘Xanadu’, a gradual decline of BA
bination of 0.25 mg l−1 BA, 0.19 mg l−1 IAA and 0.09 mg l−1 Kn from 4.95 down to 0.45 mg l−1 was essential for obtaining healthy
treatments (Martin et al., 2003). It appears that the PGR require- plantlets otherwise BA would arrest growth and development of
ment for shoot regeneration varies from one plant species to the multiplied shoots (Gangopadhyay et al., 2004). It was observed
another. that the BA-derived shoots could successfully elongate to produce
Since the application of TDZ alone has proved successful to normal shoots in eight weeks, suggesting that the low BA concen-
induce shoots from the stem nodal explants of Philodendron, the trations used in the present study not only allowed a high shoot
subsequent experiment was conducted to compare the efficiency multiplication rate but also allowed healthy shoots to be produced.
of different cytokinins on shoot proliferation. Cytokinins such as BA, The success of micropropagation on a commercial level relies
Kn and TDZ have frequently been used in the shoot proliferation of on adequate rooting of the shoots as well as high survival rate
different members of the Araceae family (Sreekumar et al., 2001; of the acclimatized plantlets. IBA has commonly been employed
Dewir et al., 2006; Jo et al., 2008; Kozak and Stelmaszczuk, 2009; for the in vitro rooting of many Araceae species, such as 0.5 mg l−1
Azza and Khalafalla, 2010; Mariani et al., 2011). This study showed IBA for nine Dieffenbachia cultivars (Zhu et al., 1999), 1.5 mg l−1 of
that both the cytokinin type and concentration had a significant IBA for D. compacta (Azza and Khalafalla, 2010), and 3 mg l−1 of
impact on shoot regeneration in Philodendron. For ‘Imperial Green’ IBA for Aglaonema ‘Cochin’ (Mariani et al., 2011). The rooting of
and ‘Imperial Red’, the BA and Kn treatments showed a higher shoot BA-derived shoots was evaluated in the present study using IBA
formation percentage than the TDZ treatment, whereas no sig- at 0.1–1 mg l−1 . Results showed 100% of rooting on all the media
nificant difference was observed between the different cytokinin (including the control) four weeks after the treatment. In general,
treatments for ‘Imperial Rainbow’. In terms of the number of shoot the frequency of rooting was higher in 0.5–1 mg l−1 IBA-treated
produced per explant, BA proved superior to Kn and TDZ in all three shoots compared to 0–0.1 mg l−1 IBA-treated shoots. However, the
cultivars. No difference was noticed between the 0.5 and 1 mg l−1 control treatment produced longer roots than with the different IBA
BA treatments in two of the three cultivars. The superiority of BA treatments, except for ‘Imperial Rainbow’ where no difference in
over other cytokinins has already been reported in Spathiphyllum root length was observed between the different treatments. Longer
cannifolium (Dewir et al., 2006), Zantedeschia aethiopica (Kozak and roots were also produced in the IBA-free treatment in Diffenbachia
Stelmaszczuk, 2009), Caladiums bicolor (Ali et al., 2007), Dieffen- compacta (Azza and Khalafalla, 2010). In the present study, all the
bachia compacta (Azza and Khalafalla, 2010), and six Philodendron shoots were successfully acclimatized with a survival rate of 100%
cultivars (Sreekumar et al., 2001). in all the three cultivars.
 F.C. Chen et al. / Scientia Horticulturae 141 (2012) 23–29 29

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