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Citation: Klanrit, P.; Kitwetcharoen,
H.; Thanonkeo, P.; Thanonkeo, S. In
Vitro Propagation of Philodendron
erubescens ‘Pink Princess’ and Ex
Vitro Acclimatization of the Plantlets.
Horticulturae 2023,9, 688. https://
doi.org/10.3390/horticulturae9060688
Received: 15 May 2023
Revised: 2 June 2023
Accepted: 8 June 2023
Published: 10 June 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
horticulturae
Article
In Vitro Propagation of Philodendron erubescens ‘Pink Princess’
and Ex Vitro Acclimatization of the Plantlets
Preekamol Klanrit 1,2 , Haruthairat Kitwetcharoen 1, Pornthap Thanonkeo 1,2 and Sudarat Thanonkeo 3, *
1Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand;
kpreek@kku.ac.th (P.K.); kharuthairat29@gmail.com (H.K.); portha@kku.ac.th (P.T.)
2Fermentation Research Center for Value Added Agricultural Products (FerVAAPs), Khon Kaen University,
Khon Kaen 40002, Thailand
3Walai Rukhavej Botanical Research Institute, Mahasarakham University, Maha Sarakham 44150, Thailand
*Correspondence: sudarat.t@msu.ac.th; Tel.: +66-862268160
Abstract:
This study describes the
in vitro
propagation and ex vitro acclimatization of Philoden-
dron erubescens pink princess, one of the most popular ornamental variegated foliage plants. For
shoot proliferation, the protocorm-like bodies of the Philodendron pink princess were cultured
on solid Murashige and Skoog (MS) media supplemented with 6-benzylaminopurine (BAP) and
1-naphthaleneacetic
acid (NAA) at different concentrations. The results revealed that supplementa-
tion with BAP alone at a concentration of 1.0 mg/L yielded the maximum number of shoots and
leaves. Furthermore, the application of BAP at 1.0 mg/L significantly enhanced the shoot prolifera-
tion of Philodendron pink princess when grown in liquid MS medium, yielding 11.2 shoots/explant
and 4.7 leaves/explant. When the established microshoots were subjected to root induction using
solid MS media supplemented with different kinds and concentrations of auxins, indole-3-butyric
acid (IBA) at 3 mg/L resulted in the highest number of roots (3.2 roots/explant) and longest root
length (1.9 cm). Three supporting materials, i.e., peat moss, vermiculite, and perlite, were used
as planting media for the ex vitro acclimatization of the Philodendron pink princess plantlets. The
results demonstrated that the
in vitro
plantlets acclimatized and exhibited a relatively high survival
frequency in all planting media without morphological abnormalities. Peat moss outperformed all
other types of planting media in terms of sustaining the vegetative growth of the plantlets. In the
future, the approach established in this study could be employed for the extensive production of
Philodendron pink princess.
Keywords: Philodendron; plant tissue culture; plant growth regulator; plant acclimatization
1. Introduction
Philodendron is the second largest and most diverse genus of the Araceae family, consist-
ing of more than 500 species [
1
]. Philodendron species are native to tropical and subtropical
regions of the Americas and West Indies [
2
], and they are widely diverse in morphological
characteristics, such as leaf size (i.e., from small to large), leaf shape (i.e., from heart-shaped
to palm-like), and leaf color (i.e., from green to red and burgundy). Furthermore, these
plants’ growth habits vary from climbing to arborescent or tree-like structures, making
them ideal for ornamental desk plants, hanging baskets, totems, and potted plants [
1
,
3
–
5
].
Due to their attractive foliage and the ability to survive interior environments, Philodendron
species are the most popular in the foliage plant market, especially in Thailand.
Philodendron erubescens, also known as Philodendron pink princess, is a hybrid species
among the most popular ornamental variegated foliage plants because it is rare and expen-
sive [
6
]. Additionally, the multicolored heart-shaped leaves of this plant, dark purplish-
green with contrasting pink variegation, make it more attractive to the grower. The con-
ventional propagation of Philodendron pink princess is mainly via stem cutting and seed.
However, because of its slow growth, short internodes, and large stems and because the
Horticulturae 2023,9, 688. https://doi.org/10.3390/horticulturae9060688 https://www.mdpi.com/journal/horticulturae
Horticulturae 2023,9, 688 2 of 10
plant seeds are also rather short-lived, these propagation procedures are not chosen for
this plant. In addition, plants developed from seed or typical stem cuttings also produce
few lateral shoots. As a result of the limitations of the conventional propagation of this
plant,
in vitro
propagation techniques for commercial production were introduced, as
they facilitate the continuous generation of plants with a high degree of uniformity and
quality in a short period [
7
]. In addition to eliminating systemic diseases caused by plant
pathogens, such as viruses, fungi, and bacteria,
in vitro
propagation produces fuller and
more compact plants than traditional methods [8].
Generally,
in vitro
propagation comprises four major steps: (1) the initiation stage, in
which the explants are surface-sterilized and transferred to the culture medium; (2) the
multiplication stage, in which the number of plant propagules is multiplied through re-
peated subcultures until the desired number is reached; (3) the rooting stage, in which
the explants are subjected to root induction; and (4) the acclimatization stage, in which
the
in vitro
plantlets are exposed to ex vitro environments [
9
]. The successful develop-
ment of
in vitro
plantlets depends on numerous parameters, including the composition of
the culture medium, the environmental conditions, and the plant species. Plant growth
regulators (PGRs) are among the most critical factors influencing plant morphogenesis;
specifically, auxin and cytokinin, the most widely used PGRs in micropropagation, play
crucial roles in shoot and root proliferation [
7
,
10
]. The types and concentrations of PGRs
used are mostly determined by the plant species, the types of plant organs and tissues used,
and the purpose of cultivation [
9
]. In addition, the types of cultivation systems also have
an impact on
in vitro
culture. Liquid culture systems are reported to be more cost-effective
for in vitro propagation than solid or gel cultures [11].
Several plant species of the genus Philodendron have been propagated via
in vitro
cultures, such as Philodendron oxycardium [
12
], Philodendron cannifolium [
13
], Philodendron
selloum [
14
,
15
], Philodendron bipinnatifidum [
16
], Philodendron xanadu [
17
], and Philodendron
birkin [
10
], but less information is available for the
in vitro
propagation of Philodendron
erubescens cv. pink princess. Only one study on the
in vitro
propagation of Philodendron
erubescens cv. red emerald has been reported since 1998 [
6
]. Therefore, this study aimed to
establish an efficient
in vitro
propagation procedure for Philodendron pink princess using
the protocorm-like body as the starting material or explant. The effects of auxins and
cytokinins on
in vitro
shoot and root proliferation were determined in solid and liquid
culture systems. In addition, the quality of the
in vitro
plantlets was also evaluated based
on their ability to adapt to ex vitro environments.
2. Materials and Methods
2.1. Chemical and Plant Materials
Murashige and Skoog (MS) basal medium was purchased from PhytoTech Labs,
Kansas, MO, USA. In addition, 6-benzylaminopurine (BAP), 1-naphthaleneacetic acid
(NAA), indole-3-butyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D) were ob-
tained from Sigma Aldrich Corporation, St. Louis, MO, USA. Perlite, vermiculite, and peat
moss were purchased from Bee Garden and Farm Co., Ltd., Khon Kaen, Thailand.
The protocorm-like bodies of Philodendron pink princess were obtained from Mrs.
Nuntipa Khumkarjorn, Udon Thani, Thailand. The plants were maintained on MS basal
medium supplemented with 2.0 mg/L BAP and 0.5 mg/L NAA and cultivated in a standard
culture room at 25
±
2
◦
C with a light intensity of 2000 Lux and a photoperiod of 16 h.
Before being used, they were subcultured on fresh solid MS basal medium supplemented
with BAP and NAA and cultivated under the abovementioned conditions for 30 days.
2.2. Effect of PGRs on Shoot Proliferation
Protocorm-like bodies of Philodendron pink princess that measured approximately
0.5 cm were selected and placed on MS basal agar medium supplemented with combina-
tions of BAP (0, 1.0, and 2.0 mg/L) and NAA (0, 0.5, and 1.0 mg/L). All of the explants
were cultured in a standard culture room at 25
±
2
◦
C with a light intensity of 2000 Lux
Horticulturae 2023,9, 688 3 of 10
and a photoperiod of 16 h. The numbers of shoots and leaves were recorded after 30 days
of cultivation.
A comparative study on the effect of PGRs on shoot proliferation between solid and
liquid culture systems was also evaluated. The selected protocorm-like bodies of Philoden-
dron pink princess were cultured using solid and liquid MS basal media supplemented
with 1.0 mg/L BAP and different concentrations of NAA (0, 0.5, and 1.0 mg/L). For the
solid culture system, the explants were cultivated in a standard culture room at 25
±
2
◦
C
with a light intensity of 2000 Lux and a photoperiod of 16 h. For the liquid culture system,
the explants were cultivated in a controlled incubator shaker at 110 rpm and 25
±
2
◦
C
with a light intensity of 2000 Lux and a photoperiod of 16 h. After 30 days of cultivation,
the numbers of shoots and leaves were recorded.
Ten explants per treatment were used, and the experiments were conducted twice.
2.3. Effect of Auxins on Root Proliferation
The microshoots established from the best shoot proliferation treatment were selected
and individually placed in a culture vessel containing 30 mL of MS basal medium sup-
plemented with NAA, IBA, and 2,4-D at 0, 0.5, 1.0, 2.0, and 3.0 mg/L. The explants were
cultivated in a standard culture room with a light intensity of 2000 Lux, a photoperiod
of 16 h, and a temperature of 25
±
2
◦
C. The number and length of the induced roots
were recorded after 30 days of cultivation. The experiment was repeated twice, each with
ten replicates.
2.4. Ex Vitro Acclimatization of the Plantlets
Well-developed plantlets from the
in vitro
culture with a height of 1.0–1.5 cm,
4–5 leaves
,
and at least three roots were selected for ex vitro acclimatization. The plantlets were
carefully collected from the culture vessel, and their roots were rinsed with tap water to
remove the adhering culture medium. The resulting plantlets were transplanted into 5.0 cm
diameter plastic pots filled with perlite, vermiculite, and peat moss. The potted plants were
placed directly in a growth chamber at 25
±
2
◦
C with a 16 h light period at approximately
250
µ
mol/m.s light intensity and 45
−
55% relative humidity. The survival rate, plant height,
number of leaves and roots, and root length were recorded after 45 days of cultivation. The
experiments were performed twice, each with ten replicates.
2.5. Experimental Design and Statistical Analysis
A completely randomized design (CRD) was used throughout this study. Experimental
data were collected and subjected to analysis of variance (ANOVA) using the SPSS program
for Windows. The results were expressed as the mean
±
standard deviation (SD), and the
means of each treatment were compared using Duncan’s Multiple Range Test (DMRT) at
the 95% level or a probability of p≤0.05.
3. Results and Discussion
3.1. Effect of PGRs on Shoot Proliferation
Cytokinins are adenine-derived, small-molecule PGRs that play significant roles in
plant cell division, cell elongation and enlargement, plant morphogenesis, nutrient allo-
cation, and plant adaptations to various abiotic stresses [
18
–
20
]. Several cytokinins are
available for plant micropropagation, and their biological functions in plant cells depend
on several factors, such as plant species, plant age, and explant type [
20
]. Previous studies
have demonstrated that BAP is more effective for shoot induction in several species of
Philodendron, such as Philodendron cannifolium [
13
], Philodendron tuxtlanum [
21
], and Philo-
dendron sp. [
7
], compared with other cytokinins. Furthermore, a recent study also reported
that combining cytokinins and auxins stimulated the elongation of shoots and yielded a
more significant shoot number, shoot length, and fresh weight of several crops, including
Philodendron bipinnatifidum, than treatments with cytokinins alone [
16
,
22
]. Thus, the effect
of cytokinin (BAP) and auxin (NAA) at different concentrations on the shoot prolifera-
Horticulturae 2023,9, 688 4 of 10
tion of Philodendron erubescens pink princess was investigated in this study. Table 1and
Figure 1
show that treatment with BAP alone at 1.0 mg/L (Treatment 4) yielded the highest
numbers of shoots and leaves, accounting for 7.7 shoots/explant and 4.1 leaves/explant,
compared with other BAP concentrations. A high concentration of BAP (2.0 mg/L) tended
to reduce the number of both shoots and leaves, consistent with the finding reported by
Thao et al. [
23
], who observed a reduction in shoot multiplication and the formation of
abnormal shoots of Alocasia sp. when cultured under a high concentration of cytokinin.
Treatment with NAA alone (Treatments 1–3) resulted in fewer shoots and leaves than the
BAP treatments, suggesting that BAP was more effective in inducing Philodendron pink
princess shoot proliferation than NAA. Generally, NAA, a member of the auxin family,
plays an essential role in plant cell growth, differentiation, and development, specifically
the formation of roots [
24
,
25
]. Although auxins have been reported to stimulate shoot
elongation [
22
], the application of NAA alone did not significantly improve the shoot for-
mation of Philodendron pink princess compared to the control without the supplementation
of PGRs (Treatment 1).
Table 1. Effects of BAP and NAA on the shoot proliferation of Philodendron pink princess.
Treatment PGRs Number of Shoots
(Shoots/Explant)
Number of Leaves
(Leaves/Explant)
BAP (mg/L) NAA (mg/L)
1 0.0 0.0 4.4 ±1.2 ab 2.4 ±0.5 b
2 0.0 0.5 5.5 ±1.9 ab 2.6 ±0.5 b
3 0.0 1.0 4.2 ±0.9 b2.4 ±0.5 b
4 1.0 0.0 7.7 ±1.1 a4.1 ±0.7 a
5 1.0 0.5 7.0 ±1.8 a3.8 ±0.6 a
6 1.0 1.0 6.6 ±1.5 ab 3.7 ±0.7 a
7 2.0 0.0 6.3 ±1.4 ab 3.5 ±0.7 a
8 2.0 0.5 6.4 ±1.8 ab 3.5 ±0.7 a
9 2.0 1.0 2.7 ±1.1 b2.3 ±0.5 b
Means
±
SDs followed by different letters within a column are significantly different at p
≤
0.05 based on
DMRT analysis.
Considering the combinations of BAP and NAA, these treatments did not exert a
synergistic effect on shoot proliferation compared with treatment using BAP at 1.0 mg/L
alone. However, among the different combinations, BAP at 1.0 mg/L and NAA at 0.5 mg/L
produced a greater number of shoots (7.0 shoots/explant) and leaves (3.8 leaves/explant)
than the other treatment combinations. Notably, the combination of BAP at 2 mg/L and
NAA at 1.0 mg/L (Treatment 9) yielded the lowest number of shoots (2.7 shoots/explant)
and leaves (2.3 leaves/explant) compared to the control and other treatments. This obser-
vation could be attributed to a hormonal imbalance, which may alter the action of another
hormone, leading to the suppression of shoot and leaf formation and growth [22,26].
Compared with other studies, the results in the current study differ from those re-
ported for Philodendron selloum [
14
] and Philodendron bipinnatifidum [
16
]. For Philodendron
selloum, the combination of 6-benzyladenine (BA) at 8 mg/L and NAA at 0.4 mg/L yielded
the highest number of shoots (10.0 shoots/explant), longest shoot lengths (9.8 cm), and
number of leaves (5.7 leaves/explant) after 12 weeks of cultivation compared with cy-
tokinin treatment alone or other combinations of BA and NAA, while a combination of BAP
at 1.0 mg/L and IBA at 0.5 mg/L produced the highest number of shoots of Philodendron
bipinnatifidum (10.9 shoots/explant) after 6 weeks of cultivation. According to Chen et al. [
8
],
BA at a concentration of 0.5 mg/L yielded the highest number of shoots of Philodendron
imperial green (48.7 shoots/explant), imperial red (47.4 shoots/explant), and imperial
rainbow (50.4 shoots/explant). These data suggest that different plant species respond
differently to different types and concentrations of PGRs. Based on the current findings,
BAP at 1.0 mg/L and combinations of BAP (1.0 mg/L) and NAA (0.5 and 1.0 mg/L) were
selected to further examine their effect on shoot proliferation in a liquid culture system.
Horticulturae 2023,9, 688 5 of 10
Horticulturae 2023, 9, x FOR PEER REVIEW 5 of 11
bipinnatifidum (10.9 shoots/explant) after 6 weeks of cultivation. According to Chen et al.
[8], BA at a concentration of 0.5 mg/L yielded the highest number of shoots of Philodendron
imperial green (48.7 shoots/explant), imperial red (47.4 shoots/explant), and imperial rain-
bow (50.4 shoots/explant). These data suggest that different plant species respond differ-
ently to different types and concentrations of PGRs. Based on the current findings, BAP
at 1.0 mg/L and combinations of BAP (1.0 mg/L) and NAA (0.5 and 1.0 mg/L) were se-
lected to further examine their effect on shoot proliferation in a liquid culture system.
Figure 1. Shoot proliferation of Philodendron pink princess on MS media supplemented with differ-
ent concentrations of BAP and NAA after 30 days of cultivation.
Table 2 and Figure 2 summarize the effects of PGRs on the shoot proliferation of Phil-
odendron pink princess in liquid MS medium. The MS medium without PGRs produced a
relatively low number of shoots and leaves, while the MS medium supplemented with 1.0
mg/L BAP yielded the highest number of shoots (11.2 shoots/explant) and leaves (4.7
leaves/explant). Furthermore, the combinations of BAP and NAA produced fewer shoots
and leaves than the treatment with BAP alone. Notably, the combination of BAP at 1.0
mg/L and NAA at 1.0 mg/L produced the lowest numbers of shoots (4.9 shoots/explant)
and leaves (3.1 leaves/explant) when cultured in liquid medium, and the established mi-
croshoots displayed abnormal and hyperhydricity symptoms, similar to the observation
in Alkanna tinctoria cultured in medium supplemented with high concentrations of cyto-
kinins and auxins [27]. Another study conducted by Ziv and Ariel [28] discovered that
Philodendron hastatum burgundy leaves developed necrosis during the proliferation stage
in agitated liquid culture.
When the shoot proliferation of Philodendron pink princess was compared between
solid and liquid culture systems, the liquid culture system produced a greater number of
shoots and leaves than solid culture, except when the liquid culture used a combination
of 1.0 mg/L BAP and 1.0 mg/L NAA (Tables 1 and 2). This finding revealed that the liquid
Figure 1.
Shoot proliferation of Philodendron pink princess on MS media supplemented with different
concentrations of BAP and NAA after 30 days of cultivation.
Table 2and Figure 2summarize the effects of PGRs on the shoot proliferation of
Philodendron pink princess in liquid MS medium. The MS medium without PGRs produced
a relatively low number of shoots and leaves, while the MS medium supplemented with
1.0 mg/L BAP yielded the highest number of shoots (11.2 shoots/explant) and leaves
(4.7 leaves/explant
). Furthermore, the combinations of BAP and NAA produced fewer
shoots and leaves than the treatment with BAP alone. Notably, the combination of BAP at
1.0 mg/L and NAA at 1.0 mg/L produced the lowest numbers of shoots (4.9 shoots/explant)
and leaves (3.1 leaves/explant) when cultured in liquid medium, and the established mi-
croshoots displayed abnormal and hyperhydricity symptoms, similar to the observation in
Alkanna tinctoria cultured in medium supplemented with high concentrations of cytokinins
and auxins [
27
]. Another study conducted by Ziv and Ariel [
28
] discovered that Philo-
dendron hastatum burgundy leaves developed necrosis during the proliferation stage in
agitated liquid culture.
When the shoot proliferation of Philodendron pink princess was compared between
solid and liquid culture systems, the liquid culture system produced a greater number of
shoots and leaves than solid culture, except when the liquid culture used a combination of
1.0 mg/L BAP and 1.0 mg/L NAA (Tables 1and 2). This finding revealed that the liquid
culture system was more effective for shoot multiplication than the solid culture system,
possibly due to suitable aeration and the continuous contact of the explants with the culture
medium, which allows for a continual supply and high absorption of nutrients [
16
]. The
growth enhancement of explants using a liquid culture system has also been reported for
several Araceae species, such as A. amazonica [29] and Spathiphyllum cannifolium [30].
Horticulturae 2023,9, 688 6 of 10
Table 2. Effect of BAP and NAA on the shoot proliferation of Philodendron pink princess cultured in
liquid MS medium for 30 days.
BAP (mg/L) NAA (mg/L)
Liquid MS Medium
Number of Shoots
(Shoots/Explant)
Number of Leaves
(Leaves/Explant)
0.0 0.0 5.0 ±0.9 c3.3 ±0.8 c
1.0 0.0 11.2 ±2.0 a4.7 ±0.5 a
0.5 8.1 ±2.4 b4.0 ±0.7 b
1.0 4.9 ±1.2 c3.1 ±0.7 c
Means
±
SDs followed by different letters within a column are significantly different at p
≤
0.05 based on
DMRT analysis.
Horticulturae 2023, 9, x FOR PEER REVIEW 6 of 11
culture system was more effective for shoot multiplication than the solid culture system,
possibly due to suitable aeration and the continuous contact of the explants with the cul-
ture medium, which allows for a continual supply and high absorption of nutrients [16].
The growth enhancement of explants using a liquid culture system has also been reported
for several Araceae species, such as A. amazonica [29] and Spathiphyllum cannifolium [30].
Table 2. Effect of BAP and NAA on the shoot proliferation of Philodendron pink princess cultured in
liquid MS medium for 30 days.
BAP (mg/L)
NAA (mg/L)
Liquid MS Medium
Number of Shoots
(Shoots/Explant)
Number of Leaves
(Leaves/Explant)
0.0
0.0
5.0 ± 0.9 c
3.3 ± 0.8 c
1.0
0.0
11.2 ± 2.0 a
4.7 ± 0.5 a
0.5
8.1 ± 2.4 b
4.0 ± 0.7 b
1.0
4.9 ± 1.2 c
3.1 ± 0.7 c
Means ± SDs followed by different letters within a column are significantly different at p ≤ 0.05 based
on DMRT analysis.
Figure 2. Shoot proliferation of Philodendron pink princess in solid and liquid MS medium supple-
mented with 1.0 mg/L BAP and different concentrations of NAA after 30 days of cultivation.
Figure 2.
Shoot proliferation of Philodendron pink princess in solid and liquid MS medium supple-
mented with 1.0 mg/L BAP and different concentrations of NAA after 30 days of cultivation.
3.2. Effect of Auxins on Root Proliferation
Auxins are among the most critical PGRs since they are involved in all physiological
processes in plants. They promote shoot elongation and initiate the formation of adven-
titious and lateral roots [
22
]. Different plant species have diverse responses to different
kinds and concentrations of auxins. This study investigated the effect of auxins, including
NAA, IBA, and 2,4-D, on the root induction of Philodendron pink princess. The microshoots
Horticulturae 2023,9, 688 7 of 10
established from the explants cultured in liquid MS medium supplemented with 1.0 mg/L
BAP were tested for their ability to induce roots. As shown in Table 3, no root formation
was observed when the explants were cultured on solid MS medium without the supple-
mentation of auxins (control treatment) and MS medium supplemented with 0.5 mg/L
NAA. Among the different auxins tested, IBA was more effective for the root induction
of Philodendron pink princess than the other auxins, consistent with the findings of Bartel
et al. [
31
], Chen et al. [
8
], and Hassan et al. [
14
]. This observation might be correlated
with the indole ring of the IBA molecule, which allows it to be efficiently absorbed by
plant cells [
32
]. IBA has been applied for the
in vitro
rooting of many Araceae species,
including Dieffenbachia compacta [
33
] and Aglaonema cochin [
34
]. In contrast, some species
of Araceae also respond well to different forms of auxins. For instance, the
in vitro
rooting
of Philodendron bipinnatifidum was significantly enhanced when the plant was treated with
1.0 to 2.0 mg/L NAA [16].
Table 3. Effect of auxins on the root induction of Philodendron pink princess.
Treatment Concentration
(mg/L)
Number of Roots
(Roots/Explant)
Root Length
(cm)
Control 0.0 0.0 e0.0 c
NAA 0.5 0.0 e0.0 c
1.0 1.5 ±0.8 cd 0.8 ±0.3 bc
2.0 1.6 ±0.9 cd 1.2 ±0.4 ab
3.0 1.4 ±0.5 cd 0.7 ±0.3 bc
IBA 0.5 1.4 ±0.5 cd 1.6 ±0.8 ab
1.0 2.2 ±1.2 bc 1.6 ±0.9 ab
2.0 2.6 ±0.5 ab 1.7 ±1.0 ab
3.0 3.2 ±0.8 a1.9 ±0.6 a
2,4-D 0.5 1.4 ±0.5 cd 0.8 ±0.3 bc
1.0 1.0 ±0.0 d0.8 ±0.2 bc
2.0 1.0 ±0.0 d0.8 ±0.3 bc
3.0 0.8 ±0.4 de 1.0 ±0.3 b
Means
±
SDs followed by different letters within a column are significantly different at p
≤
0.05 based on
DMRT analysis.
Notably, treatment with IBA at 3.0 mg/L resulted in the highest number of roots
(3.2 roots/explant) and longest root lengths (1.9 cm) compared to the other treatments,
which is consistent with the findings reported for Aglaonema cochin [
34
]. However, the
current results contrast with previous findings in other Philodendron species. For instance,
Chen et al. [
8
] demonstrated that IBA at 0.5 and 1.0 mg/L favored the root formation of
Philodendron imperial green, imperial red, and imperial rainbow, and Hassan et al. [
14
]
noted that 1.0 mg/L IBA yielded the maximum root number (31.67 roots/explant) in
Philodendron selloum. Notably, some species of Philodendron root easily in MS medium
without supplementation with PGRs [7].
3.3. Ex Vitro Acclimatization of the Plantlets
In vitro
plantlets require an ex vitro acclimatization process to ensure plant growth
and survival when transferred to soil or field environments. Several supporting materials
have been used as planting media for ex vitro acclimatization, and peat moss, vermiculite,
and perlite are the most commonly used materials [
8
,
14
,
16
,
35
]. This study investigated the
ex vitro acclimatization of micropropagated Philodendron pink princess using peat moss,
vermiculite, and perlite as planting materials. As shown in Table 4and Figure 3, the
in vitro
plantlets of Philodendron pink princess were successfully acclimatized with morphologies
comparable to those of the mother plants. Peat moss and vermiculite yielded 100% survival,
while a slight reduction in survival was observed when the
in vitro
plantlets were acclima-
tized in perlite (80% survival), similar to the findings reported by Alawaadh et al. [
16
]. The
Horticulturae 2023,9, 688 8 of 10
highest values of plant height (2.48 cm) and number of leaves (10.9 leaves/plantlet) were
observed when the
in vitro
plantlets were grown in peat moss, followed by vermiculite and
perlite. On the other hand, vermiculite and perlite yielded the highest number of roots and
root length values, respectively. Although the acclimatized plantlets grown in peat moss
had fewer roots and shorter root lengths than the other treatments, the roots that formed in
this planting material were thicker than those that formed in vermiculite and perlite.
Table 4.
Effect of planting media on the survivability of Philodendron pink princess plantlets after
45 days during the acclimatization stage.
Planting Material Survival (%) Plant Height (cm) Number of Leaves
(Leaves/Plantlet)
Number of Roots
(Roots/Plantlet) Root Length (cm)
Peat moss 100 2.48 ±0.4 a10.9 ±1.3 a4.1 ±1.6 b5.5 ±1.0 b
Vermiculite 100 1.82 ±0.3 b7.1 ±1.7 b8.0 ±1.5 a6.7 ±0.3 a
Perlite 80 1.75 ±0.4 b6.5 ±1.3 b7.5 ±1.2 a7.2 ±0.6 a
Means
±
SDs followed by different letters within a column are significantly different at p
≤
0.05 based on
DMRT analysis.
Horticulturae 2023, 9, x FOR PEER REVIEW 8 of 11
and perlite are the most commonly used materials [8,14,16,35]. This study investigated the
ex vitro acclimatization of micropropagated Philodendron pink princess using peat moss,
vermiculite, and perlite as planting materials. As shown in Table 4 and Figure 3, the in
vitro plantlets of Philodendron pink princess were successfully acclimatized with morphol-
ogies comparable to those of the mother plants. Peat moss and vermiculite yielded 100%
survival, while a slight reduction in survival was observed when the in vitro plantlets
were acclimatized in perlite (80% survival), similar to the findings reported by Alawaadh
et al. [16]. The highest values of plant height (2.48 cm) and number of leaves (10.9
leaves/plantlet) were observed when the in vitro plantlets were grown in peat moss, fol-
lowed by vermiculite and perlite. On the other hand, vermiculite and perlite yielded the
highest number of roots and root length values, respectively. Although the acclimatized
plantlets grown in peat moss had fewer roots and shorter root lengths than the other treat-
ments, the roots that formed in this planting material were thicker than those that formed
in vermiculite and perlite.
Table 4. Effect of planting media on the survivability of Philodendron pink princess plantlets after 45
days during the acclimatization stage.
Planting Material
Survival (%)
Plant Height (cm)
Number of Leaves
(Leaves/Plantlet)
Number of Roots
(Roots/Plantlet)
Root Length
(cm)
Peat moss
100
2.48 ± 0.4 a
10.9 ± 1.3 a
4.1 ± 1.6 b
5.5 ± 1.0 b
Vermiculite
100
1.82 ± 0.3 b
7.1 ± 1.7 b
8.0 ± 1.5 a
6.7 ± 0.3 a
Perlite
80
1.75 ± 0.4 b
6.5 ± 1.3 b
7.5 ± 1.2 a
7.2 ± 0.6 a
Means ± SDs followed by different letters within a column are significantly different at p ≤ 0.05 based
on DMRT analysis.
Figure 3. The plantlets of Philodendron pink princess after ex vitro acclimatization for 45 days using
(A) peat moss, (B) vermiculite, and (C) perlite as planting material.
Considering the chemical and physical properties of the planting materials used in
this study, peat moss contained higher concentrations of nutrients than vermiculite and
perlite. Furthermore, peat moss and vermiculite also have a higher water-holding capacity
than perlite [35–37]. Thus, the better growth of Philodendron pink princess plantlets in peat
moss and vermiculite in terms of plant height and the number of leaves might be associ-
ated with these planting materials’ nutrient and moisture supplies. Unlike peat moss, ver-
miculite and perlite are porous supporting materials, allowing oxygen diffusion to stim-
ulate root proliferation and respiration. Therefore, the in vitro plantlets grown in vermic-
ulite and perlite exhibited a higher number of roots and longer root lengths than those
grown in peat moss. These results agree with the findings of Hoang et al. [35].
The current study demonstrated that peat moss, vermiculite, and perlite can be used
as planting materials for the ex vitro acclimatization of Philodendron pink princess plant-
lets, with peat moss enhancing vegetative development and vermiculite and perlite
Figure 3.
The plantlets of Philodendron pink princess after ex vitro acclimatization for 45 days using
(A) peat moss, (B) vermiculite, and (C) perlite as planting material.
Considering the chemical and physical properties of the planting materials used in this
study, peat moss contained higher concentrations of nutrients than vermiculite and perlite.
Furthermore, peat moss and vermiculite also have a higher water-holding capacity than
perlite [
35
–
37
]. Thus, the better growth of Philodendron pink princess plantlets in peat moss
and vermiculite in terms of plant height and the number of leaves might be associated with
these planting materials’ nutrient and moisture supplies. Unlike peat moss, vermiculite
and perlite are porous supporting materials, allowing oxygen diffusion to stimulate root
proliferation and respiration. Therefore, the
in vitro
plantlets grown in vermiculite and
perlite exhibited a higher number of roots and longer root lengths than those grown in peat
moss. These results agree with the findings of Hoang et al. [35].
The current study demonstrated that peat moss, vermiculite, and perlite can be used
as planting materials for the ex vitro acclimatization of Philodendron pink princess plantlets,
with peat moss enhancing vegetative development and vermiculite and perlite promoting
root growth. However, based on a literature review, a mixture of peat moss and vermiculite
or perlite has been shown to promote the vegetative and root growth of several crops [
14
,
35
].
Thus, the ex vitro acclimatization of Philodendron pink princess plantlets employing a variety
of these planting materials at different ratios should be evaluated in the future.
4. Conclusions
As demonstrated in this study, BAP alone at a concentration of 1.0 mg/L promoted
the shoot proliferation of Philodendron pink princess more than treatment using NAA and a
Horticulturae 2023,9, 688 9 of 10
combination of BAP and NAA, yielding the maximum numbers of shoots and leaves in both
solid and liquid culture systems. Among the auxins tested, IBA at 3.0 mg/L was the best
condition for the root induction of Philodendron pink princess, yielding a maximum number
of roots (3.2 roots/explant) and root length (1.9 cm). The plantlets of Philodendron pink
princess acclimatized with a relatively high survival frequency in all planting materials
without morphological abnormalities. Peat moss promoted the vegetative growth of
Philodendron pink princess plantlets more than the other growth media. Liquid MS medium
supplemented with 1.0 mg/L BAP was the best culture medium for shoot proliferation,
while solid MS medium supplemented with 3 mg/L IBA was the best culture medium
for the root formation and proliferation of Philodendron pink princess. The established
protocol can be used for large-scale production using protocorm-like bodies as explants.
Additionally, peat moss alone could also be utilized as planting material for the ex vitro
acclimatization of the plantlets.
Author Contributions:
Conceptualization, P.K., P.T. and S.T.; methodology, P.K., H.K. and S.T.;
software, H.K. and P.T.; validation, P.K. and S.T.; formal analysis, P.K. and S.T.; investigation, P.K.,
H.K. and S.T.; resources, P.K. and S.T.; data curation, P.K. and S.T.; writing—original draft preparation,
P.K., H.K. and S.T.; writing—review and editing, P.K., P.T. and S.T.; visualization, H.K. and P.T.;
supervision, P.K. and S.T.; project administration, P.K. and S.T.; funding acquisition, P.K. and S.T. All
authors have read and agreed to the published version of the manuscript.
Funding:
This research was supported by the Research and Graduate Studies and Fermentation
Research Center for Value Added Agricultural Products, Khon Kaen University, under Research
Program (RP-FerVAAP-66).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors thank Pornprom Klawikkam and Chotpipat Thongkote for their
technical support.
Conflicts of Interest: The authors declare no conflict of interest.
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