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The Influence of Thidiazuron on Direct Somatic Embryo Formation from

Various Types of Explant in Phalaenopsis amabilis (L.) Blume Orchid

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DOI: 10.1016/j.hjb.2017.11.005

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 HAYATI Journal of Biosciences 24 (2017) 201e205

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HAYATI Journal of Biosciences

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Original Research Article

The Influence of Thidiazuron on Direct Somatic Embryo Formation from

Various Types of Explant in Phalaenopsis amabilis (L.) Blume Orchid
Windi Mose,1 Ari Indrianto,1 Aziz Purwantoro,2 Endang Semiarti1*
1
Graduate Study Program, Faculty of Biology, Gadjah Mada University, Yogyakarta, Indonesia.
2
Faculty of Agriculture, Gadjah Mada University, Yogyakarta, Indonesia.

a r t i c l e i n f o a b s t r a c t

Article history: Phalaenopsis amabilis is an important national flower of Indonesia as a parent for orchid breeding, so that
Received 7 October 2017 needs a good strategy to produce high number of plants. The objective of this research is to analyze the
Received in revised form use of thidiazuron (TDZ) for producing high number of plantlets, through directly induction of somatic
29 November 2017
embryos (SEs) from various explants. The method was used 20 each of protocorms, leaves, stems and
Accepted 29 November 2017
Available online 11 December 2017
roots as explants. The explants were dissected transversely, then put on various culture media: New
Phalaenopsis (NP) and NP + (1, 2, 3) mgL
1 TDZ. Cultures were maintained at 25 C with continous white
light. The formation of SEs was observed every week for 8 weeks. The results showed that SEs formation
KEYWORDS:
New Phalaenopsis medium,
increased inline with the addition of TDZ concentration to the NP medium, for both velocity and amount
Phalaenopsis amabilis orchid, of SEs formation. In NP0, SEs were formed at (26.07 ± 0.73) days after inoculation of protocorm, whereas
somatic embryo, on NP + (1, 2, and 3 mgL
1) TDZ, SEs were formed at (17.85 ± 0.67) days, (15 ± 0.64) days, and (11 ± 0.64)
thidiazuron, days, respectively. All types of explants formed SEs on NP + TDZ (1e3 mgL
1), whereas only 14 of 20
various explants protocorms produced SEs (70%), and 8 of 20 stems formed SEs (40%) in NP0. In roots, SEs was formed on
NP + 2 mgL
1 TDZ and NP + 3 mgL
1 TDZ. For stems, the highest amount of SEs (28.25 ± 1.07) was
reached on NP + 3 mgL
1 TDZ, followed by protocorm (23.30 ± 1.13) SEs and roots (8.25 ± 0.68) SEs. In
contrast, in NP0, the amount of SEs was very low (1.25 ± 0.46) from stem and (1.50 ± 0.65) from pro-
tocorms, there was no evidence of SEs formation in the leaves and roots.
Copyright © 2017 Institut Pertanian Bogor. Production and hosting by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction (Bhattacharyya et al. 2016; Kaewubon and Meesawat, 2016),

Cymbidium (da Silva and Winarto, 2016), Oncidium (Mayer et al.
Somatic embryogenesis is defined as a process in which a bi- 2010), Cattleya (Cueva-Agila et al. 2016), Vanda (Hardjo et al.
polar structure, resembling a zygotic embryo, develops from a non- 2016) and Phalaenopsis (Winarto et al. 2016). However, the selec-
zygotic cell without vascular connection with the original tissue tion of suitable types and sources of explant are critical factors for
(Arnold et al. 2002). In that process, somatic cell differentiate into a obtaining a successful culture in somatic embryogenesis system
plant without the involvement of fertilization or gamete fusion (Feng and Chen, 2014).
(Smertenko and Bozhkov, 2014). Somatic embryogenesis has Currently, some techniques of propagation have also been
emerged as a powerfull tool for studying plant development developed for a number of orchid species through somatic
because somatic embryos (SEs) resemble zygotic embryos and embryogenesis from various types of explant including leaves
undergo almost the same developmental stages (Elhiti et al. 2013). (Jainol and Gansau, 2017), roots (Meilasari and Iriawati, 2016),
Therefore, somatic embryogenesis has been considered to be a shoot tips (Van et al. 2012), stem nodal (Hong et al. 2010), seed-
suitable system for plant mass propagation and for regeneration of derived protocorms (Mahendran and Bai, 2016), and protocorm-
transgenic plants (Bhattacharyya et al. 2016). like bodies (PLBs) (Li and Xu, 2009). Furthermore, the proccess of
In recent years, somatic embryogenesis protocols have been somatic embryogenesis could be induced either directly from
successfully developed in several orchids, including Dendrobium epidermal and sub-epidermal cells of explants (Moradi et al. 2017)
or indirectly via intervening callus (Niknejad et al. 2011). However,
* Corresponding author.
plant regeneration from callus is often associated with genetic and
E-mail address: [email protected] (E. Semiarti). cytological variation making the strategy less desirable for large-
Peer review under responsibility of Institut Pertanian Bogor. scale clonal multiplication (Anjaneyulu and Giri, 2011). Direct

https://doi.org/10.1016/j.hjb.2017.11.005
1978-3019/Copyright © 2017 Institut Pertanian Bogor. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
202 W. Mose, et al

somatic embryogenesis is beneficial with its reduced time for plant flask at temperature of 25 ± 1ºC with 1000 lux of continuous light.
propagation as well as with minimized culture-induced genetic Four-week-old protocorms (developing orchid embryos)
changes (Jayanthi et al. 2011). (Figure 1C) and 6-month-old orchid seedlings (Figure 1D) were
Generally, somatic embryogenesis is considered to occur in used as the source of explants.
response to modifications in the level of available growth regula- Roots and leaves of the 6-month-old seedlings and 4-week-old
tors, especially auxins and cytokinins in tissue culture media protocorms that were cut transversely, and stems were used as
(Moradi et al. 2017). Thidiazuron (TDZ) is a potent regulator for explants. The explants were put on NP solid medium supplemented
in vitro propagation system in a wide variety of plants (Guo et al. with TDZ (0, 1, 2, 3 mgL
1), and cultures were maintained at tem-
2011). For some years, TDZ has been generally used to culture perature of 25 ± 1 ºC with 1000 lux intensity of continuous light.
orchid tissue, which could induce organogenesis and high- Subcultures were conducted every two weeks. Detailed observa-
frequency of direct somatic embryogenesis, either alone or in tion on morphology of SEs formation was conducted every day and
combination with other growth regulators (Mahendran and Bai, photographs were taken once a week for eight weeks using dis-
2016; Wu et al. 2012). Moreover, many studies reported that the secting microscope (Eschenbach, Germany).
use of TDZ alone could induced direct SE in Phalaenopsis orchid
(Feng and Chen, 2014). TDZ displayed primaryly cytokinin-like ac- 2.2. Histological analysis on the differentiation of SEs
tivity and can be used as a subtitute for both auxin and cytokinin Histological analysis on the differentiation of SEs was observed
(Kou et al. 2016). Besides, Bhataccharyya et al. (2016) reported that for each developmental stage of SEs in the surface of explants by
TDZ showed better efficacy over other purine-type cytokinins (BA anatomic preparation using paraffin method according to Ruzin
or Kinetin) even at low concentrations. (1999). The anatomic samples in the glass slides were observed
Phalaenopsis amabilis is a native Indonesian orchid and one of using light microscope (Olympus, Japan).
the most important parental species of Phalaenopsis hybrids
(Semiarti et al. 2007). Semiarti et al. (2013) reported that the 2.3. Data analysis
number of propagated plants of this orchid using seeds was still A total samples of 320 explants grouped randomly into 16
limited because of highly dependent on the existance and the treatment groups. Each group consisted of 20 explants cultured in 4
quality of siliques that resulted from pollination. Moreover, the Petri dishes (diameter 100 mm  height 15 mm), each Petri dish
continuity of mass propagation of this orchid to produce large contained 5 explants. All treatment means were compared by
number of uniform seedlings in a relatively short time to meet the following Duncan’s Multiple Range Test. Significant differences
market demands is also still limited. Therefore, to solve that between means were presented at the level of P  0.05.
problem, this study was taken to evaluate the influence of TDZ on
direct SEs formation from various types of organs of P. amabilis as
explants. 3. Results

3.1. Induction of SEs

2. Materials and Methods Based on percentage data of SEs formation after 8 weeks of
culture, it was known that SEs could be induced in all treatments,
2.1. Plant materials and culture conditions except on leaf and root explants that cultured on hormone-free
A silique of P. amabilis (Java ecotype) plant (Figures 1A and 1B) medium and root explants cultured on NP medium supplemented
following 120 days of pollination was collected from Titi Orchids with 1 mgL
1 TDZ (Table). Within four weeks some explants turned
Nursery, Harjobinangun-Pakem, Sleman, Yogyakarta. The silique to yellow and became necrotic. However, only 70% of protocorms
was wiped with 70% alcohol then passed over a fire and waited and 40% of stem explants were successfully formed SEs in TDZ-free
until the fire went out. This work is done for three times. After medium.
sterilized, the seeds were taken from the silique and sown on a In the presence of TDZ in culture medium, 100% of SEs were
solid New Phalaenopsis (NP) medium (Islam et al. 1998; Semiarti successfully formed from protocorm and stem explants. The initial
et al. 2010). Thereafter, the cultures were incubated in 100 mL embryos emerged as protruding nodular masses from the surface of

Figure 1. Phenotype of P. amabilis. (A) A bunch of flowers for the material of self-pollination to get silique. (B) 4-month-old silique. (C) 4-week-old protocorms. (D) 6-month-old
seedling (bars a, b, d ¼ 1 cm; bar c ¼ 1 mm).
 Direct somatic embryogenesis in orchid 203

Table. Effects of various concentrations of TDZ on the formation of SEs from various types of explant of P. amabilis

Types of Concentrations of TDZ Percentage of SEs formation (%) after 8 weeks of culture SEs induction time Number of SEs formed after 8 weeks of
explant (mgL
1) (n ¼ 20) (days) culture

Protocorm 0 70 26.07 ± 0.73f 1.50 ± 0.65b

1 100 17.85 ± 0.67d 9.85 ± 0.93g
2 100 15.00 ± 0.64c 16.05 ± 0.89i
3 100 11.00 ± 0.64b 23.30 ± 1.13k
Leaf 0 0 0.00 ± 0.00a 0.00 ± 0.00a
1 75 35.00 ± 0.65j 3.20 ± 0.68c
2 90 31.78 ± 0.73i 4.83 ± 0.38e
3 100 27.05 ± 0.68g 7.75 ± 0.44f
Root 0 0 0.00 ± 0.00a 0.00 ± 0.00a
1 0 0.00 ± 0.00a 0.00 ± 0.00a
2 65 36.36 ± 1.15k 4.21 ± 1.05d
3 80 32.25 ± 0.93i 8.25 ± 0,68f
Stem 0 40 31.00 ± 0.76h 1.25 ± 0.46b
1 100 22.15 ± 0.98e 13.90 ± 0.91h
2 100 18.30 ± 0.86d 21.05 ± 0.89j
3 100 14.95 ± 0.75c 28.25 ± 1.07l

Note: Data in the same column followed by the same letters are not signficantly different by Duncan’ multiple range test at P  0.05.

the wounding part of explants. This structure appeared to be pro- By the time of culture, globular embryo developed into a scu-
duced directly without intervening of callus phase (Figure 2A). tellar embryo which consisted of two protuberances at the apical
After two weeks of subcultured, the embryos become progressively part and a notch between the protuberances (Figure 3E). In sub-
enlarges, and more embryos were formed. In addition, numerous sequent development, a shoot meristem was formed from the
white absorbing hair formed in the basal region (Figure 2B). It notch. The shoot meristem was enclosed by two leaf primordia and
indicated that the embryos continued to form a striking structure coleoptile (Figure 3F). In this stage, we also noticed the formation of
on the apical region (Figure 2C) before producing a shoot root meristem at the basal part of embryo. This structure became
(Figure 2D). prominent by its dense cytoplasm and distinct nuclei. Next, the
Under periodical observation it was known that inline with the embryo elongated followed by the formation of procambium in the
high concentration of TDZ, both the induction time and the number middle part of embryo (Figure 3G). Eventually, leaf primordia
of SEs produced were also increased (Table). The results showed elongated indicating a mature embryo development (Figure 3H).
that NP medium supplemented with 3 mgL
1 TDZ was the most
suitable medium for embryos formation, which resulted in the
4. Discussion
fastest SEs formation on protocorm explants (11.00 ± 0.64 days) and
the highest SEs number on stem explants (28.25 ± 1.07). In contrast,
4.1. Induction of SEs
protocorms and stems produced the lowest number of SEs when
Plant regeneration via SEs from various vegetative parts of
cultured on NP basal medium, that is 1.25 ± 0.46 SEs from stems
orchid are essential and has been done in many species (Chugh
and 1.50 ± 0.65 from protocorms.
et al. 2009). Meilasari and Iriawati (2016) reported that SEs were
successfully induced from roots and leaves of Phalaenopsis ‘Join
3.2. Histological analysis on the differentiation of SEs Angle X Sogo Musadian’ cultured in half-strength Murashige and
The results of histological analysis showed that SEs originated Skoog (MS) medium supplemented with various combinations of
from the epidermal and sub-epidermal cells of explants as pro- plant growth regulators (PGRs). While, Hong et al. (2010) reported
embryonic masses that consisted of small, thick-walled cells with that about 55,5% of stem nodal segments of Zygopetalum mackayi
large and densely stained nuclei (Figures 3A and 3B). There were no orchid were formed SEs when planted on MS medium enriched
vascular connections between the proembryo and mother tissue. At with 4,54 mM TDZ. Here, SEs were successfully induced from stem,
four weeks after initiation of culture, some proembryos enlarged root, leaf, and protocorm explants of P. amabilis. Among those types
and formed a globular-shape embryo with a protoderm (Figure 3C) of explant, stem and protocorm showed the best response by
that surrounds the embryo. The structure consisted of a layer of production of the highest number of SEs. Similar results were re-
cells, arranged regularly and thightly. The globular embryo further ported by Moradi et al. (2017) when they assesed the potential of
elongated and formed a suspensor in the basal region (Figure 3D). different types of explant of Epipactis veratrifolia orchid. They found

Figure 2. Developmental stages of SEs of P. amabilis. (A) SEs (arrow) were formed from the surface area of the wounding part. (B) SEs enlarged and formed absorbing hair (arrow) at
the basal region. (C) SEs formed a striking structure on the apical region. (D) SEs with a shoot (arrow) (bars ¼ 50 mm).
204 W. Mose, et al

Figure 3. Anatomy of direct SE development from stem of P. amabilis. (A) The structure resemble to proembryo (arrow) originated from epidermal and (B) sub-epidermal cells
layers. (C) Globular embryo, surrounded by protoderm (arrow). (D) Globular embryo elongated with a suspensor (arrow) on the basal region. (E) The embryo formed two pro-
tuberances (p1, p2) and a scutellar notch (sn) between two protuberances. (F). SE consisted of shoot meristem (sm), leaf primordia (lp), enclosed by the coleoptile, and root
meristem (rm) at the basal part of embryo. (G). The formation of procambium. (H) Leaf primordia elongated (bars ¼ 100 mm).

that protocorm had the highest embryo induction frequency (100% epidermal cells of explant. This result also reported in C. bicolor
embryogenesis) among leaf segment, apical bud, single node, and (Mehendran and Bai, 2012) and D. crumenatum (Kaewubon and
crown. SEs were also formed from protocorm and stem explants Meesawat, 2016). Although the process of SEs formation has
when cultured on hormone-free medium. This result suggests that occured earlier at the cellular level, but visually SEs were seen as a
the endogenous hormone inside the explants might still have a role proembryo mass that rapidly differentiated to formed a globular-
in embryo formation. Consistent with our result, Kaewubon and shape embryo. This embryo undergo differentiation by formed a
Meesawat (2016) also reported that bisected protocorm of Pigeon terminal notch. The notch marks the future location in the shoot
orchid (Dendrobium crumenatum) cultured on PGRs-free medium apex and represents the earliest morphological sign of coleoptile
could formed SEs. development (Alcantara et al. 2014). At this stage, cell divisions
However, leaf and root explants cultured on TDZ-free medium were most frequent in the terminal cells that eventually forms
did not formed any embryos. Similar results were also reported by shoot and root meristem. Matsumoto et al. (1996) mentioned that
Khoddamzadeh et al. (2011) in Phalaenopsis bellina, in which leaf the formation of these features designated the bipolarity of SE and
explants planted on basal medium failed to form embryos and the fulfill the anatomical requirements of a true SE.
explants tend to be necrotic. As we used various concentrations of In conclusion, NP medium supplemented with 3 mgL
1 TDZ was
TDZ (0, 1, 2, 3 mgL
1), we found that, inline with the higher con- the best medium to induce direct SEs from all various explants of
centration of TDZ, the number of embryos increased. Similar results P. amabilis. While protocorm and stem appeared to be the most
were obtained by Balilashaki et al. (2015) using leaf tip segments of responsive explants due to the fastest and the highest number of
P. amabilis cv. ‘Surabaya’ planted on MS medium supplemented SEs produced. The easy procedure for SEs production using TDZ-
with TDZ (0.5, 1, 2, and 3 mgL
1). Moradi et al. (2017) also reported containing medium will support and give high benefit for orchid
that addition of 3 mgL
1 TDZ produced the highest number of SEs, conservation as well as orchid industry.
both in protocorms (2,5 embryos) and leaves (0,33 embryos) of
E. veratrifolia. Acknowledgements
TDZ is a diphenylurea derivative with influential cytokinin ac-
tivity and have been suggested as one of the most effective PGRs for The authors express gratitude thanks to Ms. Hj. Sri Suprih Les-
SEs induction in comparison to other cytokinins (Gantait and tari the owner of TITI Orchid nursery-Pakem, Sleman for providing
Sinniah, 2012; Sujjaritthurakarn and Kanchanapoom, 2011). TDZ P. amabilis parental plants. This work was supported by the Min-
affected plant physiology, such as cellular, nutrient, transport and istry of Research, Technology and Higher Education of the Republic
alters the endogenous levels of PGRs (Ouyang et al. 2016). Wu et al. of Indonesia through PUPT UGM research grant (2016-2017) to ES
(2012) demonstrated that TDZ was more effective to induce direct and PMDSU research grant (2016-2017) to ES and WM.
SEs from leaf explants of Renanthera Tom Thumb‘Qilin’ orchid,
which was significantly higher than BAP, kinetin, and NAA. Conflict of Interest Statement
TDZ was generally effective in inducing SEs at concentration
ranging between 0.01 to 5 mgL
1 (Gantait and Sinniah, 2012). We wish to confirm that there are no known conflicts of interest
Whereas, the use of TDZ with concentration of 1 mgL
1 failed to associated with this publication and there has been no significant
formed SEs in root explants. Lang and Hang (2006) also examined financial support for this work that could have influenced its
various concentrations of TDZ affecting the production of SEs from outcome.
root explants of Vanda coerulea orchid found that, concentration of
TDZ below 3 mgL
1 did not produced any embryos.
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