Dynamic expression of POH1 gene in shoot development during in vitro culture of
Dynamic expression of POH1 gene in shoot development during in vitro culture of
Phalaenopsis orchid
Endang Semiarti, Agus Slamet, Rinaldi Rizal, and Ixora Sartika Mercuriani
Citation: AIP Conference Proceedings 1744, 020019 (2016); doi: 10.1063/1.4953493
View online: http://dx.doi.org/10.1063/1.4953493
View Table of Contents: http://scitation.aip.org/content/aip/proceeding/aipcp/1744?ver=pdfcov
Published by the AIP Publishing
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Dynamic Expression of POH1 Gene in Shoot Development
During In Vitro Culture of Phalaenopsis Orchid
Endang Semiarti1, 2, a), Agus Slamet2, Rinaldi Rizal2 and Ixora Sartika Mercuriani3
1
Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Jalan Teknika Selatan,
Sekip Utara, Yogyakarta 55281, Indonesia.
2
Study Program of Magister, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta.
3
Study Program of Biotechnology, Graduate School of Universitas Gadjah Mada, Yogyakarta.
a)
Corresponding author: endsemi@ugm.ac.id
Abstract. Phalaenopsis Orchid Homeobox1 (POH1) gene is a KNOTTED1 homologous gene from Indonesian wild
orchid Phalaenopsis amabilis. As a member of homeobox gene family in orchid, POH1 may function in the initiation and
development of shoot in orchid. To understand the roles of POH1 gene, we analyzed the expression and function of
POH1 gene during in vitro culture of P. amabilis orchids. The method was conducted by growing seeds of P. amabilis
orchids on New Phalaenopsis (NP) medium to grow protocorms, and then in turned it grew into plantlets. Phenotypic
characters such as a number of shoots, number and size of leaves were analysed in (4, 8, 16 and 24) wk after sowing
(WAS) plantlets. Molecular analysis including the expression of POH1 gene was analysed by Reverse Transcriptase
(RT)-PCR using POH1F1R1 primers on various ages of (4, 8, 16 and 24) WAS plantlets following by protein profile
analysis using 10 % SDS-PAGE. The results showed that during in vitro condition, embryos were developed into a single
and multiple shoots with percentation of 60 % and 40 %, respectively. Accumulation of POH1mRNA (1086 bp) was
detected in (4 to 16) WAS-shoots and disappear at 24 WAS plant. Interestingly, POH1mRNA appeared again on 48
WAS and 96 WAS plantlets. These data were consistent with the result of protein profile analysis that putative POH1
protein (40.2 kDa) can be detected in almost all stages of plantlets in 10 % SDS-PAGE. These data indicated there was a
dynamic expression of POH1 gene during in vitro culture of orchid shoots to maintain shoot development.
Keywords: In vitro, orchid, POH1, shoot development.
INTRODUCTION
Phalaenopsis orchids are the most popular orchid both for decoration and floral arrangements for indoor and
outdoor ceremonial events, so people are looking for these orchids for trade. Indonesia, a tropical country is the
home of various species of orchids, including the moth orchid Phalaenopsis amabilis (L.) Blume. This orchid was
served as a national flower because of the beauty of the flowers that is not easily withered, with very attractive plant
body. Based on these advantages, the mass production of this orchid is needed. Unfortunately, the mass propagation
of orchids is not easy and needs a long period of times. In vitro culture technique will become an effective and
efficient tool for the large production of this orchid. This technique produces a large number of plants with identic
character to the parental plant. The plasticity and totipotency of plant cells and the influence of phytohormones as
plant growth regulators play important roles to determine the development of plant cells and tissues in culture
medium and to regenerate it into the whole plant [1]. The high concentration of cytokinin promotes shoot
regeneration and high concentration promotes root initiation and development [2, 3].
In orchid, mass propagation usually generated by seed germination in vitro, that produce a large number of
seedlings. Orchids can also be rapidly propagated through tissue culture by using shoot tips [4], leaves [5] and stem
nodes [6] on a suitable basal medium with the addition of plant growth regulators (PGRs). Since the orchid seeds are
a lack of endosperm, the in vitro system provides an artificial nutrition as a medium which is needed for the seeds to
Towards the sustainable use of biodiversity in a changing environment: From basic to applied research
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germinate and grow to become whole plants [1, 7]. Under aseptic conditions on an artificial medium, orchid seeds
will grow into protocorms [8, 9]. Protocorm is a tubercle cell mass which then can grow into seedlings and
eventually into whole plants [10, 11].
It is well known that the phenotypic character of the plant is encoded by a group of genes in a polygenic manner.
During its life cycle, plant growth and development consists of three phases, namely, the embryonic phase, the phase
of vegetative and reproductive phase [12]. Each phase was escorted by a group of genes that work together to form
specific proteins that are organized to form a protein complex that plays a role in producing organs of plants in this
phase and then sequentially will hold a working network with a group of genes in the next phase by inducing the
next phase of the group of key genes. Gene products of the next phase will hold a negative feedback suppresses the
activity of the gene pool before and so on. Up-regulation of genes will switch on the next gene and down-regulated
gene will switch off the group of genes which work at previous phase. Genes work in spatial and temporal [12].
Therefore, information about the specific function of the gene would be useful in manipulating plant cells under in
vitro conditions, thus in vitro culture can be used as a good tool to study the gene function in growth and
developmental process of orchids [8].
In plants, homeobox genes were categorized into five class [13], one of which is the class-1 KNOTTED1-like
homeobox (knox) genes that have been detected as transcriptional factors for the maintenance of the SAM and the
development of aboveground organs [14–16]. Scofield et al. [16] showed that members of class-1 KNOX gene
function were discrete and overlapped in the stem cell pool of the shoot apical meristem (SAM) during vegetative
growth in Arabidopsis, but STM gene is essential for both SAM and carpel development.
Semiarti et al. [17] had isolated a putative member of homeobox gene from P. amabilis, namely Phalaenopsis
Orchid Homeobox1 (POH1) gene. To understand the genetic regulation in shoot development of orchids, we
analysed the expression and function of POH1gene during shoot development in orchid in vitro. It is interesting that
in Phalaenopsis, a new shoot that often emerges from the node of the flower stalk may be induced by environmental
signals that turn on the homeobox gene in the meristem. Why is this not the case for other orchids? In this paper, we
report the function of POH1 gene from several weeks’developmental stages of the shoot during in vitro
development and from emerged shoot from adult ex vitro plant.
MATERIALS AND METHODS
Plant Materials. Seeds from self-pollinated flowers were used as plant materials. The seeds were sown on New
Phalaenopsis (NP) medium supplemented with 15 % of coconut water. The culture was maintained at 25 ºC; white
continues light with a photoperiodism 8 h light/16 h dark. Protocorms (developing orchid embryo) were transferred
onto NP medium with and without the addition of plant growth regulators Benzyladenine [(1, 3, 5) mg · L–1)] +
Gibberellin [GA3; (1, 3, 9) mg · L–1)] (mg · L–1 equal ppm = parts per million) as shoot induction media. Growing
shoots and plantlets were cultivated and analysed for morphological characters (lengths and number of leaves and
roots occurrences) and statistical analysis with SPSS and DMRT at 5 % significance level. Molecular characters
were analysed at the level of RNA and protein related to the expression of POH1 gene.
Isolation of mRNA and cDNA synthesis. A population of mRNA was isolated from (4-96) WAS of P. amabilis in
vitro cultivated plants by using RNeasy Kit (Qiagen). The cDNA was synthesized by iScript cDNA synthesis Kit
(Bio-Rad). Synthesized cDNA were used to analyze the expression of POH1 gene and the housekeeping gene Actin
was used as an internal control for Reverse transcriptase-PCR (RT-PCR). RT-PCR was conducted using a pair of
specific primers of POH1 gene according to the method described in Semiarti et al. [17].
Analysis of Protein. About 0.2 g leaves were grind into a fine powder within liquid N2 (in 20 mM Tris-Cl pH 7.5
and150 mM NaCl), centrifugated at 10 000 g for 20 min at 4 oC, kept the supernatant at -40 oC. Protein was
uploaded into SDS-PAGE with the composition of 5 % stacking gel and 10 % of running gel. The gel was immersed
thoroughly with 0.1 % coomassie blue buffer for overnight and then washed firmly to show the sharp bands of
proteins. The size of the prediction protein was conducted by converting the length of cDNA into the molecular
weight (kDa) using the link http://www.molbiol.ru/eng/scripts/01_06.html.
020019-2
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RESULTS AND DISCUSSION
Shoot Growth and Development of P. amabilis
The emergence and development of shoots from protocorm in P. amabilis were observed and followed every
week, starting from sowing seeds on medium. At week-4, the shape of protocorm became a dorsiventral structure
and color of protocorm changed from yellow to greenish. After it had transferred to a new medium, the growth rate
of protocorm increased rapidly, and at week-6 on cultivation medium, the shoot emerged and then grew up. At
week-24, shoots grew into plantlet with two leaves to three leaves and one root to two roots. The leaves and roots
grew for elongation, at week-48, the plantlets/seedlings of orchids became larger and strong enough to be transferred
ex vitro in a pot and maintained in the greenhouse. At week-72 to week-96 the orchid became an adult plant and
started to flowering that produced a flower stalk. Interestingly, one out of 10 plants showed a shoot emerged from
the third node of the flower stalk, which then grew into a complete plant (Fig. 1).
A
C
C
D
FIGURE 1. The shoot growth of P. amabilis orchid. (A) Protocorms (4 WAS); (B) In vitro plantlet (24WAS); (C) Ex
vitro seedling (48 WAS); (D) Plant with shoot emerged from the node of inflorescence stem (96 WAS). Bars: 1 cm in (A)
and (B); 5 cm in (C) and (D).
TABLE 1. The growth of leaves and roots of P. amabilis on two layers medium with various concentration of BA and GA3
Parameters
Leaf Length
(cm)
Leaf Numbers
(pieces)
Root Length
(cm)
Root Numbers
Age
(wk)
BA + GA3 Treatment
A0
*)
A1*)
A3*)
A9*)
18
2,53 ± 0.1b
2,83 ± 0.2b
3,36 ± 0.2c
4,16± 0.1d
24
3,16 ± 0.1bc
3,63 ± 0.2c
3,9 ± 0.1d
4,73± 0.2d
30
4,43 ± 0.3
d
d
18
2 ± 0.6 ab
3 ± 0.6ab
4 ± 0.6de
5± 1.0de
24
4 ± 0.6de
5 ± 0.6def
6 ± 1.2f
6± 0.6f
30
5 ± 0.6f
6 ± 1.0f
6 ± 1.0f
9± 1.0h
18
3,33± 0.2c
1,33 ± 0.2a
1,10 ± 0.1 a
1,10 ± 0.1 a
24
4,16± 0.2d
2,16 ± 0.2b
2,46 ± 0.1b
1,76 ± 0.1 a
30
5,23± 0.1e
3,86 ± 0.2c
3,23 ± 0.1c
3,33 ± 0.1c
18
2 ± 0.6b
2 ± 0.6b
3 ± 0.6c
4± 0.6d
24
3 ± 0.6
c
c
d
4± 0.6d
30
4 ± 0.6d
5 ± 0.6e
6± 0.6f
4,23 ± 0.2
3 ± 0.6
5 ± 0.6e
4,36 ± 0.1
4 ± 0.6
d
5,23± 0.1e
Notes:
Values in columns preceding the same letter are not significantly different by Duncan test at 5 % significance level.
*) value ± standard deviation
The growth rate of shoots became faster after week-18. The number of leaf and root increased significantly, as
well as the length of leaves and roots (Tab.1). The best treatment of BA+GA3 for shoot and root induction in in
vitro culture of P. amabilis is A9 treatment (5 mg · L–1 BA and 9 mg · L–1 GA3), that produced (9 ± 1.0) leaves and
(6 ± 0.6) roots that much higher than the number of leaves in other treatments and the control experiment
020019-3
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(phytohormone-free medium). The length of leaves was also the best in A9 treatment, although the lengths of roots
were not significantly different to other treatments.These results were consistent with the work of Sakamoto et al.
[18] that showed KNOX homeodomain protein directly suppresses the expression of a gibberellin biosynthetic gene
in the SAM of tobacco and found that gibberellin and other phytohormones pathway mediate KNOTTED1-type
homeobox function in plants. Yanai et al. [19] obtained that the cytokinin levels are reduced by the absence of
KNOX1 and in parallel KNOX1 protein repress GA biosynthesis. Class-1 KNOX genes are the central balancers of
hormone levels to keep indeterminacy of SAM and the maintenance of a stable organization of the meristem while
continuously producing organs from its margins resulting in the growth and development of shoots and aboveground
organs of plants. For genetic regulation, giving a treatment of phytohormones (cytokinin, auxin and GA) in the
culture medium is likely to induce the homeobox genes and its activation receptor in explant cells [12].
The Expression of POH1 Gene and Detection of Putative POH1 Protein during the Growth
of Shoots In Vitro
The high accumulation of 800 bp POH1 transcripts was detected in the leaves of growing shoots of 4 weeks after
sowing (WAS) plantlets as the result of RT-PCR (Fig. 2). The accumulation of POH1 transcripts decreased in line
with the aging of the plants. At the age of 24 wk, POH1 transcripts were almost undetectable (very weak intensity).
But at the age of 48 wk and 96 wk, the POH1 transcripts were gradually detected again. This fact could explain the
mechanism of the emergence of adventive shoot from the node of flower stalk at 96 WAS plant; this phenomenon
often occurs in Phalaenopsis orchids. The analysis of protein profile from the concerned plantlets has also
confirmed the results of RT-PCR. We detected the production of putative POH1protein with a size of 40.2 kDa in
(4 to 48) WASP plants.This shows that the dynamics of gene expression POH1 accordance with the growth of
shoots. After being treated with various concentrations of BA and GA, production of putative POH1 protein showed
equal phenomenon to the formation of POH1 transcripts that the putative POH1 protein was very low produced in
24 WAS plants. It is consistent with the observations of the protein profiles of P. amabilis leaves of plants of the
same age as the transcript analysis of POH1. The high intensity of POH1 protein in 48 WAS and 96 WAS and 24
wk of plants that treated by BA and GA3 in various concentration suggests that the growth regulators (BA and GA3)
is likely could act as the trigger for the activation of POH1 gene in the leaves/shoots of P. amabilis.
A
B
(A0)
M
4
A0
8 16 24 48
96 (A1) (A3) (A9)
(A1) (A3)
(A9)
40,2 kDa
FIGURE 2. Accumulation of POH1 transcripts and Protein profile in leaves of developing shoots of P. amabilis. A. POH1
transcripts in leaves of (4, 8, 16, 24, 48, and 96) WAS of normal/untreated plants and 24 WAS of BA+GA3-treated plants. Actin
was used as an internal control. The number below the electrophoresis gel indicated weeks after sowing (WAS).
B) Protein profile in leaves of plants at the same age as in (A). In (A) and (B) BA-GA3- = untreated plants, A0, A1, A3, A9
(A0=Kontrol/Untreated, A1 = BA 1ppm + GA3 5ppm, A3 = BA 3ppm GA3 10ppm, A9 = BA9 ppm+GA3 15 ppm). (ppm = parts
per million equal mg · L–1) (FIGURE 2A was after Semiarti et al. [20], reprinted from AIP Conf. Proc.1677, 090005 (2015).
Copyright 2015. American Institute of Physics.)
This data suggests that POH1 gene maintains shoot development in plant and keep it in normal conditions.
However, we also found that 40 % of protocorms produced multi shoots (2 shoots to 3 shoots) from one germinated
seed on NP culture medium, and another 60% grew into the single shoot from each seed. This is in accordance with
the opinion of Arditti and Ernst [8] that the embryo of Phalaenopsis orchid encountered more than one bud
emergence from one seed in the seed germination. This is likely related to the activities of homeobox genes such as
POH1, STM homologous gene and others in the shoot apical meristem during the growth of embryo and protocorm.
020019-4
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Yu et al. [14] were also got multishoot from the development of transgenic Dendrobium orchid that inserted by
antisense of DOH1 gene, a class 1 knox/homeobox, and they stated that DOH1 gene is required for maintenance of
basic plant architecture and floral transition in orchid. POH1 might have analogous function with DOH1 on the
maintenance of shoot development in Phalaenopsis orchids.
CONCLUSION
There is a dynamic expression of POH1 gene during in vitro culture of orchid shoots to maintain shoot
development from shoot apical meristem into juvenile (4 WAS to 16 WAS) and adult shoots (after 48 WAS) that
continues in ex vitro shoot development.
ACKNOWLEDGEMENTS
The authors thank the Directorate General of Higher Education (DGHE), Ministry of Education and Culture of
RI for STRANAS research grant for 3 years (2012–2014) in the contract no: 089/sp2h/pl/dit.litabmas/v/2012–2013.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
A. H. Naing, J. D. Chung, K. B. Lim, AJPS 2, 262–267 (2011). Doi:10.4236/ajps.2011.22028
A. Indrianto, “Peningkatan mutu anggrek dengan kultur jaringan: teknik embriogenesis mikrospora [Orchid
quality improvement with the tissue culture: microspore embryogenesis technique], in Prosiding Seminar
Nasional Anggrek-2003 [Proceedings of The Orchid National Seminar-2003], edited by E. Semiarti et al.
(Universitas Gadjah Mada, Indonesia, 2003), pp. 35–43.
E. F. George, M. A. Hall and G-J. de Klerk, Plant Propagation Tissue Culture, 3rd ed (Springer, The
Netherlands, 2008), pp. 1, 2, 175–187, 205–216.
G. V. S. Saiprasad, P. Raghuveer and R. Polisetty, J. Ornamental Hort. N.S. 5, 72–73 (200;2).
J. T. Chen, W. C. Chang J. K. Chan and W. C. Chang, J. Plant Growth Regulation 34 (2), 229–232 (2001).
N. S. Pathania, O. P. Sehgal, P. Debojit, B. S. Dilta and D. Paul, J. Orchid Soc. India 12 (1-2), 35–38 (1998).
A. Kumar, S. K. Nandi, N. Bag and L. M. S. Palni, “Tissue culture studies in two important orchid taxa:
Rhynchostylis retusa (L.) Bl. and Cymbidium elegans Lindl.“ in Role of Plant Tissue Culture in Biodiversity
Conservation and Economic Development, edited by S. K. Nandi, et al. L. M. S. Palni and A. Kumar
(Gyanodaya Prakashan, Nainital, India, 2002), pp. 113–124.
J. Arditti and R. Ernst, Micropropagation of Orchids. 1st ed (John Willey & Son Inc. New York, 1993), pp. 6–
8, 25–65.
E. Semiarti, A. Indrianto, A. Purwantoro, S. Isminingsih, N. Suseno, T. Ishikawa, Y. Yoshioka, Y. Machida,
and C. Machida. J. Plant Biotechnology 24, 265–272 (2007).
Y. Veyret, “Development of The Embryo and the Young Seedling Stages of Orchids” in The Orchids Scientific
Studies, edited by C. L. Withner (John Wiley & Sons, Inc., New York, 1974), pp. 223–265.
M. Suryowinoto, Mengenal Anggrek Spesies [Introduction to Orchid Species] 1st ed. (Fakultas Biologi UGM,
Yogyakarta, 1984), pp. 1–10 [Bahasa Indonesia].
S. H. Howell, Molecular Genetics of Plant Development 1st ed. (Cambridge University Press, U. K., 1998), pp.
103–190.
Y. L. Chan, K. H. Lin, Sanjaya, L. J. Liao, W. H. Chen and M. T. Chan, J.Transgenic Res. 14, 279–288 (2005).
H. Yu, S. H. Jang and C. J. Goh, J. Plant Cell 12, 213–219 (2000).
M. K. Ritter, C. M. Padilla, and R. J. Schmidt, American Journal of Botany 89 (2), 203–210 (2002).
S. Scofield, W. Dewitte and J. A. H. Murray, Plant J. 50, 767–781 (2007).
E. Semiarti, T. Ishikawa, Y. Yoshioka, M. Ikezakki, Y. Machida and C. Machida, “Isolation and
characterization of Phalaenopsis Orchid Homeobox1(POH1) cDNAS, knotted1-like homeobox family of
genes in Phalaenopsis amabilis orchid”, in AIP Conference Proceedings of The 2nd International Conference
on Mathematics and Natural Sciences (ICMNS) ITB, edited by R. M. G. Simanjuntak et al. (American Institute
of Physics, Melville, NY, 2008), pp. 289-294 .
T. Sakamoto, N. Kamiya, M. Ueguchi-Tanaka, S. Iwahori, and M. Matsuoka, Genes Dev. 15, 581–590 (2001).
020019-5
Reuse of AIP Publishing content is subject to the terms at: https://publishing.aip.org/authors/rights-and-permissions IP: 202.43.93.104 On: Fri, 17 Jun 2016 07:19:12
19. O. Yanai, E. Shani, K. Dolezai, Tarkowski, R. Sablowski, G. Sanberg, A. Samach and N. Ori, Current Biology
15, 1566–1571 (2005).
20. E. Semiarti, I. S. Mercuriani, R. Rizal, A. Slamet, B. S. Utami, I. A. Bestari, Aziz-Purwantoro,
S. Moeljopawiro, Y. Machida and C. Machida, “Overexpression of PaFT gene in the wild orchid Phalaenopsis
amabilis (L.) Blume”, in AIP Conference Proceedings 1677 of The 5th International Conference on
Mathematics and Natural Sciences (ICMNS) ITB, edited by A. Purqon et al. (American Institute of Physics,
Melville, NY, 2015), pp. 090005-1- 090005-4, doi: 10.1063/1.4930750.
020019-6
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Phalaenopsis orchid
Endang Semiarti, Agus Slamet, Rinaldi Rizal, and Ixora Sartika Mercuriani
Citation: AIP Conference Proceedings 1744, 020019 (2016); doi: 10.1063/1.4953493
View online: http://dx.doi.org/10.1063/1.4953493
View Table of Contents: http://scitation.aip.org/content/aip/proceeding/aipcp/1744?ver=pdfcov
Published by the AIP Publishing
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Reuse of AIP Publishing content is subject to the terms at: https://publishing.aip.org/authors/rights-and-permissions IP: 202.43.93.104 On: Fri, 17 Jun 2016 07:19:12
Dynamic Expression of POH1 Gene in Shoot Development
During In Vitro Culture of Phalaenopsis Orchid
Endang Semiarti1, 2, a), Agus Slamet2, Rinaldi Rizal2 and Ixora Sartika Mercuriani3
1
Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Jalan Teknika Selatan,
Sekip Utara, Yogyakarta 55281, Indonesia.
2
Study Program of Magister, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta.
3
Study Program of Biotechnology, Graduate School of Universitas Gadjah Mada, Yogyakarta.
a)
Corresponding author: endsemi@ugm.ac.id
Abstract. Phalaenopsis Orchid Homeobox1 (POH1) gene is a KNOTTED1 homologous gene from Indonesian wild
orchid Phalaenopsis amabilis. As a member of homeobox gene family in orchid, POH1 may function in the initiation and
development of shoot in orchid. To understand the roles of POH1 gene, we analyzed the expression and function of
POH1 gene during in vitro culture of P. amabilis orchids. The method was conducted by growing seeds of P. amabilis
orchids on New Phalaenopsis (NP) medium to grow protocorms, and then in turned it grew into plantlets. Phenotypic
characters such as a number of shoots, number and size of leaves were analysed in (4, 8, 16 and 24) wk after sowing
(WAS) plantlets. Molecular analysis including the expression of POH1 gene was analysed by Reverse Transcriptase
(RT)-PCR using POH1F1R1 primers on various ages of (4, 8, 16 and 24) WAS plantlets following by protein profile
analysis using 10 % SDS-PAGE. The results showed that during in vitro condition, embryos were developed into a single
and multiple shoots with percentation of 60 % and 40 %, respectively. Accumulation of POH1mRNA (1086 bp) was
detected in (4 to 16) WAS-shoots and disappear at 24 WAS plant. Interestingly, POH1mRNA appeared again on 48
WAS and 96 WAS plantlets. These data were consistent with the result of protein profile analysis that putative POH1
protein (40.2 kDa) can be detected in almost all stages of plantlets in 10 % SDS-PAGE. These data indicated there was a
dynamic expression of POH1 gene during in vitro culture of orchid shoots to maintain shoot development.
Keywords: In vitro, orchid, POH1, shoot development.
INTRODUCTION
Phalaenopsis orchids are the most popular orchid both for decoration and floral arrangements for indoor and
outdoor ceremonial events, so people are looking for these orchids for trade. Indonesia, a tropical country is the
home of various species of orchids, including the moth orchid Phalaenopsis amabilis (L.) Blume. This orchid was
served as a national flower because of the beauty of the flowers that is not easily withered, with very attractive plant
body. Based on these advantages, the mass production of this orchid is needed. Unfortunately, the mass propagation
of orchids is not easy and needs a long period of times. In vitro culture technique will become an effective and
efficient tool for the large production of this orchid. This technique produces a large number of plants with identic
character to the parental plant. The plasticity and totipotency of plant cells and the influence of phytohormones as
plant growth regulators play important roles to determine the development of plant cells and tissues in culture
medium and to regenerate it into the whole plant [1]. The high concentration of cytokinin promotes shoot
regeneration and high concentration promotes root initiation and development [2, 3].
In orchid, mass propagation usually generated by seed germination in vitro, that produce a large number of
seedlings. Orchids can also be rapidly propagated through tissue culture by using shoot tips [4], leaves [5] and stem
nodes [6] on a suitable basal medium with the addition of plant growth regulators (PGRs). Since the orchid seeds are
a lack of endosperm, the in vitro system provides an artificial nutrition as a medium which is needed for the seeds to
Towards the sustainable use of biodiversity in a changing environment: From basic to applied research
AIP Conf. Proc. 1744, 020019-1–020019-6; doi: 10.1063/1.4953493
Published by AIP Publishing. 978-0-7354-1401-3/$30.00
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germinate and grow to become whole plants [1, 7]. Under aseptic conditions on an artificial medium, orchid seeds
will grow into protocorms [8, 9]. Protocorm is a tubercle cell mass which then can grow into seedlings and
eventually into whole plants [10, 11].
It is well known that the phenotypic character of the plant is encoded by a group of genes in a polygenic manner.
During its life cycle, plant growth and development consists of three phases, namely, the embryonic phase, the phase
of vegetative and reproductive phase [12]. Each phase was escorted by a group of genes that work together to form
specific proteins that are organized to form a protein complex that plays a role in producing organs of plants in this
phase and then sequentially will hold a working network with a group of genes in the next phase by inducing the
next phase of the group of key genes. Gene products of the next phase will hold a negative feedback suppresses the
activity of the gene pool before and so on. Up-regulation of genes will switch on the next gene and down-regulated
gene will switch off the group of genes which work at previous phase. Genes work in spatial and temporal [12].
Therefore, information about the specific function of the gene would be useful in manipulating plant cells under in
vitro conditions, thus in vitro culture can be used as a good tool to study the gene function in growth and
developmental process of orchids [8].
In plants, homeobox genes were categorized into five class [13], one of which is the class-1 KNOTTED1-like
homeobox (knox) genes that have been detected as transcriptional factors for the maintenance of the SAM and the
development of aboveground organs [14–16]. Scofield et al. [16] showed that members of class-1 KNOX gene
function were discrete and overlapped in the stem cell pool of the shoot apical meristem (SAM) during vegetative
growth in Arabidopsis, but STM gene is essential for both SAM and carpel development.
Semiarti et al. [17] had isolated a putative member of homeobox gene from P. amabilis, namely Phalaenopsis
Orchid Homeobox1 (POH1) gene. To understand the genetic regulation in shoot development of orchids, we
analysed the expression and function of POH1gene during shoot development in orchid in vitro. It is interesting that
in Phalaenopsis, a new shoot that often emerges from the node of the flower stalk may be induced by environmental
signals that turn on the homeobox gene in the meristem. Why is this not the case for other orchids? In this paper, we
report the function of POH1 gene from several weeks’developmental stages of the shoot during in vitro
development and from emerged shoot from adult ex vitro plant.
MATERIALS AND METHODS
Plant Materials. Seeds from self-pollinated flowers were used as plant materials. The seeds were sown on New
Phalaenopsis (NP) medium supplemented with 15 % of coconut water. The culture was maintained at 25 ºC; white
continues light with a photoperiodism 8 h light/16 h dark. Protocorms (developing orchid embryo) were transferred
onto NP medium with and without the addition of plant growth regulators Benzyladenine [(1, 3, 5) mg · L–1)] +
Gibberellin [GA3; (1, 3, 9) mg · L–1)] (mg · L–1 equal ppm = parts per million) as shoot induction media. Growing
shoots and plantlets were cultivated and analysed for morphological characters (lengths and number of leaves and
roots occurrences) and statistical analysis with SPSS and DMRT at 5 % significance level. Molecular characters
were analysed at the level of RNA and protein related to the expression of POH1 gene.
Isolation of mRNA and cDNA synthesis. A population of mRNA was isolated from (4-96) WAS of P. amabilis in
vitro cultivated plants by using RNeasy Kit (Qiagen). The cDNA was synthesized by iScript cDNA synthesis Kit
(Bio-Rad). Synthesized cDNA were used to analyze the expression of POH1 gene and the housekeeping gene Actin
was used as an internal control for Reverse transcriptase-PCR (RT-PCR). RT-PCR was conducted using a pair of
specific primers of POH1 gene according to the method described in Semiarti et al. [17].
Analysis of Protein. About 0.2 g leaves were grind into a fine powder within liquid N2 (in 20 mM Tris-Cl pH 7.5
and150 mM NaCl), centrifugated at 10 000 g for 20 min at 4 oC, kept the supernatant at -40 oC. Protein was
uploaded into SDS-PAGE with the composition of 5 % stacking gel and 10 % of running gel. The gel was immersed
thoroughly with 0.1 % coomassie blue buffer for overnight and then washed firmly to show the sharp bands of
proteins. The size of the prediction protein was conducted by converting the length of cDNA into the molecular
weight (kDa) using the link http://www.molbiol.ru/eng/scripts/01_06.html.
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RESULTS AND DISCUSSION
Shoot Growth and Development of P. amabilis
The emergence and development of shoots from protocorm in P. amabilis were observed and followed every
week, starting from sowing seeds on medium. At week-4, the shape of protocorm became a dorsiventral structure
and color of protocorm changed from yellow to greenish. After it had transferred to a new medium, the growth rate
of protocorm increased rapidly, and at week-6 on cultivation medium, the shoot emerged and then grew up. At
week-24, shoots grew into plantlet with two leaves to three leaves and one root to two roots. The leaves and roots
grew for elongation, at week-48, the plantlets/seedlings of orchids became larger and strong enough to be transferred
ex vitro in a pot and maintained in the greenhouse. At week-72 to week-96 the orchid became an adult plant and
started to flowering that produced a flower stalk. Interestingly, one out of 10 plants showed a shoot emerged from
the third node of the flower stalk, which then grew into a complete plant (Fig. 1).
A
C
C
D
FIGURE 1. The shoot growth of P. amabilis orchid. (A) Protocorms (4 WAS); (B) In vitro plantlet (24WAS); (C) Ex
vitro seedling (48 WAS); (D) Plant with shoot emerged from the node of inflorescence stem (96 WAS). Bars: 1 cm in (A)
and (B); 5 cm in (C) and (D).
TABLE 1. The growth of leaves and roots of P. amabilis on two layers medium with various concentration of BA and GA3
Parameters
Leaf Length
(cm)
Leaf Numbers
(pieces)
Root Length
(cm)
Root Numbers
Age
(wk)
BA + GA3 Treatment
A0
*)
A1*)
A3*)
A9*)
18
2,53 ± 0.1b
2,83 ± 0.2b
3,36 ± 0.2c
4,16± 0.1d
24
3,16 ± 0.1bc
3,63 ± 0.2c
3,9 ± 0.1d
4,73± 0.2d
30
4,43 ± 0.3
d
d
18
2 ± 0.6 ab
3 ± 0.6ab
4 ± 0.6de
5± 1.0de
24
4 ± 0.6de
5 ± 0.6def
6 ± 1.2f
6± 0.6f
30
5 ± 0.6f
6 ± 1.0f
6 ± 1.0f
9± 1.0h
18
3,33± 0.2c
1,33 ± 0.2a
1,10 ± 0.1 a
1,10 ± 0.1 a
24
4,16± 0.2d
2,16 ± 0.2b
2,46 ± 0.1b
1,76 ± 0.1 a
30
5,23± 0.1e
3,86 ± 0.2c
3,23 ± 0.1c
3,33 ± 0.1c
18
2 ± 0.6b
2 ± 0.6b
3 ± 0.6c
4± 0.6d
24
3 ± 0.6
c
c
d
4± 0.6d
30
4 ± 0.6d
5 ± 0.6e
6± 0.6f
4,23 ± 0.2
3 ± 0.6
5 ± 0.6e
4,36 ± 0.1
4 ± 0.6
d
5,23± 0.1e
Notes:
Values in columns preceding the same letter are not significantly different by Duncan test at 5 % significance level.
*) value ± standard deviation
The growth rate of shoots became faster after week-18. The number of leaf and root increased significantly, as
well as the length of leaves and roots (Tab.1). The best treatment of BA+GA3 for shoot and root induction in in
vitro culture of P. amabilis is A9 treatment (5 mg · L–1 BA and 9 mg · L–1 GA3), that produced (9 ± 1.0) leaves and
(6 ± 0.6) roots that much higher than the number of leaves in other treatments and the control experiment
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(phytohormone-free medium). The length of leaves was also the best in A9 treatment, although the lengths of roots
were not significantly different to other treatments.These results were consistent with the work of Sakamoto et al.
[18] that showed KNOX homeodomain protein directly suppresses the expression of a gibberellin biosynthetic gene
in the SAM of tobacco and found that gibberellin and other phytohormones pathway mediate KNOTTED1-type
homeobox function in plants. Yanai et al. [19] obtained that the cytokinin levels are reduced by the absence of
KNOX1 and in parallel KNOX1 protein repress GA biosynthesis. Class-1 KNOX genes are the central balancers of
hormone levels to keep indeterminacy of SAM and the maintenance of a stable organization of the meristem while
continuously producing organs from its margins resulting in the growth and development of shoots and aboveground
organs of plants. For genetic regulation, giving a treatment of phytohormones (cytokinin, auxin and GA) in the
culture medium is likely to induce the homeobox genes and its activation receptor in explant cells [12].
The Expression of POH1 Gene and Detection of Putative POH1 Protein during the Growth
of Shoots In Vitro
The high accumulation of 800 bp POH1 transcripts was detected in the leaves of growing shoots of 4 weeks after
sowing (WAS) plantlets as the result of RT-PCR (Fig. 2). The accumulation of POH1 transcripts decreased in line
with the aging of the plants. At the age of 24 wk, POH1 transcripts were almost undetectable (very weak intensity).
But at the age of 48 wk and 96 wk, the POH1 transcripts were gradually detected again. This fact could explain the
mechanism of the emergence of adventive shoot from the node of flower stalk at 96 WAS plant; this phenomenon
often occurs in Phalaenopsis orchids. The analysis of protein profile from the concerned plantlets has also
confirmed the results of RT-PCR. We detected the production of putative POH1protein with a size of 40.2 kDa in
(4 to 48) WASP plants.This shows that the dynamics of gene expression POH1 accordance with the growth of
shoots. After being treated with various concentrations of BA and GA, production of putative POH1 protein showed
equal phenomenon to the formation of POH1 transcripts that the putative POH1 protein was very low produced in
24 WAS plants. It is consistent with the observations of the protein profiles of P. amabilis leaves of plants of the
same age as the transcript analysis of POH1. The high intensity of POH1 protein in 48 WAS and 96 WAS and 24
wk of plants that treated by BA and GA3 in various concentration suggests that the growth regulators (BA and GA3)
is likely could act as the trigger for the activation of POH1 gene in the leaves/shoots of P. amabilis.
A
B
(A0)
M
4
A0
8 16 24 48
96 (A1) (A3) (A9)
(A1) (A3)
(A9)
40,2 kDa
FIGURE 2. Accumulation of POH1 transcripts and Protein profile in leaves of developing shoots of P. amabilis. A. POH1
transcripts in leaves of (4, 8, 16, 24, 48, and 96) WAS of normal/untreated plants and 24 WAS of BA+GA3-treated plants. Actin
was used as an internal control. The number below the electrophoresis gel indicated weeks after sowing (WAS).
B) Protein profile in leaves of plants at the same age as in (A). In (A) and (B) BA-GA3- = untreated plants, A0, A1, A3, A9
(A0=Kontrol/Untreated, A1 = BA 1ppm + GA3 5ppm, A3 = BA 3ppm GA3 10ppm, A9 = BA9 ppm+GA3 15 ppm). (ppm = parts
per million equal mg · L–1) (FIGURE 2A was after Semiarti et al. [20], reprinted from AIP Conf. Proc.1677, 090005 (2015).
Copyright 2015. American Institute of Physics.)
This data suggests that POH1 gene maintains shoot development in plant and keep it in normal conditions.
However, we also found that 40 % of protocorms produced multi shoots (2 shoots to 3 shoots) from one germinated
seed on NP culture medium, and another 60% grew into the single shoot from each seed. This is in accordance with
the opinion of Arditti and Ernst [8] that the embryo of Phalaenopsis orchid encountered more than one bud
emergence from one seed in the seed germination. This is likely related to the activities of homeobox genes such as
POH1, STM homologous gene and others in the shoot apical meristem during the growth of embryo and protocorm.
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Yu et al. [14] were also got multishoot from the development of transgenic Dendrobium orchid that inserted by
antisense of DOH1 gene, a class 1 knox/homeobox, and they stated that DOH1 gene is required for maintenance of
basic plant architecture and floral transition in orchid. POH1 might have analogous function with DOH1 on the
maintenance of shoot development in Phalaenopsis orchids.
CONCLUSION
There is a dynamic expression of POH1 gene during in vitro culture of orchid shoots to maintain shoot
development from shoot apical meristem into juvenile (4 WAS to 16 WAS) and adult shoots (after 48 WAS) that
continues in ex vitro shoot development.
ACKNOWLEDGEMENTS
The authors thank the Directorate General of Higher Education (DGHE), Ministry of Education and Culture of
RI for STRANAS research grant for 3 years (2012–2014) in the contract no: 089/sp2h/pl/dit.litabmas/v/2012–2013.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
A. H. Naing, J. D. Chung, K. B. Lim, AJPS 2, 262–267 (2011). Doi:10.4236/ajps.2011.22028
A. Indrianto, “Peningkatan mutu anggrek dengan kultur jaringan: teknik embriogenesis mikrospora [Orchid
quality improvement with the tissue culture: microspore embryogenesis technique], in Prosiding Seminar
Nasional Anggrek-2003 [Proceedings of The Orchid National Seminar-2003], edited by E. Semiarti et al.
(Universitas Gadjah Mada, Indonesia, 2003), pp. 35–43.
E. F. George, M. A. Hall and G-J. de Klerk, Plant Propagation Tissue Culture, 3rd ed (Springer, The
Netherlands, 2008), pp. 1, 2, 175–187, 205–216.
G. V. S. Saiprasad, P. Raghuveer and R. Polisetty, J. Ornamental Hort. N.S. 5, 72–73 (200;2).
J. T. Chen, W. C. Chang J. K. Chan and W. C. Chang, J. Plant Growth Regulation 34 (2), 229–232 (2001).
N. S. Pathania, O. P. Sehgal, P. Debojit, B. S. Dilta and D. Paul, J. Orchid Soc. India 12 (1-2), 35–38 (1998).
A. Kumar, S. K. Nandi, N. Bag and L. M. S. Palni, “Tissue culture studies in two important orchid taxa:
Rhynchostylis retusa (L.) Bl. and Cymbidium elegans Lindl.“ in Role of Plant Tissue Culture in Biodiversity
Conservation and Economic Development, edited by S. K. Nandi, et al. L. M. S. Palni and A. Kumar
(Gyanodaya Prakashan, Nainital, India, 2002), pp. 113–124.
J. Arditti and R. Ernst, Micropropagation of Orchids. 1st ed (John Willey & Son Inc. New York, 1993), pp. 6–
8, 25–65.
E. Semiarti, A. Indrianto, A. Purwantoro, S. Isminingsih, N. Suseno, T. Ishikawa, Y. Yoshioka, Y. Machida,
and C. Machida. J. Plant Biotechnology 24, 265–272 (2007).
Y. Veyret, “Development of The Embryo and the Young Seedling Stages of Orchids” in The Orchids Scientific
Studies, edited by C. L. Withner (John Wiley & Sons, Inc., New York, 1974), pp. 223–265.
M. Suryowinoto, Mengenal Anggrek Spesies [Introduction to Orchid Species] 1st ed. (Fakultas Biologi UGM,
Yogyakarta, 1984), pp. 1–10 [Bahasa Indonesia].
S. H. Howell, Molecular Genetics of Plant Development 1st ed. (Cambridge University Press, U. K., 1998), pp.
103–190.
Y. L. Chan, K. H. Lin, Sanjaya, L. J. Liao, W. H. Chen and M. T. Chan, J.Transgenic Res. 14, 279–288 (2005).
H. Yu, S. H. Jang and C. J. Goh, J. Plant Cell 12, 213–219 (2000).
M. K. Ritter, C. M. Padilla, and R. J. Schmidt, American Journal of Botany 89 (2), 203–210 (2002).
S. Scofield, W. Dewitte and J. A. H. Murray, Plant J. 50, 767–781 (2007).
E. Semiarti, T. Ishikawa, Y. Yoshioka, M. Ikezakki, Y. Machida and C. Machida, “Isolation and
characterization of Phalaenopsis Orchid Homeobox1(POH1) cDNAS, knotted1-like homeobox family of
genes in Phalaenopsis amabilis orchid”, in AIP Conference Proceedings of The 2nd International Conference
on Mathematics and Natural Sciences (ICMNS) ITB, edited by R. M. G. Simanjuntak et al. (American Institute
of Physics, Melville, NY, 2008), pp. 289-294 .
T. Sakamoto, N. Kamiya, M. Ueguchi-Tanaka, S. Iwahori, and M. Matsuoka, Genes Dev. 15, 581–590 (2001).
020019-5
Reuse of AIP Publishing content is subject to the terms at: https://publishing.aip.org/authors/rights-and-permissions IP: 202.43.93.104 On: Fri, 17 Jun 2016 07:19:12
19. O. Yanai, E. Shani, K. Dolezai, Tarkowski, R. Sablowski, G. Sanberg, A. Samach and N. Ori, Current Biology
15, 1566–1571 (2005).
20. E. Semiarti, I. S. Mercuriani, R. Rizal, A. Slamet, B. S. Utami, I. A. Bestari, Aziz-Purwantoro,
S. Moeljopawiro, Y. Machida and C. Machida, “Overexpression of PaFT gene in the wild orchid Phalaenopsis
amabilis (L.) Blume”, in AIP Conference Proceedings 1677 of The 5th International Conference on
Mathematics and Natural Sciences (ICMNS) ITB, edited by A. Purqon et al. (American Institute of Physics,
Melville, NY, 2015), pp. 090005-1- 090005-4, doi: 10.1063/1.4930750.
020019-6
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