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AGRIVITA Journal of Agricultural Science. 2017. 39(1): 1-10

Embryogenic Callus Induction from Leaf Tip Explants and Protocorm-Like Body
Formation and Shoot Proliferation of Dimorphorchis lowii: Borneon Endemic Orchid
Juddy E. Jainol and Jualang Azlan Gansau*)
Tissue Culture Laboratory, Faculty of Science and Natural Resources,
University Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia
*) Corresponding author E-mail: azlanajg@ums.edu.my

Received: April 17, 2016/ Accepted: September 13, 2016

ABSTRACT orchid possess unique and beautiful dimorphic

feature, which means it has two distinct flowers
Dimorphorchis lowii is a rare epiphytic orchid on one inflorescent and has the prospect to be
endemic to Borneo and very popular due to its marketed as a valuable basket or potted flowers.
ornamental value. For the purpose of mass The increasing popularity, high degree of rarity
propagation, procedures for in vitro propagation and its difficulty-to-locate have put this species
through callus were developed. The objectives of among the most highly sought after species.
this study were to determine the effects of plant Therefore, it is imperative to develop a method for
growth regulators (PGRs) on callus induction from multiplication to reproduce a great number of
leaf tip explant and the effects of complex plantlets in order to fulfill the floriculture demand
additives on PLBs formation and shoot develop- as well as for conservation purposes.
ment. The best combination of PGR was 3.0 mg Dimorphorchis orchids, just like other
L-1 Thidiazuron (TDZ) with 0.046 mg L-1 NAA monopodial orchids are asexually propagated
supplemented in half-strength medium (½ MS) using the top cuttings of the mother plants.
(Murashige & Skoog, 1962). The percentage of Nevertheless, reproduction through this conven-
survival and callus formation obtained were 96.0 % tional technique can be detrimental to the stock
± 19.8 and 52.0 % ± 16.5 respectively. Maximum plants as it poses threat due to the removal of
shoot proliferation from PLBs was observed in meristematic tips (Gantait, Bustam, & Sinniah
Knudson C (KC) medium enriched with 15 % (v/v) (2012). Conventional method through seed
coconut water. In this treatment, 10.2 ± 6.2 propagation is sometimes problematic as seed
shoots were produced from one callus explant. germination in natural environment is a slow
Shoots were rooted on KC medium containing 15 process and it requires mycorrhizal or fungal
% (v/v) coconut water and transferred in a partners’ association for its germination (Pant &
medium mixture containing sphagnum moss, Thapa, 2012) and any disturbance in the habitat
charcoal, brick and coco peat with the ratio of or physical environment during germination will
1:1:1:1. After 2 months being acclimatized in the destroy the whole population (Mathews & Rao,
glasshouse, 78 % of plantlets survived. Histology 1980). In vitro propagation of orchids by shoot tip
observations showed that embryogenic callus culture is a well-developed method. However, by
derived from leaf tip explants might originate from using shoot tip as explant means the most
epidermis and mesophyll cells and was capable significant part that is actively growing has to be
of developing into complete plantlets. sacrificed. Therefore, a technique of reproduction
using other insignificant parts such as leaves of
Keywords: callus, in vitro, orchid, protocorm like- the plant would be beneficial (Chugh, Guha, &
bodies (PLBs) Rao, 2009).
INTRODUCTION Successful culture of plantlet from leaf
culture of many orchid species has been reported.
Dimorphorchis lowii is an endemic orchid Among them are Aerides (Devi, Devi, & Singh,
of Borneo and is one of the rarest epiphytic 2013), Aranda (Gantait, Bustam, & Sinniah,
orchids, which commonly inhibiting the area with 2012), Phalaenopsis (Kuo, Chen, & Chang, 2005;
the elevation of 600-1800 meter above sea level Khoddamzadeh et al., 2011), Vanda and
(m asl) on Kinabalu, Tombuyukon and Trus Madi Dendrobium (Martin & Madassery, 2006; Tee,
mountains in Sabah (Cribb & Bell, 2008). This Wong, Lam, & Maziah, 2010), and Oncidium

Cite this as: Jainol, J. E., & Gansau, J. A. (2017). Embryogenic callus induction from leaf tip explants and protocorm-
like body formation and shoot proliferation of Dimorphorchis lowii: Borneon endemic orchid. AGRIVITA Journal of
Agricultural Science, 39(1), 1-10. http://doi.org/10.17503/agrivita.v39i1.895
Accredited: SK No. 81/DIKTI/Kep/2011
Permalink/DOI: http://dx.doi.org/10.17503/agrivita.v39i1.895
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Juddy E. Jainol and Jualang Azlan Gansau: Embryogenic Callus Induction from Leaf Tip Explants……………………..

(Mayer, Stancato, & Appezzato-Da-Glória, 2010). germination, ½ MS was used as basal medium
In leaf cultures, for example in Renanthera (Wu for protocorm development into seedlings in
et al., 2012) and Vanilla (Janarthanam & Seshadri, which the leaves were used as the source of
2008), wound surface of leaf explants had been explants in experiment I. ½ MS was also used for
reported to produce callus. Wu et al., (2012) basal medium in experiment I, and KC was used
reported that 99.67 % plantlet formation was as basal medium in experiment II. All media were
obtained from callus originated from leaf segments supplemented with 2 % sucrose and solidified
of Renanthera orchid. Protocorm-like bodies with 0.8 % agar powder (Sigma, USA). ½ MS was
(PLBs) generally have high potency to proliferate also enriched with 100 mg L-1 myo-inositol, 0.5
rapidly and regenerate into a whole plantlet mg L-1 pyridoxine-HCl, 0.1 mg L-1 thiamine-HCl,
(Bustam, Sinniah, Kadir, Zaman, & Subramaniam, 0.5 mg L-1 nicotinic acid and 2 mg L-1 glycine.
2013). Therefore, they are the most common Different PGRs (TDZ or NAA) and concentrations
target tissues used for mass propagation or were added to ½ MS medium in as treatments
producing a large number of uniform plantlets for experiment I, and different complex additives and
commercial purposes. concentrations (15 % (v/v) coconut water, 0.2 %
This study investigated (I) the effects of (w/v) peptone or 0.2 % (w/v) yeast extracts) were
PGR on callus induction from leaf tip explants and added to KC medium as treat-ments in
(II) the effect of complex additives on the PLBs experiment II.
formation and shoot development of D. lowii. The pH of the media was adjusted to 5.2-
Histology and scanning electron microscope 5.3 and agar powder was weighed and added
observations were also conducted to study the prior to autoclaving for 20 minutes at 121 oC and
callus induction from leaf tip explants. 15 kPa. The autoclaved medium was poured
aseptically into sterile plastic petri dishes (60 mm
MATERIALS AND METHODS x 15 mm, Sterilin, UK) at 20 ml for each petri dish.
The mother plant was collected in early These petri dishes were allowed to cool down,
2009 from a local nursery in Sabah, Malaysia. covered and then sealed with parafilm M®
This plant was brought to the Orchid Unit nursery (American National Cam™, Menasha, WI) before
at University Malaysia Sabah, grown and used. For callus induction experiment, cultures
maintained in the glasshouse conditions. In were maintained at 25 ± 2 oC under continuous
December 2009, seeds were obtained and darkness. For PLB formation and shoot
cultured in Vacin & Went (VW) (Vacin & Went, development experiment, cultures were
1949) medium added with 10 % (v/v) potato maintained at 25 ± 2 oC under continuous cool-
homogenate. Protocorms developed from these white fluorescent light with photon flux density
seed cultures were then transferred to half- (PFD) of 15-25 μmol m-2 s-1.
strength MS (½ MS) (Murashige & Skoog, 1962) Effect of PGRs on Embryogenic Callus Induc-
medium containing 2.0 % (w/v) sucrose and 0.2 tion from Leaf Tip Explants
% (w/v) yeast extract. This served as the staging PGR tested in this investigation included
medium for explants source. During series of TDZ at 0.22, 0.33, 1.0, 3.0 and 4.0 mg L-1 and NAA
subcultures, protocorms grew into multiple shoots. at 0.046, 0.5 and 1.0 mg L-1. Both TDZ and NAA
Multiple shoots bearing 3-4 leaves each were were applied individually or in combination. ½ MS
chosen as source for leaf explants. The first two medium supplemented with 2.0 % (w/v) sucrose
leaves from the top with the diameter of 2-5 mm was used as basal medium.
were chosen and dissected with a sterile sharp
scalpel and the tips were cut approximately 5-7 Effects of Complex Additives on PLBs
mm in length. Development and Shoot Proliferation from
Embryogenic Callus Derived from Leaf Tip
Medium Preparation and Culture Condition Explants
In this study, three media formulations were Green embryogenic calli was cut aseptically
used as basal media: Vacin and Went (VW), into clumps of approximately 3-5 mm in diameter
modified MS containing half-strength macro and and the clumps were used as explants. In this
micro nutrients (½ MS), and Knudson C (KC) study, three types of complex additives namely
(Yam & Arditti, 2009). VW enriched with 10 % coconut water at 15 % (v/v), peptone at 0.2 %
(v/v) potato homogenate was used for seed
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Juddy E. Jainol and Jualang Azlan Gansau: Embryogenic Callus Induction from Leaf Tip Explants……………………..

(w/v) and yeast extract at 0.2 % (w/v) were used Austria), affixed to aluminium stubs and coated
to investigate the PLBs development and sub- with gold palladium in a sputter coater
sequent shoot formation from embryogenic callus (EMITECH, K550X, England). The mounted
of D. lowii. KC medium supplemented with 2.0 % specimens were examined with scanning electron
(w/v) sucrose was used as basal medium. Once microscope (Zeiss EVO MA10, U.K).
shoots were established, they were transferred
into 100 ml Erlenmeyer flask containing 20 ml of RESULTS AND DISCUSSION
KC medium supplemented with 15 % (v/v) Effect of PGRs on Embryogenic Callus Induc-
coconut water for shoot elongation and root tion from Leaf Tip Explants
formation. The effects of PGRs is shown in Table 1.
Data Analysis Incorporation of TDZ and NAA at a concen-tration
All experiments were performed in a ranges from 1.0 to 3.0 mg L-1 and 0.046 to 1.0 mg
Complete Randomized Design (CRD) with five L-1, respectively in ½ MS medium was crucial in
replicates. For embryogenic callus induction promoting the percentages of survival of explants
experiment, one petri dish represented one and embryogenic callus formation. The highest
replicate and in each replicate contained 20 leaf tip percentage of explant survival was observed in
explants. Data on percentage of survival, medium containing 3.0 mg L-1 TDZ and 0.046 mg
percentage of callus formation, callus density and L-1 NAA at 96.0 % ± 19.8 and 1.0 mg L-1 TDZ and
color were recorded after 60 days of culture. For 0.046 mg L-1 NAA at 92.0 % ± 15.3. There were
PLBs shoot proliferation from embryogenic callus no significant differences between these two
derived from leaf tip explants, one petri dish treatments. However, there were significant
represented one replicate and in each replicate differences observed when compared to the
contained 20 callus clumps. Data on percentage of other concentrations of TDZ and NAA as shown
survival rate, percentage of PLB formation, number in Table 1. For embryogenic callus induction, it
of shoots, and number of leaves were recorded was observed that medium containing 3.0 mg L-1
after 60 days and 120 days of culture. Significance TDZ and 0.046 mg L-1 NAA produced the highest
of treatment effects on these data were analyzed percentage of callus formation (52.0 % ± 16.5),
using ANOVA, p<0.05 and comparison between while other PGR treatments produced only 18-34
mean values of treatments were made by Duncan % of the explants forming callus.
Multiple Range Test using SPSS version 20 (IBM The control treatment produced only 2 % ±
Corporation, New York, USA) 1.4 of explants forming callus. These results
indicated that the presence of PGR was not
Histology and Scanning Electron Microscope essential for callus induction; however, addition
Observation on Embryogenic Callus Derived of either TDZ or NAA or both PGRs in the medium
from Leaf Tip Explants was beneficial, and the best medium for callus
induction was MS + 3.0 mg L-1 TDZ and 0.046 mg
Light Microscopy L-1 NAA. White embryogenic calli was formed and
Tissues for histological observation were covered almost all the surface of the leaf tip
secured in PAA solution (15 Picric acid: 1 Acetic explants (Figure 1A and 1B). Similar study was
acid), dehydrated in an ethanol series, and lodged done by Chen & Chang (2006) with leaf tip
in paraffin wax. The materials embedded in paraffin explants of Phalaenopsis amabilis. Chen &
were sectioned at 5 μm and stained with Chang (2006) reported that when leaf explants of
hematoxylin-eosin. The observation and Phalaenopsis amabilis were cultured on
photography were done using light microscope hormone-free medium or medium added with
(Leica ICC50 HD, UK). NAA alone at 0.1 or 1.0 mg L-1, the explants
Scanning Electron Microscopy (SEM) turned necrotic and no embryo was formed.
The samples were fixed in 2 % (v/v) However, in the presence of TDZ at 0.1, 1.0 and
glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) 3.0 mg L-1 either alone or in combination with
and dehydrated in an ethanol series. The NAA at 0.1 or 1.0 mg L-1, somatic embryos (SE)
samples were dried to the critical point with liquid directly formed at the surface of explants, without
CO2 in a critical point dryer (CPD) (Leica EM300, any interfering callus formation.
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Juddy E. Jainol and Jualang Azlan Gansau: Embryogenic Callus Induction from Leaf Tip Explants……………………..

Table 1. Effects of plant growth regulator types and concentrations on embryogenic callus induction from
leaf tip explants of D. lowii cultured in ½ MS medium supplemented with 2.0 % (w/v) sucrose
under continuous darkness at temperature of 25 ± 2 oC after 60 days of culture
Cytokinin/Auxin
Percentage of Percentage of Density of
(mg L-1) Color of callus
Survival (%) callus formation (%) callus
TDZ NAA
Control 24.0±8.0g 2.0±1.47d* + Greenish-white
0.22 0.00 64.0±28.5cd 18.0±10.4bc + Greenish-white
0.33 0.00 60.0±24.5cde 22.0±11.5bc + Yellowish-white
1.0 0.00 48.0±20.5def 24.0±13.1bc + Greenish-white
3.0 0.00 90.0±30.3ab 28.0±17.4bc ++ Greenish-white
0.0 0.046 75.0±43.9bc 28.0±17.4bc ++ Yellowish-white
0.0 0.5 65.0±48.3cd 25.0±13.9bc + Greenish-white
0.0 1.0 42.0±29.8efg 0.0±0.0e - -
1.0 0.046 92.0±15.3a 34.1±25.8bc ++ White
3.0 0.046 96.0±19.8a 52.0±16.5a +++ White
3.0 0.5 34.0±8.9fg 12.0±8.8bc + Yellowish-white
3.0 1.0 86.0±21.1ab 28.0±17.9bc ++ White
4.0 0.5 62.0±25.0cd 18.0±10.4bc + Greenish-white
4.0 1.0 50.0±26.0def 14.0±9.1bc + Greenish-white
Remarks: *Mean values within a column followed by the same letters are not significantly different at p < 0.05 according
to Duncan’s Multiple Range Test. Note. +: callus at tip, ++: callus at tip and midsection, +++: callus covered
all surfaces, - : no callus

Figure 1. Embryogenic callus induction from leaf explant. A. Callus induction from leaf tip explant cultured
on ½ MS medium supplemented with 3.0 mg L-1 TDZ and 0.046 mg L-1 NAA after 30 days of
culture (DOC). B. Callus induction from leaf tip explant cultured on ½ MS medium supple-mented
with 3.0 mg L-1 TDZ and 0.046 mg L-1 NAA after 60 DOC and C. after 90-100 DOC when
transferred under 24hr light. (Scale bars = 5 mm).

The highest frequency of SE formations PGRs-free medium but the rate was very low at
were found in media containing 3 mg L-1 TDZ only 2.0 % ± 1.4. Gantait, Bustam, & Sinniah
without NAA, or in combination with 0.1 or 1.0 mg (2012) found that in control medium, 32.9 % of
L-1 NAA, i,e., 93.8 %, 87.5 % and 81.3 %, leaf explants were able to induce embryogenic
respectively. In line with these results, Tariq, Ali, calli even without any PGRs support. The best
& Abbasi (2014) also reported that 2.0 mg L-1 TDZ concentration of TDZ in this study was similar to
in combination with 1.0 mg L-1 NAA produced the result reported by Khoddamzadeh et al.,
optimum result in callus formation from leaf (2011), where leaf explants produced the highest
explant of Artemisia absinthium. In this study, embryogenic calli or PLBs at 71.87 % when
greenish-white callus development was seen on
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Juddy E. Jainol and Jualang Azlan Gansau: Embryogenic Callus Induction from Leaf Tip Explants……………………..

cultured in ½ MS medium supplemented with 3.0 Callus from leaf tip explant cultured on ½ strength
mg L-1 TDZ alone. MS medium turned green when transferred under
PGR(s) proportions have been known to 24hr light after 90-100 DOC (Figure 1C).
have species-specific effect on callus and PLB
induction from leaf explants (Shimura & Koda, Effect of complex additive on PLB develop-
2004; Gantait, Bustam, & Sinniah, 2012). TDZ is a ment and shoot proliferation from embryo-
substituted phenyl urea with cytokinin-like activity genic callus derived from leaf tip explants
(van Staden, Zažímalová, & George, 2008; Table 2 shows the result obtained after 120
Jitsopakul, Thammasiri, & Ishikawa, 2013). The days of cultures. 15 % (v/v) coconut water
ascendancy of TDZ in inducing somatic embryos presented the highest percentage of survival and
(infant PLBs) from leaf explants was recounted in PLBs formation both at 72.0 % ± 11.9 followed by
numerous orchid species including Oncidium 0.2 % (w/v) yeast extract at 62.0 % ± 13.5 in both
(Mayer, Stancato, & Appezzato-Da-Glória, 2010), percentages of survival and PLBs formation.
Doritaenopsis (Park, Yeung, Chakrabarty, & Paek, There were no significant differences
2002), Dendrobium and Phalaenopsis (Kuo, between the effects of these two complex
Chen, & Chang, 2005). TDZ is also useful for rapid additives. However, significant differences were
plant regeneration through organogenesis (Kishor observed between these two complex additives in
& Devi, 2009; Jitsopakul, Thammasiri, & Ishikawa, terms of number of PLBs per explant with 15 %
2013), somatic em-bryogenesis (Jheng, Do, (v/v) coconut water produced the highest 16.9±6.5
Liauh, Chung, & Huang, 2006; Gow, Chen, & and 0.2 % (w/v) yeast extract produced 9.9±2.9. In
Chang, 2009), or PLB formation of orchids (Mayer, both media, the emergence of nodular structures
Stancato, & Appezzato-Da-Glória, 2010; from the callus was seen after 20-30 days of
Khoddamzadeh et al., 2011; Roy, Sajeev, culture (Figure 2A). These nodular tissues later
Pattanayak, & Deka, 2012). NAA is a synthetic progressed further and developed into aggregates
auxin plant growth regulator that is routinely used of PLBs after 60 days of culture (Figure 2B). After
in in vitro propagation of orchid to promote callus 120 days of culture, 15 % (v/v) coconut water
induction such as in Dendrobium (Mei, Danial, produced a significantly higher percentage of
Mahmood, & Subramaniam, 2012; Salam, Salam, PLBs forming shoot at 70.0 % ± 42.0 with 8.9±2.9
& Mohanty, 2013), Vanilla (Tan, Chin, & Alderson, shoots and 5.5±2.6 leaves (Figure 2C) followed by
2011), Oncidium (Mayer, Stancato, & Appezzato- 0.2 % (w/v) peptone at 40.0 % ± 20.2 with 4.5±1.2
Da-Glória, 2010), Phalaenopsis (Khoddamzadeh shoots and 2.7±1.1 leaves.
et al., 2011), and Renanthera (Wu et al., 2012).

Table 2. Effect of complex additives types and concentrations on PLBs development and shoot proliferation
from callus culture of D. lowii cultured on KC medium supplemented with 2.0 % (w/v) sucrose
under 24-hour light photoperiod with PFD of 15-25 μmol m-2 s-1 at 25 ± 2 oC after 60 and 120
days of culture
Complex Percentage Percentage of Number of Percentage Number of Number of
additive of proliferated new PLBs of PLBs shoots**) leaves**)
Type and survival (%)*) explant (%)*) formed* formed
concentration shoot (%)**)
(w/v, v/v)
Control 40.0 ± 20.5b 40.0 ± 20.5b 5.8 ± 1.9c 2.0 ± 1.6b 1.8 ± 0.1b 1.1 ± 0.5c
0.2 % peptone 48.0 ± 20.2b 48.0 ± 20.2b 6.5 ± 4.0c 40.0 ± 20.2a 4.5 ± 1.2b 2.7 ± 1.1b
0.2 % YE 62.0 ± 13.5a 62.0 ± 13.5a 9.9 ± 2.9b 46.0 ± 20.1a 3.1 ± 1.9b 1.7 ± 0.4b
15 % CW 72.0 ± 11.9a 72.0 ± 11.9a 16.9 ± 6.5a 70.0 ± 42.0a 8.9 ± 2.9a 5.5 ± 2.6a
Remarks: Mean values within a column followed by the same letters are not significantly different at p<0.05 according
to Duncan’s Multiple Range Test; * Data collected after 60 days of culture; ** Data collected after 120 days
of culture
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Juddy E. Jainol and Jualang Azlan Gansau: Embryogenic Callus Induction from Leaf Tip Explants……………………..

Figure 2. Plantlet development from callus: A. Callus cultured on KC medium added with 15 % (v/v) coconut
water. B. PLBs with shoots after 50-60 DOC and C. Shootlets after 120 DOC. (Scale bars = 5
mm). D. Plantlets formed when cultured on KC medium supplemented with 15 % (v/v) coconut
water after 150-180 DOC. E. Plantlets were separated from clumps and prepared for
acclimatization. F. Plantlets acclimatization after 2 months. (Scale bars = 10 mm)

Overall, coconut water at 15 % (v/v) showed of PLBs in Phalaenopsis gigantean is improved

the superiority compared to the other two complex with the addition of coconut water in the culture
additives (peptone & yeast extract). Coconut medium. The PLB and callus induction in
water is extensively used in the plant micropro- Cymbidium hybrids is also promoted by the
pagation due to its growth enhancer substance coconut water in the culture media (Teixeira da
and cytokinin-type action that assist cell division Silva & Tanaka, 2006). Coconut water has also
and stimulate fast growth (Arditti, 2008; Yong, Ge, been reported to enhance shoot multiplication in
Ng, & Tan, 2009; Prando, Chiavazza, Faggio, & Vanilla planifolia (Kalimuthu, Senthilkumar, &
Contessa, 2014). Coconut water contains high Murugalatha, 2006).
levels of Z-type cytokinin (trans-zeatin riboside, Rooted shoots (Figure 2D & Figure 2F)
trans-zeatin O-glucoside, dihydrozeatin O-gluco- were obtained after culturing cluters of shoots on
side, trans-zeatin, dihydrozeatin, trans-zeatin KC medium containing 15 % (v/v) coconut water
riboside-5 -monophosphate), iP-type cytokinin for 4 to 5 months and these plantlets were
(N6-isopentenyladenine), kinetin, kinetin riboside transferred in a medium mixture containing
and ortho-topolin in addition to sugars, vitamins, sphagnum moss, charcoal, bricks and coco peat
minerals and amino acids (Yong, Ge, Ng, & Tan, with the ratio of 1:1:1:1. After 2 months, 78 % of
2009; Tan, Cheng, Bhat, Rusul, & Easa, 2014). plantlets aged between 1-1½ year survived after
Murdad et al., (2006) reported that the production being transferred to the glasshouse (Figure 2F).
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Juddy E. Jainol and Jualang Azlan Gansau: Embryogenic Callus Induction from Leaf Tip Explants……………………..

Figure 3. Scanning electron microscope (SEM) and histology view on A. SEM view on embryogenic callus
formed on the abaxial part of leaf tip explant, scale bar = 400 μm, 77 x. B. Light microscopic
views of leaf tip showed longitudinal section of explant showing cell dedifferentiation in callus that
produced unorganized cell mass, upper & lower epidermis (UE) & (LE), mesophyll cell (M), callus
(CL), scale bar = 50 μm, 40x. C. Single cells divide and developed into PLBs of different sizes,
scale bars = 40 μm, 1.00K x. D. Cross section of calli with multi-globular compact callus cells,
scale bar = 50 μm, 40x (Jainol, 2016).

Histology and Scanning Electron Microscope showed densely lower epidermis and mesophyll
Observations on Embryogenic Callus Derived cells underwent dedifferentiation. Most of the
from Leaf Tip Explants time, callus developed at the cut ends of leaf tip
Embryogenic callus derived from leaf tip explants, suggesting that the wounding might
explants shows cells dedifferentiation (Figure 3A- have triggered dedifferentiation of cells to form
B). Begum, Tamaki, Tahara, & Kako (1994) callus. Kuo, Chen, & Chang (2005) reported that
reported that callus formation from orchids used the highest ability of Phalaenopsis leaf explants
explants that contained meristematic cells or to form embryos was found at adaxial surfaces
epidermal cells, suggesting that callus cells arise near cut ends on TDZ-containing medium. Figure
from these types of cells. Figure 3B shows the 3C-D show that the callus masses composed of
cross-section through the leaf tip explants which spherical, compact and loose aggregates of cells.
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Juddy E. Jainol and Jualang Azlan Gansau: Embryogenic Callus Induction from Leaf Tip Explants……………………..

Spherical single cells can be seen embedded F. Q., & Subramaniam, S. (2013).
within the friable callus. These cells started to Selection of optimal stage for protocorm-
divide and gave rise to somatic embryos or PLBs like bodies and production of artificial
of different sizes. Globular-stage somatic embryo seeds for direct regeneration on different
or PLBs might originate either from the cells media and short term storage of
within the callus or from the callus surface. These Dendrobium Shavin White. Plant Growth
PLBs developed into shoots that would formed Regulation, 69(3), 215–224. http://doi.
complete plantlets when cultured on KC medium org/10.1007/s10725-012-9763-6
supplemented with 15 % (v/v) coconut water. Chen, J. T., & Chang, W. C. (2006). Direct
somatic embryogenesis and plant
CONCLUSION regeneration from leaf explants of
The present study demonstrated that the Phalaenopsis amabilis. Biologia
success of embryogenic callus induction from Plantarum, 50(2), 169-173. http://doi.
leaf tip explants of Dimorphorchis lowii is greatly org/10.1007/s10535-006-0002-8
influenced by plant growth regulators. Leaf tip Chugh, S., Guha, S., & Rao, I. U. (2009).
explants were able to produce embryogenic Micropropagation of orchids: A review on
callus in medium free of PGR but the frequency the potential of different explants.
was very low (2 %). In this study, the best result Scientia Horticulturae, 122(4), 507–520.
in embryogenic callus formation on leaf tip http://doi.org/10.1016/j.scienta.2009.07.
explants was obtained at combination of 3.0 mg 016
L-1 TDZ and 0.046 mg L-1 NAA Furthermore, 15 Cribb, P., & Bell, A. (2008). The Bornean endemic
% (v/v) coconut water was found to be the best genus Dimorphorchis (Vandeae: Aeridinae).
complex additive to promote PLBs development Malesian Orchid Journal, 2, 77-92.
and subsequent shoot formation from embryo- Devi, H. S., Devi, S. I., & Singh, T. D. (2013). High
genic callus of D. lowii. Histology observations frequency plant regeneration system of
revealed that, embryogenic callus might have Aerides odorata Lour. through foliar and
originated from epidermis and mesophyll cells shoot tip culture. Notulae Botanicae Horti
and these calli have the capability to multiply and Agrobotanici Cluj-Napoca, 41(1), 169–
formed complete plantlets. However, further 176. Retrieved from https://www.
study needs to be conducted to study cell de- researchgate.net/publication/236949935
differentiation, and the structural and biological _High_Frequency_Plant_Regeneration_
functions of cells involved in the initiation and System_of_Aerides_odorata_Lour_Thro
formation of embryogenic callus. ugh_Foliar_and_Shoot_Tip_Culture
Gantait, S., Bustam, S., & Sinniah, U. R. (2012).
ACKNOWLEDGMENT Alginate-encapsulation, short-term storage
and plant regeneration from protocorm-
We thank Tissue culture laboratory, Uni- like bodies of Aranda Wan Chark Kuan
versity Malaysia Sabah for providing the facilities “Blue” Vanda coerulea Grifft. ex. Lindl.
and equipment for this research. (Orchidaceae). Plant Growth Regulation,
68(2), 303–311. http://doi.org/10.1007/
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