Vol 5|Issue 4| 2015 |253-256.

Asian Journal of Pharmaceutical Science & Technology

e-ISSN: 2248 – 9185
www.ajpst.com Print ISSN: 2248 – 9177

IN VITRO CALLOGENESIS FROM BUD AND STEM EXPLANTS OF

DRAGON FRUIT (HYLOCEREUS UNDATUS)
A. Thinesh and T. H. Seran
Department of Crop Science, Faculty of Agriculture, Eastern University, Chenkalady, Sri Lanka

ABSTRACT
This study was carried out to find out the effect of thidiazuron (TDZ) and 6-benzyl aminopurine (BAP) on callus
formation of dragon fruit under in vitro conditions for vegetative propagation. Therefore, bud and stem segments of dragon fruit
were cultured on Murashige and Skoog (MS) media with two different concentrations of BAP or TDZ for callogenesist under in
vitro conditions. The bud explants in MS medium containing 3 mg/l TDZ and 0.5 mg/l NAA exhibited the highest in vitro
response compared to other media. Further, result revealed that most responsible plant part for callus formation would be the stem
segment. TDZ concentration at 3 mg/l showed the best for callus formation in cultured stem explants of dragon fruit. In this
study, TDZ is more effective plant growth regulator than BAP for friable callus production from stem explants of dragon fruit.

Key words: BAP, dragon fruit, callus, in vitro culture, TDZ.

INTRODUCTION
Dragon fruit (Hylocereus undatus) is a perennial
climbing cactus with green stems and it grows in tropical
and subtropical countries. It has the potential to increase
demand in export market [1] and also be a profitable fruit
crop for farmers in Sri Lanka. Fruit pulp is an edible part of
dragon fruit. It is generally used in fruit salad and is a
natural source of antioxidants [2]. Dragon fruit is
propagated by seeds or stem cuttings. Cross pollination is
necessary for setting of fruits [3]. Hence, superior planting
materials are not sufficient for large scale cultivation of
dragon fruit. In vitro culture technique is an alternative way
to rapidly multiplicate plants in order to obtain disease-free
improved planting materials and also it is a faster and
efficient process compared to other conventional plant
propagation methods [4, 5].
Micropropagation of the cacti facilitate for the
production of large numbers of plants from a relatively
small stock [6]. Direct shoots regenerated from dragon fruit
explants which were obtained from in vitro germinated
seedlings and cultured in BAP and NAA [5]. Seeds are not
true to type [7] and it has low seed viability [8]. Suitable
initial explant and correct plant growth regulators are
significant features for better callus production in order to
develop protocol for indirect organogenesis or somatic
embryogenesis for a large scale production of healthy
planting materials. However, there is lack of information on
callogenesis of dragon fruits. Therefore, this study was
Corresponding Author: T. H. Seran E-mail: thayaseran@yahoo.com
aimed to develop a protocol for callus formation from bud
and stem segments of dragon fruits which were vegetatively
propagated by using stem cuttings.
MATERIALS AND METHODS
This experiment was carried out in 2013 at the
Tissue Culture Laboratory, Eastern University of Sri Lanka
to select the most suitable expant and medium for callus
production from dragon fruit explants.
Plant material
Stem cuttings were collected from one year old
healthy plant and they were vegetatively grown in polybags
containing soil: cattle manure (2:1 v/v) at a net house.
Newly formed shoots were obtained from vegetatively
propagated mother plants (2 months old) to excise bud and
stem explants.
Sterilization of explants
The newly formed shoots were collected from the
healthy mother plants of dragon fruit. The axillary buds and
stem segments were excised from the shoots. They were
rinsed thoroughly in running water and were then washed in
distilled water. Subsequently they were dipped in 70%
ethanol for 1 min and surface sterilized using 30% of
CloroxTM (Sodium hypochlorite, 5.25% active ingredient)
with 1 ml of tween 20 for 20 min. Thereafter

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they were washed four times thoroughly in sterilized
distilled water to avoid presentence of CloroxTM residues.
Culture medium
In this experiment, MS [9] media containing two
different concentrations (2.0 mg/l, 3.0 mg/l) of BAP (6-
benzyl- amino- purine) or TDZ (thidiazuron) with 0.5 mg/l
NAA (α-naphthalene acetic acid) were prepared for culture
establishment. Agar (8 g/L) was added to solidify the
medium and the pH was adjusted to 5.8 with 1N HCL or 1
N NAOH. Ten ml of medium was poured into each culture
vessel (125 ml capacity) and they were covered by using
plastic lid. These culture bottles were autoclaved at 121 0C
for 20 min at 15 psi subsequently they were kept to cool
before the inoculation of explants.
Inoculation of explants
The sterilized axillary buds from shoots of dragon
fruit as well as stem segments (0.5 cm long) from the
middle portion of the stem were excised under sterile
laminar flow with the sterilized scalpel and forceps.
Subsequently those excised explants were cultured on MS
media containing the above mentioned plant growth
regulators and 30 g/l sucrose. Each culture bottle containing
four explants were labeled and sealed with parafilm strips.
Culture environment
The cultured bottles containing explants were
incubated at 25±20C under white fluorescent light in
photoperiod of 16 hrs light and 8 hrs dark. In first weeks,
the cultures were observed daily for contamination and then
once in two days for in vitro response. This experiment was
repeated thrice.
Data collection
Data were recorded at regular interval. The
collected data were analyzed using Statistical Analysis
System (SAS) software (SAS Institute, Cary, North
Carolina, USA). The treatment means were compared using
Turkey’s honestly significant difference test at 5%
significant level.
RESULTS AND DISCUSSION
Successful in vitro vegetative propagation through
organogenesis or somatic embryogenesis depends on
concentration and kind of plant growth regulators, explant
selection, morphogenic response and ex vitro plantlet
survival [10]. The selection of suitable explants and medium
are the most important aspect for the better in vitro response
of dragon fruit. In the present study, the bud explants and
stem segments of dragon fruit were cultured on different
media supplemented with two different concentrations (2
and 3 mg/l) of BAP or TDZ in combination with 0.5 mg/l
NAA to determine the most suitable medium for
callogenesis.
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Survival of cultured explants
Survival rate was over 60 % in two types of
explants at the first week of culture (Figure 1). High
percentage (85%) of survival was recorded in stem
segments. Contamination percentage was higher (31%) in
cultured bud explants than stem segments. Further, it was
noted that the survival percentage of stem explants was 65%
at the second week afterward it reduced to 58% at the third
week. Stem explants exhibited lower contamination than
bud explants. The survival rate of bud explants was less
than 50% at fourth week. The microbes present in plant
tissues commonly resulted in decreasing survival rate of the
cultured explants, in vitro morphogenic response and their
variable growth [11]. Browning of explants was noted in a
few cultures during their incubation. It is generally due to
phenolic compounds that are released from the cut surfaces
of the explants and these compounds may lead to death of
the explants [12].
In vitro response of bud explants
The bud explants were cultured on different media
to find out the in vitro response and result showed that
colour of the explants turned light green to dark green
within three weeks of culture. Slight swelling of the bud
explants was also observed. In vitro response was recorded
in all tested combinations of plant growth regulators (Table
1). Greenish yellow nodules were first noticed on bud
explants cultured on MS medium containing TDZ (3.0 mg/l)
and NAA (0.5 mg/l) after two weeks of culture (Plate 1).
MS medium with BAP (3.0 mg/l) and NAA (0.5 mg/l) also
exhibited these structures which developed on the surface of
the bud explants.
In vitro response percentage was significantly high
(P<0.05) in the explants cultured on MS medium
supplemented with 3 mg/l TDZ or BAP while it was
relatively low in 2 mg/l TDZ or BAP at the fourth week of
culture. There was remarkable variation (P<0.05) in
explants showing in vitro response between TDZ and BAP
at 3 mg/l and about 60% of cultured explants exhibited
nodulation. Nodule colour changed from greenish yellow to
greenish or reddish colour. It was observed in the explants
cultured on MS medium supplemented with 3 mg/l TDZ or
BAP (Plate 2).
In vitro response of stem segments
Yellowish white friable callus was initiated at the
cut end of the stem explants cultured on different media
(Plate 3). Dahanayake and Ranawake reported that stem
explants showed high ability for plant regeneration [6].
Wound in in vitro cultured explants generally stimulate
callus formation [13]. More than 50% of the cultured stem
segments on MS medium with 3 mg/l TDZ and 0.5 mg/l
NAA exhibited in vitro response after three weeks of culture
(Table 2). The frequency of response was low on the MS
medium containing 2 mg/l BAP and 0.5 mg/l NAA. TDZ is
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function as an effective bioregulant in tissue cultures in
most of plant species [14].
Callus formation from stem segments
The result revealed that rate of callus formation
was considerably varied between the tested media (Table 2).
Friable callus was first formed on the stem segments
cultured on MS medium supplemented with TDZ (3.0 mg/l)
and NAA (0.5 mg/l). MS medium containing BAP (3.0
mg/l) and NAA (0.5 mg/l) also exhibited whitish - yellow
friable callus however frequency of cultured explants
showing callogenesis was relatively low. Significant
difference (P<0/01) was observed in callogenesis among the
treatments. The percentage of callus formation (65%) from
the cultured stem segments was comparatively high
(P<0.05) in MS medium supplemented with TDZ (3.0 mg/l)
and NAA (0.5 mg/L). In this study, a combination of 3.0
mg/l TDZ and 0.5 mg/l NAA was most suitable for
production of friable callus from stem explants of dragon
fruit. Iram and Anis [15] stated that TDZ exhibited stronger
effect than other cytokinins commonly used in in vitro
culture study.

Table 1. In vitro response of bud explants on different media at the four weeks of culture
Plant growth regulators (mg/l) Explant colour In vitro response %
2 BAP + 0.5 NAA Dark green 14.2 c
2 TDZ + 0.5 NAA Dark green 18.7 c
3 BAP + 0.5 NAA Dark green 48.4 b
3 TDZ + 0.5 NAA Dark green 62.0 a

Table 2. In vitro response of stem segments on different media at the four weeks of culture.
Plant growth regulators (mg/l) Explant colour In vitro response Callogenesis %
2 BAP + 0.5 NAA Light yellow Friable callus 11.7 d
2 TDZ + 0.5 NAA Light yellow Friable callus 22.2 c
3 BAP + 0.5 NAA Yellowish white Friable callus 45.4 b
3TDZ + 0.5 NAA Yellowish white Friable callus 65.0 a
Means with the same letter are not significantly different using Tukey’s honesty significant difference Test at 5% significant
level. Total number of explants cultured was 48 in each treatment.

Fig 1. The survival of two types of dragon fruit explants cultured in 3 mg/l BAP

Plate 1. Bud explants and in vitro cultured explants on MS Plate 2. In vitro response of bud explants cultured on
media (x 3) [A: Newly formed shoot of dragon fruit; B: MS media at fourth week of culture (×3) [Cultured bud
Axillary bud; C: Bud explants; D: Cultured bud explant in 3 explant in 3 mg/l TDZ (A) and BAP (B); a- reddish
mg/l BAP (E) at the second week of culture]. nodules; b- greenish nodules].

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Plate 3. In vitro response of stem explants cultured on MS media [A: Viny shoot of dragon fruit plant; B: Stem
segments (0.5 cm long); C-D: Callus formation in 3 mg/l BAP (C) and TDZ (D) at the third week of culture (×3); E-F:
Callus formation in 3 mg/l BAP (E) and TDZ (F) at the fifth week of culture (×3).

CONCLUSION
The results could be concluded that the most
responsible plant part for the friable callus formation was
the stem segment cultured on MS medium supplemented
with 3 mg/l TDZ and 0.5 mg/l NAA in which the
percentage of callogenesis was 65% at the four weeks of
culture. TDZ at 3 mg/l with NAA was the most effective
plant growth regulator for the initial culture establishment
and callus production of dragon fruit explants than BAP
among tested concentrations of BAP and TDZ. Uniform
good quality callus is required for a large scale production
of planting materials through organogenesis or somatic
embryogesis in dragon fruit.

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