In Vitro Cell.Dev.Biol.
—Plant (2012) 48:469–477
DOI 10.1007/s11627-012-9439-y
MICROPROPAGATION
In vitro propagation of purple pitahaya (Hylocereus costaricensis
[F.A.C. Weber] Britton & Rose) cv. Cebra
María Viñas & Mainor Fernández-Brenes &
Alvaro Azofeifa & Víctor M. Jiménez
Received: 18 September 2011 / Accepted: 3 April 2012 / Published online: 16 May 2012 / Editor: John W. Forster
# The Society for In Vitro Biology 2012
Abstract Limitations to large-scale propagation of purple
pitahaya (Hylocereus costaricensis [F.A.C. Weber] Britton
& Rose), a potential source of betalains for the food indus-
try, can be overcome through utilization of in vitro culture
technologies. In this work, successful in vitro propagation
from areoles of adult purple pitahaya plants is reported.
Factors affecting culture initiation, bud sprouting and
growth, shoot multiplication, rooting, and acclimatization
were studied. Best results for culture initiation were
obtained from the central region of new joints by disinfec-
tion of large (5–7 cm in length) explants that were subse-
quently divided. Explants were sequentially treated with
Extran® alkaline detergent for 10 min, followed by immer-
sion in 70 % (v/v) ethanol for 15–30 s, a mixture of the
fungicide Benomyl and the bactericide Agrymicin (2 gl−1
each) for 30 min, and disinfection in sodium hypochlorite
(1.0 %w/v) for 15 min. Culture of sectioned individual
areoles, without removing thorns, on Murashige and Skoog
medium supplemented with 15 or 30 μM N6-benzylamino-
purine (BAP) for 3 mo with monthly subcultures, followed
by transfer to the same medium with reduced BAP
(0–2 μM), induced bud sprouting in over 80 % of explants,
adequate growth of the shoots, with production of lateral
shoots, and spontaneous rooting within 160 d. These plants
were successfully acclimatized in vermiculite and peat moss
(1:1), or perlite and peat moss (2:1) in the greenhouse, with
over 90 % survival rate. One hundred percent of the in vitro-
derived plants were successfully transferred to the field.
Furthermore, these plants showed higher survival rates,
M. Viñas : M. Fernández-Brenes : A. Azofeifa :
V. M. Jiménez (*)
CIGRAS, Universidad de Costa Rica,
2060, San Pedro, Costa Rica
e-mail: victor.jimenez@ucr.ac.cr
larger height and increase in stem diameter than equivalent
plants from the same genotype, derived from stem segments
(the common clonal propagation system utilized for this
species) that were simultaneously planted.
Keywords Acclimatization . Climbing cactus . Clonal
propagation . Dragon fruit . Pitahaya
Introduction
Pitahayas (Hylocereus spp.) are perennial climbing cactus
plants native to tropical areas of North, Central, and South
America (Morton 1987). Main countries producing pitahaya
fruit (commonly known as dragon fruit) are Vietnam,
Colombia, Mexico, Costa Rica, and Nicaragua and, to a
lesser degree, cultivation occurs in Australia, Israel, and
Reunion Island. The European Union and Asia, especially
China, are the largest import markets (Le Bellec et al. 2006).
Fruits from most Hylocereus species have red-purple pig-
mented skin, while the pulp color ranges from white (in H.
undatus) to red and purple (in H. polyrhizus and H. costar-
icensis; Esquivel et al. 2007a).
Red-fleshed pitahaya fruits have gained importance over
recent years through increased fresh fruit consumption and
also as a source of natural pigments in food processing, due
to their high content of betalains. This nitrogen-rich group
of pigments, derived from tyrosine, has advantages over
anthocyanins (plant pigments with similar color variations)
especially in low-acidic and neutral-pH foods, due to their
higher stability in pH range 3–7 (Stintzing and Carle 2007).
Usually, propagation of pitahaya is conducted by using
cuttings from field plants. However, multiplication rates are
very low and it is difficult to obtain enough planting mate-
rial because of the large size (∼50 cm lengths) of the cuttings
470 VIÑAS ET AL.
required (Le Bellec et al. 2006). Despite acceptable seed
germination efficiencies of between 71 and 83 % for H.
undatus (El Obeidy 2006), such propagation is not commer-
cially feasible, because seed-derived plants have a long
juvenile period, delaying fruit production for several years
(Le Bellec et al. 2006). Vegetative propagation of pitahaya
can be improved, however, using biotechnological tools
such as in vitro tissue culture. This technique could enable
production of large numbers of clonal plants in relatively
short time periods using very little starting material (Rubluo
et al. 1993).
Many studies evaluating adequate in vitro conditions for
propagation of several cactus species have been conducted
using in vitro-germinated plants, to avoid disinfection of
explants, because of extreme sensitivity to common disin-
fection procedures and subsequent low survival rates (Pérez-
Molphe-Balch and Dávila-Figueroa 2002; Santos-Díaz et al.
2003). However, clonal propagation from adult material,
through activation of areoles, should be the preferred way
to reproduce genotypes with promising characteristics for
commercial plantations (Brasil et al. 2005). This has been
successfully conducted with members of the following cac-
tus genera: Mammillaria (Rubluo et al. 1993; Ramirez-
Malagon et al. 2007), Schlumbergera and Rhipsalidopsis
(Sriskandarajah and Serek 2004), Nopalea (Brasil et al.
2005), and Opuntia (García-Saucedo et al. 2005; Khalafalla
et al. 2007), among others.
To our knowledge, there are two reports for in vitro micro-
propagation of Hylocereus spp. from somatic tissues (Drew
and Azimi 2002; Mohamed-Yasseen 2002), but both refer to
H. undatus (white-fleshed pitahaya), a different species to that
used in this study. Developing efficient micropropagation
procedures for particular species generally requires detailed
studies to define specific composition of mineral salts, plant
growth regulators, and organic compounds in the culture
medium. Therefore, the aim of this study was to develop a
protocol for in vitro establishment and vegetative propagation
of Hylocereus costaricensis cv. Cebra, a violet-fleshed pita-
haya rich in betalains (Esquivel et al. 2007b), using areoles as
explants. Moreover, it describes an efficient procedure for
successful acclimatization and transfer to the field of in
vitro-generated plants.
Materials and Methods
Plant material. Stem segments (ca. 50 cm in length) from
adult pitahaya plants (Hylocereus costaricensis [F.A.C.
Weber] Britton & Rose cv. Cebra), grown organically in
Barranca, Puntarenas, Costa Rica (N 9° 57.566′; W 84°
43.217′), were rooted and cultivated in pots containing a
mixture of peat moss (V-J Centroamerica, San Jose, Costa
Rica) and Andisol soil (1:1) in a greenhouse, until they
produced new joints. At least 3 wk before each experiment,
the plants were sprayed weekly with an equal mixture of
2 gl−1Agrymicin (bactericide with active ingredients strep-
tomycin and oxytetracycline; Pfizer, Mexico City, Mexico),
and 2 gl−1Benomyl (fungicide with active ingredient beno-
myl; Piscis, San Jose, Costa Rica).
Preparation of explant material. For Experiment 1, young
joints (10–20 cm in length) were collected from sprayed
greenhouse plants and cut into lengths of 1–2 cm (with only
one areole per section). Explants were subsequently washed
with running tap water for 15 min. Pre-treatment to disin-
fection started with immersion in an alkaline solution (0.1 %
w/v) of Extran® MA 01 (Merck, Darmstadt, Germany) for
10 min, followed by 15–30 s in ethanol (70 %v/v) and,
finally, in the mixture of Agrymicin and Benomyl described
above, for either 15 or 30 min. Disinfection as such was
conducted with either 1.0 or 1.5 %w/v sodium hypochlorite
(NaOCl), supplemented with one drop of Tween 20 (Sigma,
St. Louis, MO) per 100 ml, for 15 min. Finally, the explants
were washed three times with sterile distilled water in a
laminar flow cabinet. Explant borders, usually damaged by
the disinfection procedure, were removed by trimming,
leaving individual areoles with little surrounding tissue.
Eighteen areoles for each treatment were used.
Experiment 2 followed the same sequence of steps as
for Experiment 1, but used larger tissue sections (5–
7 cm lengths and with five to six areoles per section),
only one pre-treatment (Agrymicin and Benomyl for
30 min) followed by disinfection with 1.0 %w/v NaOCl
for 15 min. Explants were subsequently dissected, after
sterilization, into smaller sections, similar in size to
those of Experiment 1.
Culture conditions. Explants were cultured upright on basal
medium composed of Murashige and Skoog (1962) mineral
salts (MS), supplemented with thiamine·HCl (0.1 mg l−1),
pyridoxine·HCl (0.5 mg l−1), nicotinic acid (0.5 mg l−1),
glycine (2 mg l−1), myo-inositol (100 mg l−1), sucrose (5 %
w/v), and plant growth regulators as described below for
each experiment. The pH was adjusted to 5.80 (±0.01) with
KOH, and the medium was gelled with 0.8 % (w/v) agar
(Riedel-de Haën, Seelze, Hannover), poured into 25 ×
150 mm culture tubes, sealed with polypropylene closures
(wall thickness: 0.79 mm, Sigma), and autoclaved
(1.05 kg cm−2; 20 min). Explant cultures were placed under
a photoperiod of 12 h (10.2–20.5 μmol m−2 s−1, Sylvania
Supersaver Cool White, 32 W, F48T12/CW/SS) at 24–25°C.
Basal medium in Experiment 1 was supplemented with
0.5 μM naphthalene acetic acid (NAA) and 0.5 μM thidia-
zuron (Mohamed-Yasseen 2002). Due to lack of response (as
described below), 45 d after establishment, explants in this
experiment were transferred to basal medium supplemented
 IN VITRO PROPAGATION OF PURPLE PITAHAYA
with 10 μM N6-benzylaminopurine (BAP), based on previous
results of Escobar et al. (1986) with Opuntia spp.
Following results from Experiment 1, explants from Ex-
periment 2 were cultured directly on basal medium supple-
mented with BAP (0, 5, 15, 30, 45, or 60 μM). They were
transferred monthly to the corresponding culture medium
for 3 mo. Every 7 d, the number of initiated areoles was
recorded, as well as the shoot height at day 77. Ten areoles
for each treatment were used.
Effect of areole position in the joint. In order to study the
effect of the position of the areoles on bud sprouting, joints
from donor plants were cut into three sections: distal, central
and basal and used as explants. These sections were initially
prepared and disinfected as described for Experiment 2, and
the areoles of each section were cultured on basal medium
supplemented with 30 μM BAP. Explants were transferred
to the same medium every month for 3 mo. After 80 d of
culture, the number of sprouted buds and calli developed
were evaluated. Eighty-four areoles for each treatment were
used.
Effect of lower BAP concentrations on shoot prolifera-
tion. To determine the effect of lowering BAP concentra-
tions during the shoot proliferation phase, a new batch of
explants was disinfected and only central sections were
cultured, as described for Experiment 2, but the basal me-
dium was supplemented with only 15 or 30 μM BAP. After
3 mo culture, sprouting explants were transferred, without
any further dissection, to basal medium supplemented with
0, 1, 2, 5, 15, or 30 μM BAP contained in baby food jars
(5×7 cm). Between 16 and 64 explants were used in each
case, depending on the treatment. All explants were subcul-
tured monthly to the corresponding culture medium for
3 mo. After a further 80 d culture on media with lower
BAP levels, the number of shoots per areole, shoot height,
development of apical necrosis, and callus formation were
evaluated.
Acclimatization of plants in the greenhouse. For acclimati-
zation, agar was carefully removed from roots with tap
water and the plants were placed in 24-well trays (each well
measuring 6×6 cm; V-J Centroamerica) under mist irriga-
tion (4 s every 15 min). The following substrates were
tested: vermiculite (medium coarse, J. P. Austin, Inc., Bea-
ver Falls, PA) + peat moss (V-J Centroamerica; 1:1), perlite
(W. R. Grace & Co., Cambridge, MA) + peat moss (2:1),
perlite + peat moss (1:1), and perlite alone. After 60 d in the
greenhouse, the number of surviving plants and plant sizes
were evaluated. After 4 mo, the plants were transferred to
polyethylene bags (13×18 cm) in a substrate composed of
peat moss + Andisol soil (1:1). A total of 192 plants were
acclimatized in the greenhouse.
471
Transfer to the field. After 4 mo in polyethylene bags,
plants were transferred to the field in Cañas, Guanacaste,
Costa Rica (N 10° 19.580′; W 85° 08.342′). Due to the
climbing behavior of pitahaya, plants were planted in
mounds next to a living support (approximately 2.0–
2.5 m in height) of Gliricidia sepium or Erythrina berter-
oana (frequently used for this purpose in the region).
Planting distance was 2×2 m and each plant was tied to
the corresponding living support. For comparison purposes,
rooted stem cuttings 30–70 cm in length, derived from field
growing plants of the same Cebra genotype [clonally prop-
agated using conventional methods (Merten 2003; Le Bel-
lec et al. 2006)], were planted simultaneously following the
same procedure. After 4 mo in the field, the number of
surviving plants, plant height and stem diameter were
evaluated. Twenty in vitro-derived and 20 conventionally
propagated plants were transferred to field conditions in
this experiment.
Statistical analysis. Percentage of areoles that produced
shoots, explants that formed callus, explants showing apical
death, and explants surviving in vitro conditions were eval-
uated as presence or absence (frequencies), by chi-square
analysis using Infostat statistical program, version 2008
(Universidad Nacional de Córdoba, Córdoba, Argentina).
Shoot height, number of shoots per explant, size of the
plants, and stem diameter were analyzed with one-way
analysis of variance, followed by Tukey’s test for mean
comparison, using the statistical program Statistica 6.0
(StatSoft Inc., Tulsa, Oklahoma).
Results
Culture initiation and axillary bud growth. In Experiment
1, 100 % contamination was observed when the
explants were pretreated with the mixture of Agrymicin
and Benomyl for 15 min, irrespective of the NaOCl
concentration employed for subsequent disinfection.
Complete disinfection of the explants was only achieved
with 30 min washes in Agrymicin and Benomyl solu-
tion (Table 1). However, 61 % of explants were severe-
ly damaged, presumably due to the disinfection process.
Therefore, in Experiment 2, larger explants (5–7 cm
length) were sterilized with the most successful treat-
ment used for Experiment 1 as follows: alkaline deter-
gent for 10 min, followed by immersion in 70 % (v/v)
ethanol for 15–30 s, a mixture of fungicide and bacte-
ricide (2 g l−1 each) for 30 min, and disinfection in
sodium hypochlorite (1.0 % w/v) for 15 min. This
resulted in a significant reduction in the level of
472 VIÑAS ET AL.

Table 1 Contamination and tissue damage suffered by explants after pre-treatment and disinfection, 1 mo after in vitro culture initiation

Experiment Explant length (cm) Total no of explants Agrymicin+Benomyl (min) NaOCl (%) Contamination (%) Damage (%)

1 1–2 20 15 1.0 100 n.e.

1.5 100 n.e.
20 30 1.0 0 61
1.5 0 61
2 5–7 18 30 1.0 0 16

n.e., not evaluated (due to contamination)

damage, to just 16 % of explants, and no contamination The effect of varying BAP concentrations (0, 5, 15, 30,
was observed (Table 1). 45 or 60 μM) on bud sprouting is presented in Fig. 1A.
Buds from surviving explants from Experiment 1 had not Areoles cultured on a medium free of plant growth regula-
sprouted on basal medium supplemented with NAA and tors failed to initiate shoots during the 84-day evaluation
thidiazuron after 45 d in culture. It was only after they were period. On the contrary, explants on medium containing 15,
transferred to medium containing 10 μM BAP that sprouting 30, or 60 μM BAP behaved similarly, with over 60 % of the
initiated. Therefore, several alternative BAP concentrations areoles sprouting. An intermediate group (5 and 45 μM
were tested in Experiment 2. In all experiments, buds sprouted BAP) producing 20–30 % of sprouting buds was also iden-
directly from intact areoles, without an intermediate callus tified. The first axillary shoots were observed approximately
phase. Using areoles without thorns or eliminating the thorns 28 d after culture initiation in treatments using 15, 30, or
of the areoles prevented bud sprouting (data not shown). 60 μM BAP. While in the treatment with 45 μM BAP,
Fig. 1 Effect of BAP
concentration on A the
percentage of areoles that
produced shoots over time and
B on shoot height and callus
formation after 84 d in culture.
Values followed by the same
letter are not significantly
different at the p≤0.05
according Chi-square analysis
(A) and Tukey’s test (B).
 IN VITRO PROPAGATION OF PURPLE PITAHAYA 473

Table 2 Effect of the position of the areole in the original joint on
areole sprouting and callus formation after 80 d of culture
Position Number of Areoles that produced
non-contaminated shoots (%)
explants
Distal 84 61.9b
Central 63 82.5a
Basal 65 67.7b
Values followed by the same letter within a column are not significant-
ly different at the p≤0.05, according Chi-square analysis
axillary shoot growth was observed after 42 d, and in the
treatment with 5 μM BAP shoots developed later, approxi-
mately 63 d after culture initiation (Fig. 1A).
Only one shoot per areole was observed with treatments
using 5, 15, or 30 μM BAP. However, medium containing
45 or 60 μM BAP eventually produced two to three growing
shoots per areole, but they displayed abnormal morphology
and apical necrosis. From day 77 onwards, no new shoots
were produced from areole explants and the height of the
sprouted shoots was assessed at that time. Shoots growing
on medium containing 15 or 30 μM BAP were longer than
those on medium without BAP or with 45 μM BAP
(Fig. 1B). Callus frequently developed at the base of the
explants in all treatments supplemented with BAP (Fig. 1B).
However, such callus formation did not affect the growth of
shoots already formed.
Explants from the central section of the joints sprouted
more profusely than either basal or distal sections on medi-
um containing 30 μM BAP after 80 d in culture (Table 2).
The position of the explant did not, however, influence the
degree of callus formation.
Effect of reduced BAP concentrations on shoot proliferation.
As indicated above, shoots that grew continuously on high
concentrations of BAP (15 or 30 μM) resulted in apical
death. Therefore, to test whether further growth of formed
shoots was possible, explants were transferred to the basal
medium containing lower doses of BAP. For this experiment
new explants were initially grown on medium supplemented
Callus with 15 or 30 μM BAP for a period of 3 mo, and transferred
(%) to medium containing 0, 1, 2, 5, 15, or 30 μM BAP for a
further 80 d culture. In media with the lowest BAP concen-
52.4a
trations (0, 1 or 2 μM), actively growing shoots, without
abnormalities or apical necrosis, were produced. Further-
46.0a
more, reduced BAP supplementation did not affect the rate
40.0a
of new shoot development (ranging between two and five),
or shoot height (1.3 to 2.3 cm) at the end of the culture period
(Table 3). BAP supplementation, however, promoted callus
formation at the base of the explants, compared to the control
without BAP. Callus growth was more substantial at higher
BAP concentrations, which coincided with abnormal appear-
ance of developed plantlets (Fig. 2), and most of these
plantlets eventually died. More remarkable is the fact that
the lowest BAP concentrations (0, 1, and 2 μM) significantly
reduced apical death (Table 3), and produced normal and
healthy plantlets.
Shoot clumps developing from a single areole were
maintained together during all subcultures. Root production,
in the form of basal and aerial roots, initiated spontaneously
for 100 % of explants. They were subsequently considered
as true plants. In the last subculture, all callus at the base of
the shoots was carefully removed, without damaging the
roots, and the plants were maintained in vitro for an additional
30 d before acclimatization commenced. No additional
growth or new shoots were observed during this period.
Acclimatization of plants in the greenhouse. After 60 d in
the greenhouse, more than 75 % of the in vitro-derived
plants had survived. Acclimatization was especially suc-
cessful with the combination of vermiculite and peat moss
(1:1) or perlite and peat moss (2:1), showing 92.9 and
96.4 % survival rates, respectively. Largest plants were also
obtained with these soil substitutes (Table 4).
Transfer of plants to field conditions. Greenhouse plants
were transferred to the field when they were approximately

Table 3 Effect of reduced BAP supplementation on several growth and development parameters of pitahaya explants after 80 d culture on media
with lower BAP concentrations

BAP concentration Number of surviving Number of new shoots Shoots height Explants with callus Explants with apical
(μM) explants per areole (cm) (%) death (%)

0 39 2±0.2b 2.3±0.2a 41.0b 0.0b

1 23 3±0.3ab 1.7±0.2a 87.0a 0.0b
2 46 2±0.3b 2.0±0.1a 78.3a 8.7b
5 10 4±0.5ab 1.9±0.1a 80.0a 100.0a
15 3 4±1.2ab 1.9±0.2a 100.0a 66.7a
30 8 5±0.8a 1.3±0.2a 75.0ab 75.0a

Values followed by the same letter within a column are not significantly different at the p≤0.05, according Chi-square analysis and Tukey’s test
474 VIÑAS ET AL.

Fig. 2 Appearance of pitahaya

shoots on different BAP
concentrations after 80 d in
culture. Bars01 cm.

30–40 cm in height. Simultaneously, rooted stem cut- in vitro culture of pitahaya is illustrated in Fig. 3,
tings derived from field grown plants were planted for including the approximate time required for each step.
comparison. In the field, the main stems of all in vitro-
derived plants dried out, but were soon replaced by
growth of new lateral shoots. After 4 mo in the field,
the in vitro cultured plants showed the highest survival
rate (100 %) and were larger in size than the plants Discussion
derived from stem cuttings (Table 5). The final diameter
of the stem was similar in both groups (3.1–3.4 cm), Culture initiation and axillary shoot growth. Explant disin-
indicating a greater recovery of in vitro-derived plants, fection of cacti is difficult due to the softness of the tissue
which were initially thinner (∼1–2 cm in diameter) and the presence of thorns, which entrap air bubbles and
compared with conventionally propagated plants which prevent good contact with the disinfecting agent (Skirvin et
were 3–4 cm. After 17 mo in the field, some in vitro- al. 1999). Thorns cannot be removed from explants other-
derived plants had started to flower, while none of the wise axillary shoot growth does not occur, as observed in
conventionally propagated plants had reached that level this study. Drew and Azimi (2002) reported that for Hylo-
of maturity (data not shown). The complete process for cereus undatus only intact areoles produced axillary shoots.
As demonstrated in the present study, pitahaya tissue is very
sensitive to disinfection treatment with NaOCl. To reduce
damage, it was necessary to initially treat larger explants,
Table 4 Effect of soil substitute on size and survival of pitahaya in with five to six areoles each, prior to cutting into smaller
vitro plants after 60 d in the greenhouse segments for in vitro culture. Large explants were also
Substrate Number of Survival Size successfully employed for other cactus species with good
plants (%) (cm) disinfection and bud sprouting rates (Brasil et al. 2005;
Ramirez-Malagon et al. 2007), and their use has been the
Vermiculite + peat moss (1:1) 60 92.9a 1.9±0.2a preferred way to initiate H. undatus in vitro cultures as well
Perlite + peat moss (2:1) 58 96.4a 1.7±0.2a (Drew and Azimi 2002; Mohamed-Yasseen 2002). Contam-
Perlite + peat moss (1:1) 24 87.5ab 0.6±0.3b ination rates with our disinfection method (producing over
Perlite 24 75.0b 0.3±0.1b 80 % healthy explants) are comparable with best results
Values followed by the same letter within a column are not significant-
published for in vitro cactus bud cultures using field or
ly different at the p≤0.05, according Chi-square analysis and Tukey’s greenhouse explants (Juárez and Passera 2002), and are
test considerably better than others in which only 20 % of
 IN VITRO PROPAGATION OF PURPLE PITAHAYA 475

Table 5 Growth performance of pitahaya plants 4 mo after transfer to the field, according to their propagation method

Propagation method Number of plants Survival (%) Height (cm) Stem diameter (cm)

In vitro culture 21 100.0a 46.1±5a 3.1±0.3az

Field cuttings 19 78.9b 29.7±4b 3.4±0.3ay

Values followed by the same letter within a column are not significantly different at the p≤0.05 level, according Chi-square analysis and Tukey’s test
z
Stem diameter when initially transferred to the field was 1–2 cm for in vitro cultured plants
y
Stem diameter when initially transferred to the field was 3–4 cm for field cuttings

explants were successfully established in vitro (Ramirez-
Malagon et al. 2007).
In the present work, axillary shoot growth did not occur
when the basal medium was supplemented with NAA plus
thidiazuron, but only when BAP was used. However, the
use of NAA plus thidiazuron for shoot initiation was
reported to be successful for H. undatus cultures
(Mohamed-Yasseen 2002). There are many published exam-
ples in which closely related species, and even different
cultivars from a single species, respond differently to the
same culture medium and conditions, probably due to ge-
netic conditioning (Machakova et al. 2008; Shen et al.
2008). According to Aliyu and Mustapha (2007), sprouting
of cactus axillary bud requires low levels or no auxin, but
always high levels of cytokinins. This was also observed in
Nopalea cochenillifera (Brasil et al. 2005), in several Mam-
millaria species (Ramirez-Malagon et al. 2007), and in
Opuntia ficus-indica (Khalafalla et al. 2007). In the present
study, inclusion of BAP for axillary bud sprouting of pita-
haya cv Cebra was essential (Fig. 1).
BAP concentrations, similar to the best found in this
study for bud sprouting and shoot growth and proliferation
(15 and 30 μM, respectively), were also used by Giusti et al.
(2002) and Khalafalla et al. (2007), with good results for
other cactus species (Escobaria minima, Mammillaria
pectinifera, Pelecyphora aselliformis, and O. ficus-indica).
However, the use of BAP below 10 μM has been also
reported for other cacti (Brasil et al. 2005; García-Saucedo
et al. 2005), revealing species-specific requirements, as ob-
served in other plant families (Machakova et al. 2008).
The shortest time required to initiate bud development,
in the present study, was 28 d after explant establishment
in treatments supplemented with 15, 30, or 60 μM BAP.
Only 20 d were required for bud sprouting of H. undatus
areoles with 5 μMN6(Δ2-isopentenyl) adenine, which
extended to 26 d on hormone-free medium (Drew and
Azimi 2002), while after 25 d Opuntia amyclaea buds
had already sprouted on media with 10 μM BAP (Escobar et
al. 1986). Faster response (1 wk after establishment) was
observed for the cactus Schlumbergera (Sriskandarajah and
Serek 2004).
With BAP concentrations of 5, 15, or 30 μM in the media
only one shoot per areole was observed, however with 45 or
60 μM of BAP two to three shoots per areole were observed
(data not shown). Growth of only one shoot per areole was
also reported in Opuntia lanigera and H. undatus
(Mohamed-Yasseen 2002; Estrada-Luna et al. 2008), while
in other cactus species, such as Schlumbergera and Rhipsa-
lidopsis, 6 and 15 shoots were produced per areole, respec-
tively (Sriskandarajah and Serek 2004).

Fig. 3 Complete process for in Plants in greenhouse Establishment (0 months) Bud induction (+2 ½ months)
vitro propagation of pitahaya
from the source of explant
material to establishment of in
vitro-derived plants in the field.

Transfer to field conditions

(+4 months) Acclimatization (+8 months)
Proliferation of shoots (+2 ½ months)
476 VIÑAS ET AL.
More areoles from the central region of the joint pro-
duced shoots, reaching 82.5 % in contrast to 61.9 % and
67.7 % from the apical and basal sections, respectively.
Utilization of the central section of Sulcorebutia alba cla-
doles was also employed by Dabekaussen et al. (1991) to
obtain explants, because basal and distal sections showed
irregular bud sprouting. This might be related to distribution
of endogenous hormones within the stem, as noted by
Brukhin and Morozova (2011).
Effect of lower BAP concentrations on shoot proliferation.
Transfer of growing shoots, sprouted on medium containing
15 or 30 μM BAP for 3 mo, to medium with low concen-
trations of BAP (0–2 μM) promoted development of normal
shoots without symptoms of apical necrosis, and also induced
development of more than one shoot per areole (Table 3).
Successive subcultures on medium with high levels of BAP
(15 or 30 μM) promoted callus formation at the base of the
explants, possibly at the expense of the formation of new
shoots from each areole. Callus formation on BAP-
supplemented medium has been reported for other cactus
species (Wyka et al. 2006; Shishkova et al. 2007). Even low
BAP concentrations (8.8 μM) have promoted callus formation
at the expense of shoot proliferation in P. aselliformis (Santos-
Díaz et al. 2003).
Spontaneous formation of roots has been reported for H.
undatus (Drew and Azimi 2002) and Notocactus magnificus
(de Medeiros et al. 2006). Reduction of BAP levels during
the shoot proliferation phase might have been the trigger for
rooting, as observed in other studies (Brasil et al. 2005).
Spontaneous rooting is especially important to accelerate
acclimatization thereby avoiding an extended in vitro
rooting-phase.
Acclimatization of plants in the greenhouse. In general, in
vitro cactus plants acclimatize easily (Ramirez-Malagon et
al. 2007; Estrada-Luna et al. 2008). According to Malda et
al. (1999b) plants with succulent stems and crassulacean
acid metabolism (CAM) are positive characteristics that
minimize water stress during acclimatization. In this study,
more than 90 % survival was observed when vermiculite
plus peat moss (1:1) or perlite plus peat moss (2:1) were
used as soil substitutes. Similar high success rates, have
been reported in several other cactus species (Giusti et al.
2002; Sriskandarajah and Serek 2004; Orea and Medrano
2007; Quiala et al. 2009).
Substrates with high water retention behaved better for
acclimatization in this work (Table 4). Perlite retains little
water (Heiskanen 1995) and, therefore, promotes dryer con-
ditions. Despite the fact that cacti are known to withstand
drought, substrates with some water retention are advanta-
geous during acclimatization of in vitro-derived plants,
which are usually very sensitive to water stress (Orea and
Medrano 2007; Chandra et al. 2010).
Transfer of plants to field conditions. Survival rate and
average plant height were higher in in vitro-derived
plants than for plants derived from traditional field cut-
tings (Table 5). It has been observed that in vitro con-
ditions can enhance growth of cactus plants, probably
due to alterations of the CAM metabolism and because
the plants can fix CO2 continuously, in the light and dark
(Malda et al. 1999a, b). Continuation of an altered CAM
metabolism after the in vitro phase might have afforded
an advantage due to the high plasticity of CAM plants
for carbon assimilation (Kluge et al. 2001), and was
reflected by increased acclimatization values.
Having developed reliable protocols for in vitro culture
and acclimatization of purple-fleshed pitahaya cv. Cebra, the
next step will be to evaluate whether these procedures also
work with other purple-fleshed pitahaya genotypes, already
studied for their desired physico-chemical characteristics,
such as cv’s Rosa, San Ignacio, Lisa and Orejona (Esquivel
et al. 2007a).
Acknowledgments This investigation was partially funded by the
University of Costa Rica (project number VI-734-A5-029). Authors
wish to thank R. Crespo, F. Brenes-Alvarez, and E. Arias-Bustos for
kindly providing the starting plant material used in this work. More-
over, we are grateful to D. González from the National Technical
University in Costa Rica for providing facilities for field experiments.
Assistance of A. Murillo-Williams, A. Monge and E. Rodríguez during
field work is also acknowledged. Authors specially wish to thank
journal editor for the critical reading of the manuscript, very valuable
comments and improvements.
References
Aliyu B, Mustapha Y (2007) Effect of different media on the in vitro
growth of cactus (Opuntia ficus-indica) explants. Afr J Biotechnol
6:1330–1331
Brasil J, Jereissati E, Santos M, Campos F (2005) In vitro micro-
propagation of Nopalea cochenillifera (Cactaceae). J Appl Bot
Food Qual 79:160–162
Brukhin V, Morozova N (2011) Plant growth and development—basic
knowledge and current views. Math Model Nat Phen 6:1–53
Chandra S, Bandopadhyay R, Kumar V, Chandra R (2010) Acclimati-
zation of tissue cultured plantlets: from laboratory to land. Bio-
technol Lett 32:1199–1205
Dabekaussen MAA, Pierik RLM, Van der Laken JD, Hoek Spaans J
(1991) Factors affecting areole activation in vitro in the cactus
Sulcorebutia alba Rausch. Sci Hortic 46:283–294
de Medeiros LA, de Ribeiro RCS, Gallo LA, de Oliveira ET, Demattê
MESP (2006) In vitro propagation of Notocactus magnificus.
Plant Cell Tissue Organ Cult 84:165–169
Drew RA, Azimi M (2002) Micropropagation of red pitaya (Hyloce-
reous undatus). Acta Horticult 575:93–98
 IN VITRO PROPAGATION OF PURPLE PITAHAYA
El Obeidy AA (2006) Mass propagation of pitaya (dragon fruit). Fruits
61:313–319
Escobar AHA, Villalobos VM, Villegas AVM (1986) Opuntia micro-
propagation by axillary proliferation. Plant Cell Tissue Organ Cult
7:269–277
Esquivel P, Stintzing FC, Carle R (2007a) Comparison of morphological
and chemical fruit traits from different pitaya genotypes (Hylocereus
sp.) grown in Costa Rica. J Appl Bot Food Qual 81:7–14
Esquivel P, Stintzing FC, Carle R (2007b) Pigment pattern and expres-
sion of colour in fruits from different Hylocereus sp. genotypes.
Innov Food Sci Emerg Technol 8:451–457
Estrada-Luna AA, JdJ M-H, Torres-Torres ME, Chablé-Moreno F (2008)
In vitro micropropagation of the ornamental prickly pear cactus
Opuntia lanigera Salm-Dyck and effects of sprayed GA3 after
transplantation to ex vitro conditions. Sci Hortic 117:378–385
García-Saucedo PA, Valdez-Morales M, Valverde ME, Cruz-
Hernández A, Paredes-López O (2005) Plant regeneration of three
Opuntia genotypes used as human food. Plant Cell Tissue Organ
Cult 80:215–219
Giusti P, Vitti D, Fiocchetti F, Colla G, Saccardo F, Tucci M (2002) In
vitro propagation of three endangered cactus species. Sci Hortic
95:319–332
Heiskanen J (1995) Physical properties of two-component growth
media based on Sphagnum peat and their implications for plant-
available water and aeration. Plant Soil 172:45–54
Juárez MC, Passera CB (2002) In vitro propagation of Opuntia ellisiana
Griff. and acclimatization to field conditions. Biocell (Mendoza)
26:319–324
Khalafalla M, Abdellatef E, Mohameed-Ahmed MM, Osman M (2007)
Micropropagation of cactus (Opuntia ficus-indica) as strategic
tool to combat desertification in arid and semi arid regions. Int J
Sustain Crop Prod 2:1–8
Kluge M, Razanoelisoa B, Brulfert J (2001) Implications of genotypic
diversity and phenotypic plasticity in the ecophysiological suc-
cess of CAM plants, examined by studies on the vegetation of
Madagascar. Plant Biol 3:214–222
Le Bellec F, Vaillant F, Imbert E (2006) Pitahaya (Hylocereus spp.): a
new fruit crop, a market with a future. Fruits 61:237–250
Machakova I, Zazimalova E, George EF (2008) Plant Growth Regu-
lators I: Introduction; Auxins, their Analogues and Inhibitors. In:
George EF, Hall MA, De Klerk G-J (eds) Plant propagation by
tissue culture, 3rd edn. Springer, Dordrecht, pp 175–204
Malda G, Backhaus RA, Martin C (1999a) Alterations in growth and
crassulacean acid metabolism (CAM) activity of in vitro cultured
cactus. Plant Cell Tissue Organ Cult 58:1–9
Malda G, Suzán H, Backhaus R (1999b) In vitro culture as a potential
method for the conservation of endangered plants possessing
crassulacean acid metabolism. Sci Hortic 81:71–87
477
Merten S (2003) A review of Hylocereus production in the United
States. J Prof Assoc Cactus Dev 5:98–105
Mohamed-Yasseen Y (2002) Micropropagation of pitaya (Hylocereus
undatus Britton et Rose). In Vitro Cell Dev Biol Plant 38:427–429
Morton J (1987) Strawberry pear. In: Morton J. F. (ed) Fruits of warm
climates. Miami, FL, pp 347–348
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Orea CDP, Medrano VA (2007) Pitahaya (Hylocereus undatus) accli-
matization: a pedagogical model. Acta Horticult 748:195–198
Pérez-Molphe-Balch E, Dávila-Figueroa CA (2002) In vitro propaga-
tion of Pelecyphora aselliformis Ehrenberg and P. strobiliformis
Werdermann (Cactaceae). In Vitro Cell Dev Biol Plant 38:73–78
Quiala E, Matos J, Montalvo G, de Feria M, Chávez M, Capote A,
Pérez N, Barbón R, Kowalski B (2009) In vitro propagation of
Pilosocereus robinii (Lemaire) Byles et Rowley, endemic and
endangered cactus. J Prof Assoc Cactus Dev 11:18–25
Ramirez-Malagon R, Aguilar-Ramirez I, Borodanenko A, Perez-
Moreno L, Barrera-Guerra J, Nuñez-Palenius HG, Ochoa-Alejo
N (2007) In vitro propagation of ten threatened species of Mam-
millaria (Cactaceae). In Vitro Cell Dev Biol Plant 43:660–665
Rubluo A, Chávez V, Martínez AP, Martínez-Vázquez O (1993) Strat-
egies for the recovery of endangered orchids and cacti through in-
vitro culture. Biol Conserv 63:163–169
Santos-Díaz MDS, Méndez-Ontiveros R, Arredondo-Gómez A,
Santos-Díaz MDL (2003) In vitro organogenesis of Pelecyphora
aselliformis Erhenberg (Cactaceae). In Vitro Cell Dev Biol Plant
39:480–484
Shen X, Kane ME, Chen J (2008) Effects of genotype, explant source,
and plant growth regulators on indirect shoot organogenesis in
Dieffenbachia cultivars. In Vitro Cell Dev Biol Plant 44:282–288
Shishkova S, García-Mendoza E, Castillo-Díaz V, Moreno NE,
Arellano J, Dubrovsky JG (2007) Regeneration of roots from
callus reveals stability of the developmental program for de-
terminate root growth in Sonoran Desert Cactaceae. Plant Cell
Rep 26:547–557
Skirvin RM, Motoike S, Norton MA, Ozgur M, Al-Juboory K,
McMeans OM (1999) Establishment of contaminant-free peren-
nial plants in vitro. In Vitro Cell Dev Biol Plant 35:278–280
Sriskandarajah S, Serek M (2004) Regeneration from phylloclade
explants and callus cultures of Schlumbergera and Rhipsalidopsis.
Plant Cell Tissue Organ Cult 78:75–81
Stintzing FC, Carle R (2007) Betalains - emerging prospects for food
scientists. Trends Food Sci Technol 18:514–525
Wyka TP, Hamerska M, Wroblewska M (2006) Organogenesis of
vegetative shoots from in vitro cultured flower buds of Mammil-
laria albicoma (Cactaceae). Plant Cell Tissue Organ Cult 87:27–
32