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In vitro plant regeneration of Passiflora setacea D.C. (Passifloraceae): the

influence of explant type, growth regulators, and incubation conditions

Article  in  In Vitro Cellular & Developmental Biology - Plant · November 2014

DOI: 10.1007/s11627-014-9650-0

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In Vitro Cell.Dev.Biol.—Plant
DOI 10.1007/s11627-014-9650-0

MICROPROPAGATION

In vitro plant regeneration of Passiflora setacea D.C.

(Passifloraceae): the influence of explant type, growth
regulators, and incubation conditions
Lorena Melo Vieira & Diego Ismael Rocha & Mariana Futia Taquetti &
Luzimar Campos da Silva & José Marcello Salabert de Campos &
Lyderson Faccio Viccini & Wagner Campos Otoni

Received: 21 December 2013 / Accepted: 2 October 2014 / Editor: John Finer

# The Society for In Vitro Biology 2014

Abstract The present study aimed to establish a protocol for comparison with seed-derived plantlets (2C=2.60 pg). This
in vitro organogenesis of Passiflora setacea and to determine is the first report on the in vitro regeneration of P. setacea.
the genetic stability of regenerated plants. Three types of
explants (leaf, hypocotyl, and root), four growth regulator Keywords Adventitious bud . Cytokinins .
combinations [Murashige and Skoog (MS) salts, MS + 6- Micropropagation . Passion fruit . Shoot organogenesis .
benzyladenine (BA), MS + thidiazuron (TDZ), and MS + Tissue culture
BA + TDZ], and two light regimes (16-h photoperiod and
continuous darkness) were tested. After 30 d on induction
medium, the percentage of explants forming shoots was eval-
Introduction
uated. Direct and indirect organogenesis was evident from
hypocotyl- and root-derived explants, whereas only indirect
The cultivation of passion fruit has shown strong growth and
organogenesis was observed from leaf explants. The presence
expansion due to great popularity in different consumer mar-
of BA was essential for shoot formation from leaf explants and
ket segments, including both fresh-fruit and juice production
improved the response of hypocotyl segments under a 16-h
and the cosmetics industry. The most widely cultivated spe-
photoperiod compared to the cytokinin-free control. However,
cies of Passiflora are Passiflora edulis Sims (yellow passion
after transfer to shoot elongation medium, the greatest number
fruit) and Passiflora alata Curtis (sweet passion fruit).
of elongated shoots was obtained from hypocotyl segments
However, the susceptibility of the plants to pathogens such
that had been induced on BA + TDZ medium under a 16-h
as Fusarium oxysporum Schl. f. sp. passiflorae (Flores et al.
photoperiod, as was also observed for root explants. Flow
2012) and Cowpea aphid-borne virus has limited fruit pro-
cytometry analysis confirmed the genetic stability of the
duction and expansion of the growing area for this crop
regenerants based on DNA quantity (2C = 2.57 pg) in
(Trevisan et al. 2006; Zerbini et al. 2008).
For these reasons, a number of Passiflora spp. that are not
grown commercially have been evaluated in breeding pro-
L. M. Vieira : D. I. Rocha : M. F. Taquetti : L. C. da Silva :
W. C. Otoni
grams because of valuable traits that may improve fruit yield
Plant Biology Department, Federal University of Viçosa, University and quality and disease resistance (Vieira and Carneiro 2004;
Campus, Peter Henry Rolfs Avenue, 36570-900 Viçosa, MG, Brazil Faleiro et al. 2008; Zerbini et al. 2008). Passiflora setacea
D.C. (synonym: Passiflora sururuca Vell.), popularly known
J. M. S. de Campos : L. F. Viccini
as ‘sleep passion fruit,’ has a vigorous growth habit that shows
Institute of Biological Sciences, Department of Biology, Federal
University of Juiz de Fora, 36036-900 Juiz de Fora, MG, Brazil genetic resistance to some pathogens (Oliveira and Ferreira
1991; Braga et al. 2006). In addition, it produces fruits that are
W. C. Otoni (*) widely used for fresh consumption, production of confections,
Departamento de Biologia Vegetal/BIOAGRO, Universidade
Federal de Viçosa, Av. P. H. Rolfs s/n, Ed. CCBII, Campus
esthetic purposes, and pharmacological purposes (Oliveira
Universitário, 36570-900 Viçosa, MG, Brazil and Ruggiero 2005). Propagation of P. setacea has been
e-mail: wotoni@ufv.br traditionally done by seeds, although nonuniform germination
 VIEIRA ET AL.
and difficult rooting have been commonly reported (Manica
et al. 2005; Pádua et al. 2011), which limits the large-scale
production of this species.
The establishment of tissue culture protocols for P. setacea
could be usefully applied to various aspects of biotechnology
such as micropropagation, germplasm conservation, and pro-
duction of secondary metabolites. In vitro propagation sys-
tems have been achieved for a wide range of passion fruit
species, although none has yet been described for P. setacea.
Passiflora primarily regenerates via organogenesis, using a
variety of plant growth regulators and explant types (Otoni
et al. 2013).
Cytokinins are one of the main plant growth regulators
involved in the control of growth and development of adven-
titious buds, and they have shown efficient results in inducing
organogenic responses in Passiflora (Nhut et al. 2007; Dias
et al. 2009; Dias et al. 2010; Garcia et al. 2011; Silva et al.
2011). The cytokinin 6-benzyladenine (BA) is often used for
passion fruit shoot–bud induction (Otoni et al. 2013), al-
though thidiazuron (TDZ) alone or in combination with BA
has been used (Trevisan and Mendes 2005; Pinto et al. 2010;
Garcia et al. 2011). Despite the advantages of in vitro propa-
gation systems, genetic instability has been commonly ob-
served in plants derived from such systems. Instability may
limit the use of a regeneration system, particularly in massive
plant propagation and genetic transformation (Evans et al.
1984; Karp 1995; Smulders and de Klerk 2011). Methods to
assess and monitor the genetic stability of in vitro plants are
desirable, and flow cytometry analysis has been used to detect
changes in ploidy (Souza et al. 2004; Loureiro et al. 2005;
Paim Pinto et al. 2010; Silva et al. 2011; Escobedo-
GraciaMedrano et al. 2014).
As previously mentioned, there have been no published
reports of in vitro regeneration of P. setacea to date. The
characteristics of P. setacea, such as its potential application
as a rootstock, genetic resistance to premature death, tolerance
to some bacterial and fungal diseases, and difficulty of prop-
agation through seeds, justify the investigation of in vitro
regeneration protocols for this species. The aim of this study
was to evaluate the effects of explant type, addition of growth
regulators to the culture medium, and incubation conditions
on the in vitro organogenesis of P. setacea and to determine
the genetic stability of the regenerants.
Materials and Methods
Plant material. Mature seeds of P. setacea D.C. were obtain-
ed from mature fruits harvested from natural populations in
the Janaúba municipality, Brazil (15° 48′ 09′′ S, 43° 18′ 32′′
W). The seeds were washed in running water to remove arils.
These seeds were placed on absorbent paper and dried in the
shade at room temperature (25°C) for approximately 3 d, and
the remaining arils were manually removed.
The seeds were mechanically decoated using a minivice as
suggested by Reis et al. (2007). The decoated seeds were
surface sterilized by immersion (1 min) in 70% (v/v) ethanol,
followed by immersion (20 min) in 2.5% (v/v) commercial
sodium hypochlorite with three drops of Tween 20 per
100 mL solution, and rinsed five times in deionized,
autoclaved water. The seeds were subsequently transferred
to 250-mL culture flasks (30 flasks with 10 seeds per flask;
Vidrolabor®, Paulínia, Brazil) containing 60 mL half-strength
MS medium (Murashige and Skoog 1962) supplemented with
B5 vitamins (Gamborg et al. 1968), 1.5% (w/v) sucrose, and
0.25% (w/v) Phytagel® (Sigma Chemical Co., St. Louis, MO)
and adjusted to pH 5.8±0.1. Unless otherwise stated, media
were sterilized by autoclaving at 121°C and 1.1 Pa for 15 min.
The flasks were sealed with rigid polypropylene closures with
two orifices (10 mm) that were covered with 0.45-μm (pore
size) adhesive membranes (Milliseal®, AVS-045 Air Vent,
Tokyo, Japan).
The flasks were kept in the dark for 15 d until the seeds
germinated. The seedlings were then transferred to a
temperature-controlled growth environment with a 16/8 h
(light/dark) light regime at an irradiance of 36 μmol m−2 s−1
(provided by fluorescent tubes, 20 W, Osram GmbH,
Germany) at 27±2°C for 15 d.
In vitro organogenesis. Under aseptic conditions, hypocotyl
and root segments (10 to 20 mm in length) and leaf segments
(10×10 mm with the midrib) were excised from in vitro-
germinated seedlings after 30 d and used as explants. These
explants were transferred to 90-mm×15-mm Petri dishes (J.
Prolab, Curitiba, Brazil) containing 25 mL culture medium
consisting of MS salts, B5 vitamins, and 3% (w/v) sucrose.
The media were then supplemented with 4.43 μM 6-
benzyladenine (BA), 2.27 μM thidiazuron (TDZ), or BA
(4.43 μM) + TDZ (2.27 μM). These media addenda were
based on the report of P. alata organogenesis by Pinto et al.
(2010). MS medium without growth regulators was used as a
control. In all treatments, 0.25% (w/v) Phytagel® was added as
a gelling agent. The pH was adjusted to 5.8, and the media
were autoclaved at 120°C and 1.1 Pa for 20 min. The plates
were sealed with acrylic adhesive tape (3 M Nexcare®
Micropore, Nova Veneza, Brazil), and the leaf segments were
inoculated with the adaxial surface facing the culture medium.
The plates were incubated at 27°C under a 16-h photoperiod
(36 μmol m−2 s−1) or in the dark (plates covered with alumi-
num foil). Five plates were used per treatment, containing ten
explants each. The experiments were evaluated 30 d after the
induction period, and the number of shoots and presence of
callus formation on the explants were assessed with the aid of
a stereomicroscope (Olympus Model SZX7, Olympus
Optical, Tokyo, Japan).
 REGENERATION OF PASSIFLORA SETACEA
Elongation and acclimatization. To induce elongation, regen-
erated structures (shoots with leaves 5–10 mm in length) were
transferred to 250-mL flasks containing approximately 50 mL
of MS medium supplemented with 2.88 μM gibberellic acid
(GA3), B5 vitamins, 3% (w/v) sucrose, and 0.25% (w/v)
Phytagel®. The pH was adjusted to 5.8, and the medium
was autoclaved at 120°C and 1.1 Pa for 20 min. The flasks,
containing four explants each, were sealed with transparent
polypropylene lids and maintained at 27°C and a 16-h photo-
period. The number of elongated shoots was determined after
30 d of incubation. Shoots longer than 30 mm were excised,
and the proximal portions of the stems were immersed in a
solution containing 4.9 μM indole-3-butyric acid (IBA) for
10 s to induce rooting. Shoots were transferred to plastic cups
containing a 1:1 mixture of Plantmax® (DDL Agroindústria,
Betel Paulínia, SP, Brazil) and coconut fiber and were gradu-
ally acclimatized in the greenhouse at 25°C.
Microscopy. The histological evaluation of the in vitro regen-
eration process was carried out using leaf segments, hypo-
cotyls, and roots collected after 30-d culture in induction
medium. Samples were fixed in modified Karnovsky solution
(2.5% glutaraldehyde, 4% paraformaldehyde, and 5 mM
CaCl2 in 0.1 M cacodylate buffer, pH 6.8; Karnovsky 1965)
for 72 h at 4°C.
For light microscopy, the samples were dehydrated in an
ethanol series and embedded in an acrylic resin (HistoResin
Leica®, Wehrheim, Germany). The embedded samples were
sectioned with an automatic rotary microtome (Leica
RM2155, Nussoloch, Germany). The sections (7-μm
thickness) were adhered to slides and stained with 0.05%
toluidine blue (pH 6.8) (O'Brien and McCully 1981) for
10 min. After drying, the permanent slides were mounted
using Permount synthetic resin. Images were captured using
a light microscope (Olympus AX70TRF, Olympus Optical)
equipped with a digital camera (Diagnostic Instruments Inc.,
Spot 3.2.0 Insight Color, Sterling Heights, Michigan).
For scanning electron microscopy, the fixed samples were
dehydrated through an acetone series, critical-point dried
(CPD 030; Bal-Tec, Balzers, Liechtenstein), mounted on alu-
minum stubs, and coated with gold (FDU 010; Bal-Tec).
Examinations and photography were performed using a
SEM (Leo 1430VP; Zeiss, Heidelberg, Germany).
Flow cytometry analysis. The genetic stability of the plants
regenerated from leaves, hypocotyls, and roots, cultivated
under a 16-h photoperiod light regime and acclimatized, were
assessed by flow cytometry. Control plants were obtained by
means of in vitro germination of decoated mature seeds of
P. setacea, as previously described, and further acclimatized.
Samples for each treatment (n=5) were collected, maintained
in moistened paper, and analyzed within a maximum period of
2 d. Approximately, 20–30 mg fresh leaf tissue from each
plant was finely chopped with a disposable steel razor blade in
1 mL LB01 buffer to release nuclei (Doležel et al. 1989).
Lycopersicon esculentum ‘Stupicke polni tyckove rane’ (2C
DNA content of 1.96 pg) was used as an internal reference
standard (Doležel et al. 2007). The macerated tissues were
sieved through two layers of cheesecloth using a plastic pi-
pette, filtered through a 50-μm nylon mesh, and collected in
polystyrene tubes. The suspension was stained with 25 μL
propidium iodide solution (1 mg mL−1; Sigma), and 5 μL
RNase (Amresco, Solon, OH) was added to each sample.
Samples were incubated at 4°C in the dark and examined after
1–2 h.
At least 10,000 nuclei were analyzed in each sample.
Analyses were performed using a FACS Calibur flow
cytometer (Becton–Dickinson, Franklin Lakes, NJ) at the
Biological Sciences Institute of the Federal University of
Juiz de Fora (ICB/UFJF). Cytometric histograms were gener-
ated and analyzed using Cell Quest and WinMDI 2.8 software
(available at http://www.cyto.purdue.edu/flowcyt/software/
Winmdi.htm). Nuclear DNA content (pg) was estimated by
the following equation:
G1 peak channel of sample
Sampleð2C DNAÞ ¼
G1 peak channel sample of L: esculentum
 1:96pg ðL: esculentum DNA contentÞ
Statistical analyses. The experimental design was completely
randomized in a 2×4 factorial with five replicates. Each
replicate was a Petri dish containing 10 explants, totaling 50
explants per treatment. The collected data were tested for
normality with the Kolmogorov–Smirnov test, and non-
normal distributions (p<0.05) were transformed into the
arcsin of the square root of x/100. The transformed data were
subjected to analysis of variance (ANOVA), and the Tukey
test at 5% probability was used when differences between F
test treatments were observed. ANOVA was used to observe
the effects of treatment type and explant type on the ploidy
variation of plants that were subjected to flow cytometry. SAS
software (SAS Institute 2010) was used for analysis.
Results
Effects of growth regulators (BA and TDZ) and light condi-
tions on the in vitro organogenesis of P. setacea. Adventitious
buds were induced on MS medium supplemented with differ-
ent cytokinins, under two different light regimes, using leaf-,
hypocotyl-, and root-derived explants (Table 1). Data were
collected after 30 d of culture on induction medium. For leaf-
derived explants, the presence of BA was necessary for
 VIEIRA ET AL.

Table 1. Mean values (%) for shoot formation in leaf, hypocotyl, and light condition, the BA + TDZ treatment resulted in approx-
root explants of Passiflora setacea on different culture media under 16-h
imately twice the number of buds obtained from media sup-
photoperiod or continuous dark
plemented with either BA or TDZ alone (Table 1).
Type of explant Culture medium Shoot development (%)
Shoot elongation and proliferation. Transfer of leaf-, hypo-
16-h light Dark
cotyl-, and root-derived explants containing developing ad-
Leaf MS 0.0 Ab 0.0 Ab ventitious buds to elongation medium resulted in shoot devel-
BA 50.0 Aa 14.0 Aab opment from some buds. For each explant type, the treatments
TDZ 0.0 Bb 0.0 Bb that showed the highest number of adventitious buds in the
BA + TDZ 20.0 Aab 50.0 Aab induction medium also showed the greatest number of elon-
Hypocotyl MS 46.0 Ab 47.0 Aa gated shoots developed after 30 d on the elongation medium
BA 98.0 Aa 47.0 Ba (Fig. 1G). Leaf-derived explants showed a higher rate of
TDZ 63.0 Ab 2.50 Bb elongated shoots when grown on induction medium supple-
BA + TDZ 85.0 Aab 67.0 Aa mented with BA under a 16-h photoperiod or with BA + TDZ
Root MS 20.0 Ab 13.0 Aab
in the dark (25% for both treatments) (Table 2). We did not
BA 47.0 Ab 2.50 Bb
observe shoot elongation from leaf-derived explants induced
TDZ 41.0 Ab 40.0 Aa
on cytokinin-free medium or on TDZ medium under either
light condition (Table 2). The highest percentages of elongat-
BA + TDZ 93.0 Aa 52.0 Ba
ed shoots were observed in hypocotyl- and root-derived ex-
Means followed by the same uppercase letter within a line and lowercase plants cultured on induction medium supplemented with BA +
letter within a column do not differ from each other by Tukey’s test (p≤ TDZ (52 and 80%, respectively). Shoots induced from hypo-
0.05)
cotyls on TDZ medium in the dark did not elongate.
Additionally, no shoot regeneration was observed from root
adventitious shoot–bud regeneration. Organogenic calli were segments cultured on either cytokinin-free medium under a
induced from all BA-containing treatments but not from the 16-h photoperiod or on BA induction medium in the dark
cytokinin-free control or medium with TDZ alone (Table 1). (Table 2). Elongated shoots were rooted in substrate (Fig. 1H)
Calli were green and compact across the edge of the leaf with rooting efficiency of up to 80%. Plants were successfully
segments, and no morphological differences were observed acclimatized and developed normally under greenhouse con-
among the treatments (Fig. 1A, B). The highest regeneration ditions (Fig. 1I). Morphological abnormalities were not visu-
efficiencies were observed from explants cultured on BA- ally detected in adult plants.
containing medium under a 16-h photoperiod and on BA +
TDZ in the dark. No shoot organogenesis was observed from Histological characterization. Histological analyses of sam-
leaf explants cultured on medium supplemented with TDZ ples collected after 30 d on the induction medium revealed
alone under either light condition (Table 1). intense callus development along the cut edge areas of the
For hypocotyl and root explants, adventitious bud regener- leaf- and hypocotyl-derived explants. Meristems and shoot
ation was observed in all treatments, including the cytokinin- buds with leaf primordia were observed on the callus surface
free controls (Table 1). Direct shoot organogenesis was ob- (Fig. 2A–D). The meristematic cells had dense cytoplasm and
served after 30 d, although indirect plant regeneration was also reduced volume compared to adjacent, vacuolated cells
observed from callus on the surfaces of explants (Table 1; (Fig. 2B, D). In root segments, direct regeneration was ob-
Fig. 1C, D). In most cases, the regeneration percentage from served (Fig. 2E, F). The vascular system of these buds was
hypocotyl segments was higher than from the leaf and root connected with vascular tissue of the explant, consistent with
explants. For hypocotyl-derived explants, the highest number the pattern of organogenic regeneration (Fig. 2F).
of shoot buds was observed in cultures grown under a 16-h
photoperiod. Under this light condition, the BA and BA + Genetic stability. Flow cytometry analyses were performed
TDZ treatments resulted in approximately 68% more shoot on leaf samples from the regenerated plants from hypocotyl-
buds than the TDZ and cytokinin-free control treatments and root-derived explants grown on each of the four shoot
(Table 1). induction media under a 16-h photoperiod, and from in vitro
The combination of BA and TDZ had also a positive effect seed-derived plants. These data were used to compare the
on the frequency of adventitious bud induction from root- stability of the ploidy level of regenerants to that of the seed-
derived explants (Fig. 1E, F). Under both 16-h photoperiod derived control plants. The amount of DNA in G1 resulted in
and dark conditions, BA + TDZ promoted the development of histograms with a suitable resolution to determine the ploidy
more buds than the other treatments, although the number of of the examined plants. The results showed that the regener-
shoot buds was higher under a 16-h photoperiod. Under this ated plants had DNA amounts (average 2C=2.57 pg) similar
 REGENERATION OF PASSIFLORA SETACEA

Figure 1. In vitro organogenesis

of Passiflora setacea after 30 d of
culture in induction medium. (A,
B) Leaf-derived explants with
shoot formation on medium sup-
plemented with BA under a 16-h
photoperiod (A) and indirect bud
formation on BA + TDZ medium
in the dark (B). ca callus, asterisk
shoot buds. C, D Hypocotyl ex-
plants with indirect formation of
shoots on BA + TDZ and BA
media (D), both cultured under
16-h photoperiod. E, F Root ex-
plants with indirect organogenesis
on TDZ medium (E) and direct
organogenesis on BA + TDZ
medium (F), both under 16-h
photoperiod. G Shoot cultures in-
duced from hypocotyl explants on
MS medium supplemented with
GA3 for elongation. H Regener-
ated plants from leaf- (1),
hypocotyl- (2), and root-derived
(3) explants. I Regenerated
1-year-old plant growing in
greenhouse. Bars=1 mm (A, E),
2 mm (B, C, D, F), 40 mm (G),
25 mm (H), and 100 mm (I).

to the control (2C=2.60 pg), i.e., diploid levels, with no
significant variation in ploidy regardless of the explant type
or treatment conditions (Fig. 3).
Discussion
This study was conducted to determine the in vitro morpho-
genic potential of P. setacea leaf, hypocotyl, and root explants
derived from in vitro-germinated seedlings. Many authors
have reported in vitro organogenesis for several species from
the Passiflora genus, including the effects of growth regula-
tors in the culture medium, type of explants (Dornelas and
Vieira 1994; Monteiro et al. 2000; Pinto et al. 2010; Silva
et al. 2011; Pacheco et al. 2012; Rosa and Dornelas 2012),
and light conditions (Appezzato-da-Glória et al. 1999; Pinto
et al. 2010; Garcia et al. 2011). However, to our knowledge,
this is the first reported regeneration protocol for P. setacea.
The light regime can influence morphogenic competence
(Moreira-Dias et al. 2000; Garcia et al. 2011; Siegien et al.
2013). In Passiflora, different morphogenic pathways have
been obtained by modulating light regime conditions and
plant growth regulators. Although somatic embryogenesis
 VIEIRA ET AL.

Table 2. Mean values (%) for shoot elongation obtained from leaf-, hypocotyl-, and root-derived explants of Passiflora setacea cultured under 16-h
photoperiod or continuous dark during shoot induction followed by 30 d on elongation medium

Induction medium Elongation medium Type of explant

Leaf Hypocotyl Root

16-h light Dark 16-h light Dark 16-h light Dark

MS MS + GA3 0.0 c 0.0 c 20.0 abc 12.5 abc 0.0 c 2.5 bc

BA MS + GA3 25.0 a 8.0 b 32.5 ab 7.5 bc 47.5 ab 0.0 c
TDZ MS + GA3 0.0 c 0.0 c 7.5 bc 0.0 c 5.0 bc 12.5 bc
BA + TDZ MS + GA3 12.0 b 25.0 a 52.5 a 12.5 abc 80.0 a 42.5 abc

Means followed by the same lowercase letter within a column do not differ from each other by Tukey’s test (p≤0.05)

has been reported in a few species cultured in the dark (Silva
et al. 2009; Paim Pinto et al. 2010; Rocha et al. 2012b),
adventitious buds often are not formed in the absence of light
(Moran-Robles 1979; Appezzato-da-Glória et al. 1999; Pinto
et al. 2010; Garcia et al. 2011). P. setacea seems to be less
sensitive to light regime conditions since we have observed
the emergence of shoots under both light (16-h photoperiod)
and dark conditions, although the percentages of adventitious
bud induction and shoot elongation were generally higher
under the 16-h photoperiod condition.
Shoot–bud differentiation depends on the endogenous hor-
monal balance present in the explants (George et al. 2008;
Müller and Leyser 2011). Cytokinin supplementation was not
essential for the induction of adventitious buds in hypocotyl-
and root-derived explants of P. setacea since we observed bud
formation from these tissues on cytokinin-free medium.
However, cytokinins promoted an increase in the percentage
of adventitious buds. BA proved to be the best cytokinin for
adventitious bud induction from leaf- and hypocotyl-derived
explants of P. setacea cultured under a 16-h photoperiod. The
effect of BA in promoting differentiation of buds formed from
these explant types has also been reported for other Passiflora
spp., including Passiflora mollissima, Passiflora giberti,
Passiflora maliformis, Passiflora amethystina (Dornelas and
Vieira 1994), P. edulis (Appezzato-da-Glória et al. 1999;
Fernando et al. 2007), Passiflora suberosa (Monteiro et al.
2000), Passiflora cincinnata (Lombardi et al. 2007), and
Passiflora foetida (Soares et al. 2012). TDZ promoted the

Figure 2. Scanning electron

microscopy (A, C, E) and light
microscopy (B, D, F) of in vitro
adventitious organogenesis in
Passiflora setacea explants 30 d
after the start of induction. A, B
Adventitious bud (asterisk) and
leaf primordium (lp) formation
from calli (ca) obtained from leaf-
derived explants (le). C, D
Hypocotyl segments bearing
shoot buds (asterisk), leaf
primordia (lp), meristemoids
(me), and callus (ca). E, F Root
segments showing direct organo-
genesis with shoot-bud formation
(asterisk) and vascular connection
with the explant (arrow). Bars=
300 μm (A, B, D, F), 1000 μm
(C), and 500 μm (E).
 REGENERATION OF PASSIFLORA SETACEA
Figure 3. Flow cytometry analysis of leaf samples derived from accli-
matized in vitro-regenerated plants of Passiflora setacea using
Lycopersicon esculentum ‘Stupicke polni tyckove rane’ (1.96 pg DNA)
as an internal reference standard.
development of fewer buds than did BA, except in root
segments cultured in the dark (Table 1). However, TDZ had
a positive effect used in combination with BA. The combina-
tion of BA and TDZ has been proposed to optimize in vitro
shoot organogenesis induction (Fratini and Ruiz 2002; Pinto
et al. 2010). The synergistic effect of two combined cytokinin
sources on multiplication and elongation of buds in Passiflora
was tested for leaf discs and hypocotyl segments of P. alata
cultured on media containing BA and TDZ, but this treatment
did not differ from the other tested media in number of shoots
produced (Pinto et al. 2010). In the present study, the presence
of BA or BA + TDZ increased the formation of shoot buds in
hypocotyl-derived explants, particularly under a 16-h photo-
period. In root-derived explants, the combination of BA +
TDZ under a 16-h photoperiod provided the highest number
of shoots.
Adventitious buds developed indirectly on the leaf-
derived explants of P. setacea, while both direct and
indirect organogenesis was observed from hypocotyl and
root segments. In leaf and hypocotyl explants, meristemoids
differentiated in the periphery of the proliferating callus
tissue. These observations are in agreement with the
morphogenic pattern described by Fernando et al. (2007)
for P. edulis, although we did not observe the abnormal
shoot structures on leaf discs as observed in that study and
in P. alata (Pinto et al. 2010). In Passiflora, in vitro
regeneration from roots has been reported in P. cincinnata
(Lombardi et al. 2007; Silva et al. 2011) and in P. edulis
(Silva et al. 2011; Rocha et al. 2012a) via both direct and
indirect organogenesis. In these species, buds formed di-
rectly from the procambial cells and had a vascular con-
nection between the adventitious shoot and the root explant
(Lombardi et al. 2007; Rocha et al. 2012a), as also ob-
served in the present study.
Although some of the P. setacea regenerated plants were
obtained indirectly from callus, flow cytometric analysis
showed that plants derived from seeds contained 2.60 pg of
nuclear DNA (2C), and all of the regenerated plants from leaf,
hypocotyl, and root explants maintained this ploidy.
The results of this work showed that in vitro-cultured
P. setacea explants respond differently according to the type
of explant used, the growth regulators added to the culture
medium, and the lighting conditions. The highest frequency of
organogenesis in this study was obtained for hypocotyl ex-
plants cultured on medium with BA and maintained under a
16-h photoperiod. These results, which are the first for this
species, showed no variation in genetic stability, thus demon-
strating the usefulness of this protocol for micropropagation of
P. setacea.
Acknowledgments The authors would like to thank Prof. Francisco
Tanaka for use of the electron microscope facility at NAP/MEPA-
ESALQ/USP, CAPES (Brasília, DF, Brazil), CNPq (Brasília, DF, Brazil),
and FAPEMIG (Grant CRA-APQ-01451-12; Belo Horizonte, MG, Bra-
zil). We also acknowledge Prof. J. Doležel for making available
L. esculentum ‘Stupicke.’
References
Appezzato-da-Glória B, Vieira MLC, Dornelas MC (1999) Anatomical
studies of in vitro morphogenesis in leaf explants of passionfruit.
Pesq Agropec Bras 34:2007–2013
Braga MF, Santos EC, Junqueira NTV, Sousa AATC, Faleiro FG,
Rezende LN, Junqueira KP (2006) Enraizamento de estacas de
três espécies silvestres de Passiflora. Rev Bras Frutic 28:284–288
Dias LLC, Santa-Catarina C, Ribeiro DM, Barros RS, Floh ELS, Otoni
WC (2009) Ethylene and polyamine production patterns during
in vitro shoot organogenesis of two passion fruit species as affected
by polyamines and their inhibitor. Plant Cell Tissue Organ Cult 99:
199–208
Dias LLC, Ribeiro DM, Santa-Catarina C, Barros RS, Floh EIS, Otoni
WC (2010) Ethylene and polyamine interactions in morphogenesis
of Passiflora cincinnata: effects of ethylene biosynthesis and action
modulators, as well as ethylene scavengers. Plant Growth Regul 62:
9–19
Doležel J, Binarová P, Lucretti S (1989) Analysis of nuclear DNA content
in plant cells by flow cytometry. Biol Plant 31:113–120
Doležel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA content
in plants using flow cytometry. Nat Protoc 2:2233–2244
Dornelas MC, Vieira MLC (1994) Tissue culture studies on species of
Passiflora. Plant Cell Tissue Organ Cult 36:211–217
Escobedo-GraciaMedrano RM, Maldonado-Borges JI, Burgos-Tan MJ,
Valadez-González N, Ku-Caiuch JR (2014) Using flow cytometry
and cytological analyses to assess the genetic stability of somatic
embryo-derived plantlets from embryogenic Musa acuminata Colla
(AA) ssp. malaccensis cell suspension cultures. Plant Cell Tissue
Organ Cult 116:175–185
Evans DA, Sharp WR, Medina-Filho HP (1984) Somaclonal and
gametoclonal variation. Am J Bot 77:759–774
Faleiro FG, Junqueira NTV, Fávero AP, Lopes MA (2008) Pré-
melhoramento de plantas: experiências de sucesso. In: Faleiro FG,
Farias Neto AL, Ribeiro Junior WQ (eds) Pré-melhoramento,
 VIEIRA ET AL.
melhoramento e pós-melhoramento: Estratégias e desafios.
Embrapa Cerrados, Planaltina, pp 43–62
Fernando JA, Vieira MLC, Machado SR, Appezzato-da-Glória B (2007)
New insights into the in vitro organogenesis process: the case of
Passiflora. Plant Cell Tissue Organ Cult 91:37–44
Flores PS, Otoni WC, Dhingra OD, Souza Diniz SPS, Santos TM,
Bruckner CH (2012) In vitro selection of yellow passion fruit
genotypes for resistance to Fusarium vascular wilt. Plant Cell
Tissue Organ Cult 108:37–45
Fratini R, Ruiz MN (2002) Comparative study of different cytokinins in
the induction of morphogenesis in lentil (Lens culinaris Medik.). In
vitro Cell Dev Biol Plant 38:46–51
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of
suspension cultures of soybean root cells. Exp Cell Res 50:151–158
Garcia R, Pacheco G, Falcão E, Borges G, Mansur E (2011) Influence of
type of explant, plant growth regulators, salt composition of basal
medium, and light on callogenesis and regeneration in Passiflora
suberosa L. (Passifloraceae). Plant Cell Tissue Organ Cult 106:47–54
George EF, Hall MA, DeKlerk GJ (2008) Plant propagation by tissue
culture. Volume 1: The Background, 3rd edn. Springer, Dordrecht
Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high
osmolality for use in electron microscopy. J Cell Biol 27:137–138
Karp A (1995) Somaclonal variation as a tool for crop improvement.
Euphytica 85:295–302
Lombardi SP, Passos IRS, Nogueira MCS, Appezzato-da-Glória B
(2007) In vitro shoot regeneration from roots and leaf discs of
Passiflora cincinnata Mast. Braz Arch Biol Technol 50:239–247
Loureiro J, Pinto G, Lopes T, Doležel J, Santos C (2005) Assessment of
ploidy stability of the somatic embryogenesis process in Quercus
suber L. using flow cytometry. Planta 221:815–822
Manica I, Brancher A, Sanzonowicz C, Icuma IM, Aguiar JLP, Azevedo
JA, Vasconcellos MAS, Junqueira NTV (2005) Maracujá-doce:
Tecnologia de produção, pós-colheita, mercado, 1ªth edn. Cinco
Continentes, Porto Alegre
Monteiro ACBA, Nakasawa GT, Mendes BMJ, Rodriguez APM (2000)
Regeneração in vitro de Passiflora suberosa a partir de discos
foliares. Sci Agric 57:571–573
Moran-Robles MJ (1979) Potentiel morphogénétique dês entrenoeuds de
Passiflora edulis var. flavicarpa Deg. et P. mollissima Bailey en
culture in vitro. Turrialba 29:224–228
Moreira-Dias JM, Molina RV, Bordón Y, Guardiola JL, Garcia-Luiz A
(2000) Direct and indirect shoot organogenic pathways in epicotyl
cuttings of Troyer citrange differ in hormone requirements and in
their response to light. Ann Bot 85:103–110
Müller D, Leyser O (2011) Auxin, cytokinin and the control of shoot
branching. Ann Bot 107:1203–1212
Murashige T, Skoog F (1962) A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol Plant 15:
473–497
Nhut DT, Khiet BLT, Thi NN, Thuy DTT, Duy N, Hai NT, Huyen PX
(2007) High frequency shoot formation of yellow passion fruit
(Passiflora edulis f. flavicarpa) via thin cell layer (TCL) technology.
In: Jain SM, Haggman H (eds) Protocols for micropropagation of
woody trees and fruits. Springer, Dordrecht, pp 417–426
O’Brien TP, McCully ME (1981) The study of plant structure principles
and selected methods, 1st edn. Termarcarphi Pty, Melbourne
Oliveira JC, Ferreira FR (1991) Melhoramento genético do
maracujazeiro. In: São José AR, Ferreira FR, Vaz RL (eds) A cultura
do maracujá no Brasil. FUNEP, Jaboticabal, pp 211–246
Oliveira JC, Ruggiero C (2005) Espécies de maracujá com potencial
agronômico. In: Faleiro FG, Junqueira NTV, Braga MF (eds)
Maracujá: germoplasma e melhoramento genético. Embrapa
Cerrados, Planaltina, pp 142–158
Otoni WC, Paim Pinto DL, Rocha DI, Vieira LM, Dias LLC, Silva ML,
Silva CV, Lani ERG, Silva LC, Tanaka FAO (2013) Organogenesis
and somatic embryogenesis in passionfruit (Passiflora sps.). In:
View publication stats
Aslam J, Srivastava OS, Sharma MP (eds) Somatic embryogenesis
and gene expression. Narosa Publishing House, New Delhi, pp 1–17
Pacheco G, Garcia R, Lugatto D, Vianna M, Mansur E (2012) Plant
regeneration, callus induction and establishment of cell suspension
cultures of Passiflora alata Curtis. Sci Hortic 144:42–47
Pádua JG, Schwingel LC, Mundin RC, Salomão AN, Roverijosé SCB
(2011) Germinação de sementes de Passiflora setacea e dormência
induzida pelo armazenamento. Rev Bras Sementes 33:80–85
Paim Pinto DL, Barros BA, Viccini LF, Campos JMS, Silva ML, Otoni
WC (2010) Ploidy stability of somatic embryogenesis-derived
Passiflora cincinnata Mast plants as assessed by flow cytometry.
Plant Cell Tissue Organ Cult 103:71–79
Pinto APC, Monteiro-Hara ACBA, Stipp LCL, Mendes BMJ (2010) In
vitro organogenesis of Passiflora alata. In Vitro Cell Dev Biol Plant
46:28–33
Reis LB, Silva ML, Lima ABP, Oliveira MLP, Pinto DLP, Lani ERG,
Otoni WC (2007) Agrobacterium rhizogenes-mediated transforma-
tion of passionfruit species: Passiflora cincinnata and P. edulis
flavicarpa. Acta Hortic 738:425–431
Rocha DI, Vieira LM, Tanaka FAO, Silva LC, Otoni WC (2012a)
Anatomical and ultrastructural analyses of in vitro organogenesis
from root explants of commercial passion fruit (Passiflora edulis
Sims). Plant Cell Tissue Organ Cult 111:69–78
Rocha DI, Vieira LM, Tanaka FA, Silva LC, Otoni WC (2012b) Somatic
embryogenesis of a wild passion fruit species Passiflora cincinnata
Masters: histocytological and histochemical evidences. Protoplasma
249:747–758
Rosa YBJ, Dornelas MC (2012) In vitro plant regeneration and de novo
differentiation of secretory trichomes in Passiflora foetida L.
(Passifloraceae). Plant Cell Tissue Organ Cult 108:91–99
SAS Institute (2010) SAS/STAT® 9.1 Users guide. SAS Institute Inc, Cary
Siegien I, Adamczuk A, Wróblewska K (2013) Light affects in vitro
organogenesis of Linum usitatissimum L. and its cyanogenic poten-
tial. Acta Physiol Plant 35:781–789
Silva ML, Paim Pinto DL, Guerra MP, Floh EIS, Bruckner CH, Otoni WC
(2009) A novel regeneration system for wild passion fruit species
(Passiflora cincinnata Mast.) based on somatic embryogenesis from
mature zygotic embryos. Plant Cell Tiss Organ Cult 99:47–54
Silva CV, Oliveira LS, Loriato VAP, Silva LC, Campos JMS, Viccini LF,
Oliveira EJ, Otoni WC (2011) Organogenesis from root explants of
commercial populations of Passiflora edulis Sims and a wild
passionfruit species, P. cincinnata Masters. Plant Cell Tissue
Organ Cult 107:407–416
Smulders MJM, de Klerk GJ (2011) Epigenetics in plant tissue culture.
Plant Growth Regul 63:137–146
Soares WS, Rêgo MM, Rêgo ER, Barroso PA, Nascimento KS,
Ferreira KT (2012) Estabelecimento in vitro e micropropagação
de maracujá silvestre (Passiflora foetida L.). Rev Bras Plantas
Med 14:138–142
Souza MM, Palomino G, Pereira TNS, Pereira MG, Viana AP (2004)
Flow cytometric analysis of genome size variation in some
Passiflora species. Hereditas 141:131–138
Trevisan F, Mendes BMJ (2005) Optimization of in vitro organogenesis
in passion fruit (Passiflora edulis f. flavicarpa). Sci Agricola 62:
346–350
Trevisan F, Mendes BMJ, Maciel SC, Vieira MLC, Meletti LMM,
Rezende JAM (2006) Resistance to Passion fruit woodness virus
in transgenic passionflower expressing the virus coat protein gene.
Plant Dis 90:1026–1030
Vieira MLC, Carneiro MS (2004) Passiflora spp., passionfruit. In: Litz
RE (ed) Biotechnology of fruit and nut crops. CABI Publishing,
Wallingford, pp 435–453
Zerbini FM, Otoni WC, Vieira MLC (2008) Transgenic passionfruit. In:
Kole C, Hall TC (eds) Compendium of transgenic crop plants:
tropical and subtropical fruits and nuts, 1st edn. John Wiley and
Sons, Berlin, pp 213–234