ORIGINAL PAPER
Anatomical and ultrastructural analyses of in vitro organogenesisfrom root explants of commercial passion fruit(Passiflora edulis Sims)
Diego Ismael Rocha • Lorena Melo Vieira •
Francisco Andre Ossamu Tanaka •
Luzimar Campos da Silva • Wagner Campos Otoni
Received: 5 December 2011 / Accepted: 5 May 2012 / Published online: 17 May 2012
� Springer Science+Business Media B.V. 2012
Abstract This study aimed to characterize the anatomical
events and ultrastructural aspects of direct and indirect in
vitro organogenesis in Passiflora edulis. Root explants
were cultured on induction medium, supplemented with
4.44 lM 6-benzyladenine. Roots at different stages of
development were collected and processed for observation
by light microscopy and scanning and transmission elec-
tron microscopy. Patterns of direct and indirect regenera-
tion were observed in the explants. During direct
organogenesis, the organogenic buds and nodules, formed
from meristemoids, originated from the pericycle regions
distant from the cut surface. Completely differentiated buds
were observed after 20 days of culture. During indirect
organogenesis, bud formation occurred via meristemoids at
the periphery of the calli, which differentiated from the
cortical region of the initial explant. Regardless of the
regeneration pattern, the meristemoids had similar ultra-
structural characteristics; however, differences were
reported in the nuclear shape of the cells of the meri-
stemoids formed directly and indirectly. This study
provides important information for enhancing the under-
standing and characterization of the organogenic process in
non-meristematic explants and provides information on the
use of roots as explants in genetic transformation protocols
for this important tropical species.
Keywords Histology � Meristemoids � Passiflora �Pericycle � Root culture � Shoot regeneration
Introduction
The economic importance of passion fruit is growing
worldwide. In Brazil, the world’s largest producer of pas-
sion fruit, Passiflora edulis Sims is the predominantly
cultivated species. Moreover, approximately 97 % of all
passion fruits commercially grown are planted with this
genus (Junqueira et al. 2005; FNP and COMERCIO 2009),
for both fresh-fruit and juice markets. The plants are cul-
tivated throughout Brazil. However, the low productivity
of commercial orchards and the susceptibility of the plants
to pathogens such as Fusarium oxysporum Schl. f. sp.
passiflorae (Flores et al. 2012) and Cowpea aphid-borne
virus (Nascimento et al. 2006) have limited the fruit pro-
duction and expansion of the growing area for this crop.
These constraints have been attributed to the lack of
breeding programs for this species (Zerbini et al. 2008).
Tissue culture techniques have been effectively used to
assist the genetic improvement of several crop species.
Most plant transformation procedures require the prior
establishment of efficient systems for gene transfer,
selection, and regeneration of transgenic plants (Yang et al.
2010). Tissue culture studies involving Passiflora began in
the 1960s, and since then, a number of techniques have
been established for several species within the genus
D. I. Rocha � L. M. Vieira � W. C. Otoni (&)
Plant Tissue Culture Laboratory/BIOAGRO, Plant Biology
Department, Federal University of Vicosa, University Campus,
P.H. Rolfs Avenue, Vicosa, MG 36570-000, Brazil
e-mail: [email protected]
F. A. O. Tanaka
Plant Pathology and Nematology Department, Research Support
Center/Electron Microscopy Applied to Agriculture, University
of Sao Paulo, Campus ‘Luiz de Queiroz’, Padua Dias Avenue,
Piracicaba, SP 13418-900, Brazil
L. C. d. Silva
Plant Anatomy Laboratory, Plant Biology Department, Federal
University of Vicosa, University Campus, P.H. Rolfs Avenue,
Vicosa, MG 36570-000, Brazil
123
Plant Cell Tiss Organ Cult (2012) 111:69–78
DOI 10.1007/s11240-012-0171-4
(Vieira and Carneiro 2004; Vieira et al. 2005; Zerbini et al.
2008; Alexandre et al. 2009). The organogenesis pathway
is the prevailing regeneration system used in passion fruit.
Organogenesis-based regeneration of P. edulis has been
achieved from shoot tips (Faria and Segura 1997a), leaf
discs (Otahola 2000; Becerra et al. 2004; Trevisan and
Mendes 2005), hypocotyls (Faria and Segura 1997b; Fer-
nando et al. 2007; Dias et al. 2009), nodal segments
(Kantharajah and Dodd 1990), internodal segments (Biasi
et al. 2000), and most recently, from root explants (Silva
et al. 2011).
Roots provide an excellent source of explants for mass
propagation because of their ease of maintenance and
manipulation in vitro (Vinocur et al. 2000). Passiflora root
explants have shown great regenerative potential because
of the high rate of shoot-bud formation as compared to
other non-meristematic explants (Lombardi et al. 2007;
Silva et al. 2011). Roots are also particularly advantageous
for studies involving Agrobacterium rhizogenes-mediated
transformation (Reis et al. 2007).
Histological studies describing the origin and different
stages of in vitro morphogenesis have contributed signifi-
cantly to the understanding and optimization of various
regeneration systems (Almeida et al. 2006; Fernando et al.
2007; Varshney et al. 2011; Rocha et al. 2012; Rosa and
Dornelas 2012) as well as to the selection and validation of
the techniques used in genetic transformation programs.
The anatomical and ultrastructural characterization of
meristemoids formed from hypocotyls and leaf discs of P.
edulis are crucial for understanding the low regeneration
rates of these explants (Fernando et al. 2007). The supe-
riority of hypocotyledonary explants has been demon-
strated, and their use for genetic transformation of the
species has been recommended (Fernando et al. 2007).
However, to the best of our knowledge, no such studies
have been performed with P. edulis root explants.
This study describes the anatomical events and ultra-
structural aspects associated with in vitro organogenesis of
commercial passion fruit (P. edulis) root explants. The
study identifies the cells and/or tissues involved in the
morphogenetic process of both direct and indirect regen-
eration pathways. This study provides the basic, relevant
knowledge on this potential regeneration system as well as
information for genetic manipulation of Passiflora.
Materials and methods
Plant material and in vitro induction of organogenesis
In vitro induction of organogenesis from P. edulis root
explants was performed according to Silva et al. (2011). Seeds
of P. edulis from the Maguary ‘‘FB-100’’ population were
kindly supplied by Flora Brasil, Ltda (Araguari, MG,
http://www.viveiroflorabrasil.com.br). The seed coats were
removed, and the seeds were surface sterilized and rinsed in
deionized sterile water. The seeds were subsequently trans-
ferred to 250-mL glass jars (10 seeds per jar; a total of 200
seeds) containing 40 mL half-strength MS medium (Mu-
rashige and Skoog 1962) supplemented with vitamin B5
(Gamborg et al. 1968), myo-inositol (0.01 % w/v), sucrose
(3 % w/v), and Phytagel� (0.25 % w/v) (Sigma Chemical
Co., USA); the pH of the medium was adjusted to 5.7 ± 0.1.
The jars were sealed with vented rigid polypropylene lids with
two 10 mm-diameter holes covered with 0.45-lm-pore size
polytetrafluoroethylene membranes (MilliSeal� Air Vent,
Tokyo, Japan). All jars were kept in the dark for 15 days, until
the seeds germinated. The seedlings were transferred to a
temperature-controlled growth room (27 ± 2 �C) equipped
with 2 fluorescent tubes (20 W; Osram Luz do Dia, Sao Paulo,
Brazil) that provided a 16-h photoperiod (36 lmol m-2 s-1
irradiation) for 15 days.
Root segments (10–20 mm) from 30-day-old seedlings,
excluding those adjacent to the hypocotyl, were excised
and incubated in 90 9 15-mm polystyrene Petri dishes (J.
Prolab, Brazil) containing 25 mL MS medium supple-
mented with B5 vitamins, myo-inositol (0.01 % w/v),
sucrose (3 % w/v), Phytagel� (0.25 % w/v), and 4.44 lM
6-benzyladenine; the pH of the medium was adjusted to
5.7 ± 0.1. The plates were sealed with Nexcare Micropore
tape (3 M, Brazil) and kept in the same light and temper-
ature conditions as described previously. A total of 40
plates were inoculated, with 12 root explants in each plate.
Microscopy sample preparation
For anatomical and ultrastructural analyses, P. edulis root
explants were collected on a daily basis for the first 4 days
of culture; thereafter, these explants were collected every
2 days over a period of 30 days. The samples were fixed in
Karnovsky (1965) solution [2.5 % glutaraldehyde and 4 %
paraformaldehyde in monobasic potassium phosphate
buffer (pH 7.2) and 5 mM calcium chloride].
Light microscopy (LM)
For anatomical studies, fixed samples were dehydrated in a
graded ethanol series and embedded in methacrylate
(Historesin, Leica Instruments, Germany). Cross and lon-
gitudinal sections (5-lm thick) were obtained using an
automatic rotary microtome (RM 2155, Leica Microsys-
tems Inc., USA) and stained with toluidine blue (O’Brien
and McCully 1981). The specimens were mounted in
Permount on glass slides. Photographs were taken using a
light microscope (Olympus AX70TRF; Olympus Optical,
70 Plant Cell Tiss Organ Cult (2012) 111:69–78
123
Japan) equipped with a digital camera (Spot Insight Color
3.2.0; Diagnostic Instruments Inc., USA).
Scanning electron microscopy (SEM)
Fixed samples were dehydrated through an acetone series,
critical point dried (CPD 030; Bal-Tec, Balzers, Liech-
tenstein), mounted on aluminum stubs, and coated with
gold (FDU 010; Bal-Tec, Balzers, Liechtenstein). Exam-
inations and photography were performed using a SE
microscope (Leo 1430VP; Zeiss, Heidelberg, Germany)
equipped with a digital camera.
Transmission electron microscopy (TEM)
Fixed samples were post-fixed with 1 % osmium tetroxide
in 0.05 M phosphate buffer, dehydrated through an acetone
series, and embedded in Spurr epoxy resin (Spurr 1969).
The specimen blocks were prepared for ultramicrotomy by
using a trimmer (EM Trim; Leica Microsystems Inc.,
USA). Ultrathin sections (70-nm thick) were cut using an
ultramicrotome (Leica UC6; Leica Microsystems Inc.,
USA) and stained with uranyl acetate and lead citrate
(Reynolds 1963). Examinations and photography were
performed using a TE microscope (EM900; Zeiss, Ger-
many) coupled to a digital camera, at 80 kV.
Results
Morphogenetic responses of root explants
Regeneration of P. edulis root explants occurred via both
direct and indirect organogenesis (Fig. 1). The first mor-
phological changes were recorded during the first week of
culture, when the root explants became slightly swollen.
After 10–12 days of culture, organogenic sectors were seen
as small green structures emerging on the surface of the
explants in regions located far from the cut surface
(Fig. 1a). These structures developed into buds (Fig. 1b, c)
or organogenic nodules (Fig. 1d). Completely formed buds
emerged after 20 days of culture (Fig. 1c).
Callus formation started 3 or 4 days after inoculation
(on to the induction medium) in regions near the cut sur-
face of the explants (Fig. 1e). The calli showed continuous
increase in volume, exposing the inner tissues (Fig. 1f) and
giving rise to adventitious buds 20–22 days after inocula-
tion (Fig. 1g).
Origin of direct and indirect organogenesis in P. edulis
During direct organogenesis, initial morphogenetic
responses were observed in the pericycle, regardless of the
developmental stage of the explants. The pericycle of the
initial explants consisted of a single cell layer (Fig. 2a–c).
The first periclinal divisions of the pericycle cells occurred
in regions distant from the cut surface of the explant 3 or
4 days after inoculation on the induction medium (Fig. 2d,
e). This process gave rise to zones of proliferation con-
taining several cell layers after 8 days of culture (Fig. 2f).
After 10 days on the induction medium, cells in the pro-
liferation zone became meristematic and produced separate
meristemoids by repeated divisions (Fig. 2g). Meristemoid
cells contained dense cytoplasm with large nuclei containing
prominent nucleoli. The development of meristemoids
(Fig. 2h) resulted in the disruption of the cortex and root
epidermis (Fig. 2i); subsequently, these meristemoids differ-
entiated into buds (Fig. 2j). Buds arose endogenously with the
vascular system connected to the main vascular tissue of the
parent explant (Fig. 2j), confirming the organogenic regen-
eration pattern. Alternatively, the meristemoids also contin-
ued to divide and form organogenic nodules (Fig. 3a).
Organogenic nodules were formed by a group of parenchymal
cells and were covered by continuous uniseriate epidermis
(Fig. 3a). The leaf primordia differentiated from the periph-
eral cells of the nodules (Fig. 3a, b).
In the indirect organogenesis pathway, the callus formed
on the cut surface from the cortical cells (Fig. 3c). After
successive divisions, the cortical cells became hypertrophic
and divided multiple times, giving rise to a group of
daughter cells that remained enclosed in the parent cell
wall (Fig. 3c, d). Meristemoids developed within the
peripheral layers of the calli from cells derived from the
inner layers of the cortex (Fig. 3c, d) and differentiated into
buds (Fig. 3e), which was similar to that observed in the
direct organogenic pathway.
The organogenic process in P. edulis root explants was
asynchronous. Buds and organogenic nodules in various
stages of development were observed in the same explant as
well as in different explants at different times during culture.
Cytological characterization of meristemoids
Meristemoids that formed directly from the pericycle
(Fig. 4a–d) showed a dense cytoplasm with large, round
nuclei containing prominent, occasionally multiple,
nucleoli (Fig. 4d). Most vacuoles were small and under-
developed (Fig. 4a). The mitochondria were numerous and
mostly globose (Fig. 4a). The Golgi apparatus, containing
a few cisternae, was positioned near the cell wall and was
involved in the active production of dictyosome vesicles
(Figs. 4a, c). Fusion of Golgi apparatus-derived vesicles
formed the cell plates in the meristemoids (Fig. 4b). Plas-
modesmata were distributed throughout the newly formed
cell walls between adjacent cells of the meristemoids
(Fig. 4c).
Plant Cell Tiss Organ Cult (2012) 111:69–78 71
123
Meristemoids that originated indirectly from the calli were
ultrastructurally similar to those formed via the direct path-
way; however, some cells showed nuclear differences. In
these cells, the nucleus had numerous, evident nuclear pores
(Fig. 4e, f), and progressive formation of membrane invag-
inations that resulted in irregularities in the nuclear envelope
(Fig. 4g). Mitochondria and the rough endoplasmic reticulum
were occasionally found within these invaginations (Fig. 4h,
i). Lobe-shaped nuclei were also observed in callus cells that
were not involved in the regeneration process.
Discussion
This study elucidated the ultrastructural characteristics of
the cells and provided a structural description of the events
involved in the regeneration of P. edulis from root explants
cultured on a semi-solid induction medium. The use of root
explants for in vitro regeneration is limited to a few species
(Parveen and Shahzad 2011). In Passiflora, in vitro
regeneration from roots has been successfully reported in
P. cincinnata via both direct organogenesis (Lombardi
et al. 2007; Silva et al. 2011) and indirect organogenesis
(Lombardi et al. 2007) and recently in P. edulis via direct
organogenesis only (Silva et al. 2011).
The buds formed on the roots of P. edulis via direct
organogenesis originated directly from the pericycle cells; the
pericycle has been proposed to be an extended meristem in the
plant body (Evert 2006; Smet et al. 2006). A number of studies
have reported the involvement of the pericycle in the regen-
eration of shoots from root explants (Vinocur et al. 2000; Vila
et al. 2005; Lombardi et al. 2007; Arora et al. 2011), which is
consistent with the current knowledge on the molecular
mechanisms involved in the induction of shoot meristems in
roots. Atta et al. (2009) demonstrated the ability of pericycle
cells to rapidly re-enter the cell cycle on induction with
cytokinin and maintain diploidy after several cycles of divi-
sion, thus maintaining the genetic fidelity of the regenerated
plants. In fact, the stability of the ploidy level of P. edulis
regenerants obtained by direct organogenesis from root seg-
ments has been previously confirmed by nuclear DNA flow
cytometry (Silva et al. 2011). Such experiments have shown
the potential use of this type of explant for the clonal propa-
gation of this commercial species.
Pericycle cells are lateral root initiation sites (Casimiro
et al. 2003; Smet et al. 2006); however, under proper
conditions, they may also be involved in the formation of
shoot meristems. Regeneration systems, such as those in
the present study, in which root explants are cultured on
media supplemented with only cytokinin, molecular studies
have shown that the competence to form buds is acquired at
sites where spontaneous formation of lateral roots occurs
(Atta et al. 2009; Motte et al. 2011). When root segments
of Arabidopsis thaliana were cultured under these condi-
tions, shoot regeneration via direct organogenesis occurred
at sites where lateral root formation from pericycle cells
occurred; these structures were termed lateral root meri-
stem (LRM)-like protuberances (Atta et al. 2009). Simi-
larly, we report the regeneration of shoot meristems and
leaf primordia from nodular structures that originated from
the P. edulis root pericycle cells, suggesting that the LRM-
like protuberances in Arabidopsis are equivalent to the
organogenic nodules described in this study.
Differentiation of organogenic nodules has been repor-
ted in regeneration systems for different types of explants
in different plant species (Fortes and Pais 2000; Moyo et al.
2009; Ferreira et al. 2009; Sahai et al. 2010). In some cases,
these structures were interpreted to be abnormal embryos
because they usually had a globular morphology, as dis-
cussed by Haensch (2004). However, the vascular con-
nections with the parent explant, the lack of bipolarity, and
the differentiation of leaf primordia and/or buds on their
surfaces do not support this hypothesis. In P. edulis, the
formation of organogenic nodules was previously descri-
bed in leaf and hypocotyl explants and was referred to as
protuberances (Fernando et al. 2007); the histological
organization of the structures formed from roots was sim-
ilar to that observed in nodules formed from hypocotyl
explants.
Callus formation from the division of cortical cells in
root explants was also reported in Curcuma zedoaria (Melo
et al. 2001) and Cleome rosea (Simoes et al. 2009). In these
species, the cortical cells became hypertrophic, and the
buds were formed from the meristemoids at the periphery
of the calli. The calli derived from differentiated tissues,
such as cortical parenchyma, may give rise to a high degree
of somaclonal variation (Bordallo et al. 2004). The irreg-
ular shape of the nucleus and the occurrence of invagin-
ations and nuclear pore complexes in the calli cells and
meristemoids of indirect origin corroborate this hypothesis.
Nuclear irregularity is a sign of possible amitoses (Tylicki
et al. 2002; Appezzato-da-Gloria and Machado 2004). This
process is related to nuclear fragmentation, which may
compromise the genetic stability of regenerated buds. This
would make the use of the indirect pattern of regeneration
impracticable for clonal propagation and genetic transfor-
mation systems; additional flow cytometry analyses are
required to confirm the ploidy stability of P. edulis plants
regenerated via the indirect pathway.
Fig. 1 In vitro organogenesis of Passiflora edulis root explants.
a Acquisition of competence to form buds or organogenic nodules
(arrowhead) 10 days after induction. b–d Direct organogenesis. b,
c Bud formation after 16 and 20 days of culture. d Organogenic
nodule. e–g Indirect organogenesis after 4, 12, and 20 days of culture.
Bars = 1 mm
b
Plant Cell Tiss Organ Cult (2012) 111:69–78 73
123
Ultrastructural characteristics, including dense cyto-
plasm, numerous mitochondria, numerous Golgi apparatus,
and prominent nuclei, observed in the meristemoids of
P. edulis were similar to those described in the meristem-
oids of Glycine max and Bauhinia forficata (Appezzato-da-
Gloria and Machado 2004). A large number of mitochon-
dria indicate a high level of energy utilization by these cells
and is characteristic of tissues undergoing differentiation
(Pihakaski-Maunsbach et al. 1993). In the meristemoids of
G. max, the increased number of Golgi apparatus, typically
involved in the secretion of substances into the apoplast,
was associated with an accelerated synthesis of cell wall
components (Appezzato-da-Gloria and Machado 2004). A
similar interpretation was made for P. edulis because cell
plate formation was usual in these regions. The numerous
Fig. 3 In vitro organogenesis of Passiflora edulis. a, c, d Light
micrographs. b, e Scanning electron micrographs. a, b Organogenic
nodules. Leaf primordia formed at the periphery of the nodules
(arrows). c–e Indirect organogenesis. c, d Meristemoids, formed at
the periphery of the callus that originated from the cortical region of
the initial explant. e Indirect organogenesis. Note the occurrence of
buds (*). ca callus, ep epidermis, hp hypertrophy, me meristemoids,
vc vascular cylinder. In a, b, e, bars = 200 lm; in c,
d bars = 100 lm
Fig. 2 Direct organogenesis from root explants of Passiflora edulis.
a, b, d Cross sections. c, e–j Longitudinal sections. a–c Root segment
used for initial explants. d–f Periclinal divisions of the pericycle
(arrowhead) after 4 (d–e) 8 (f) days of culture. g, h Formation and
early development of meristemoids (*). i Disruption of the cortex and
epidermis caused by the development of meristemoids. j Developed
bud. Note the vascular connection with the explant (arrow). co cortex,
en endoderm, ep epidermis, pe pericycle, px protoxylem, lp leaf
primordial, x xylem. In a, b, d, bars = 50 lm; in c, e, j,bars = 100 lm
b
Plant Cell Tiss Organ Cult (2012) 111:69–78 75
123
Fig. 4 Transmission electron microscopy of meristemoids and callus
of Passiflora edulis. a–d Meristemoids formed via the direct pathway.
a Cells with dense cytoplasm, mitochondria, and Golgi apparatus.
b Cell plate formation. Note the occurrence of vesicles (blackarrowheads). c Plasmodesmata (white arrows). d Regular-shaped
nucleus with prominent nucleoli. e–i Meristemoids formed via the
indirect pathway. e Irregular-shaped nucleus with nuclear pore
complex (detail). f Nuclear pores (white arrowheads). g Nuclear
membrane invaginations (black arrows). h, i Endoplasmic reticulum
and mitochondria within the nuclear invaginations. ga Golgi appa-
ratus, m mitochondria, nu nucleolus, er rough endoplasmic reticulum,
v vacuole. In a, e, bars = 1 lm; in b, c, h, i, bars = 0.5 lm; in d, g,
bars = 2 lm; and in f, bars = 0.2 lm
76 Plant Cell Tiss Organ Cult (2012) 111:69–78
123
cytoplasmic connections, also called plasmodesmata,
present in adjacent meristemoid cells indicated intense
symplastic communication. The frequency of plasmodes-
mata has been reported to increase in meristematic cells
because these connections are essential for the intercellular
transport of signaling molecules involved in controlling the
differentiation pathway of these cells (Verdeil et al. 2007).
In this study, we described the process of in vitro
organogenesis of P. edulis root explants. We also presented
an ultrastructural analysis of the cells involved in the dif-
ferent patterns of regeneration. Our results corroborate the
morphogenetic model previously described for hypocotyls
of this species (Fernando et al. 2007). The study also
demonstrated the similarities in the organogenic responses
and cytological characteristics of the cells involved in both
the direct and indirect regeneration patterns from the main
sources of non-meristematic explants of P. edulis, i.e., leaf
discs and hypocotyls (Fernando et al. 2007). A detailed,
cell-level characterization of plant regeneration systems
from different explants is not available in the literature,
suggesting that P. edulis may be a possible model for such
studies. In addition, these findings allow a better under-
standing of the organogenic pathway in Passiflora, which
provides insight into the characterization of molecular
events associated with cell totipotency. This study also
provides increased knowledge on the use of root explants
for genetic transformation of this important tropical spe-
cies, which represents almost entire commercial passion
fruit production worldwide.
Acknowledgments The authors would like to thank CAPES
(Brasılia, DF), CNPq (Brasılia, DF), and FAPEMIG (Belo Horizonte,
MG) for financial support; Viveiros Flora Brasil Ltda. (Araguari, MG)
for kindly supplying seeds of the Maguary ‘FB 100’ population; the
NMM-UFV (Vicosa, MG) and NAP/MEPA-ESALQ/USP (Piraci-
caba, SP) for use of the electron microscope facilities; and Dr. Andrea
Dias Koehler for her critique and valuable suggestions.
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