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: wotoni@ufv.br

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

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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).

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72 Plant Cell Tiss Organ Cult (2012) 111:69–78

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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

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74 Plant Cell Tiss Organ Cult (2012) 111:69–78

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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

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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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