Morphogenetic potential of native passion fruit (Passiflora gibertiiN. E. Brown.) calli

STRUCTURAL BIOLOGY

Morphogenetic potential of native passion fruit (Passiflora gibertiiN. E. Brown.) calli

Milene Alves de Figueiredo Carvalho • Renato Paiva • Eduardo Alves •

Raırys Cravo Nogueira • Vanessa Cristina Stein • Evaristo Mauro de Castro •

Patrıcia Duarte de Oliveira Paiva • Daiane Peixoto Vargas

Received: 10 December 2012 / Accepted: 17 May 2013 / Published online: 13 July 2013

� Botanical Society of Sao Paulo 2013

Abstract Some species of non-cultivated passion fruit

plant have important contributions to genetic improvement.

However, there are few studies concerning about embryo-

genic and organogenic calli mainly related with structural

alterations during their development. The objective of this

work was to characterize, structurally the callogenesis on

leaf explants of Passiflora gibertii N. E. Brown. The coty-

ledons were inoculated in MS culture medium, containing

half salt concentration, supplemented with sucrose (3 %),

and picloram?kinetin for the calli formation. Different calli

colors were obtained and used for structural analyses. The

calli colors were translucent, white, dark-yellow, white-

brown, light-yellow, and white-yellow. After 30 days of

cultivation, the calli were fixed in Karnovsky and prepared

for the visualization under the scanning and transmission

electron microscope and optic microscope. Translucent

and light-yellow calli did not present morphogenic

characteristics. The cells had different shapes forming non-

organized cellular system and the absence or reduced starch

content. On the other hand, white and dark-yellow calli

manifested embryogenic characteristics such as small iso-

diametric cells, an organized cellular, dense cytoplasm rich

in mitochondria and endoplasmic reticulum, small vacuole

and significant starch contend. The culture medium supple-

mented with 4.14 lM of picloram ?0.46 lM of kinetin is the

most suitable to induce embryogenic cells.

Keywords Callogenesis � Cell � Picloram � Starch grain �Ultrastructure � Vacuole

Introduction

The wide diversity existing among the different Passiflora

spp. species is a potential sources of disease resistance,

M. A. de Figueiredo Carvalho (&)

Departamento de Biologia, Embrapa Cafe, Universidade Federal

de Lavras, Setor de Fisiologia Vegetal, Caixa Postal 3037,

Lavras, MG CEP 37200-000, Brazil

e-mail: [email protected]

R. Paiva

Departamento de Biologia, Universidade Federal de Lavras,

Campus Universitario, Setor de Fisiologia Vegetal, Caixa Postal

3037, Lavras, MG CEP 37200-000, Brazil

E. Alves

Departamento de Fitopatologia, Universidade Federal de Lavras,

Campus Universitario, Caixa Postal 3037, Lavras, MG CEP

37200-000, Brazil

R. C. Nogueira

Universidade Federal do Para, Campus Universitario de

Altamira, Rua Coronel Jose Porfırio, 2515, Sao Sebastiao,

Altamira, PA CEP 68372-040, Brazil

V. C. Stein

Universidade Federal de Goias, Campus Jataı—Unidade Jatoba,

Rodovia BR-364, km 192, 3800, Setor Industrial, Jataı, GO CEP

75801-615, Brazil

E. M. de Castro

Departamento de Biologia, Universidade Federal de Lavras,


37200-000, Brazil

P. D. de Oliveira Paiva

Departamento de Agricultura, Universidade Federal de Lavras,


37200-000, Brazil

D. P. Vargas

Embrapa Clima Temperado, Rodovia BR-392, km 78, Caixa

Postal 403, Pelotas, RS CEP 96010-971, Brazil

123

Braz. J. Bot (2013) 36(2):141–151

DOI 10.1007/s40415-013-0015-4

providing evidence to both: genetic breeding programs and

rootstocks for commercial varieties (Roncatto et al. 2004;

Silva Paula et al. 2010) The Passiflora gibertii N.

E. Brown. presents resistance to the main passion fruit

diseases (Meletti and Bruckner 2001; Cunha et al. 2002;

Fischer 2003; Oliveira et al. 2004; Silva Paula et al. 2010)

and is promising for future breeding by possibility of

giving rise to new hybrid, as well as, rootstocks for yellow

passion fruit.

The in vitro culture development of native Passiflora

spp. is important for the conservation and multiplication of

clones with superior features (Santana et al. 2011). The

somatic embryogenesis is tissue culture technique that is a

feasible strategy to study the physiologic embryo devel-

opment, to obtain high multiplication rate possibility,

besides possible gene transference by genetic transforma-

tion (Moura Barros 1999). In vitro Passiflora spp. regen-

eration can be acquired by somatic embryogenesis and

there are a consensus regarding about the growth regulators

role on the embryogenic inducing. The auxins and cyto-

kinins are involved on the activation and regulation of

cellular division and differentiation (Chen et al. 2010;

Souza et al. 2011). Considering these categories of growth

regulators, the exogenous application of auxins, like 2,

4-dichlorophenoxyacetic (2,4-D), picloram, and dicamba,

is well documented to induce the transition of somatic cells

into embryogenic cells (Prakash and Gurumurthi 2010;

Palmer and Keller 2011), and the picloram auxin is a

interesting regulator to be studied, because it promotes high

calli induction (Figueiredo et al. 2000; Rosal 2004; Stella

and Braga 2002).

However, the lack knowledge of somatic embryogenesis

control factors and the asynchrony in the somatic embryos

development are the main reasons for the marginal appli-

cation of this technique (Stein et al. 2010). The compre-

hension of plant organogenesis and embryogenesis, in the

early development stages of the meristematic cells,

requires the study of the subcellular changes and correla-

tions with biochemical alterations (Pihakashi-Maunsbach

et al. 1993). The application of this methodology is

promising to gain information associated to the morpho-

logic and biochemical parameters of the viable cells.

Despite the significant importance of ultrastructural and

morphological (Villalobo et al. 2012) studies of Passiflora

spp. calli induction, not many reports have been published.

Research has been conducted to characterize the cellular

alterations and the organelle activity on the explant regions

potentially morphogenic and to verify the in vitro regen-

eration pathway (Monteiro et al. 2000; Fernando et al.

2003). The embryogenic cells have several common

characteristics which could be evaluated by ultrastructural

studies (Williams and Maheswaran 1986). This research

aimed to characterize the morphogenetic potential of

P. gibertii N. E. Brown. calli with different colors by

conducting morphological and ultrastructural analyses.

Materials and methods

P. gibertii N. E. Brown seeds (access CPAC MJ-22-01)

were obtained from the Embrapa Cerrados (CPAC) germ-

plasm collection, Planaltina-DF. To obtain the young

mother plants (2 months old), the seeds were germinated

in vivo and maintained in growth chamber, at temperature

of 25 ± 2 �C, 43 lmol m-2 s-1 of photon irradiance and

16 h of photoperiod.

The calli were obtained using cotyledons previously

sterilized under aseptic conditions by immersed in sodium

hypochlorite solution (NaClO), containing 0.5 % active

chlorine and Tween 20 (one drop per 100 ml hypochlorite),

for 10 min and washed, three times, with autoclaved dis-

tilled water. After sterilization, the cotyledons were excised

(&1 cm2 diameters) and inoculated with the abaxial sur-

face in contact with the medium.

For calli induction, the explants were inoculated in MS

medium (Murashige and Skoog, 1962) half salt concen-

tration supplemented with 3 % sucrose, 0.5 % agar and

different picloram concentrations (0, 2.07, 4.14, 6.21 and

8.28 lM) combined with kinetin (0 and 0.46 lM). After

inoculation, the explants were maintained in darkness at a

temperature of 25 ± 2 �C for 30 days.

For morphological and ultrastructural analyses different

colors of calli were used. To analyze the calli under

scanning and transmission electron microscopy (TEM),

they were fixed in modified Karnovsky [glutaraldehyde

(2.5 %) and paraformaldehyde (2.5 %) in cacodylate buf-

fer, pH 7.2], for at least 24 h, at room temperature. The

calli were then washed in 0.05 M cacodylate buffer (three

times every 10 min). Subsequently, the calli were fixed in a

solution containing 1 % osmium tetroxide and 0.05 M

cacodylate buffer for 4 h. The calli were then dehydrated in

an ascending acetone gradient (25, 50, 75 and 90 %), for

10 min, and in 100 % acetone three times, for 10 min.

For the scanning electron microscopy (SEM) analysis,

after dehydration, the calli were dried in the critical point

dryer CPD 030, using liquid C02. The samples were sput-

tered with gold prior to SEM analyzes. The observations

were made using electron microscopy (LEO Evo 040),

operating between 10 and 20 kV. For TEM analysis,

immediately after dehydration, the calli were put in an

ascending gradient acetone/Spurr resin 30 % for 8 h, 70 %

for 12 h and finally twice at 100 % with a 24 h interval. To

polymerize, the tissues were molded in pure silicon resin

and dried in a forced-air oven at 70 �C, for 48 h. The

blocks obtained were subjected to thinning using a razor

blade to section the excessive resin. Subsequently, the

142 M. A. de Figueiredo Carvalho et al.

123

blocks were cut into semi-thin (1 lm) and ultrathin

(100 nm) sections using a Reichrt-Jung (ultracut) ultrami-

crotome, with a diamond blade. The semi-thin sections

were collected with a gold ring and put on glass slides. The

sections were later stained with toluidine blue (1 g tolui-

dine blue, 1 g sodium borate and 100 mL water purified in

a 0.2 lM Millipore filter) and permanently mounted in

Permount medium. The ultra-thin sections, on the other

hand, were collected on the formvar-coated slot grids

(Rowley and Moran 1975). The sections were post-stained

with uranyl acetate, followed by lead acetate for 3 min and

later examined in a Zeiss transmission electron microscope

(EM 902 to 80 kV model).

Results

During the callus induction, different calli colors were

observed depending of the picloram ? kinetin concentra-

tion. The colors observed were translucent, white, dark-

yellow, (4.14 lM of picloram ?0.46 lM of kinetin),

white-brown (6.21 lM of picloram ?0.46 lM kinetin),

light-yellow, and white-yellow (8.28 lM of picloram

?0.46 lM of kinetin) (Fig. 1).

In relation to the structural studies, the calli colors

showed different structures (Table 1). The translucent calli

(Fig. 1) presented large cells isodiametric (Fig. 7), oblique

and elongated (Fig. 8) completely disorganized on the

calli. About the cells content, they presented narrow

cytoplasm (Fig. 11), mitochondria (Fig. 12), endoplasmic

reticulum (Figs. 10, 12), and large vacuole (Fig. 12). In

general, the cells also presented thin walls (Fig. 11) cor-

roborating with the anatomical analysis that showed large

cells and thin walls (Fig. 9).

Likewise, in light-yellow calli (Fig. 2) were observed

large and elongated cell with thick walls and disorganized

on the calli (Figs. 13, 14, 16) showing prominent intercel-

lular space (Fig. 15). It was also observed that the cellular

content was low with narrow cytoplasm, mitochondria,

endoplasmic reticulum (Figs. 16, 17) a large vacuole

(Fig. 18) and low starch content (Fig. 15).

On the other hand, the white calli (Fig. 3) and dark-yellow

calli (Fig. 4) presented predominance of well-organized small

isodiametric cells (Figs. 19, 21, 25, 26); however, the white

calli also showed elongated cells (Fig. 20). The cells of these

two colors calli had dense cytoplasm (Figs. 23, 24, 28, 29),

large number of mitochondria (Figs. 22, 28), and endo-

plasmic reticulum (Figs. 22, 29) as well as, small vac-

uole (Figs. 22, 28) and significant starch grain content

(Figs. 21, 24, 27). The rounded nucleus with prominent

nucleolus (Fig. 28) and secretory vesicles (Fig. 30) were

also visualized only on dark-yellow calli.

The white-yellow calli (Fig. 5) showed an extended

cellular disarrangement (Fig. 32), but also occurs clusters

Figs. 1–6 Different calli colors of Passiflora gibertii N. E. Brown: (1) translucent, (2) light-yellow, (3) white, (4) dark-yellow (5) white-yellow,

(6) white-brown. Bar 5 mm

Morphogenetic potential of native passion fruit calli 143

123

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123

of organized small isodiametric cells (Fig. 31) with small

intercellular spaces (Fig. 33) and the white-brown calli

(Fig. 6) manifested well-organized small cells, which were

predominantly isodiametric shape (Figs. 37, 38). Those

cells showed dense cytoplasm (Figs. 34, 41), large number

of mitochondria and endoplasmic reticulum (Figs. 35, 36, 42)

and significant starch grains content (Figs. 33, 36, 39), but

also large vacuole (Figs. 35, 41). The white-yellow had

nucleus with prominent nucleolus (Fig. 35) and secretory

vesicles (Fig. 34) while the white-brown (Fig. 6) had iso-

diametric nucleus (Fig. 40) and low phenolic compound

(Fig. 39).

Discussion

Despite many efforts, there is not an efficient protocol for

somatic embryogenesis of P. gibertii N. E. Brown. More-

over, cytological evaluations of the events that lead or not

to embryogenesis from the tissue of explants had not been

previously performed for this specie.

Interestingly, the differentiations of the cotyledonary

explants generate some structures with no-embryogenic

and embryogenic characteristics correlated with the calli

color. According Shang et al. (2009), the embryogenic and

non-embryogenic calli differ, not only in the morphological

Figs. 7–12 Scanning electron micrographs (7, 8), photomicrograph

(9) and transmission electron micrographs (10, 11) of Passiflora

gibertii N. E. Brown. translucent calli cells. M mitochondria, ER

endoplasmic reticulum, Cy cytoplasm, V vacuole. Bar 100 lm (7, 8),

50 lm (9), 2 lm (10–12)


123

structure and embryogenic behavior, as well as in the

cellular characteristics, as was observed on the P. gibertii

N. E. Brown. calli colors. The translucent and light-yellow

calli presented large cells on different cell shapes and

disorganized cellular system, with few organelles that can

be interpreted as signals of low metabolic activity and non-

embryogenic features, as verified in other species (Moura

et al. 2010; Zienkiewicz et al. 2011).

Fernando (1999) reported that a disorganized cellular

proliferation and accentuated vacuolization of the soybean

cotyledon mesophyll cells are not cellular standards nor-

mally related to embryogenic process (Williams and

Maheswaran 1986). Nogueira et al. (2007), also observed,

during the first callus culture of Byrsonima intermedia

A. Juss, non-embryogenic cells with dimensions of

140 9 30 lm2, featuring elongated shape. Schumann et al.

(1995) related non morphogenic features to the irregular

calli shaped, with elongated and large cells. Steiner et al.

(2005), working with Araucaria angustifolia (Bertol.)

Kuntze callus, observed some elongated and vacuolated

non-embryogenic cells. Therefore, according with the

structural characteristics observed on the P. gibertii

N. E. Brown. translucent and light-yellow calli are not

recommended to obtain embryogenic line.

The white and dark-yellow calli was induced on the

same culture medium (4.14 lM of picloram ?0.46 lM of

Figs. 13–18 Scanning electron micrographs (13, 14) photomicro-

graph (15) and transmission electron micrographs (16–18) of

Passiflora gibertii N. E. Brown. light-yellow calli. M mitochondria,

ER endoplasmic reticulum, Cy cytoplasm, V vacuole, S starch, TW

thickened wall. Starch grains arrow tips. Bar 100 lm (13, 14), 50 lm

(15), 2 lm (16–18)


123

kinetin) and both showed embryogenic characteristics as the

acquisition of embryogenic competence has been attributed

to the cells that show meristematic traits during the induction

phase (Feher 2005). Auxins and cytokinins are the two

growth regulators most commonly employed for the acti-

vation and regulation of cellular division and differentiation

(Feher et al. 2003; Carvalho et al. 2011). The type of auxin

added to the culture medium has a marked effect on

embryogenic competence and the picloram can be used to

induce the formation of embryogenic calli and, in some

cases, can be more effective than 2,4-D (George 1993).

The structural characteristics of the white and dark-

yellow calli, such as, isodiametric shape, dense cytoplasm,

large amount of mitochondria, and starch grains are similar

to those characteristics described for cells with embryo-

genic competence. According to Canhoto et al. (1996), pro-

embryo cells have a rich cytoplasm, made up of many

ribosomes, some starch grains, small sections of rough

endoplasmic reticulum and numerous mitochondria. The

mitochondria show the high energetic demand for the dif-

ferentiation. In those cells, the starch grains are the primary

source of energy, and they will be probably rapidly

mobilized for continuous cellular proliferation (Cangahu-

ala-Inocente et al. 2004).

Second Silva Guedes et al. (2011), the starch grains

accumulated primarily in cells close to sites of intense cell


graph (21) and transmission electron micrographs (22–24) of

Passiflora gibertii N. E. Brown. white calli cells. M mitochondria,

ER endoplasmic reticulum, Cy cytoplasm, V vacuole, S starch. Starch

grains arrow tips. Bar 100 lm (19, 20), 50 lm (21), 2 lm (22–24)


123

division. Reserves, like starch grain and protein body, are

crucial for the morphogenic events, and several studies

have correlated the mobilization dynamics of these com-

pounds with somatic embryogenesis patterns (Rocha et al.

2012), supporting the idea that reserve components are

necessary for cellular reorganization and differentiation

(Zienkiewicz et al. 2011).

Fernando et al. (2001), working with Carica papaya L.

and Portillo et al. (2012), working with Agave tequilana

Weber, described the presence of starch grains, evidenced

by PAS staining, in embryogenic callus. Indeed, it has been

shown that before becoming morphogenic (organogenesis

or embryogenesis), the cells synthesize and store consid-

erable starch content (Williams and Maheswaran 1986).

The white calli also showed secretary vesicles that may

probably contain lipids as reported by Rocha et al. (2012)

for cotyledons of P. cincinnata Mast. These authors iden-

tified the lipids as an initial reserve source for embryogenic

process. These lipids apparently are later replaced by starch

grains, as the synthesis of starch can be directly linked to

the mobilization of lipid content via the glyoxylate cycle.

According with the structural characteristics observed

on the white and dark-yellow calli the medium with

4.14 lM of picloram ?0.46 lM of kinetin is recommend


graph (27), and transmission electron micrographs (28–30) of

Passiflora gibertii N. E. Brown. dark-yellow calli cells.

M mitochondria, ER endoplasmic reticulum, Cy cytoplasm, V vacuole,

N nucleus, Nu nucleolus, SV secretory vesicles. Starch grains arrow

tips. Bar 100 lm (25, 26), 50 lm (27), 2 lm (28–30)


123

to obtain embryogenic line. This medium can induce

embryogenic cells with small size, dense cytoplasmatic,

large nucleus with prominent nucleolus, small vacuoles and

an abundance of starch grains (Fernando et al. 2001;

Mikuła et al. 2005).

Sane et al. (2006) described that the secondary calli

presented a friable granular aspect (embryogenic cells),

with cells rich in soluble proteins in the cytoplasm, small

vacuoles, large nucleus, and easily visible nucleolus and

some cells contained starch grains. In the banana calli, the

embryogenic cells also appear to be similar to meristematic

cells, with isodiametric shape, dense cytoplasm, starch

grains, and isodiametric mitochondria (Oliveira Ribeiro

et al. 2012).

Corroborating with this, Rocha et al. (2012), working

with somatic embryogenesis of P. cincinnata Mast.

observed that the zygotic embryos formed protodermal

cells, with isodiametric shape and fundamental meristem

tissue showing periclinal divisions. According Portillo

et al. (2012), the cells undergo a series of divisions which

contribute to pro-embryo formation. In the somatic

embryogenesis of Passiflora cincinnata Mast., protodermal

and fundamental meristematic cells had large nuclei, as

observed on white and dark-yellow P. gibertii N.

E. Brown. calli.

Moreover, the white-yellow and white-brown calli

although have presented meristematic characteristics, such

as, isodiametric small cells and dense cytoplasm, presented

Figs. 31–36 Scanning electron micrographs (31, 32), photomicro-


Passiflora gibertii N. E. Brown. white and yellow calli cells.


N nucleus, Nu nucleolus, S starch, SV secretory vesicles. Starch grains

arrow tips. Bar 100 lm (31, 32). Bar 50 lm (33), 2 lm (34–36)


123

large vacuole and the vacuolation has been defined by

Filonova et al. (2000) as an early marker of cell death.

The results obtained in the present study yield

detailed structural information about the P. gibertii N.

E. Brown. calli. Understanding, the background of the

cell differentiation will be useful for morphogenetic

manipulation.

Acknowledgments This work was financial supported by the fol-

lowing Brazilian agencies: Fundacao de Amparo a Pesquisa do Estado

de Minas Gerais (FAPEMIG), Coordenacao de Aperfeicoamento de

Pessoal de Nıvel Superior (CAPES), and Conselho Nacional de

Desenvolvimento Cientıfico e Tecnologico (CNPq).

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