Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 150:637–650

https://doi.org/10.1007/s11240-022-02318-0

ORIGINAL ARTICLE

Novel avenues for passion fruit in vitro regeneration from endosperm

culture, and morpho‑agronomic and physiological traits of triploid
Passiflora cincinnata Mast. emblings
Marcelo Dias Machado1   · Claudinei Silva Souza1   · Mariana Machado2   · Aryane Campos Reis3   ·
Saulo Marçal de Sousa3   · Elyabe Monteiro Matos3   · Lyderson Facio Viccini3   · Wagner Campos Otoni2   ·
Ilio Fealho de Carvalho4   · Diego Ismael Rocha5   · Maurecilne Lemes da Silva1,4 

Received: 6 March 2022 / Accepted: 24 April 2022 / Published online: 12 May 2022
© The Author(s), under exclusive licence to Springer Nature B.V. 2022

Abstract
The present study describes a new regeneration system based on somatic embryogenesis from mature endosperm Pas-
siflora cincinnata Mast. cultures. Moreover, the morpho-agronomic and phenological traits, as well as enzymatic activity
of regenerated triploid emblings are compared to those of diploids. Mature endosperms were cultured on Murashige and
Skoog medium supplemented with various concentrations (4.5–45.2 µM) of 2,4-dichlorophenoxyacetic acid (2,4-D) and
4.5 μM 6-benzylaminopurine (BA). No plant growth regulators were included in the control group. Embryogenic calli were
observed only in treatments supplemented with 13.6 and 18.1 µM 2,4-D + 4.5 µM BA, with the highest number of somatic
embryos per explant and regenerated plants (emblings) obtained with 18.1 µM 2,4-D. Most emblings (70%) were triploid
(2n = 3x = 27), with a DNA amount (4.38 pg) similar to that of endosperm and 1.5 times greater than in diploid P. cincinnata
seedlings (2n = 2x = 18), that contained 2.98 pg of DNA. While the number of organs and/or structures was akin to that in
diploids, triploid emblings generally exhibited larger and longer vegetative and floral structures. The flower lifespan was also
slightly altered by triploidy, nectar concentration was 27% higher, and the activity of plant defense enzymes β-1,3-glucanase
and polyphenol oxidase was 29.8% and 22.1% higher. This study describes a new regeneration system for the production of
phenotypic variants of this ornamental passion fruit species, opening new perspectives for future studies on genetic passion
fruit breeding.

Key message 
Mature endosperms of Passiflora cincinnata display high embryogenic competence when cultivated in an auxin-rich induction
medium and the regenerated triploid emblings are morphologically distinct from their diploid counterparts.

Keywords  Endosperm · Floral traits · Plant defense enzymes · Passion flower · Triploidy · Somatic embryogenesis

Communicated by Mohammad Reza Abdollahi.

3
* Diego Ismael Rocha Departamento de Biologia, Universidade Federal de Juiz de
diego.rocha@ufv.br Fora, Juiz de Fora, MG 36036‑900, Brazil
4
* Maurecilne Lemes da Silva Departamento de Biologia, Universidade do Estado de Mato
maurecilne.carvalho@unemat.br Grosso, Tangará da Serra, MT 78300‑000, Brazil
5
1 Departamento de Agronomia, Universidade Federal de
Programa de Pós‑Graduação em Genética e Melhoramento
Viçosa, Viçosa‑MG 36570‑900, Brazil
de Plantas, Universidade do Estado de Mato Grosso,
Tangará da Serra, MT 78300‑000, Brazil
2
Departamento de Biologia Vegetal, Universidade Federal de
Viçosa, Viçosa, MG 36570‑900, Brazil

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Introduction
Polyploidy is a strategic tool in plant breeding programs
(Sattler et al. 2016; Touchell et al. 2020). Polyploid plants
have more than two sets of chromosomes, and this condi-
tion often promotes desirable morpho-agronomic charac-
teristics, favoring the development of more productive and/
or more adaptable cultivars (Osborn et al. 2003). Polyploid
organisms can arise in natural populations (Soltis and
Burleigh 2009; Van de Peer et al. 2017) or be induced via
somatic hybridization (Grosser and Gmitter 2011). The lat-
ter can occur from in vitro cultures of tissues with different
ploidy levels (Thomas and Chaturvedi 2008; Wang et al.
2016; Abdullah et al. 2021) or from in vitro and ex vitro
tissues treated with antimitotic agents (Dhooghe et al. 2011;
Sumarji and Suparno 2017).
Genome duplication can cause significant genetic and
epigenetic changes, possibly leading to phenological, phe-
notypic, and physiological alterations (Adams and Wendel
2005; Otto and Whitton 2007; Yang et al. 2011; Van de Peer
et al. 2017). Triploid plants often have larger vegetative and
reproductive organs, thicker, darker-colored leaves, and
longer-lasting flowers. In addition, they display improved
vigor and greater tolerance to biotic and abiotic factors,
which confers superior adaptation to a wide range of envi-
ronmental stresses; however, they are generally sterile owing
to gamete imbalance (Comai 2005; Ranney 2006; Sun et al.
2011; Padoan et al. 2013; Santana-Vieira et al. 2016; Wang
et al. 2016).
Given the remarkable features displayed by triploid
individuals, plant regeneration systems based on in vitro
endosperm culture have been established to obtain genotypes
with larger vegetative and reproductive organs (ornamental
plants) and seedless fruits (fruit crops) (Kumar et al. 2015;
Wang et al. 2016; Iannicelli et al. 2020). The endosperm is
a nutrient-rich reserve tissue found in angiosperm seeds,
which is closely related to the growth and development of
zygotic embryos. It forms following the fusion of a hap-
loid spermatic nucleus with the two nuclei from the central
cell of the embryo sac during fertilization. This step results
in a single, naturally triploid tissue (Cailleau et al. 2010),
explaining why endosperm in vitro culture offers a direct
regeneration system for triploid plants (Thomas and Chatur-
vedi 2008; Wang et al. 2016).
Plant regeneration from the endosperm requires that
parenchyma storage cells acquire competence to reprogram
themselves and take on a new developmental route. Cellular
competence is acquired by hormonal signals from auxins,
such as 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphtha-
leneacetic acid, or indole-3-butyric acid, and cytokinins,
such as 6-benzylaminopurine (BA) or kinetin, which are
added to the culture medium (Hoshino et al. 2011). De novo
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Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 150:637–650
shoot organogenesis (DNSO) constitutes the main regenera-
tion pathway during in vitro endosperm culture (Hoshino
et al. 2011). However, regeneration can proceed also from
somatic embryos. Owing to the complexity of this morpho-
genetic process, whereby two meristematic poles (shoot and
root apical meristems) need to be established synchronously,
few studies have reported the induction of somatic embry-
ogenesis from endosperm culture (Kosmiatin et al. 2014;
Sukmara et al. 2014).
In Passiflora (Passifloraceae), a tropical genus valued for
the production of passion fruit and its ornamental potential,
regeneration of triploid plants by endosperm culture was
reported in 1996 by Mohamed et al. However, it is only in
recent years that this biotechnological tool has been tried
in P. edulis Sims (Antoniazzi et al. 2018), the main com-
mercial species of the genus, as well as P. cincinnata Mast.
(Silva et al. 2020) and P. foetida L. (Mikovski et al. 2021),
two ornamental species (Bernacci et al. 2005; Mikovski
et al. 2019). In all these systems, regeneration occurred
from adventitious buds in mature endosperms cultivated in
cytokinin-rich induction medium.
Passion fruit somatic embryogenesis was induced suc-
cessfully from in vitro cultures of zygotic embryos (Rocha
et al. 2020). Based on the established regeneration system
for P. cincinnata, in which somatic embryos were formed in
induction medium supplemented with 18.1 µM 2,4-D and
4.5 µM BA (Silva et al. 2009), embryogenic cultures were
optimized for various Passiflora spp. using 8.8–72.4 μM
2,4-D and 4.5 μM BA (Paim Pinto et al. 2011; Rosa et al.
2015; Prudente et al. 2017). P. cincinnata is a model species
for passion fruit morphogenetic studies (Dias et al. 2009;
Otoni et al. 2013; Vieira et al. 2018; Silva et al. 2020). Inter-
estingly, the anthers of this species display high embryo-
genic competence (Silva et al. 2021) when cultivated in
induction medium supplemented with 18.1 µM 2,4-D and
4.5 µM BA. Given the close ontogenetic, physiological,
and functional relationship between zygotic embryos and
endosperm, as well as the high embryogenic responsive-
ness of P. cincinnata tissues when cultured in medium con-
taining a 4:1 ratio of auxin (2,4-D) to cytokinin (BA), we
hypothesized that these culture conditions could induce the
formation of somatic embryos from P. cincinnata endosperm
storage cells.
In the present study, we describe a system for the regener-
ation of P. cincinnata triploid plants, based on new morpho-
genetic pathways. Moreover, to facilitate the use of specific
genotypes in passion fruit breeding programs, we compare
the morpho-agronomic, phenological, and physiological
traits of diploid and triploid plants.
Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 150:637–650 639
Materials and methods
Plant material
P. cincinnata seeds were isolated from ripe fruits and dried
at room temperature. The seed coat was removed with the
aid of a mini-vise. Then, the seeds were immersed in 70%
(v/v) alcohol for 3 min and disinfected for 25 min with
100 mL of 2.5% (v/v) commercial NaClO containing three
drops of Tween-20. The seeds were rinsed four times in dis-
tilled, autoclaved water and immersed for 10 h to facilitate
endosperm isolation.
Induction of somatic embryogenesis
For the induction of somatic embryogenesis, endosperms
were aseptically isolated from zygotic embryos and cultured
in 90 × 15 mm polystyrene Petri dishes containing 30 mL
of induction medium composed of MS basic salts (Calsson
Labs, Smithfield, UT, USA) (Murashige and Skoog 1962),
0.01% (w/v) myo-inositol (Synth, Diadema, SP, Brazil), 3%
(w/v) sucrose (Exodo Cientifica Ltda., Sumaré, SP, Brazil),
and 8 g/L agar ­(Acumedia®; Neogen, Lansing, MI, USA).
Culture medium was supplemented with different concentra-
tions of 2,4-D (4.5, 9.0, 13.6, 18.1, 22.6, 27.1, 31.7, 36.2,
and 45.2 µM) and 4.5 µM BA. No plant growth regula-
tors (PGRs) were added to the control group. The pH was
adjusted to 5.7 ± 0.1, and the culture medium was autoclaved
at 121 °C and 1.1 atm for 20 min. Each treatment consisted
of five Petri dishes with 10 endosperms each. The dishes
were kept in a culture room in the absence of light and at
25 ± 2 °C for 30 days.
Embryogenic calli were transferred to Petri dishes
containing maturation medium consisting of MS salts as
described above, but without PGRs. The dishes were kept in
a growth room under the same environmental conditions as
described above, but with a 16-h photoperiod and 36 μmol/
m2/s irradiance (Special Day Light, 20 W; Osram, Barueri,
SP, Brazil) for 45 days.
Cotyledonary stage somatic embryos were isolated and
subcultured in flasks containing MS-half strength medium,
0.01% (w/v) myo-inositol, 3% (w/v) sucrose, 8 g/L agar, and
no PGRs. Emblings of roughly 10 cm in length, obtained
after 90 days of cultivation, were transferred to plastic cups
containing Plantmax ­HT® substrate (Eucatex Agro, Paulínia,
SP, Brazil). The plastic cups were covered with transparent
plastic bags and kept under a 16-h photoperiod and 25 ± 2 °C
for 20 days.
Embling acclimatization
P. cincinnata emblings (n = 10), obtained from different
embryogenic lines, measuring approximately 30 cm were
transferred to 30-dm3 plastic pots filled with a 3:1 mix-
ture of soil and substrate, and grown in a greenhouse. To
compare the morpho-agronomic features of diploid and
triploid plants, ten seeds of P. cincinnata were germinated
in black polyethylene plastic bags of 28 × 14 cm containing
Plantmax ­HT® substrate, kept in a greenhouse, and irri-
gated daily. After germination, three diploid plants were
also cultivated under the same conditions and used as con-
trols. The pots were spaced 1.5 m between rows and 4 m
between plants. The conduction system was in a vertical
spreader with a smooth wire at a height of 1.8 m, without
elimination of terminal buds and lateral branches.
The foliar application of fertilizer was carried out
monthly in the vegetative phase and every 15 days in the
reproductive phase, at a dosage of 3–9 L/ha. The fertilizer
consisted of N = 10%; ­P2O5 = 10%; ­K2O = 10%; Fe = 0.1%;
Zn = 0.1%; Cu = 0.1%; S = 0.3%; and Mn = 0.5%.
Flow cytometry
For f low cytometry, approximately 20–30  mg of
endosperm tissue, young and fresh leaves from diploid
seedlings (control), and endosperm-derived plants regen-
erated by somatic embryogenesis (emblings) were chopped
with a disposable steel razor blade in 1 mL WPB buffer
to release the nuclei (Loureiro et al. 2007). Pisum sati-
vum (2C DNA content = 1.96 pg) was used as an internal
reference standard (Doležel 1997). Ten samples of each
material were analyzed.
Previously macerated tissues were filtered through a
50-μm nylon mesh and collected in a polystyrene tube.
The filtrate was stained with 25 μL propidium iodide solu-
tion (1 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) sup-
plemented with 2.5 µL RNase (20 mg/L). At least 10,000
nuclei per sample were analyzed in triplicate. Data were
acquired using a CytoFLEX instrument (Beckman Coulter
Life Sciences, Indianapolis, IN, USA) at the Institute of
Biological Sciences of the Federal University of Juiz de
Fora, and then plotted and analyzed using CytExpert 2.0.1
software. Nuclear DNA content (pg) was estimated using
the following equation (Doležel and Bartos 2005): Sample
(2C DNA) = G1 peak channel of sample × 9.09 (P. sativum
DNA content) / G1 peak channel of P. sativum.
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Cytogenetic analyses
Cytogenetic analyses were performed in seedlings (dip-
loid control) and those emblings (n = 3) that, based on
flow cytometry analysis, contained more DNA than the
seedlings. Slides were prepared from young root tips pre-
treated with 3 mM 8-hydroxyquinoline at 4 °C for 8 h and
fixed in 3:1 (v/v) ethanol/acetic acid for 24 h at -20 °C.
The root tips were submitted to enzymatic maceration in
4% (w/v) cellulase (Onozuka R-10; Serva, Heidelberg,
Germany) and 40% (v/v) pectinase (Sigma-Aldrich) for
3 h at 37 °C. Cytological preparations were performed as
described by Carvalho and Saraiva (1993), and stained
with 4,6-diamidino-2-phenylindole. Slides were imaged
using a BX51 fluorescence microscope with CellSens soft-
ware (Olympus, Tokyo, Japan).
Morpho‑agronomic characterization
Comparison of the morpho-agronomic characteristics of
seed-derived diploid plants and endosperm-derived triploid
P. cincinnata plants (n = 3) was carried out following the
instructions for distinguishability, homogeneity, and stability
assays proposed by the National Service for the Protection
of Cultivars (Brasil 2016) for wild ornamental species and
interspecific hybrids of the genus Passiflora, as well as the
descriptors proposed by Fonseca et al. (2017) and Nobrega
et al. (2017). The triploid plants were obtained from differ-
ent explants. Comparisons were carried out only on plants
that were already in the reproductive stage, and included 29
morpho-agronomic descriptors. The descriptors were evalu-
ated based on the structures found in the middle third of each
plant. Ten leaves and flowers from each plant were analyzed.
Plant dimensions were measured with the aid of a digital
caliper and an analog refractometer (Akrom, São Leopoldo,
RS, Brazil) to quantify the Brix of the floral nectar. Values
reported here represent the average of ten repetitions per
genotype (diploid or triploid), and each repetition corre-
sponds to the average of ten measurements of vegetative or
reproductive structures per plant.
Phenological observations were also carried out within
a period of 30 days to determine the average opening and
closing times of the flowers. In total, three flowers per plant
were evaluated, with 30 flowers per genotype.
Enzymatic evaluation
The activities of β-1,3-glucanases, polyphenol oxidase and
peroxidase were quantified due to their involvement with
disease resistance mechanisms in plants. To evaluate the
enzymatic activity of diploid and triploid plants, we col-
lected two leaves per plant (n = 3) from the middle third of
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Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 150:637–650
the plant, placed them in labeled aluminum envelopes, snap-
froze in liquid nitrogen, and stored at −80 °C.
The activity of β-1,3-glucanases was determined by col-
orimetric quantification of reducing sugars released from
laminarin (Lever 1972). The protein extract was obtained
by adding 2 mL of extraction medium containing 50 mM
sodium phosphate buffer (pH 6.5), 1 mM phenylmethylsul-
fonyl fluoride, and 200 mg polyvinylpolypyrrolidone. For
the reaction, 230 μL of 0.1 M sodium acetate buffer (pH
5.0), 250 μL laminarin substrate solution (4 mg/mL) and
20 μl plant extract were mixed and incubated at 45 °C for
30 min. Then, the reaction was stopped by the addition of
500 μL of 3,5-dinitrosalicylic acid, heated to 100 °C for
5 min, and cooled to 30 °C. Absorbance was read at 540 nm.
The amount of reducing sugars formed was quantified using
a standard curve of glucose concentrations. Results were
expressed in mg glucose/min/mg protein.
Polyphenol oxidase activity was determined according to
Duangmal and Apenten (1999), and the protein extract was
obtained by macerating the leaf sample in 0.1 M sodium
acetate buffer (pH 5.0). For the reaction, 100 μL enzymatic
extract, 900 μL of 0.1 M sodium phosphate buffer (pH 6.8),
and 20 mM catechol were mixed, readings were taken at
420 nm, and values were expressed as absorbance/min/mg
fresh weight.
For the determination of peroxidase activity, the extract
was obtained by maceration of leaf samples in liquid nitro-
gen followed by extraction in 0.1 M sodium acetate buffer
(pH 5.2) containing 1 mM ethylenediaminetetraacetic acid.
For the reaction, 2.9 mL of 50 mM potassium phosphate
buffer (pH 6.0), 20.2 mM guaiacol, 90 mM ­H2O2, and 10 μL
protein extract were mixed (Chance and Maehly, 1955).
Optical density values at 470 nm were obtained using a spec-
trophotometer (FEMTO, São Paulo, SP, Brazil), and were
expressed as absorbance/min/mg fresh weight.
Experimental design
The induction of somatic embryogenesis followed a com-
pletely randomized experimental design with five Petri
dishes containing ten endosperms representing an experi-
mental unit. The experiment included nine treatments (2,4-D
concentrations) and a control (without PGRs). The evalu-
ated parameters were explant responsiveness after 30 days of
culture, mean number of somatic embryos per explant after
45 days in maturation medium, and mean number of regen-
erated plants per explant after 90 days of culture. Tukey’s
test with a 5% significance level was used.
The morpho-agronomic descriptors and mean enzymatic
activities of diploid (seed-derived) and triploid plants were
submitted to a normality and homogeneity test (Bartlett),
and evaluated by Student’s t-test to verify significant dif-
ferences between the means of the two genotypes. A value
Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 150:637–650 641
of P < 0.05 was considered statistically significant. In case
of an asymmetric pattern, data were subjected to logarithmic
transformation and submitted to a non-parametric test using
the Genes Program (Cruz, 2016). In addition, morpho-agro-
nomic descriptors of diploid and triploid P. cincinnata plants
were evaluated by principal component analysis (PCA) using
the pcaMethods package in R (Stacklies et al. 2007), fol-
lowing z-score transformation of the data set (Worley and
Powers 2013).
Results
Somatic embryogenesis is induced by specific
auxin:cytokinin ratios
P. cincinnata endosperms were responsive to auxin-rich
induction medium (Fig.  1a). However, yellowish pro-
embryogenic masses were observed only in treatments
supplemented with 13.6 and 18.1 µM 2,4-D + 4.5 µM BA
(Fig. 1b). Differentiation of somatic embryos in the early
stages of development could be observed after 15 days of
in vitro culture (Fig. 1a). The highest number of somatic
embryos (8.8 ± 1.3) was detected in induction medium sup-
plemented with 18.1 µM 2,4-D + 4.5 µM BA (Fig. 1b). Con-
trol explants showed no response after 30 days of culture
(data not shown).
Emblings were obtained from somatic embryos after
90 days of maturation in the absence of PGRs and germina-
tion in half-strength medium (Fig. 1a). The highest mean
number of regenerated plants (7.0 ± 1.2) was obtained from
explants previously cultivated in induction medium supple-
mented with 18.1 μM 2,4-D and 4.5 μM BA (Fig. 1c).
Even though the regenerated plants were acclimatized
to greenhouse conditions, only 30% of them survived.
Endosperm-derived emblings grown in the greenhouse
entered the flowering phase 210 days after initiation of the
somatic embryogenesis induction process (Fig. 1a).
Most endosperm‑derived emblings are triploids
Flow cytometry analysis revealed differences in the amount
of DNA between seedlings and emblings of P. cincinnata
(Fig. 1d). P. cincinnata seedlings contained 2.98 pg of DNA,
with a coefficient of variation of 2.6% (Fig. 1d). Instead, 70%
of tested P. cincinnata emblings had on average 4.38 pg of
DNA, or roughly 1.5 times more than diploid plants, and
a coefficient of variation of 2.8% (Fig. 1d). The remaining
30% of emblings contained a similar amount of DNA as
seedlings (data not shown).
P. cincinnata seedlings displayed 18 chromosomes
(2n = 2x = 18) (Fig.  1e); whereas emblings with higher
DNA content by flow cytometry exhibited 27 chromosomes
(2n = 3x = 27) (Fig. 1f), confirming triploidy.
Morpho‑agronomic characterization of P. cincinnata
diploid and triploid plants
The greatest morpho-agronomic differences between dip-
loid and triploid plants were observed with respect to the
dimensions of vegetative and reproductive organs, while
the number of organs or structures was the same (Table 1).
Both genotypes displayed pentalobed leaves in the middle
third of the plants (Fig. 2a). The leaves of triploid plants had
larger petioles (Fig. 2b) and longer and wider leaf blades
(Fig. 2c, d), but the number of extrafloral nectaries in the
petiole and number of leaf blades were analogous (Table 1).
The branches were cylindrical and predominantly dark green
in diploid plants, but ranged in color from dark to purplish
green (data not shown) and displayed a 17% larger diameter
in triploid emblings (Fig. 2e).
Morphological differences between diploid and triploid
plants were even more evident in their reproductive organs
(Fig. 3a). All quantitative descriptors, with the exception
of style length, anther width, nectar chamber width, ovary
length, ovary diameter, and peduncle length, were signifi-
cantly higher in triploid plants (Fig. 3b–k; Table 1). Color
intensity of floral organswas apparently greater in triploid
plants (Fig. 3a), although it has not been quantified.
Flower lifespan and floral nectar concentration are
altered by triploidy
Flowering of diploid and triploid plants occurred after
210 days of culture. It took 22–26 days for diploid plants
and 17–21 days for triploids to move from the production of
flower buds to anthesis. Anthesis of diploid flowers occurred
at approximately 5:20 a.m. and they remained open for 14 h.
In triploid emblings, anthesis occurred at 05:00 a.m. and the
flowers remained open for up to 15 h. Nectar concentration
was 27% higher in triploid emblings (39.26 ± 1.88% Brix)
than in diploid plants (30.91 ± 1.4% Brix).
The activity of plant defense enzymes is affected
by ploidy level
The activities of β-1,3-glucanase and polyphenol oxidase
were 29.8% and 22.1% higher in triploid emblings, respec-
tively, compared to diploid P. cincinnata (Fig. 4a, b). A simi-
lar trend was observed for peroxidase activity, although the
difference was not significant (Fig. 4c).
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Fig. 1  Somatic embryogenesis, nuclear DNA content, and chromo-
some assessment of Passiflora cincinnata emblings. a Overview
of the regeneration process via somatic embryogenesis. From left
to right: endosperm (en) (bar = 5  mm); embryonic callus showing a
heart-shaped somatic embryo (h) (bar = 5  mm); torpedo (t)  embryo
stage with callus (ca) (bar = 2.5 mm); cotyledonary (co) embryo stage
(bar = 5  mm); young embling (bar = 50  mm); and embling in the
reproductive phase in the field (bar = 110 mm). b Number of somatic
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Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 150:637–650
embryos per explant after 45 days in maturation medium. c Number
of regenerated plants per explant. d Histograms comparing nuclear
DNA content of P. cincinnata seedlings (upper panel), emblings
(lower panel), and Pisum sativum (internal standard) determined
using propidium iodide (PI) staining. e Metaphase plate showing 18
chromosomes of P. cincinnata seedlings. f Metaphase plate showing
27 chromosomes of P. cincinnata emblings. Bars (e, f) = 5 µm
Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 150:637–650 643

Fig. 2  Vegetative organs of diploid and triploid Passiflora cincinnata Asterisks refer to statistical differences between diploid and triploid
plants. a Leaf morphology. b Petiole length. c, d Leaf blade length plants within each parameter (t-test). Bar = 50 mm.
(c) and width (d). e Stem width. Error bars denote the standard error.

Table 1  Floral features of diploid and triploid Passiflora cincinnata Diploid and triploid P. cincinnata plants are
plants morpho‑physiologically distinct
Floral features Diploid Triploid
Based on morphological characteristics, nectar concentra-
Anther length (mm) 11.99 ± 0.40 14.00 ± 0.48* tion, and enzymatic activity, PCA analysis revealed that the
Anther width (mm) 5.36 ± 0.46 5.75 ± 0.18 first two principal components (PC1 and PC2) accounted for
Bract length (mm) 32.59 ± 1.39 38.87 ± 0.96* 84.2% of total variation (Fig. 5), with PC1 largely explain-
Bract width (mm) 17.11 ± 1.78 21.27 ± 0.58* ing the separation between triploids and diploids. Almost
Corona colored rings length (mm) 18.40 ± 0.91 22.65 ± 1.31* all evaluated traits displayed higher values in triploid plants.
Nectar chamber width (mm) 4.73 ± 0.38 5.19 ± 0.89
Number of anthers 5.00 ± 0.00 5.00 ± 0.00
Number of bracts 3.00 ± 0.00 3.00 ± 0.00 Discussion
Number of corona colored rings 3.00 ± 0.10 3.80 ± 0.26*
Number of extrafloral nectaries – leaf 3.00 ± 0.00 3.00 ± 0.00 The DNSO-based regeneration of P. cincinnata triploid
blade plants has been previously reported for endosperms culti-
Number of extrafloral nectaries – 2.00 ± 0.00 2.00 ± 0.00 vated in a cytokinin-rich medium (Silva et al. 2020). In the
petiole
present study, we describe an alternative in vitro regenera-
Number of petals 5.00 ± 0.00 5.00 ± 0.00
tion system capable of inducing somatic embryogenesis by
Number of sepals 5.00 ± 0.00 5.00 ± 0.00
altering the relationship between exogenously added PGRs
Number of stigmas 3.00 ± 0.00 3.00 ± 0.00
and the light regimen. Whereas DNSO was previously
Ovary length (mm) 6.15 ± 0.93 8.25 ± 1.88
induced by cultivating mature endosperms in medium
Peduncle length (mm) 28.86 ± 8.27 36.76 ± 9.50
supplemented with 9 µM BA under a 16:8 h photoperiod
Values correspond to the mean (mm) and standard error. Asterisks (Silva et al. 2020); here, somatic embryos were obtained
indicate statistical differences between diploid and triploid plants through cultivation in medium supplemented with a higher
within each floral feature according to the F-test
auxin:cytokinin ratio (18.1 µM 2,4-D + 4.5 µM BA) in the
absence of light (Fig. 6).

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644 Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 150:637–650

13
 Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 150:637–650 645

◂Fig. 3  Flower features of diploid and triploid Passiflora cincinnata and floral structures than their diploid counterparts, but
plants. a Flower organ morphology highlighting corona filaments an equal number of lateral organs. Higher ploidy induces
(cf), androgynophore (a), ovary (ov), sepal (se), petal (pe), stamen fil-
ament (stf), and style (sty). b Hypanthium diameter. c Corona diam-
an increase in cell volume to accommodate polyploid tis-
eter. d Androgynophore length. e Ovary diameter. f Sepal length. g sues (Malladi and Hirst 2010; Wu et al. 2013), leading to
Sepal width. h Petal length. i Petal width. j Stamen filament length. larger and more voluminous leaves, flowers, stems, and
k Style length. Error bars denote the standard error. Asterisks refer to roots (Dhooghe et al. 2011; Kumar et al. 2015). These
statistical differences between diploid and triploid plants within each
parameter (t-test). Bars = 20 mm
events can contribute to the development of new genotypes
and phenotypes and, consequently, to greater intraspecific
genetic diversity (Padoan et  al. 2013; Serapiglia et  al.
The regeneration of endosperm-derived triploid 2015; Spoelhof et al. 2017).
emblings is uncommon, and existing reports point to the In addition to greater floral size, triploidy changed also
need to combine two or more PGRs to induce somatic the phenology of P. cincinnata emblings. Floral phenology is
embryogenesis. In most of these studies, auxins are frequently altered by polyploidy. According to Rezende et al.
required for the induction of embryogenic calli in in vitro (2020), the flower traits most frequently affected by hybridi-
endosperm cultures (Thomas and Chaturvedi 2008) and zation and polyploidy were floral morphology (83.8%), color
2,4-D is the main PGR used for this purpose, regardless (59.46%), and phenology (27.02%). In addition to morpho-
of explant type or plant species (Yang and Zhang, 2010; logical changes, the flower buds of triploid plants developed
Nic-Can and Loyola-Vargas 2016). Interestingly, the high- more quickly, showed a longer flower opening period, and
est number of somatic embryos and P. cincinnata emblings higher Brix values than diploids. These modifications can
regenerated from endosperm culture was observed under affect the communication between flowers and pollinators,
the same growth conditions previously described for the as well as accessibility of the pollinator to the floral reward,
induction of embryogenic cultures from zygotic embryos compromising the plant-pollinator match (Cortis et al. 2009;
of this species (Silva et al. 2009; Paim Pinto et al. 2011; Van der Niet et al. 2014; Rezende et al. 2020). Future studies
Vieira et al. 2018). Endosperms and zygotic embryos are
embryonic structures with a shared origin linked to double
fertilization and similar physiology. The endosperm plays
an important role in supporting embryo growth by sup-
plying nutrients and producing signals to regulate embryo
development (Yan et al. 2014). These results suggest the
conservation of hormonal signaling and similar endog-
enous conditions for the expression of totipotency during
induction of somatic embryogenesis. Accordingly, Passi-
flora embryonic tissues could be used as simple and effi-
cient systems for the regeneration of diploid and triploid
plants (Fig. 6).
Determining the amount of nuclear DNA and the num-
ber of chromosomes is essential for the validation of
polyploid production/regeneration systems. In the pre-
sent study, 70% of endosperm-derived emblings had a
1.5 times higher DNA content (2n = 3x = 27) than their
respective diploids (2n = 2x = 18). Compared to the DNSO
pathway (Silva et al. 2020), the number of triploid plants
obtained via somatic embryogenesis is slightly smaller,
although still satisfactory, as no mixoploidy, aneuploidy,
or ploidy above the 3 × level was observed among regener-
ated emblings. In Actinidia deliciosa, for example, most
endosperm-derived plants were aneuploids (Góralski et al.
2005); whereas in Azadirachta indica, both diploid and
triploid plants were obtained from endosperm-derived
calli (Chaturvedi et al. 2003), as observed here. Fig. 4  Enzymatic activity of diploid and triploid Passiflora cincin-
nata plants. a β-1,3-glucanase. b Polyphenol oxidase. c Peroxidase.
Vegetative and reproductive organs of P. cincinnata
Abs, absorbance.  FW, fresh weight.  Glu, glucose. Error bars denote
were differently sized with respect to ploidy level. Trip- the standard error. Asterisks refer to statistical differences between
loid plants exhibited generally larger and longer vegetative diploid and triploid plants within each parameter (t-test)

13

646
Fig. 5  Principal component analysis score plot constructed using data from diploid and triploid Passiflora cincinnata plants. PC1, principal
component 1; PC2, principal component 2
will determine if the floral changes observed in endosperm-
derived plants have any specific effect on the P. cincinnata
plant-pollinator interaction.
Triploid plants of P. cincinnata exhibited higher glu-
canase and polyphenol oxidase activities compared to dip-
loid plants. These oxidative enzymes are important plant
defense mechanisms directly associated with cell wall deg-
radation by pathogens, disease susceptibility, and senescence
(Thipyapong et al. 2004; Araji et al. 2014). At present, we
cannot confirm that P. cincinnata triploid plants are more
tolerant to pathogens compared to their respective diploids,
as such experiments were beyond the scope of the present
study. However, numerous studies have demonstrated that
increased ploidy levels could strengthen enzyme activity,
improving survival and resistance to drought (Padoan et al.
2013; del Pozo and Ramirez-Parra 2014), cold (Deng et al.
2012; Bertrand et al. 2015), salt stress (Chao et al. 2013;
Elmaghrabi et al. 2013; Nezhad and Mansouri 2019), and
even pathogens (Hias et al. 2018).
Stronger enzymatic activity in polyploid organisms is
accompanied by upregulation of the respective genes (Caver-
zan et al. 2012; Wu et al. 2013), although it is not necessarily
proportional to the DNA content of these organisms. In this
13
Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 150:637–650
study, the enzymatic activities of glucanase and polyphe-
nol oxidase were 20% to 30% higher in triploid organisms.
Chromosomal duplication may increase enzymatic activity,
although it may not necessarily double it; some enzymes
may not be affected at all and others may actually exhibit
lower expression (Xing et al. 2011). Even though duplication
does not introduce new genetic material and only gener-
ates additional copies of genes and chromosomes, genome
alterations could disturb gene or enzyme patterns (Ranney
2006). The effects of neofunctionalization, mutations, and
transposable elements in coding regions of regulatory genes,
as well as epigenetic mechanisms, which are common in
polyploids, may also contribute to altered gene expression
profiles (Osborn et al. 2003).
In summary, we describe here an alternative regeneration
system based on somatic embryogenesis that leads to triploid
P. cincinnata plants from endosperm culture. We also show
that the organs of these triploid emblings are larger and pos-
sess greater enzymatic activity than their dipoloid counter-
parts. We believe that this regeneration system will enable
the production of phenotypic variants of this species, and
stimulate future genetic studies on passion fruit breeding.
Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 150:637–650 647
Fig. 6  Schematic diagram summarizing the regeneration pathways
established from embryonic tissues of Passiflora cincinnata. (1)
Somatic embryogenesis from mature endosperm culture (present
Acknowledgements  This work was supported by Fundação de Amparo
à Pesquisa do Estado de Mato Grosso (FAPEMAT) (Cuiabá, MT);
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Bra-
zil (CAPES)—Finance Code 001; and Conselho Nacional de Desen-
volvimento Científico e Tecnológico (CNPq) (420913/2018-1). We also
thank the CNPq for granting a scholarship to MM (DCR-314905/2018-
9). We would like to thank Editage (www.​edita​ge.​com) for English
language editing.
Author contributions  MLS, DIR, and MDM designed the study; MDM
and CSS established embryonic cultures; MM performed statistical
analyses; ACR, EMM, SMS, and LFV performed cytogenetics and flow
cytometry analyses; MDM, CSS, and DIR performed morphometric
evaluations; IFC and MDM carried out enzymatic analyses; MDM,
DIR, MLS, and WCO wrote the manuscript; MM, IFC, ACR, SMS,
and LFV revised the final version of the manuscript.
Funding  This work was supported by Fundação de Amparo à Pesquisa
do Estado de Mato Grosso (FAPEMAT) (Cuiabá, MT), Fundação de
Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (Belo Hor-
izonte, MG; Grants APQ-00772–19 and APQ-02581-21), Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Brasília,
DF; Finance Code 001), and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) (Brasília, DF; Grant 420913/2018–1).
study). (2) De novo shoot organogenesis from mature endosperm
culture (Silva et  al. 2020). (3) Somatic embryogenesis from mature
zygotic embryo culture (Silva et al. 2009)
Data availability  Not applicable.
Code availability  Not applicable.
Declarations 
Conflicts of interest  The authors declare that they have no conflict of
interest.
Ethical approval  Not applicable.
Consent to participate  Not applicable.
Consent for publication  All co-authors approved the final version of
the manuscript.
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