Physiol Mol Biol Plants

https://doi.org/10.1007/s12298-017-0501-4

RESEARCH ARTICLE

Morphological analyses and variation in carbohydrate content

during the maturation of somatic embryos of Carica papaya
Ellen Moura Vale1,2 • Ricardo Souza Reis1,2 • Lucas Zanchetta Passamani1,2 •

Claudete Santa-Catarina3 • Vanildo Silveira1,2

Received: 20 July 2017 / Revised: 15 November 2017 / Accepted: 26 December 2017

Ó Prof. H.S. Srivastava Foundation for Science and Society 2018

Abstract Efficient protocols for somatic embryogenesis of endogenous carbohydrate profile may be a valuable
papaya (Carica papaya L.) have great potential for parameter for developing optimized protocols for the
selecting elite hybrid genotypes. Addition of polyethylene maturation of somatic embryos in papaya.
glycol (PEG), a nonplasmolyzing osmotic agent, to a
maturation medium increases the production of somatic Keywords Somatic embryogenesis  Polyethylene glycol 
embryos in C. papaya. To study the effects of PEG on Maturation  Histomorphology
somatic embryogenesis of C. papaya, we analyzed somatic
embryo development and carbohydrate profile changes
during maturation treatments with PEG (6%) or without Introduction
PEG (control). PEG treatment (6%) increased the number
of normal mature somatic embryos followed by somatic The use of somatic embryogenesis, an in vitro culture
plantlet production. In both control and PEG treatments, technique in which single cells or small groups of somatic
pro-embryogenic differentiation to the cotyledonary stage cells give rise to embryos (Tautorus et al. 1991), represents
was observed and was significantly higher with PEG a valuable tool for developing cultures of economic
treatment. Histomorphological analysis of embryonic cul- importance, since it has great potential for the mass prop-
tures with PEG revealed meristematic centers containing agation of elite plants (Gupta et al. 1993; Kim et al. 2005).
small isodiametric cells with dense cytoplasm and evident Maturation is a crucial step for the success of somatic
nuclei. Concomitant with the increase in the differentiation embryogenesis. Important processes of this stage include
of somatic embryos in PEG cultures, there was an increase cell expansion, differentiation, and accumulation of reserve
in the endogenous content of sucrose and starch, which substances essential for germination and regeneration of
appears to be related to a rising demand for energy, a key somatic embryos (Mishra et al. 2012).
point in the conversion of C. papaya somatic embryos. The Since the early studies of somatic embryogenesis in
Carica papaya L. (Caricaceae) (Bruijne et al. 1974; Yie
and Liaw 1977), several studies have been conducted to
& Vanildo Silveira
[email protected]
develop efficient protocols to enable its use on a com-
mercial scale. Recently, reports have been published on
1
Laboratório de Biotecnologia, Centro de Biociências e somatic embryogenesis of C. papaya with induction
Biotecnologia (CBB), Universidade Estadual do Norte (Malabadi et al. 2011; Anandan et al. 2012) and maturation
Fluminense Darcy Ribeiro (UENF), Av. Alberto Lamego
2000, Campos dos Goytacazes, RJ 28013-602, Brazil
cultures (Heringer et al. 2013; Vale et al. 2014), the latter
2
demonstrating the importance of polyethylene glycol
Unidade de Biologia Integrativa, Setor de Genômica e
(PEG) in the maturation of C. papaya somatic embryos.
Proteômica, UENF, Av. Alberto Lamego 2000,
Campos dos Goytacazes, RJ 28013-602, Brazil PEG is a non-plasmolyzing osmoticum that induces
3 hydric deficit; it has been used to stimulate somatic embryo
Laboratório de Biologia Celular e Tecidual, CBB-UENF, Av.
Alberto Lamego 2000, Campos dos Goytacazes, maturation in several species, such as Hevea brasiliensis
RJ 28013-602, Brazil (Linossier et al. 1997), Picea abies (Svobodová et al.

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1999), Glycine max (Walker and Parrott 2001), Aesculus Induction and multiplication of embryogenic
hippocastanum (Troch et al. 2009), and C. papaya (Mishra cultures
et al. 2010; Heringer et al. 2013; Vale et al. 2014). The
effects of PEG action include the modulation and regula- Induction of embryogenic cultures was performed as
tion of genes (Stasolla et al. 2003a, b) and proteins (Her- described by Heringer et al. (2013) and Vale et al. (2014).
inger et al. 2013; Vale et al. 2014). The addition of 6% Briefly, immature fruit were disinfected in 70% ethanol
PEG to MS culture medium increases somatic embryo (Merck, Darmstadt, Germany) for 2 min and then in 50%
formation and protein synthesis in C. papaya hybrid commercial bleach (2–2.5% sodium hypochlorite) for
UENF/CALIMAN 01 (Heringer et al. 2013). In addition to 30 min, followed by three washes with autoclaved distilled
promoting somatic embryo development, using PEG for water. Then, immature seeds were removed and sorted in a
maturation treatments leads to changes in protein abun- laminar flow cabinet; and immature embryos were isolated
dance. Most of the proteins stimulated by PEG are related to be used as explants. These immature embryos were
to carbohydrate and energy metabolism (18.4%) and stress inoculated into test tubes (25 9 150 mm) containing
response (18.4%) (Vale et al. 2014). Furthermore, PEG 10 mL MS culture medium (Murashige and Skoog 1962)
treatments induce differential expression of enolase, (Phytotechnology Lab, Shawnee Mission, KS, USA) sup-
esterase and ADH3 proteins, which may play important plemented with 3% sucrose (Vetec, São Paulo, Brazil),
roles in C. papaya embryo maturation (Vale et al. 2014). 20 lM 2,4-dichlorophenoxyacetic acid (2,4-D) (Sigma-
Carbohydrate contents were also changed by PEG Aldrich, St. Louis, USA) and 2.0 g L-1 Phytagel (Sigma-
(Businge et al. 2013; Hudec et al. 2016) during the matu- Aldrich). The pH of the culture medium was adjusted to 5.8
ration of Norway spruce somatic embryos. Changes in the before Phytagel was added. The culture medium was
soluble carbohydrate levels of embryogenic cultures may sterilized by autoclaving at 121 °C for 15 min. The tubes
be a response to the hydric stress induced by PEG addition. were then inoculated with explants and incubated in the
It has been shown that soluble carbohydrates regulate a dark at 25 ± 2 °C for 42 days. The induced embryogenic
range of developmental processes from embryo develop- cultures were then isolated and subcultured in culture
ment to plant senescence (Gibson 2005). media with the same composition. Before maturation
In somatic embryogenesis, variations in soluble carbo- experiments, four subcultures were made at intervals of
hydrate and starch levels may provide important informa- 21 days for embryogenic culture multiplication.
tion on the mechanisms by which embryos are converted to
plantlets (Pescador et al. 2008). They may act as signaling Maturation treatment
molecules and/or gene expression regulators (Eveland and
Jackson 2012). However, little is known about the effects Three colonies with 300 mg fresh matter (FM) were
of PEG on carbohydrate metabolism and the conversion of inoculated into Petri dishes (90 9 15 mm) containing
somatic cells to the somatic embryos, especially in C. 20 mL MS culture medium supplemented with myo-inos-
papaya. itol (Merck) (100 mg L-1), Phytagel (2.0 g L-1), sucrose
Based on these previous observations, to determine the (3%), and either 0 or 6% PEG 3350 (Sigma-Aldrich),
effects of PEG on somatic embryogenesis in C. papaya, we hereafter referred to as control and PEG treatments,
assessed somatic embryo development and carbohydrate respectively. The pH of the culture medium was adjusted to
profiles during the maturation process in media with or 5.8 before adding Phytagel; the medium was sterilized by
without (control) PEG (6%). autoclaving at 121 °C for 15 min. The cultures were
incubated in a growth chamber at 25 ± 1 °C in the dark for
the first 7 days, followed by a photoperiod with 16 h of
Materials and methods light (60 lmol m2 s1). Sixteen Petri dishes were used per
treatment, each containing three colonies. Samples from
Plant material four Petri dishes of each treatment were collected after 0, 7,
14, 21 and 28 days of culture. Sections of one colony from
To induce somatic embryogenesis in papaya, immature each culture were removed for histomorphological analy-
zygotic embryos were isolated from mature seeds of the sis, and the remaining colonies were stored at - 20 °C for
hybrid papaya UENF/CALIMAN01 and used as explants. subsequent soluble carbohydrate and starch analyses. In
Immature fruit were kindly provided by the Caliman addition, cellular growth and the number of somatic
Agricola Co., located in the city of Linhares, Espı́rito Santo embryos were evaluated after 28 days of culture.
(ES), Brazil (19°230 S 40°40 W).

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Growth analyses and the number of somatic 20 lm membrane and stored at - 20 °C until carbohydrate
embryos analysis. Carbohydrates were identified and quantified by
HPLC (Shimadzu Corporation, Kyoto, Japan), using an
For both treatments, cellular growth and the number of evaporative light scattering detector (ELSD-LT II) at 40 °C,
somatic embryos were measured at the beginning of the nitrogen gas pressure of 350 MPa, a gain of 9 and a filter
experiment (day 0) and after 7, 14, 21, and 28 days of setting of 4. A Prevail Carbohydrate ES (Alltech Associates,
culture. Cellular growth was measured in terms of the Deerfield, IL, USA) 5 lm column (250 9 4.6 mm) and pre-
increase in fresh mass (FM) from the initial value (300 mg column (7.5 9 4.6 mm) were used for separation. The
per colony). The number of somatic embryos of each gradient was achieved by mixing decreasing proportions of
developmental stage (globular, cordiform, torpedo, and absolute acetonitrile (Merck) with water. The acetonitrile
cotyledonary) were counted. After 28 days of culture, the gradient was programmed as follows: 80% for the first
number of morphologically abnormal somatic embryos was 16 min, 80–70% between 16 and 23 min, and 70% from 23
counted, including fused embryos, embryos with fused to 30 min, with a flow rate of 1 mL min-1 at 25 °C. A 5-lL
cotyledons, and mono or poly-cotyledonary embryos. sample was injected, and the peak areas and retention times
were measured by comparison with carbohydrate standards
Histomorphological analyses containing sucrose, fructose, and glucose (Sigma-Aldrich).
Starch extraction was performed according to the method
Samples were collected at the beginning of the experiment (day described by McCready et al. (1950) with modifications.
0) and after 7, 14, 21, and 28 days of culture and were then fixed Pellets from soluble carbohydrate extracts were resus-
in a solution containing 2.5% glutaraldehyde (Merck, Darm- pended in 1 mL 30% perchloric acid (PCA-Sigma-Aldrich),
stadt, Germany) and 4% paraformaldehyde (Merck) in 100 mM stirring for 1 h and centrifuging at 16,0009g for 15 min at
sodium cacodylate (pH 7.2) (Merck) for 24 h at room temper- 4 °C. Supernatants were collected, and the resulting pellet
ature. The samples were subsequently washed with 100 mM was resuspended and centrifuged with the same conditions.
sodium cacodylate (pH 7.2) for 45 min at room temperature. The supernatants were mixed, and starch quantification
Before microscopy, samples were dehydrated twice proceeded according to the method described by Colvin Jr.
through an ethanol series of 30, 50, 70, 90 and 100% for et al. (1961). Briefly, the samples (10 lL) were mixed with
1 h each. After dehydration, the samples were infiltrated a solution of 100 lL 0.05% anthrone (Sigma-Aldrich) and
with HistoResin (Leica, Wetzlar, Germany) and 100% 72% sulfuric acid (Vetec), (1:10; v/v). This mixture was
ethanol (1:1, v/v) for 12 h and, subsequently, with 100% vortexed and heated in a dry block heater at 100 °C for
HistoResin for 24 h before embedding in HistoResin. 10 min. Then, the samples were placed in water at room
Sections (approximately 5 lm thick) were cut and stained temperature for 3 min and then incubated in the dark for
with 1% toluidine blue (Sigma-Aldrich). Samples were 30 min. Readings were performed in a 96-well Microplate
examined under a Zeiss Axioplan light microscope (Carl Spectrophotometer (Bio-Tek, Winooski, VT, USA) at
Zeiss, Jena, Germany) equipped with an Axiocam MRC5 620 nm. Starch concentrations were determined using a
digital camera (Carl Zeiss), interfaced with the AxioVi- range of glucose concentrations as standards, multiplying
sionLE 4.8 software (Carl Zeiss) for image analysis. the readings by 0.9 according to McCready et al. (1950).

Determination of soluble carbohydrates and starch Statistical analysis

content
All experiments were performed using completely random-
Soluble carbohydrates were quantified using high-perfor- ized designs. Data was analysed using analysis of variance
mance liquid chromatography (HPLC), as described by (ANOVA) (P \ 0.05) followed by the Student–Newman–
Aragão et al. (2015). Samples (300 mg MF each) of C. Keuls (SNK) test or t test using the R statistical software (R
papaya embryogenic cultures were macerated with 1 mL Core Team 2014) and the easyanova package (Arnhold 2013).
extraction solution consisting of 80% ethanol (Merck), 3%
polyvinylpolypyrrolidone—PVPP (w/v) (Sigma-Aldrich),
and 1% ascorbic acid (w/v) (Sigma-Aldrich) at 4 °C. After Results
maceration, the samples were vortexed briefly and then
transferred to a water bath at 70 °C for 90 min. Following Effects of maturation treatments on somatic embryo
centrifugation at 16,0009g for 10 min, the supernatant was formation
removed, and the pellets were then re-extracted with 1 mL
extraction solution and centrifuged again at 16,0009g for Significant differences in the number and quality of
10 min. The supernatants were combined, filtered through a somatic embryo production between treatments were

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Table 1 Somatic embryo number (SE), fresh matter (FM) increment lower histomorphological organization and consequently
and abnormal mature somatic embryo (ASE) in embryogenic cultures formed fewer somatic embryos (Fig. 3a).
of C. papaya after 28 days of culture under control and PEG matu-
In addition, the embryogenic cultures showed asyn-
ration treatments
chronous differentiation during the 28 days of incubation,
Treatment SE FM (mg) ASE (%) giving rise to embryos at different maturity stages: globu-
Control 115b 600.0 a 24 a lar, cordiform, torpedo and cotyledonary (Fig. 3). Somatic
PEG 150 a 361.6 b 4b embryo differentiation started with the formation of a small
b
group of cells forming the pro-embryo (Fig. 3c), containing
Means followed by the same letters are not significantly different
cells with prominent nuclei, densely stained cytoplasm and
according to the t test (P \ 0.05). CV coefficient of variation. (n = 4;
CV SE = 9.3%; CV FM = 24.9%; CV ASE = 32.9%) cells bounded by a cell wall, which preceded the globular
embryo stage. Each globular embryo was characterized by
observed (Table 1). PEG use increased the number of protoderm formation, large suspensor, and a large meris-
somatic embryos (Table 1; Fig. 1a, b). By contrast, the tematic cell cluster on the axial portion, suggesting
control had higher FM increment (600 mg) than did PEG increased division and the formation of juxtaposed cells
treatment (361.6 mg) (Table 1). Furthermore, PEG treat- around the embryo (Fig. 3d). Cordiform embryos also
ment significantly reduced the number of abnormal somatic exhibited meristematic cells, which were more pronounced
embryos (Table 1). at the apex of the embryo, along with early cotyledon
Next, the progression of somatic embryo development development (Fig. 3e). Embryos in the torpedo stage were
was evaluated during control and PEG incubation. Somatic characterized by full cotyledon development, while
embryos were counted according to their state of devel- embryos in late torpedo and cotyledonary stages showed
opment: globular, cordiform, torpedo and cotyledonary development of meristematic tissues such as the procam-
(Fig. 1c–f), the last of which was able to germinate and bium (Fig. 3f).
regenerate plants (Fig. 1g, h).
Our results showed a significant increase in the number Soluble carbohydrate and starch content
of globular somatic embryos within the first 7 days of during maturation
incubation with PEG (63.4 embryos per colony) compared
to control treatment (11.2 embryos per colony), with a During the maturation of the embryogenic cultures, the
similar number for PEG treatment after a 14-day incuba- carbohydrates sucrose, fructose, and glucose were identi-
tion (62.4 somatic embryos per colony) (Fig. 2a). A sig- fied and quantified in both control and PEG treatments
nificant decrease in the number of globular somatic (Fig. 4). The cultures showed significant differences with
embryos was observed from 14 to 21 days of incubation in respect to endogenous sucrose content (Fig. 4a). The
PEG treatment compared to the control (Fig. 2a), while highest sucrose content was measured on the seventh day
there was a significant increase in number of somatic of incubation and was significantly higher for the PEG
embryos at the cotyledonary stage (Fig. 2d). cultures. Sucrose then decreased through the rest of the
culture period (Fig. 4a). Interestingly, this peak of sucrose
Histomorphological analysis during maturation level for PEG treatment coincided with a significant
increase in the number of globular somatic embryos
Embryogenic cultures incubated with PEG presented (Fig. 2a). For fructose and glucose, distinctive accumula-
morphological differences compared to control cultures tion patterns were identified in cultures depending on the
(Fig. 3a, b). Embryogenic cultures incubated in PEG had a treatment applied (Fig. 4b, c). Embryogenic cultures
higher abundance of cells with embryogenic characteris- without PEG (control) showed increasing amounts of
tics, i.e., small isodiametric cells with dense cytoplasm and hexoses throughout the incubation time (Fig. 4b, c). On the
conspicuous nuclei. These cells were organized into small other hand, PEG cultures exhibited increasing contents of
groups of meristematic cells called ‘‘meristemoids’’, both glucose and fructose until the 14th and the 21st day of
observed mainly in the peripheral regions of the cultures. incubation, respectively, which were followed by signifi-
These meristemoids quickly differentiated into globular cant decreases to the end of incubation on the 28th day
somatic embryos and progressed to more advanced (Fig. 4b, c).
embryonal stages (Fig. 3b–g). The highest starch content was found in embryogenic
On the other hand, the control embryogenic cultures cultures with PEG treatment, reaching a peak at day 7 of
produced large numbers of non-embryogenic cells, which incubation, and then decreased through day 28 (Fig. 4d).
were more elongated and more dispersed, with large vac- Like sucrose (Fig. 4a), the peak of starch accumulation in
uoles relative to embryonic cells (Fig. 3a). Given these cultures with PEG coincided with a significant increase in
non-embryogenic cell clusters, the control cultures showed the number of globular-stage somatic embryos, thus

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Fig. 1 Morphology of C. papaya embryogenic cultures after 28 days torpedo (e), cotyledonary (f), germinating embryo (g) and regenerated
of maturation culture without (control) (a) or with PEG (b), and plantlet (h). The arrowhead indicates an abnormal embryo. Bars: a
somatic embryo developmental stages: globular (c), cordiform (d), and b = 0.5 mm; c, d, e, f and g = 0.2 mm; h = 1.0 cm

suggesting that sucrose and starch play important roles in Discussion

somatic embryo formation in C. papaya. Moreover,
embryogenic cultures incubated on control medium The use of PEG improved somatic embryo maturation in C.
showed increasing contents of starch only in the first papaya (Table 1; Figs. 1, 2, 3). Consistently, among the
14 days of incubation, but were still lower than the starch currently used osmotic agents, PEG has been demonstrated
content observed in the first 7 days of incubation with to be important for the maturation of different species,
PEG, and they decreased throughout the rest of the incu- including H. brasiliensis (Linossier et al. 1997), G. max
bation period (Fig. 4). (Walker and Parrott 2001), A. hippocastanum (Troch et al.

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Fig. 2 Number of somatic embryos at globular (a), cordiform (b), culture for the same treatment. Means followed by different letters are
torpedo (c) and cotyledonary (d) stages of C. papaya cultures during significantly different by the SNK test (P \ 0.05). CV coefficient of
maturation, for control and PEG treatment. Lowercase letters denote variation. (n = 4; CV globular = 26.2%; CV cordiform = 62.0%;
significant differences between treatments for each day of culture. CV torpedo = 21.9%; CV cotyledonary = 34.1%)
Uppercase letters denote significant differences between days of

Fig. 3 Histomorphological aspects of C. papaya embryogenic cul- heart-shaped (e), torpedo (f) and cotyledonary (g) stages. Asterisks
tures after 28 days of maturation in control (a) and PEG groups (b), indicate embryos developing within the cultures. Arrowheads indicate
pro-embryogenic masses (c) and somatic embryos at globular (d), SAMs, black arrows indicate procambium. Bars = 20 lm

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Fig. 4 Contents (mg g-1 FM) of sucrose (a), glucose (b), fructose followed by different letters are significantly different (P \ 0.05)
(c) and starch (d) through 28 days of culture in control and PEG according to the SNK test. CV coefficient of variation. (n = 3; CV
treatments. Lowercase letters denote significant differences between sucrose = 39.8%; CV glucose = 11.4%; CV fructose = 7.5%; CV
treatments for each day of culture. Capital letters denote significant starch = 15.3%)
differences between days of culture in the same treatment. Means

2009), and C. papaya (Mishra et al. 2010; Heringer et al. We observed that the embryogenic cultures grown with
2013; Vale et al. 2014). Large PEG molecules are unable to PEG produced more cells with embryogenic characteris-
pass through cell walls, which leads to a restriction of tics, i.e., small in size with dense cytoplasm, and contain-
water absorption and reduced turgor pressure, reducing the ing large nuclei with prominent nucleoli and small
intracellular osmotic potential and ultimately leading to vacuoles (Fig. 3), resulting in the development of many
desiccation (Misra et al. 1993). meristematic centers, which subsequently led to an
Supplementation of the culture medium with 6% PEG increased number of somatic embryos (Fig. 2). Addition-
resulted in a significant improvement in the number and ally, the formation of pro-embryogenic masses (Fig. 3c),
quality of somatic embryos produced (Table 1), promoting which would later give rise to globular (Fig. 3d), cordiform
higher conversion from globular (Fig. 2a) to cotyledonary (Fig. 3e), torpedo (Fig. 3f) and cotyledonary embryos
(Fig. 2d) somatic embryos. Similarly, a previous study (Fig. 3g) was observed. Histological analyses allowed the
showed that PEG affected the maturation of P. abies observation not only of the development of somatic
embryogenic tissues by accelerating somatic embryo for- embryos and their respective structural characteristics but
mation, thus producing a higher number of mature embryos also of their quality. Such histodifferentiation studies of
(Hudec et al. 2016). In G. max, PEG addition into culture dicot somatic embryos have contributed to the under-
media enhances the development of cotyledonary embryos standing of somatic embryo differentiation in various
and the regeneration of plantlets (Walker and Parrott 2001). species (Kärkönen 2000; de Feria et al. 2003; Cangahuala-
In the maturation of genetically transformed C. papaya Inocente et al. 2004; Langhansova et al. 2005; Li et al.
somatic embryos, cultures had a maximum conversion of 2008; Correia and Canhoto 2010; Pinto et al. 2011).
globular somatic embryos into cotyledonary embryos when At the molecular level, PEG may act to regulate the
using 4.2% PEG, thereby increasing the rate of plant expression of many genes responsible for controlling cell
regeneration (Mishra et al. 2010). The same phenomenon division and differentiation, as well as the development of
was observed for the maturation of hybrid UENF/CALI- the shoot apical meristem (SAM) (Stasolla et al. 2003a).
MAN01 C. papaya somatic embryos (Heringer et al. 2013; According to Liu et al. (1993), cotyledon development
Vale et al. 2014). occurs predominantly through cell division in cotyledonary

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tissues, and then, cotyledonary slit cells stop dividing, cleavage is an essential reaction for plant growth (Sturm and
leading to the formation of heart-shaped embryos. More- Tang 1999). As such, the higher sucrose contents induced by
over, it has been proposed that the osmotic stress induced PEG (Fig. 4a) in the early stages of incubation may be
by PEG may also cause changes in DNA methylation, related to cell division and differentiation, which in turn
favoring the expression of genes important for differenti- increase the formation of C. papaya somatic embryos. These
ation (Smulders and Klerk 2011). results corroborate those observed by Stasolla et al. (2003a).
The addition of 6% PEG also improved the quality of C. These authors demonstrated that PEG increased the
papaya somatic embryos formed in culture (Table 1). El expression of sucrose synthase genes, at specific develop-
Dawayati et al. (2012) demonstrated the morpho-ontoge- mental stages, in embryogenic cultures of Picea glauca.
netic action of PEG on the normal development of somatic Therefore, increasing the sucrose content may be a cellular
embryos of Phoenix dactylifera, suggesting the involve- response to the osmolality changes caused by PEG, espe-
ment of PEG in the activation of morphogenetic processes cially during the first few days of culture. In addition, Nieves
in these cultures. The improved morphogenic responses of et al. (2003) have attributed high hexose contents to the
callus matured with PEG may be related to the control of elevated activity of neutral and acidic invertase enzymes,
SAM activity (Stasolla et al. 2003a), specifically in the which promote sucrose hydrolysis for reserve mobilization;
expression of important genes for SAM development as their activities can increase intracellular hexose contents
Fiddlehead, AGM, and Knotted-like and ZWILLE and the storage of metabolic compounds, promoting rapid
(Moussian et al. 1998; Stasolla et al. 2003b). cell proliferation (Blanc et al. 2002). Proteins related to
In addition to these morphological aspects, the reduced carbohydrate metabolism have been found to be differen-
osmotic potential caused by PEG may stimulate endoge- tially expressed during the embryogenic maturation of
nous production of abscisic acid (ABA), which is respon- papaya UENF/CALIMAN01 treated with PEG, which may
sible for synthesizing important storage molecules during play important roles in the maturation of these cultures
somatic embryo development including late embryogenesis (Vale et al. 2014). The authors indicated that enolase, a key
abundant (LEA) proteins (Stasolla and Yeung 2003). In protein in the glycolytic pathway, can be used as a marker of
addition, 6% PEG treatment increases the soluble protein papaya embryonic maturation (Vale et al. 2014), reinforcing
content of C. papaya cv. UENF/CALIMAN01 embryo- the role of carbohydrate metabolism in culture maturation
genic cultures (Heringer et al. 2013). Likewise, proteomic for this species.
analyses have found a number of proteins that are differ- In the present study, the observed reductions in glucose
entially regulated between 6% PEG and control in papaya and fructose content after 14 and 21 days of incubation,
embryogenic cultures of the same cultivar (Vale et al. respectively, in embryogenic cultures treated with PEG
2014). may be related to the increased maturation of somatic
In addition to the modulation of proteins, PEG can also embryos to the cotyledonary stage in C. papaya (Fig. 2c).
induce and modulate the production of carbohydrates This decrease in endogenous hexose contents may be an
during somatic embryogenesis (Saranga et al. 1992; Lygin important factor for the reorientation of cellular metabo-
et al. 2012; Hudec et al. 2016). In the present study, PEG lism (Blanc et al. 2002), because somatic embryos are
treatment influenced the endogenous content of sucrose, ready for germination after the formation of cotyledonary
glucose, fructose, and starch during papaya somatic embryo. Kubeš et al. (2014) found that the level of hexoses
embryo development, suggesting the importance of car- was higher than that of sucrose during the maturation of P.
bohydrates for morphogenetic responses during maturation abies somatic embryos. These authors suggested that
under osmotic stress (Fig. 4). sucrose may exert important effects on the signaling cas-
Endogenous sucrose contents were significantly cade that triggers somatic embryogenesis; however, it must
increased by PEG treatment for the first 7 days of incubation remain at low levels for a specific duration of the cultiva-
(Fig. 4a). As such, we have shown that PEG-induced tion period. In Medicago arborea, Martin et al. (2000) also
osmotic stress positively influences endogenous sucrose reported higher amounts of fructose and glucose and lower
levels and promotes the continued development of somatic amounts of sucrose in embryogenic cultures, attributing the
embryos in C. papaya (Fig. 2a). The mechanisms of signal reduced sucrose concentration to its consumption during
transduction and gene regulation involved in the metabolism somatic embryo development.
of carbohydrates in plants are not completely understood Like sucrose, the starch contents in embryogenic cul-
(Padilla-Chacón et al. 2010). However, soluble carbohy- tures increased significantly after 7 days under PEG treat-
drates, such as sucrose and glucose, have been recognized to ment (Fig. 4d). This observation suggests that greater
play roles in gene expression for a diverse range of pro- sucrose levels may promote the accumulation of starch,
cesses, such as cell cycle control, stress responses, energy since sucrose is a substrate for ADP-glucose pyrophos-
storage, cell differentiation and development. Thus, sucrose phorylase, an important enzyme for starch synthesis (James

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et al. 2003). The increase in starch contents was highest References

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