In Vitro Cell.Dev.Biol.

—Plant (2017) 53:505–513

DOI 10.1007/s11627-017-9842-5

PLANT TISSUE CULTURE

Complete germination of papaya (Carica papaya L. cv.

`Maradol Roja´) somatic embryos using temporary immersion
system type RITA® and phloroglucinol in semi-solid culture
medium
Laisyn Posada-Pérez 1 & Yenny P. Montesinos 1 & Diosdada G. Guerra 2 & Dion Daniels 3 &
Rafael Gómez-Kosky 4

Received: 6 July 2016 / Accepted: 28 June 2017 / Published online: 8 August 2017 / Editor: Ewen Mullins
# The Society for In Vitro Biology 2017

Abstract A critical factor in somatic embryogenesis proto-
cols in papaya (Carica papaya L.) has been incomplete ger-
mination of somatic embryos due to formation of a basal cal-
lus, which prevents the emission of the radicle. This work
aims to achieve complete germination of somatic embryos in
liquid and semi-solid culture media. The effect of the culture
conditions on germination of somatic embryos using the
RITA® temporary immersion system were evaluated as well
as the effect of phloroglucinol on germination of somatic em-
bryos in semi-solid culture medium. The results of using the
RITA® culture medium with a combination of 0.02 μM BAP
and 2.90 μM gibberellic acid had a good response for total
germination (100%) but somatic embryos had only partial
germination with 400 mg fresh mass. However, the optimum
inoculum density was 200 mg fresh mass of somatic embryos
which produced 100% total germination and 95% somatic
embryos with complete germination. Also, it was possible to
achieve complete germination of somatic embryos with low
callus formation (13%) using phloroglucinol at a
* Laisyn Posada-Pérez
laisyn@ibp.co.cu
1
Instituto de Biotecnología de las Plantas, Universidad Central BMarta
Abreu^ de Las Villas, Carretera a Camajuaní Km 5 ½, Santa
Clara, Villa Clara, Cuba
2
Instituto de Investigaciones de Viandas Tropicales (INIVIT),
Carretera Central km 248, Santo Domingo, Villa Clara, Cuba
3
Faculty of Science & Technology, University of Belize,
Hummingbird Avenue, Belmopan, Belize
4
Estación Territorial de Investigaciones de la Caña de Azúcar
(ETICA) Centro Villa Clara, Instituto de Investigaciones de la Caña
de Azúcar (INICA), Autopista Nacional Km 246, CP 53
100 Ranchuelo, Villa Clara, Cuba
concentration of 475.8 μM on semi-solid culture medium.
This is the first report of two biotechnological strategies for
complete germination of plants from somatic embryos in the
papaya cultivar `Maradol Roja´.
Keywords Basal callus . Papaya . Phloroglucinol . Somatic
embryogenesis . Temporary immersion system
Introduction
Papaya (Carica papaya L.) is a plant widely distributed in
tropical and subtropical regions around the world and is one
of the main fruits consumed worldwide (Teixeira da Silva
et al. 2007). That reality makes it a significant source of in-
come among exports from many countries like India, Brazil,
Nigeria, and Mexico (Fuentes and Santamaría 2014). Its high
yield, nutritive value, and being one of the few fruits of con-
tinuous production, puts it among the jewels of tropical fruits
(Jian et al. 2007) with important food and medicinal applica-
tions (Teixeira da Silva et al. 2007; Zhang et al. 2011). In
Cuba, the most commercially important cultivar is `Maradol
Roja´ and crop production exceeded 1.97 million tons in 2013
(FAOSTAT 2016).
This species essentially cross-pollinates and the fundamen-
tal way of propagation is by seed, although these are not able
to maintain some of the characteristics in the offspring, which
brings variations in the quality and size of the fruit, as well as
its productivity. At the same time, for papaya, there are con-
ventional methods of vegetative propagation such as grafting
and rooting of cuttings, but although they have had some
success, it is not a commercial practice, since the number of
plants produced by the mother plant is limited. In that sense,
the use of in vitro culture techniques is an essential alternative
506 POSADA-PÉREZ ET AL.
for multiplication, either to produce a large number of plants
(planting material of high quality) or to support breeding pro-
grams towards the introduction of new cultivars or hybrids on
a commercial scale (Farzana et al. 2008). Direct somatic em-
bryogenesis (DSE) has been the main method used for prop-
agation and genetic improvement in papaya (Malabadi et al.
2011; Anandan et al. 2012; Sekeli et al. 2013; Dhekney et al.
2016). DSE has been developed with the use of different
growth regulators and the explant mostly used to achieve,
regardless of the cultivar, is the zygotic embryo (Posada-
Pérez et al. 2007; Ascencio-Cabral et al. 2008; Malabadi et al.
2011; Bukhori et al. 2013).
Several authors reported the success of papaya embryogen-
esis culture in different cultivars (Gallardo et al. 2004; Fitch
et al. 2005; Anandan et al. 2011; Nzilani et al. 2013).
However, callus formation at the radicle end is frequently
observed during germination of somatic embryos, so complete
germination (development of shoot and root) is low
(Ascencio-Cabral et al. 2008; Sekeli et al. 2013; Dhekney
et al. 2016). This problem is very common in woody species
such as forest and fruit trees, where the formation of callus at
the base of the explant prevents root formation (Teixeira da
Silva et al. 2013). This is also the case in papaya, which is a
semi-woody species (Jimenez et al. 2014).
The development achieved by the use of different types of
temporary immersion systems has revolutionized plant bio-
technology (Ducos et al. 2007; Mallon et al. 2012). Plants
or somatic embryos cultured in liquid medium have less ex-
posure to the components of the medium, greater oxygenation
and agitation. A favorable alternative is temporary immersion
systems, especially RITA® type, which has proven benefits
for in vitro culture and especially for somatic embryo germi-
nation (Aragón et al. 2014; Heringer et al. 2014).
One product of the degradation of phloridzin and a precur-
sor in the biosynthetic pathway of lignin is phloroglucinol
(PG). This is a compound known for its properties as a plant
growth promoter but its effect usually is masked by other
similar compounds (Teixeira da Silva et al. 2013). Its positive
effect in reducing hyperhydricity (Ross and Grasso 2010) on
lignification (Ross and Castillo 2009, 2010) and also on the
removal of callus formation at the site of the radicle formation
in somatic embryos has been demonstrated (Daud et al. 2013;
Teixeira da Silva et al. 2013). Phloroglucinol also increased
the percentage of rooting and in vitro acclimatization of papa-
ya shoots under photoautotrophic conditions (Posada-Pérez
et al. 2016).
Phloridzin, a compound structurally similar to PG but ther-
molabile, less active, and more expensive, has been used suc-
cessfully as a growth regulator in several species, including
papaya (Ascencio-Cabral et al. 2008). For several reasons, the
possibility of using PG as an alternative in the complete ger-
mination (germination of shoots and root) of somatic embryos
would be very beneficial for the in vitro culture of papaya. In
fact, it has already been used in some crops (Reis et al. 2008;
George et al. 2010) with an increase in the number of somatic
embryos and germination of plants, provided that it is used in
low concentrations (Teixeira da Silva et al. 2013).
The objective of this work was the development of two
biotechnological strategies (RITA® in liquid culture medium
and phloroglucinol in semi-solid culture medium) for the ger-
mination of complete plants from somatic embryos of papaya
(Carica papaya L. cv. `Maradol Roja´), to solve the problem
of basal callus formation and the absence of the radicle in
somatic embryos of this crop.
Materials and methods
Plant material Immature fruits (obtained from hermaphrodite
elongated flowers) from papaya cv. `Maradol Roja´ were used
as initial plant material. The fruits were collected 90–120 d
after anthesis. After selecting the fruits in the field, they were
washed with detergent and an abundance of water. The fruits
were dried in a laminar flow cabinet and sectioned using a
sterile knife. The seeds were removed and cut with a scalpel
No.11 and curved tweezers used to extract the immature zy-
gotic embryos. The culture medium for formation of somatic
embryos was composed of half-strength MS basal medium
(Murashige and Skoog 1962), MS vitamins, 400 mg L−1 glu-
tamine, 100 mg L − 1 myo-inositol, 22.6 μM 2,4-
dichlorophenoxyacetic (2,4-D), 60 g L−1 sucrose and 5 g L−1
Agargel® (Sigma-Aldrich, St. Louis City, MO) according to
the protocol of Posada-Pérez et al. (2007).
At 8 wk of culture, somatic embryos were obtained directly
without callus formation. These somatic embryos were then
subcultured in a multiplication culture medium composed of
half-strength of MS basal medium, MS vitamins, 400 mg L−1
glutamine, 100 mg L−1 myo-inositol, 9.05 μM 2,4-D, 60 g L−1
sucrose, and 5 g L−1 Agargel®.
For all experiments, somatic embryos with 30 d on matu-
ration medium were used. The maturation culture medium
consisted of 100% MS basal medium, MS vitamins, 5 μM
abscisic acid (ABA), 30 g L−1 sucrose, and 5 g L−1 Agargel®.
Thirty milliliters of the culture media were put into 250-mL
glass culture vessels. These vessels were covered with plastic
polycarbonate lids. The pH was adjusted to 5.8 prior to
autoclaving for all the culture media. All the culture media
and culture vessels used were sterilized in an autoclave at
121 °C and 101.3 kPa according to manufacturer’s instruc-
tions (Sigma-Aldrich (2012).
All these phases of somatic embryogenesis were performed
under conditions of total darkness and a temperature of
27 ± 2 °C.
Description of the RITA® temporary immersion system
RITA® is a polycarbonate container (15 × 13 cm, 1-L-1
 COMPLETE GERMINATION OF PAPAYA
volume) for semi-automated temporary immersion (CJ
DIFFUSION, CIRAD, France) and is a single jar with upper
and lower compartments, and was the first temporary immer-
sion container, which reduced the risk of air leakage and con-
tamination. The immersion frequency was every 12 h with
duration of 1 min.
Effect of culture conditions in RITA® on germination
of somatic embryos. Effect of BAP concentration With the
objective to achieve complete germination of the somatic em-
bryos, 6-benzylaminopurine (BAP) at 0, 0.01, 0.02, and
0.04 μM was evaluated. Each RITA was inoculated with
400 mg FWt somatic embryos in the cotyledonal stage. A
volume of 220 mL of liquid culture medium was used per
RITA®. The germination culture medium was composed of
half-strength MS basal medium, MS vitamins, 2.90 μM
gibberellic acid (GA3), 100 mg L−1 myo-inositol and 20 g
L−1 sucrose.
Effect of the inoculum density The effect of the inoculum
density was determined using the best concentration from
the previous experiment with BAP and the lower inoculum
densities with the same culture medium that was used above.
The RITA® were inoculated with 100, 200, and 300 mg FWt
of somatic embryos in the cotyledonal stage. For each exper-
iment, 10 repetitions (RITA®) by treatment were used. Each
experiment was repeated twice.
At the conclusion of the germination stage of the somatic
embryos (after 30 d of culture), in both experiments, the fol-
lowing were evaluated: the total number of somatic embryos
germinated, the number of somatic embryos with complete
germination (development of shoot and root), the number of
embryos with partial germination (development of shoot or
root), the presence of basal callus, shoot height (from the base
where the root begins to the leaves) cm, leaf number, roots
number, and root length (cm).
Effect of phloroglucinol on germination of somatic embryos
in semi-solid culture medium In order to evaluate the effect of
PG in the complete germination of papaya somatic embryos,
two experiments were carried out. In a first experiment, four
PG concentrations were evaluated: 0, 79.3, 158.6, and
237.9 μM with BAP. A second experiment was conducted
evaluating PG at higher concentrations: 0, 317.2, 475.8, and
634.4 μM PG with BAP, using the same culture medium for
all treatments.
Somatic embryo clusters (8–10) on the maturation culture
medium after 30 d were placed to germinate in a culture me-
dium of Posada-Pérez et al. (2007) with half-strength MS
salts, MS vitamins, 0.97 μM BAP, 100 mg L−1 myo-inositol,
20 g L−1 sucrose, and solidified with 7 g L−1 Plant Agar
(Duchefa, Haarlem, the Netherlands).
507
To each 250-mL glass culture vessel with 30 mL of medi-
um, four groups of somatic embryos per treatment were used.
In both experiments, 10 glass culture vessels, with six
cotyledonal somatic embryos as repetition, were used.
After 30 d of culture for both experiments, the fol-
lowing variables were evaluated: the total number of
somatic embryos germinated, the number of somatic
embryos with partial germination, the number of so-
matic embryos with complete germination, the pres-
ence of callus at the base of the somatic embryos,
shoot height (from the base where the root begins to
the leaves) in centimeters, leaf number, root number,
and root length (cm). The experiments were repeated
twice.
Subsequently, the small plantlets were placed in a growth
culture medium without growth regulators, half-strength MS
basal medium, 1 mg L−1 thiamine, 20 g L−1 sucrose, and
solidified with 7 g L−1 Plant Agar. Groups of plants from the
different experiments were taken to ex vitro conditions
(conversion) to assess the survival at 15 d after transplanting.
Culture conditions. In vitro The RITA® systems and culture
vessels with somatic embryos were maintained at 27 ± 2 °C,
under sunlight and a 13-h photoperiod with a range of
Photosynthetic Photon Flux (PPF) from 48 to
62.5 μmol m −2 s −1 , measured with a Lux light meter,
401025 model (Exchert Instruments, Nashua City, NH).
Experiments were repeated twice.
Ex vitro The acclimatization condition was characterized and
averaged daytime temperature, which was 30 ± 2 °C, relative
humidity of 70–75%, and PPF ranged from 224 to 457 μmol
m−2 s−1 measured as described above. As substrate, 90% (v/v)
zeolite and 10% (v/v) sugarcane (Saccharum spp.) compost
was used. These components were placed in plastic pots of
500 mL total volume. First, the compost was deposited in the
bottom and on top; the zeolite was placed to ensure good
aeration of the roots of papaya plantlets. Irrigation was done
manually by spraying twice daily. The plants were covered
with a transparent glass jar for 5 d to ensure high relative
humidity greater (above 90%) and 70% shade with black
Saran netting.
Statistical analysis All statistical analyses were carried out
using the SPSS software package for Windows (version 21,
2012). For the analysis of the normality of the variables, the
Shapiro-Wilk test was used. For the comparison between
means, the nonparametric alternative analysis of variance,
Kruskal-Wallis test, and the comparison between pairs of
groups Mann-Whitney test was used. In all cases, significant
differences were set to p ≤ 0.05.
508 POSADA-PÉREZ ET AL.

Results and discussion
Effect of culture conditions in RITA® on germination of
somatic embryos. Effect of BAP concentration With the use
of temporary immersion systems RITA® type, high germina-
tion rates (greater than 65.0%) in all treatments at 30 d of
culture were obtained. In the absence of cytokinin (BAP), a
lower percentage (22%) of somatic embryos were observed
with complete germination. However, in this treatment, the
length of the shoots and plants obtained was very small com-
pared with other treatments (Table 1).
With 0.02 μM BAP, there was a superior response in all the
variables evaluated. Although this treatment did not achieve
complete germination of the somatic embryos, 100% partial
germination (only shoot development) and very low callus
formation at the base of the germinated embryos was achieved
(Table 1). Hence, this concentration was selected for the sec-
ond experiment where different inoculum densities (mg FWt
of somatic embryos) were evaluated in the RITA®.
The treatment with the lowest BAP concentration
(0.01 μM) had the lowest germination (65.0%) and almost
no leaves were formed in the shoots from the germinated
embryos. This could be attributed to the balance reached be-
tween the growth regulators present in the culture medium.
Several authors reported the synergistic effect of cytokinin
and gibberellic acid, promoting the germination of somatic em-
bryos, in several plant species (Zlenko et al. 2002; Martin 2004;
Paramageetham et al. 2004; Cangahuala-Inocente et al. 2007;
Junaid et al. 2007; Sharmin et al. 2014). The effectiveness of
gibberellic acid could be due to gene activation or synthesis of
new gene products involved in the development of somatic
embryo (Sharmin et al. 2014). Also, GA3 has been used in
the elongation of regenerated shoots (Lakshmi et al. 2013).
Overall, gibberellins can shorten the interphase of the
cell cycle since they induce DNA synthesis in cells found
in the G1 phase. This is reflected in greater growth of the
meristematic zone due to the larger number of cells
entering cell division with the application of gibberellins
(Taiz and Zeiger 2002; Iglesias and Talón 2008). It was
observed in Helianthus annuus L. that the effect of apply-
ing gibberellic acid, reduced the diameter as well as the
stem and petiole (Silva et al. 2001).
In the treatment of papaya, somatic embryos with 0.01 μM
BAP combined with GA3 showed reduced germination. The
same happened with the highest concentration (0.04 μM) of
this cytokinin. These results corroborate the fact that the ef-
fects gibberellins produced are influenced by the changes in
the concentration of growth regulator and susceptibility of
plant tissue (Ikeda et al. 2001).
Effect of the inoculum density The results showed that
there was a parabolic relationship between the inoculum
density and the number of somatic embryos with complete
germination in RITA® (Fig. 1A, B). The highest percentage
of complete germination (95.0%) of the papaya somatic
embryos was obtained with a density of 200 mg FWt of
somatic embryos (Fig. 1C, D). This treatment also had
superior performance for the rest of the variables evaluated
(Table 2). An inoculum density of 300 mg Fwt caused a
lesser height of the new plants in the RITA® systems and a
higher percentage of somatic embryos with partial
germination with respect to the density of 200 mg FWt.
This response may be attributed to the stress of the somatic
embryos caused by an inadequate amount of water, sucrose,
and nutrients available in the culture medium (Yildiz et al.
2011). These authors make reference to the term Bcompetition
between explants^ depending on the amount of
explants placed in each culture vessel or Petri dish. They
further note that the responsiveness of in vitro tissues
cultured could be increased not only by determining the cor-
rect concentration and combination of auxins or cytokinins in
the culture medium but also by adjusting the spacing between
explants in culture.

Table 1. Effect of BAP concentrations on germination of somatic embryos of papaya (Carica papaya L. cv. `Maradol Roja´) in temporary immersion
system RITA® type, at 30 d of culture

BAP GA3 Total SE with complete SE with partial Presence Shoot Leaf Roots Root
concentration concentration Germination germination germination of callus height number number length
(μM) (μM) (%) (development of (development (%) (cm) (cm)
shoot and radicle) of shoot) (%)
(%)

0 2.90 100.0 22.0 a 78.0 b 0.0 c 0.68 b 1.00 b 0.40 a 0.33 a

0.01 2.90 65.0 0.0 b 65.0 c 3.0 b 1.35 a 0.22 d 0.0 b 0.0 b
0.02 2.90 100.0 0.0 b 100.0 a 4.0 b 1.38 a 1.45 a 0.0 b 0.0 b
0.04 2.90 70.0 0.0 b 70.0 b 75.0 a 1.19 a 0.65 c 0.0 b 0.0 b

Different letters within column represent means with significant differences by Kruskall-Wallis/Mann-Whitney test (p ≤ 0.05). (n = 100). Inoculum
density of 400 mg FWt of somatic embryos
 COMPLETE GERMINATION OF PAPAYA 509

Fig. 1. Somatic embryos of

papaya (Carica papaya L.)
cultivar `Maradol Roja´
germinated in temporary
immersion system RITA® type at
30 d of culture. (A, B) Temporary
immersion system with an
inoculum density of 200 mg FWt
of somatic embryos with
complete germination. (C, D)
Aspect of the somatic embryos
with complete germination.
(Bar 0.5 cm).
A B

C D

No signs of hyperhydricity were observed in the plants
regenerated from somatic embryos at the frequency and im-
mersion time (1 min every 12 h) used in this experiment.
No results have been found thus far in the literature on the
germination of papaya somatic embryos using temporary im-
mersion systems. The only work found was from Posada-
Pérez et al. (2003) in which the authors suggest the use of a
filtration system Sartorium Co. employed as TIS, with the
operating principle similar to the RITA® system, but for re-
petitive multiplication of somatic embryos.
Several authors point out the positive effects on the germi-
nation of somatic embryos in different plant species and espe-
cially in woody species using temporary immersion systems
of the RITA® type. In guava (Psidium guajava L. cv. `Red
Dwarf´) Vilchez et al. (2001) obtained 100% of somatic em-
bryos with complete germination using RITA®, which was
not possible using semi-solid culture medium. Etienne et al.
(1999) obtained high germination rates (70–80%) of somatic
embryos in RITA® systems in coffee (Coffea arabica L.).
Also Barbón et al. (2014) in (Coffea arabica L. cv. `Red
Caturra´) indicate the effect of the inoculum density for the
germination of somatic embryos using temporary immersion
systems RITA® type, achieving a high germination percent-
age (60%).
Steinmacher et al. (2011) and Heringer et al. (2014) in
peach palm (Bactris gasipaes Kunth) also reported high ger-
mination rates using these temporary immersion systems com-
pared with semi-solid medium and another culture system. In
cocoa (Theobroma cacao L.), Niemenak et al. (2008) point
out the advantages of using temporary immersion systems of
the RITA® type for increased germination of somatic embry-
os in this crop, which support the results achieved in this work.

Table 2. Effect of inoculum density on germination of somatic embryos of papaya (Carica papaya L. cv. `Maradol Roja´) in temporary immersion
systems RITA® type at 30 d of culture

Inoculum Total SE with complete SE with partial Presence of Shoot height Leaf Roots Root length
density Germination germination germination callus (%) (cm) number number (cm)
(mg FWt) (%) (development of (development
shoot and radicle) (%) of shoot) (%)

100 100.0 85.0 b 15.0 b 4.0 a 1.38 a 1.70 a 0.40 a 0.33 a

200 100.0 95.0 a 5.0 c 2.0 b 2.19 a 1.65 a 0.40 a 0.32 a
300 100.0 80.0 c 20.0 a 2.0 b 0.88 b 1.35 a 0.23 b 0.25 a

Different letters within column represent means with significant differences by the Kruskall-Wallis/Mann-Whitney test (p ≤ 0.05) (n = 100)
510 POSADA-PÉREZ ET AL.
With temporary immersion systems, the behavior and mor-
phology of plants generally appear closer to in vivo norm than
behavior found with in vitro culture. Somatic embryos in these
systems are individualized and not in clusters or aggregates
and have a morphology resembling that of zygotic embryos
(Etienne and Berthouly 2002). In no case the presence of
hyperhydricity in the somatic embryos or plants germinated
from them was observed.
All the advantages of these temporary immersion systems
appear to be the result of the physical conditions created in the
culture vessel (Etienne and Berthouly 2002; Watt 2012), such
as (1) renewed direct contact with the culture medium during
each immersion, which means a more efficient supply of nu-
trients compared to that attained in semi-solid culture medi-
um; (2) very short immersion time, most of the time the tissues
are covered with a surface film avoiding drying out, very low
resistance to gas diffusion and hence minimal interruption of
gas exchange between the somatic embryo and the atmo-
sphere; (3) complete renewal of the atmosphere within the
vessel at regular intervals, which means no long accumulation
of harmful gases such as ethylene, and (4) agitation by air flow
during the immersion phase, causing dispersion of plant
tissues.
The results are always superior to a culture medium in semi-
solid state. The permanent presence of a surface film of liquid
culture medium on the tissues appears to be one of the main
reasons for the success of this system; hence, it ensures nutrient
availability outside the periods of immersion, and also aids gas
exchange more than total immersion. The permanent presence
of residual water also explains the absence of damage and
desiccation of somatic embryos, even in the case of short and
occasional immersion (Etienne and Berthouly 2002).
Another variable linked to the physical conditions that
seems significant is the concentration of dissolved oxygen
in the liquid culture medium whose effect on in vitro phe-
nomena is known for many years (Teisson et al. 1999). In
the temporary immersion system, this concentration
reaches saturation only 1 min after bubbling. In the case
of Erlenmeyer agitated without plant tissue and without
oxygen consumption, the concentration of this dissolved
gas never exceeds 95% saturation. All these points mean
that, in such apparatus, the physical conditions of nutrition,
gas exchange, and water relations of plant tissues are
completely different from those found in conventional
techniques. These physiological conditions lead to totally
different behaviors of the explants (Cabasson et al. 1997).
Sumaryono et al. (2008) used temporary immersion sys-
tems similar in operation to RITA® for increased germination
rates (56.8%) and quality of the plants obtained in oil palm
(Elais guineensis Jacq.). Etienne et al. (1997) in rubber plant
(Hevea brasiliensis Müll. Arg.) improved germination of so-
matic embryos using the RITA® and indicate that 60% of
somatic embryos had complete germination.
In herbaceous species such as banana (Musa AAAB cv.
`FHIA-18′) Barranco et al. (2002) report the effect of inocu-
lum density in the germination of somatic embryos obtaining
up to 85% when the RITA® were inoculated with 500 mg
FWt of somatic embryos.
A group of in vitro plants obtained from the germination of
somatic embryos at the optimum inoculum density obtained in
RITA® were placed in growth medium so that they developed
further.
Effect of phloroglucinol on germination of somatic embryos
in semi-solid culture medium Low concentrations of PG eval-
uated in the first experiment did not have the expected effect
on the control of callus formation at the base of papaya so-
matic embryos during germination. In all concentrations, there
was callus formation in 100% of the base of the shoots.
According to Michalczuk et al. (1992) and Dhekney et al.
(2016), somatic embryos have the ability to conjugate auxins,
which are subsequently excreted into the culture medium dur-
ing the germination process resulting in callus formation.
However, in the second experiment, higher concentrations
of PG had a positive effect on the complete germination of the
papaya somatic embryos with low basal callus formation in
the explants in treatments with 457.8 and 634.4 μM PG. In
both treatments, somatic embryos were found with complete
and partial germination without callus formation (Fig. 2A–C);
however, the lowest value of this variable was achieved with
the concentration of 457.8 μM PG, which is a very important
parameter for somatic embryogenesis of this crop. The best
results were achieved with the concentration of 457.8 μM PG
(Table 3).
Teixeira da Silva et al. (2013) mentions PG in protocols for
somatic embryogenesis where root elongation does not occur,
which is very common in forest and fruit species. The callus
tends to form at the base of the explant, but where the roots
never form. This situation also occurs in the somatic embryos
of papaya without PG (Fig. 2D), although in some cases,
callus did not prevent the formation of the radicle (Fig. 2E).
These are the first results of the effect of PG on germination of
somatic embryos in semi-solid culture medium in this crop.
In the specific case of papaya somatic embryogenesis, there
is only one work by Ascencio-Cabral et al. (2008) that used
phloridzin. These authors reported that 3.0 mg L−1 of
phloridzin in combination with 0.35 mg L−1 GA3, Difco®
bacto agar and placing the cultures under white light, managed
to improve their germination rates and increased root elonga-
tion; however, the germination percentage obtained was only
70%. In the present work, the germination value reached was
100% and only using 0.97 μM BAP as growth regulator with
PG.
Murali et al. (1996) reported enhanced germination of so-
matic embryos (93%) with addition of PG (0.79 mM) in cul-
ture medium on rose (Rosa hybrida L. cv. `Arizona´).
 COMPLETE GERMINATION OF PAPAYA 511

Fig. 2. Effect of phloroglucinol

on complete germination of
papaya (Carica papaya L.
cultivar `Maradol Roja´) somatic
embryos in semi-solid culture
medium at 30 d of culture. (A)
Somatic embryos with complete
germination (shoot and radicle)
and partial germination (only
shoot) in 457.8 μM PG. (B)
Somatic embryo with partial ger-
mination in 634.4 μM PG. (C)
Somatic embryo with complete
germination in 457.8 μM PG.
(D), E Somatic embryo germinat-
ed with the presence of the basal
callus in culture media without
PG (control). (F In vitro) papaya
plants obtained from somatic em-
bryos germinated in semi-solid
culture medium with 457.8 μM
PG after 30 d in growth culture
medium at 30 d of culture. (G)
In vitro plants in acclimatization
conditions at 15 d after
transplanting in the greenhouse.

No significant statistical differences were found for the
variables plant length and number of leaves between the dif-
ferent treatments and the control without PG. The variables
root number and root length reached the highest values where
somatic embryos germinated in 475.8 μM compared with
634.4 μM treatments (Table 3).
In this regard, Teixeira da Silva et al. (2013) reported that
occasionally PG has shown inhibitory effects. Find et al.
(2002) in embryogenic culture of Abies nordmanniana found
limited maturation and germination of somatic embryos with
used 40 μM PG.
Despite being a phenolic compound, it is reported by au-
thors such as Teixeira da Silva et al. (2013) regarding its influ-
ence in controlling some in vitro morphogenetic processes,
indicating that its role is far from being understood. However,
there are positive effects on somatic embryogenesis (Hanower
and Hanower 1984; Find et al. 2002; Reis et al. 2008).
These results provide the first information on the effect of
PG on the germination of somatic embryos of papaya,
`Maradol Roja´ cultivar.
Reis et al. (2008), in studying the effect and analysis of
phenolic compounds during somatic embryogenesis of

Table 3. Effect of phloroglucinol on germination of somatic embryos of papaya (Carica papaya L. cv. `Maradol Roja´), at 30 d of culture in semi-solid
culture medium

Phloroglucinol Total SE with complete ET ALSE with Presence of Shoot height Leaf Roots Root length
concentration Germination germination partial germination callus (%) (cm) number number (cm)
(μM) (%) (development of shoot (development of
and radicle) (%) shoot) (%)

0 100.0 a 30.0 ab 70.0 ab 73.0 a 1.13 a 2.07 a 0.40 ab 0.29 a

317.2 100.0 a 33.0 ab 67.0 ab 67.0 a 1.29 a 2.40 a 0.40 ab 0.32 a
475.8 95.0 a 53.0 a 47.0 b 13.0 b 1.07 a 2.00 a 0.53 a 0.35 a
634.4 90.0 b 20.0 b 80.0 a 33.0 ab 1.19 a 2.03 a 0.20 b 0.12 b

Different letters within column represent means with significant differences by the Kruskall-Wallis/Mann-Whitney test (p ≤ 0.05)
(n = 120)
512 POSADA-PÉREZ ET AL.
Feijoa sellowiana Berg., found that exogenous phenols not
only promote the formation of somatic embryos, but also ger-
mination. PG appears to be very efficient in this process.
These authors also found that in the presence of PG, total
levels of endogenous phenols peaked at the moment when
the first somatic embryos reached the globular stage.
Although, they indicate that the exact role of this compound
in promoting germination of somatic embryos needs further
evaluation.
Plants obtained in the optimum concentration of PG
(457.8 μM) had good development in this culture medium
(Fig. 2F) during 30 d of culture and then ex vitro acclimatiza-
tion conditions reaching a 95.4% survival rate 15 d after
transplanting (Fig. 2G).
Conclusions
Complete germination of papaya cv. `Maradol Roja´ somatic
embryos was achieved with the use of temporary immersion
systems RITA® in MS culture medium with 0.02 μM BAP
and an initial inoculum density of 200 mg FWt of somatic
embryos.
The use of 475.8 μM phloroglucinol in semi-solid medium
achieved complete germination of papaya somatic embryos
with low basal callus formation.
Acknowledgments We are grateful to Prof. Dr. Dion Daniels,
University of Belize, Belize, for his kind help in revising the English
language.
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