© The Authors, 2023, Published by the Universidad del Zulia*Corresponding author: jvilchezp@fa.luz.edu.ve
Keywords:
Abscisic acid
Somatic embryogenesis
Sucrose
Psidium guajava L.
Maturation and germination of somatic embryos of guava var. Cuban Red Dwarf EEA-1840
Maduración y germinación de embriones somáticos de guayabo var. Enana Roja Cubana EEA-1840
Maturação e germinação de embriões somáticos de goiaba var. Anã Vermelha Cubana EEE-1840
Jorge Vilchez-Perozo
1*
Nilca Albany Valero
2
Fernando Pliego Alfaro
3
Carolina Sánchez Romero
3
Rev. Fac. Agron. (LUZ). 2023, 40(4): e234032
ISSN 2477-9407
DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v40.n4.01
Crop production
Associate editor: Dra. Rosa Razz
University of Zulia, Faculty of Agronomy
Bolivarian Republic of Venezuela
1
Department of Botany, Faculty of Agronomy, University
of Zulia, AP 15205, Maracaibo, Zulia (4005ZU), Bolivarian
Republic of Venezuela.
2
Department of Chemistry, Faculty of Agronomy, University
of Zulia, AP 15205, Maracaibo, Zulia (4005ZU), Bolivarian
Republic of Venezuela.
3
Department of Botany and Plant Physiology, Faculty of
Science, University of Malaga, Teatinos Campus, s/n 29071
Malaga, Spain.
Received: 19-07-2023
Accepted: 22-09-2023
Published: 3
0-09-2023
Abstract
Somatic embryogenesis is an alternative for the accelerated propagation
of promising guava (Psidium guajava L.) materials of agronomic interest.
However, low maturation and germination rates of somatic embryos are some
of the aspects that limit its application in breeding programs for dierent
purposes. In this sense, the eect of three concentrations (0, 1 and 1.5 mg.L
-1
)
of abscisic acid (ABA) on embryo maturation and two concentrations of
sucrose (3 and 5 %) and Murashige and Skoog (MS) macronutrients (50
and 100 %) on the germination of somatic embryos of guava var. Cuban
Red Dwarf EEA-1840 were studied. After six weeks of culture, ABA had a
negative eect on somatic embryo maturation in culture media supplemented
with 1 or 1.5 mg.L
-1
, whereas 3.95 to 5.49 times more mature embryos
were observed in the absence of ABA. Germination of somatic embryos
was signicantly improved when the concentration of macronutrient in the
culture medium was reduced independently of the sucrose concentration [MS
50 % + 3 % of sucrose (73,3 %) and MS 50 % + 5 % de sucrose (55,0 %)].
It is concluded that the simplication of the culture media with reduction
of the macroelements MS and sucrose to standard concentration favors the
germination of mature embryos of guava var. Cuban Red Dwarf EEA-1840.
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Rev. Fac. Agron. (LUZ). 2023, 40(4): e234032. October-December. ISSN 2477-9407.
2-6 |
Resumen
La embriogénesis somática es una alternativa para la propagación
acelerada de materiales promisorios de guayaba (Psidium guajava L.)
de interés agronómico. Sin embargo, las bajas tasas de maduración y
germinación de los embriones somáticos son algunos de los aspectos
que limitan su aplicación en programas de mejoramiento genético
con diferentes propósitos. En este sentido, se estudió el efecto de tres
concentraciones (0, 1 y 1.5 mg.L
-1
) de ácido abscísico (ABA) sobre
la maduración embrionaria y de dos concentraciones de sacarosa (3 y
5 %) y macronutrientes Murashige y Skoog (MS) (50 y 100 %) sobre
la germinación de embriones somáticos de guayaba var. Enana Roja
Cubana EEA-1840. Después de seis semanas de cultivo, el ABA tuvo
un efecto negativo sobre la maduración de los embriones somáticos
en medios de cultivo suplementados con 1 o 1.5 mg.L
-1
, mientras
que se observaron de 3.95 a 5.49 veces más embriones maduros
en ausencia de ABA. La germinación de embriones somáticos
mejoró signicativamente cuando se redujo la concentración de
macronutrientes en el medio de cultivo, independientemente de la
concentración de sacarosa [MS 50 % + 3 % sacarosa (73,3 %) y MS
50 % + 5 % sacarosa (55,0 %)]. Se concluye que la simplicación del
medio de cultivo con reducción de los macroelementos MS y sacarosa
a la concentración estándar favorece la germinación de embriones
maduros de guayaba var. Enana Roja Cubana EEA-1840.
Palabras clave: ácido abscísico, embriogénesis somática, sacarosa,
Psidium guajava L.
Resumo
A embriogênese somática é uma alternativa para a propagação
acelerada de materiais promissores de goiabeira (Psidium guajava L.)
de interesse agronômico. No entanto, as baixas taxas de maturação
e germinação dos embriões somáticos são alguns dos aspectos que
limitam sua aplicação em programas de melhoramento para diferentes
nalidades. Neste sentido, estudou-se o efeito de três concentrações
(0, 1 e 1,5 mg.L
-1
) de ácido abscísico (ABA) na maturação embrionária
e de duas concentrações de sacarose (3 e 5 %) e dos macronutrientes
Murashige e Skoog (MS) (50 e 100 %) na germinação de embriões
somáticos de goiabeira var. Anã Vermelha Cubana EEE-1840. Após
seis semanas de cultura, o ABA teve um efeito negativo na maturação
de embriões somáticos em meios de cultura suplementados com
1 ou 1,5 mg.L
-1
, enquanto foram observados embriões 3,95 a 5,49
vezes mais maduros na ausência de ABA. A germinação de embriões
somáticos foi signicativamente melhorada quando a concentração de
macronutrientes no meio de cultura foi reduzida, independentemente
da concentração de sacarose [MS 50 % + 3 % de sacarose (73,3 %) e
MS 50 % + 5 % de sacarose (55,0 %)]. Conclui-se que a simplicação
do meio de cultura com redução dos macroelementos MS e sacarose
para a concentração padrão favorece a germinação de embriões
maduros de goiabeira var. Anã Vermelha Cubana EEE-1840.
Palavras-chave: ácido abscísico, embriogénese somática, sacarose,
Psidium guajava L.
Introduction
The introduction of new crop varieties requires the accelerated
multiplication of these genotypes, and in vitro culture techniques are
one of the alternatives to achieve this goal. Guava (Psidium guajava L.)
is considered the most prized fruit of the Psidium genus and one of
the most valuable in the tropics, being the Cuban Red Dwarf EEA-
1840 an elite variety for its notable stability (González et al., 2023),
characterized by being a material of low growth, very branched and
that can reach yields above 100 t.ha
-1
of fresh fruit (Ramos et al.,
2023).
Somatic embryogenesis is a promising method for vegetative
propagation of a large number of Myrtaceae species, due to the
bipolar nature of the embryo and the ease with which the entire
production process can be automated. In addition, the process allows
to get high multiplication coecients in short time periods based
on the principles of microbial kinetics, added to the possibility of
encapsulating the embryos and obtaining articial seeds (Guan et
al., 2016). This biotechnological technique is an alternative for the
accelerated eld introduction of new varieties and cultivars.
The ability of totipotent cells to properly proliferate and mature
is greatly inuenced by physicochemical aspects of in vitro culture,
including the balance of plant growth regulators (Gao et al., 2021)
and mean osmotic pressure (do Nascimento et al., 2021).
Within the process of somatic embryogenesis, the nal steps are
germination and conversion into plants. The rst signs of germination
of somatic embryos are elongation of the hypocotyl, appearance of
green color in the cotyledons and elongation of the radicle (Alemano
et al., 1997; Canhoto et al., 1999). Conversion into plants is dened
by some authors as the development of the shoot with emission of the
rst pair of true leaves (Alemano et al., 1997); while others dene it as
the survival of the propagule under ex vitro environmental conditions
(Stuart and Strickland, 1984).
Although most reports on somatic embryogenesis of woody
species do not give much information on these processes, it is known
that low rates of plant regeneration are caused by problems in the
germination and conversion of somatic embryos (Rezende et al.,
2011).
In some cases, it has been indicated the need to give several
recultures in the germination medium, to improve the germination
of somatic embryos due to the developmental asynchrony of these
structures (Cruz et al., 1990). Gómez (1998), points out that partial
or total drying of the somatic embryo increases its subsequent
germination and growth, normalizing and synchronizing germination;
besides, it facilitates the simultaneous growth of roots and shoot.
Somatic embryos of Myrtaceae species have been reported to
germinate well on simple media, usually at half salts and without
hormones (Rai et al., 2008) and the synchronization in germination
is improved when somatic embryos are recultured on medium with
a sucrose content of 2 % (Canhoto et al., 1999). Sucrose is the most
common source of carbohydrate in culture medium (Kaur et al.,
2022).
On the other hand, gibberellic acid as well as coconut water,
enhanced germination of somatic embryos in Myrciaria cauliora
(Litz, 1984a), Eugenia sp. (Litz, 1984b) and Myrtus communis
(Canhoto et al., 1999). Somatic embryos do not enter dormancy,
but treatment of somatic embryos with exogenous ABA can induce
a dormancy state similar to that of zygotic embryos. (Rai et al.,
2008). ABA has been recognized as a factor in promoting normal
development and maturation of somatic embryos (Cipriano et al.,
2018).
The objective of this research was to evaluate the eect of ABA
on embryo maturation, and sucrose and mineral salts on germination
of somatic embryos of guava (Psidium guajava L.). var. Cuban Red
Dwarf EEA-1840.
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Vilchez-Perozo et al. Rev. Fac. Agron. (LUZ). 2023 40(4): e234032
3-6 |
Materials and methods
This research was carried out in the facilities of the Biotechnology
Laboratory “Profa. Silvia León de Sierralta” of the Faculty of
Agronomy of the University of Zulia.
Plant material
For the somatic embryo maturation assay, embryogenic callus
of guava variety “Cuban Red Dwarf EEA-1840” with embryos at
the globular stage was used as plant material (gure 1a). For the
somatic embryo germination assay, mature somatic embryos at the
cotyledonary stage were used (gure 1b). Previously, the induction of
somatic embryogenesis and embryo multiplication were carried out
following the protocol described by Vilchez-Perozo et al. (2002).
following the protocol of Gómez et al. (2005). All culture media were
gelled with 4 g.L
-1
Agargel (Sigma-Aldrich®) and pH was adjusted to
5.8 with NaCl or 0.5 M HCl.
Table 1. Treatments resulting from the combination of the study
factors sucrose concentration and macronutrients of the
culture medium MS (Murashige Skoog, 1962), on the
germination of somatic embryos of guava var. Cuban
Red Dwarf EEA-1840.
Treatments Sucrose (%) MS macronutrients (%)
1 3 50
2 5 50
3 3 100
4 5 100
Twelve replicates were used per treatment. Each replicate
consisted of a 120 mL capacity glass bottle with 25 mL of culture
medium, described above, in which ve mature embryos at the
cotyledonary stage were cultured. The torpedo-cotyledonary and
cotyledonary stages have been reported as the most suitable ones
for initiating the germination phase (Rai et al., 2008; Oliveira et al.,
2022).
After eight weeks of culture, the germination percentage was
evaluated per replicate and calculated as follows: total germinated
embryos divided by total embryos seeded and this ratio multiplied
by one hundred. Somatic embryos were considered to be germinated
when they changed from white-opaque to green colour and presented
development of the caulinar axis, cotyledonary leaves and root axis
(Gómez et al., 2005).
Culture conditions
The embryo maturation and germination phases were carried
out under continuous white uorescent light with an irradiance level
of 200 μmol
-1
.m
-2
.s
-1
, temperature of 26 ± 1°C and average relative
humidity of 46 %.
Statistical analysis
Data processing was carried out using Statistix
®
version 8.0
analytical software (Analytical Software, Tallahassee, Florida, USA,
2003). Prior to statistical analysis, data obtained in each experiment
were subjected to check the assumption of a normal distribution to the
Shapiro-Wilk test a signicance level of P ≤ 0.05 (Sokal and Rohlf,
2013). Subsequently, analysis of variance (ANOVA) was executed in
order to determine the eect of the study factors with a signicance
level of P ≤ 0.05. In those cases where the eect of the study factor
and/or its interaction was statistically signicant (P ≤ 0.05), mean
comparison was performed using the Tukey’s test.
Results and discussion
Eect of abscisic acid on the maturation of guava somatic
embryos
The eect of dierent ABA concentrations on the maturation of
somatic embryos of guava var. “Cuban Red Dwart EEA-1840” table
2 shows. ANOVA revealed the existence of signicant dierences (P
≤ 0.05) among the ABA concentrations tested (0, 1 and 1.5 mg.L
-1
).
In general, higher somatic embryo production was observed at the
elongated torpedo (27.4) and cotyledonary (12.1) stages on ABA-
free maturation medium. In Olea europea, maturation of somatic
embryos on growth regulator-free medium has been reported (Mazri
et al., 2020). A signicant decrease in somatic embryo production at
Figure 1. Plant material used in maturation and germination
of somatic embryos of guava var. “Cuban Red Dwarf
EEA-1840”. (a) Embryogenic callus with embryos at
the globular stage. (b) Mature somatic embryos at the
cotyledonary stage (marked with an arrow).
Eect of abscisic acid concentration on the maturation of
guava somatic embryo
To evaluate the eect of ABA on the maturation of guava somatic
embryos, three concentrations were evaluated: 0, 1 and 1.5 mg.L
-1
.
The culture medium used was MS (Murashige and Skoog, 1962) with
half the macronutrients, 3 % sucrose and pH was adjusted to 5.8 with
NaCl or 0.5 M HCl. The culture media were gelled with 4 g.L
-1
of
Agargel (Sigma-Aldrich®). A randomized experimental design was
used with ten replicates per treatment. Each replicate consisted of a
120 mL glass bottle with 25 mL of culture medium and 25 mg of
embryogenic callus with approximately fty globular-stage embryos.
After six weeks of culture, the number of white-opaque somatic
embryos at torpedo-elongate and cotyledonary stages was evaluated.
The use of the white-opaque appearance of somatic embryos as a
criterion of somatic embryo maturation has been indicated in several
species (Corredoira et al., 2003); (Perán-Quesada et al., 2004).
Eect of sucrose concentration and MS macronutrients on the
germination of guava somatic embryos
To evaluate the eect of sucrose and MS macronutrients
concentration on the germination of guava somatic embryos, a
factorial experiment 2 x 2 was established, testing two concentrations
of sucrose (3 and 5 %) and two concentrations of MS macronutrients
(50 and 100 %), table 1. In addition, culture media were supplemented
with 0.25 mg.L
-1
N
6
-benzylaminopurine (6-BAP) and 0.01 mg.L
-1
DI-31 (a brassinosteroid analog C
27
H
42
O
5
, equivalent to Biobras 16),
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Rev. Fac. Agron. (LUZ). 2023, 40(4): e234032. October-December. ISSN 2477-9407.
4-6 |
both developmental stages was observed as the ABA concentration
increased. This behavior was probably due to the fact that evaluated
concentrations of ABA could be high for this species, aecting the
hormonal balance and triggering an inhibitory eect on the embryo
maturation process. A series of investigations evidenced a similar
behavior in the maturation of somatic embryos of Juglans regia
(Vahdati et al., 2008) and Coea arabica var. Colombia (Riviello-
Cogco et al., 2021), where ABA concentrations above 2 mg.L
-1
reduced the maturation and growth of somatic embryos.
Table 2. Eect of abscisic acid (ABA) on the maturation of guava
somatic embryos var. Cuban Red Dwarf EEA-1840.
Concentration of ABA
(mg.L
-1
)
Number of embryos per culture
Torpedo-elongated Cotyledonary
0 27.4 a 12.1 a
1 8.1 b 1.9 b
1.5 5.5 b 1.7 b
CV: 19.6 SE:1.95 SE: 1.5
Dierent letters indicate signicant dierences obtained by Tukey’s mean
comparison test (P≤0.05).
Additionally, in all treatments the characteristic asynchrony of
the culture was observed, with a predominance of somatic embryos
at the globular-heart stage, which were impossible to quantify. The
dierentiation of somatic embryos into white-opaque torpedo-
cotyledonary and cotyledonary stages was observed, being an
indication of the accumulation of reserve substances, necessary
for their subsequent germination (Vahdati et al., 2008), which was
especially evident in the ABA-free culture medium. Maturation is
a critical stage between embryo development and germination and,
for the achievement of maximum conversion of somatic embryos
to seedlings, it is necessary to use well matured embryos (Perán-
Quesada et al., 2004).
Eect of sucrose and MS macronutrients on germination of
guava somatic embryos
After 12 days of culture, the rst morphological changes in
the germination process of somatic embryos were visualized; e.g.,
changes of cotyledon coloration and epicotyl development (gure
2a), which gradually continued until the eight week of evaluation.
Most embryos showed normal germination, although several
types of anomalies could be observed, such as incomplete germination
(gure 2b), which can be caused either by a decient accumulation of
reserve substances (LEA proteins) during the maturation of somatic
embryos. Rong et al. (2023) determined in Vitis vinifera L. that the
morphological dierences in these phenotypically abnormal embryos
were likely associated with endogenous phythormones levels, how
indoleacetic acid and ABA. On the other hand, Perán-Quesada et al.
(2004) indicate that a high concentration of sucrose in the medium
or the continuous culture in relatively high level of osmotic pressure
can be a source of abnormal germination in somatic embryos. This
has also been previously reported in this guava variety (Gómez et
al., 2005). Secondary embryogenesis was also eventually observed at
the base of some embryos (gure 2c), with new embryo formation at
dierent stages of development during the evaluation period.
Figure 2. General aspects of germination of somatic embryos
of guava var. “Cuban Red Dwarf EEA-1840”. (a)
Beginning of embryo germination, note the cotyledon
(Cot) color change and the beginning of epicotyl (epic)
development. (b) Abnormal somatic embryo germination.
(c) Presence of secondary embryogenesis at the base of
the germinating somatic embryos, with development
of embryos at the globular (ESg), torpedo (ESt) and
cotyledonary (ESct) stages. Black bar = 3 mm.
The results obtained revealed signicant dierences (P ≤ 0.05)
among the treatments tested (gure 3), with higher germination rates
achieved in culture medium with MS macronutrients at half strength
and 3 % or 5 % sucrose.
Figure 3. Eect of interaction the concentration of macronutrients
in the MS medium and sucrose on the germination
of somatic embryos of guava var. “Cuban Red Dwarf
EEA-1840”. 1: MS 50 % + 3 % sucrose; 2: MS 50 % + 5 %
sucrose; 3: MS 100 % + 3 % sucrose; 4: MS 100 % + 5 %
sucrose. Values with dierent letters diered statistically (P
≤ 0.05) for Tukey’s mean comparison test. MS: Murashige
and Skoog (1962) culture medium.
In general, higher germination percentages were reached with the
lowest concentrations evaluated of MS macronutrients.
This could be explained by the fact that the concentration of mineral
elements and sucrose are determining factors in the availability of
water in the culture medium, as they increase its osmolarity (Kubeš
et al., 2014; do Nascimento et al., 2021), decreasing water potential
and thus hindering the free movement of water from the culture
medium to the somatic embryos. In this regard, George et al. (2008)
a
ab
b
b
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Rong, Y., Junduo L., Ningbo Z., Qinhan Y., & Weirong X. (2023). Phenotypically
abnormal cotyledonary Vitis vinifera embryos dier in anatomy,
endogenous hormone levels and transcriptome proles. Tree Physiology,
(43),3, 467–485, https://doi.org/10.1093/treephys/tpac129
points out that cells maintained in an environment with low osmotic
potential lose water and decrease their water potential, altering their
morphogenenic capacity. This could explain the lower germination
values at 3 and 5 % of sucrose with 100 % of the MS macronutrients.
In this sense, Shohael et al. (2013) point out that high concentrations
of nutritional elements in the culture medium could have an adverse
eect on the germination of somatic embryos, possibly due to their
toxicity; although this eect is closely related to the absorption of
elements and will also depend on the level of the nutritional element
in the plant tissue.
Rai et al. (2007 and 2008) reported that the germination percentage
of guava somatic embryos decreased as sucrose concentration
increased above 3 %. This behavior has been reported to be caused
by dormancy of somatic embryos, where the resumption of growth
depends on a specic treatment or condition, in addition to the
presence of water (Gray, 1987). On the other hand, Choi and Jeong
(2002) also reported an inhibition of germination of somatic embryos
in peanut (Arachis hypogaea L.) due to high sucrose concentrations in
the culture medium and pointed out that the induction of dormancy in
somatic embryos was related to the accumulation of ABA as sucrose
concentration increases and with it a high osmotic stress.
Conclusions
ABA concentrations tested have negative eects on the
maturation of somatic embryos of guava var. Cuban Red Draft EEA-
1840. Seventy-three per cent germination was obtained in culture
media with MS macronutrients at half strength and 3 % sucrose. The
germination percentage tends to decrease as the concentration of
macronutrients in MS medium increases.
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