Phytolacca tetramera Hauman is an endemic species of the Province of Buenos Aires (Argentina), in danger of extinction. This species has active fungicides. The methanol extract of its berries possess antifungal activity against opportunistic fungal pathogens. The somatic embryogenesis technique relies on the formation of an embryo from a somatic cell, that is, without the need for the fusion of gametes (Tisserat et al, 1979), which facilitates mass production of in vitro plants. Obtaining somatic embryos gives us the ability to automate the production process in short periods of time and in a confined space. The technique is a necessary step to generate synthetic seeds. In order to adjust a protocol of somatic embryogenesis sections young leaves of Phytolacca tetramera obtained in vitro were placed in a Murashige and Skoog medium supplemented with 2, 4-dichlorophenoxyacetic acid at different concentrations. Somatic embryos were obtained directly from the cutting edge of the blade and embryogenic callus (indirect way) from the midrib.Facultad de Ciencias Agrarias y ForestalesCentro Experimental de Propagación Vegetativ

Abedini, Walter

Adema, Marina

Basiglio Cordal, María de los Ángeles

Briones, María Valentina

Panarisi, M. H.

Sharry, Sandra Elizabeth

Villarreal, Blanca

Emirates Journal of Food and Agriculture

English

SEDICI - Repositorio de la UNLP

Emir. J. Food Agric. 2014. 26 (6): 552-557
doi: 10.9755/ejfa.v26i6.18021
http://www.ejfa.info/
552
Regular Article
PLANT TISSUE CULTURE
Induction of somatic embryogenesis in Phytolacca tetramera, medicinal species of 
Argentina
M. A. Basiglio Cordal*, M. Adema, V. Briones, B. Villarreal, M. H. Panarisi, W. Abedini and S. Sharry
Centro Experimental de Propagación Vegetativa (C.E.Pro.Ve), Facultad de Ciencias Agrarias y Forestales, Universidad 
Nacional de La Plata, CICPBA- CONICET, Diagonal 113 N° 469 (1900), La Plata, Buenos Aires, Argentina
Abstract
Phytolacca tetramera Hauman is an endemic species of the Province of Buenos Aires (Argentina), in danger of 
extinction. This species has active fungicides. The methanol extract of its berries possess antifungal activity 
against opportunistic fungal pathogens. The somatic embryogenesis technique relies on the formation of an 
embryo from a somatic cell, that is, without the need for the fusion of gametes (Tisserat et al, 1979), which 
facilitates mass production of in vitro plants. Obtaining somatic embryos gives us the ability to automate the 
production process in short periods of time and in a confined space. The technique is a necessary step to 
generate synthetic seeds. In order to adjust a protocol of somatic embryogenesis sections young leaves of 
Phytolacca tetramera obtained in vitro were placed in a Murashige and Skoog medium supplemented with 2, 4-
dichlorophenoxyacetic acid at different concentrations. Somatic embryos were obtained directly from the 
cutting edge of the blade and embryogenic callus (indirect way) from the midrib.
Key words: Somatic embryogenesis, Endemism, Ombusillo, In vitro culture, Germplasm
Introduction
The Family Phytolaccaceae includes 6
subfamilies, 18 genera and 70 species. It is 
widespread in tropical and temperate regions, 
especially in the Neotropics and S. Africa. All 
species of this family are widely used in folk 
medicine for the treatment of diseases such as 
edema, rheumatism and dermatitis (Gattuso, 
Zacchino, 1998).
Phytolacca tetrámera Hauman, commonly 
known as "Ombusillo", is a dioecious species, 
belonging to the family Phytollacaceae, up to 1.5
meters high. Is an endemic NE Bs. As geophyte 
bush, whose distribution starts near the city of La 
Plata to Ensenada Samborombón, and around, 
forming a fundamental part and endemism of the 
flora characteristic of the Parque Costero Del Sur 
(36° lat. S-. 57° 30'long OR) area declared by 
UNESCO, "World Biosphere Reserve", since 1984. 
Regarding compounds and properties, P. tetramera, 
is a natural source of active principles and
fungicides (Escalante, 2003) and probably antiviral, 
antitumor and antibacterial compounds (Abedini et 
al., 1997). The methanol extract of the berries, 
showed antifungal activity against opportunistic 
fungal pathogens. Fractionation of the butanol 
extract, allowed the isolation of three triterpenoid 
saponins monodesmosidics: phytolaccosides B [3-
O-β-d-xylopiranosyl-phytolaccagenin], E [3-O-β-d-
glucopyranosyl-(1 → 4) - β -d-xylopiranosyl-
phytolaccagenin] and F [3-O-α-l-rhamnopyranosyl-
(1 → 2) - β-d-glucopyranosyl-(1 → 2) - acid β-d-
xylopyranosyl-phytolaccagenic] (Escalante et al., 
2008).
The first of these compounds was the most 
active and also showed the broad spectrum of 
action, resulting in defects of the hyphae and the 
fungal membrane thickening in Saccharomyces 
cerevisiae (Escalante et al., 2003).
The agar dilution method showed minimum 
inhibitory concentrations (MICs) between 25 and 
250 mg/ml, Trichophyton mentagrophytes being the 
most susceptible species. The structures of the three 
saponins were elucidated by spectroscopic methods 
mainly 1D and 2D NMR (Escalante et al., 2003).
Received 30 December 2013; Revised 04 February 2014; 
Accepted 05 February 2013; Published Online 25 March 2014
*Corresponding Author
M. A. Basiglio Cordal
Centro Experimental de Propagación Vegetativa (C.E.Pro.Ve), 
Facultad de Ciencias Agrarias y Forestales, Universidad 
Nacional de La Plata, CICPBA- CONICET, Diagonal 113 N° 
469 (1900), La Plata, Buenos Aires, Argentina
Email: maribasiglio@hotmail.com
M. A. Basiglio Cordal et al.
553
Escalante et al. (2008) has shown that the 
Phytolaccosido B, alters the morphology of fungi 
and yeasts. The defects were similar to those 
produced by enfumafungin, a known inhibitor of (1
-> 3) - beta - D-glucan synthase, an enzyme that 
catalyzes the synthesis of (1 -> 3) - beta-D - glucan, 
one of the main polymers of the fungal cell wall.
However, tests revealed that this enzyme does not 
inhibit phytolaccoside (1 -> 3)-beta-D-glucan 
synthase, but produced a marked improvement in
the activity of chitin synthase and, concomitantly, 
an increase of chitin, another important polymer
cell walls of fungi. This finding was confirmed by 
fluorescence microscopy and also by measuring the
chitin (Escalante et al., 2008). Medicinal plants as 
an alternative to traditional crops are revealed as an 
important source for the development of new 
activities. 
To resolve the problem of extraction of forest 
plants, one option is asexual or vegetative 
propagation of this species, which would generate a 
large number of individuals with the same genetic 
identity. In vitro culture is an alternative for the 
breeding and conservation of species difficult to 
cultivate. The tissue culture techniques have been 
used routinely for over four decades (Murashige 
and Skoog, 1962).
In vitro culture conditions, somatic embryos or 
cells can regenerate shoots, roots and / or flowers in 
response to given stimulus (Radice, 2004). Somatic 
embryogenesis is based on the formation of an 
embryo from a somatic cell, that is, without the 
need for the fusion of gametes (Tisserat et al., 
1979), which facilitates mass production of in vitro 
plants. This technique has emerged as a new way of 
propagation and is a tool for in vitro conservation 
of germplasm (Griga, 2000) and breeding (Das et 
al., 2002). Used for increasing the multiplication 
coefficients, lower production costs and gives the 
possibility to automate the production process using 
bioreactors (De Feria, 2000).
There are two types in vitro somatic 
embryogenesis, direct and indirect. In direct 
somatic embryogenesis, embryos appear directly 
over the original explant. In indirect somatic 
embryogenesis, embryos originating from callus or 
from embryogenic cell suspension (Perez Molphe 
et al., 1999). The process of somatic embryogenesis 
involves different steps (Thorpe, 1995), starting 
from the induction, followed by histo-
differentiation, maturation and subsequent 
germination, concluding with the conversion of the 
embryo plant. The technique of somatic 
embryogenesis is a necessary step to generate 
synthetic seeds. Synthetic seed described generally 
to an encapsulated somatic embryo with an 
artificial covering and can be a technology that 
enables the extensive scaling required for 
commercial production of species of interest, even 
with high germination (http://www.cyd.conacyt.
gob.mx/258/articulos/semillassinteticas.html).
In Phytolacca tetramera, somatic 
embryogenesis, would be a vital process because, 
the species is known for being critically endangered 
threat, and being a natural source of active 
compounds.
The objective of this work was to achieve the 
induction of somatic embryogenesis in Phytolacca 
tetrámera Hauman.
Materials and Methods
The mother plants were obtained by in vitro
micropropagation from nodal and internodal 
sections, seeded in a basal culture medium of 
Murashige and Skoog (1962) (MS) supplemented 
with 3% sucrose and without the addition of plant 
growth regulators to induce the process of 
organogenesis adventitia. The leaf sections 3-5 cm 
were used as explants for the induction of somatic 
embryogenesis. The sheets were cut into 1.5 cm 
sections, retaining the midrib, and were placed on 
the abaxial side of a 5 explants per container in the 
culture medium containing Murashige and Skoog 
(MS) complete, supplemented con 2, 4 D (2, 4 -
dichlorophenoxyacetic acid) at different 
concentrations (0.5, 1 and 1.5 ppm) with the 
corresponding Witness in the absence of 2, 4 D. As 
gelling agent Difco Bacto agar was used in a range 
of 0.6 to 0.7% and the pH was adjusted between 5.8
to 6.2.
All media were autoclaved at 1 atm pressure 
and a temperature of 120°C for 20 minutes. The 
containers used were Petri dishes with 15 ml 
culture medium c / u. The explants were incubated 
in the dark at 25 + / -2°C temperature.
Results
Experiments followed for 20 days to evaluate 
the effect of different concentrations of 2, 4 D 
showed qualitative and quantitative differences. 
The response of nodal sections placed in culture 
medium containing different concentrations of 2, 4-
D are shown in Table 1. The differentiation began 
at 6 days of seeding explants, showing the winding 
sections of leaves. At 20 days of culture, callus 
formation was observed in both the cut surface of 
the sections at the level of the main rib. No callus 
formation in the control treatment was observed. 
However, root formation ranging between 2 to 4
per explant, and 3 to 5 cm in length was observed. 
The calluses formed were rib level and white 
Emir. J. Food Agric. 2014. 26 (6): 552-557
http://www.ejfa.info/
554
friable, embryogenic callus typical characteristics. 
In the same could be identified in globular stage 
embryos. They subcultured in liquid medium 
continued development.
The callus formed on the edges of the explant 
were disintegrable, and they could be observed in 
different stages of somatic embryos (globular and 
torpedo). 
Figure 1. Phytolacca tetramera in vitro.
Figure 2. Foliar Sections of Phytolacca tetramera in 
induction medium.
Figure 3. Cotyledon embryo stage, located in the midrib. 
Table 1. Effect of the concentration of 2, 4 D in the induction of callus formation from leaf segments Phytolacca 
tetramera Hauman 30 days of cultivation.
Culture Medium Concentration of growth 
regulator 2,4-D (ppm)
Number of explants with 
callus formation
Percentage of explants 
with callus formation (%)
MS 0.5 20 100%
MS     1.0 10 50%
MS                                                  1.5                                         17   85%
M. A. Basiglio Cordal et al.
555
Discussion 
In this paper, the indirect somatic 
embryogenesis was induced from leaf sections of P. 
tetramera. The similar results were obtained in 
different plant species previously (Stamp and 
Henshaw, 1987; Bespalhok Filho et al., 1993;
Robacker 1993; Jayasree 2001; Devaraju and 
Reddy 2013).
In the present study, it was found that the 
incubation conditions and culture medium 
influences the development of embryogenic callus. 
The concentration of 2,4-D and the area of the 
explant were decisive for the induction of 
embryogenic callus and embryos, which is 
consistent with the reports by Raghavan (2004). He 
found that in embryos from Arabidopsis, cultured 
in a medium containing 2, 4-D, following a brief 
period of growth by cell expansion, divisions was
initiated in the procambial cells facing the adaxial 
side at the base of the cotyledons. Cell division 
activity later spread to almost the entire length of 
the leaf cotyledons to form a callus on which 
globular and heart-shaped embryos appeared in 
about 10 d after culture. In the present 
investigation, the results on somatic embryogenesis 
have shown that auxin such as 2,4-D are essential 
for induction of somatic embryogenesis from leaf
explants of P. tetramera, where in the growth 
regulators play a primary role in the medium for 
induction of somatic embryogenesis in nature. The 
type of auxin or auxin in combination with 
cytokinin used in the medium can greatly influence 
somatic embryo frequency. Proliferated 
embryogenetic suspension cultures were 
established in this species. Reddy and Reddy 
(1983) reported the improved response of auxin like 
2,4-D in a single treatment for the induction of 
somatic embryogenesis compared to combination 
of 2,4-D and cytokines in Arachis hypogea. 
Somatic embryogenesis is generally believed to be 
triggered by an auxin and for many plants, 2, 4-D 
has been widely regarded to be effective for 
somatic embryogenesis (Ammirato 1983; Finer, 
1988). In this study, callus formation was observed 
after 20 days in culture. Bespalhok et al. (1993) 
reported that somatic embryos were first observed 
15 days after culture initiation. The majority of the 
somatic embryos appeared to form directly without 
intermediate callus formation. In fact due to the 
extremely high pressure, the species P. tetramera
has been declared as endangered (Delluchi, 2006). 
In recent years, several groups have addressed the 
need to conserve native medicinal species and to 
explore the possibilities of identifying high-yielding 
individuals or populations for the development of in 
vitro production systems (Ved, 1997; Ciddi, 2000; 
Cantelmo et al., 2013). The present work on the
regenerated plants were morphologically similar to 
seed-derived plants. Khadke and Kuvalekar (2013) 
reported similar results with the medicinal plant 
Nothapodytes foetida, a critically endangered plant 
species, and suggest that this system may prove to 
be beneficial for ex situ conservation as well as for 
amenability of this endangered plant for genetic 
manipulation. 
Conclusion
Reliable protocols useful for P. tetramera mass 
propagation through callus culture and somatic 
embryos formation had been established and 
verified. Plant regeneration through somatic 
embryogenesis has several advantages over other 
routes to in vitro plant production and appears that 
most promising area of research for large scale 
production, rapid plant propagation and support of 
conservation strategies.  This system is potentially 
useful for the micropropagation of this species, as 
well as for the production of substances with 
pharmacological interest, such as phytolaccoside B 
[3-O-β-d-xylopiranosyl-phytolaccagenin]. The 
results obtained in this study could have enormous 
significance, since this is the first report of somatic 
embryogenesis for this medicinal important and to 
the whole genus Phytolacca species. However, 
these studies should continue to improve embryo 
conversion and complete plant regeneration. This 
technique will allow us to have a lot of plants to 
introduce and domesticate wild species to establish 
crop field or restore populations in natural 
ecosystems. 
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Induction of somatic embryogenesis in Phytolacca tetramera, medicinal species of Argentina

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Induction of somatic embryogenesis in Phytolacca tetramera, medicinal species of Argentina

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