The rocoto (Capsicum pubescens Ruiz & Pav.) is a herbaceous or shrubby plant, it differs from other
species of the genus Capsicum by its purple corolla and black seeds. Peru is considered as its center of
origin, where it is widely used in medicine, pharmaceutical industry, and especially in gastronomy. In the
present work a methodology was developed to determine the effect of 2,4-dichlorophenoxyacetic (2,4-D)
concentration on in vitro induction callus from cotyledons of rocoto cv. Serrano. Rocoto seeds were
germinated in vitro, of which the cotyledons were cleaved in the first week and placed in the MS culture
media added with 2,4-D. Five treatments with different concentrations of 2,4-D were evaluated. The best
-1
results were obtained with the treatments that 0.75 and 1 mg·L were added for callus induction, achieving
72% callus proliferation at 21 days. This study aims to be the basis for future studies related to the culture
of tissue in rocoto, allowing to indicate the potential use of callus of rocoto in the induction of somatic
embryos and isolation of protoplasts.
The Biologist
(Lima)
ORIGINAL ARTICLE / ARTÍCULO ORIGINAL
EFFECT OF 2,4-DICHLOROPHENOXYACETIC CONCENTRATION ON IN VITRO CALLUS
INDUCTION USING COTYLEDONS OF ROCOTO (CAPSICUM PUBESCENS RUIZ & PAV.)
EFECTO DE LA CONCENTRACIÓN DE 2,4-DICLOROFENOXIACÉTICO EN LA INDUCCIÓN DE
CALLOS IN VITRO UTILIZANDO COTILEDONES DE ROCOTO (CAPSICUM PUBESCENS RUIZ &
PAV.)
ABSTRACT
Keywords: 2,4-D – cotyledons – Capsicum pubescens – callus – in vitro
The Biologist (Lima)
ISSN Versión Impresa 1816-0719
ISSN Versión en linea 1994-9073 ISSN Versión CD ROM 1994-9081
327
The Biologist (Lima), 201 , 1 (2), jul-dic: 9 7 327-334.
,1 1
Angel David Hernández Amasifuen* ; Alexis Argüelles Curaca ;
1 1
Anthony Apolinario Cortez Lázaro & Hermila Belba Díaz Pillasca
1Laboratorio de Biotecnología Vegetal de la Escuela Profesional de Biología con mención en Biotecnología, Universidad
Nacional José Faustino Sánchez Carrión, Av. Mercedes Indacochea N° 609, Huacho, Perú.
* Corresponding autor: adhernandz@hotmail.com
The Biologist
(Lima)
VOL. 17, Nº 2, JUL-DIC 2019
The Biologist (Lima)
Versión en Linea:
ISSN 1994-9073
Versión Impresa:
ISSN 1816-0719 Versión CD-ROM:
ISSN 1994-9081
PUBLICADO POR:AUSPICIADO POR:
ESCUELA PROFESIONAL DE BIOLOGÍA,
FACULTAD DE CIENCIAS NATURALES Y MATEMÁTICA,
UNIVERSIDAD NACIONAL FEDERICO VILLARREAL
The rocoto (Capsicum pubescens Ruiz & Pav.) is
described as a herbaceous or shrub, with woody
trunk and dichotomous branching, has alternating,
rough and pubescent leaves. It is characterized
more by its purple or purple corolla, smooth and
persistent fruit, and especially by black seeds. It is
found in the wild in the Andes of Peru, based on
proof of being its center of origin; in Peru there are
also two well-defined cultivars of rocoto: serrano
and monte. It has 24 chromosomes, of which one
pair is acrocentric, it also has a high percentage of
self-incompatibility (Guevara et al., 2000; Valdez,
2017).
The applications that are given to rocoto are
oriented to gastronomy as fresh food, sauce or
pasta, also to pharmaceutical and medicinal
products with the extraction of the enzyme
capsaicin for the preparation of drugs that are
intended to relieve gastric pain. Therefore, the
production of rocoto in Peru has been increasing
year after year, being the main destination of the
fruits for Peruvian gastronomy due to the
gastronomic boom of recent years, which is why
the rocoto has increasingly positioned itself as a
flagship product in Peru (Sánchez, 2015; Sardón,
2015; Caballero et al., 2017).
It should be taken into account that this crop has
RESUMEN
Palabras clave: 2,4-D – cotiledones – Capsicum pubescens – callos – in vitro
El rocoto (Capsicum pubescens Ruiz & Pav.) es una planta herbácea o arbustico, se diferencia de otras
especies del género Capsicum por su corola de color púrpura y semillas negras. Se considera al Perú como
su centro de origen, donde es muy empleado en la medicina, industria farmacéutica, y sobre todo en la
gastronomía. En el presente trabajo se desarrolló una metodología para determinar el efecto de la
concentración de 2,4-diclorofenoxiacético (2,4-D) en la inducción de callos in vitro a partir de cotiledones
de rocoto cv. Serrano. Se germinaron in vitro semillas de rocoto, de las cuales a la primera semana se
escindieron los cotiledones y fueron colocados en los medios de cultivo MS adicionado con 2,4-D. Se
evaluó cinco tratamientos con diferentes concentraciones de 2,4-D. Los mejores resultados fueron
-1
obtenidos con los tratamientos que se adicionaron 0.75 y 1 mg·L para la inducción de callos, logrando a
los 21 días 72% de proliferación de callo. Este estudio pretende ser base para futuros estudios relacionados
al cultivo de tejido en rocoto, permitiendo indicar potencial uso de callos de rocotos en la inducción de
embriones somáticos y aislamiento de protoplastos.
INTRODUCTION
328
susceptibility to diseases caused by Phytophthora
capsici, Fusarium oxysporum, Risotonia
solanacearum, anthracnose, wilting and root rot,
which generate damage to different plant
structures, such as lack of vigor, yellowing of the
ribs, deformations and leaf falls, abortion in the
fruits or their fall, or death of the plant (Lucana,
2012; Hernández et al., 2019b). In addition, some
of the symptoms may be related to diseases caused
by viruses, like the tomato spotted wilt virus
(TSWV), alfalfa mosaic virus (AMV), pepper mild
mottled virus (PMMoV), Peru tomato virus (MTV)
and Tomato mosaic virus (ToMV), the latter by
dissemination on contact between plants, also by
seeds from infected cultivars or seedlings (Valdez,
2017; Vallejo-Gutiérrez et al., 2019).
Being important to strengthen the rocoto value
chain is to obtain seeds or seedlings free of
pathogens, and therefore have great production.
Within the alternatives of aseptic plant material,
biotechnological tools are present, with tissue
culture techniques, being the technique of
callogenesis or embryogenesis often inducing
somaclonal variation (Orlinska & Nowaczyk,
2015).
In this way, genotypes of good quality can be
multiplied, as has been developed in the tissue
culture and regeneration of in vitro seedlings of
different species of the genus Capsicum
(Sanatombi & Sharma, 2007), but no tissue culture
The Biologist (Lima). Vol. 17, Nº2, jul - dic 2019
Hernández Amasifuen
329
work has been reported. in C. pubescens.
The present investigation was carried out with the
objective of developing a methodology to
determine the effect of 2,4-dichlorophenoxyacetic
concentration on callus induction from in vitro
cotyledons of rocoto cv. Serrano.
This research was carried out in the facilities of the
plant biotechnology laboratory of the professional
school of Biology with a mention in
Biotechnology, located at the José Faustino
Sánchez Carrión National University, Huacho,
Lima, Peru.
Disinfection of plant material
Rocoto seeds were used from the fruits present in
the greenhouse of the Plant Biotechnology
laboratory of the School of Biology. The fruits were
opened and the seeds were extracted, to which a
first wash with more detergent water was carried
MATERIALS AND METHODS
out than to remove which waste still present from
the fruit, for which a brush was used to remove any
possible contaminant, the rinsing was continued of
the seeds for detergent removal. Then the seed
disinfection process began, using the protocol
established by Argüelles et al. (2019), transferring
all the material to the laminar flow cabinet and
continuing to immerse the seeds in 70% ethanol for
60 s, then transfer the seeds to a 2% sodium
hypochlorite solution for 15 min with constant
agitation. Subsequently, they were transferred to
sterile distilled water to remove traces of sodium
hypochlorite, so the rinsing was done three times,
and planting was continued in the test tubes
containing the MS culture medium, placing 3 seeds
per test tube. They were kept in photoperiod of 16
hours of light and 8 hours dark, with a temperature
of 25 ± 1° C and relative humidity of 75 ± 1%.
The MS culture medium consisted of salts and
vitamins described by Murashige & Skoog (1962)
(Table 1), but was used at half of its concentration;
-1 -1
7 g·L agarose, 30 g·L of sucrose were added and
the pH was adjusted to 5.7. Subsequently, it was
sterilized in an autoclave at 1.2 Bar pressure and a
temperature of 121 ° C for 20 min.
Table 1. Composition of the Murashige and Skoog medium (MS).
Components
Concentration (mg·L-1)
KNO3
1900
NH4NO3
1650
CaCl2
332.2
MgSO4
180.7
KH2P O4
170
C10H14N2Na2O8. 2H2O37.26
FeSO4.7H2O 27.8
MnSO4. H2O 16.9
ZnSO4. 7H2O 8.6
H3BO36.2
KI 0.83
Na2MoO4. 2H2O 0.25
CoCl2. 6H2O 0.025
CuSO4. 5H2O 0.025
Glycine 2
Myoinositol 100
Nicotinic acid 0.5
Pyridoxine HCL 0.5
Thiamine HCL 0.1
The Biologist (Lima). Vol. 17, Nº2, jul - dic 2019
Effect of 2,4-dichlorophenoxyacetic on rocoto
330
2,4-D treatments (Table 2), in addition all
-1 -1
treatments consisted of 30 g·L sucrose and 7 g·L
agarose. All plates were incubated at 25 ° C, in total
darkness and with relative humidity of 75 ± 2% for
21 days.
Callus Induction
After seven days of sprouting, the seedlings of
rocoto split the cotyledons, placing five segments
of the cotyledons per plate with MS culture
medium (Table 1), which was used at half of its
concentration and It was supplemented with five
Table 2. Treatments for the callus induction on rocoto leaves.
Treatment 2,4-D (mg·L-1)
T10
T20.25
T30.5
T40.75
T51
2,4-D = 2,4-Dichlorophenoxyacetic acid.
In callus induction from cotyledons of rocoto,
callus formation was observed from day 11 on the
induction medium T and T (Fig. 1), From day 15
4 5
on induction media, it was observed that the first 4
treatments had callus formation, but treatments T
4
and T had a percentage of callus induction greater
5
than 50%, while treatment T did not present callus
1
induction. At day 21, the treatment with greater
callus induction was obtained and in less time the
-1
treatments with 0.75 and 1 mg·L of 2,4-D with
72% (Fig. 2) with a translucent and compact
appearance (Fig. 3).
Experimental design and data análisis
The data analyses were conducted using statistical
package agricolae of the free software R (version
3.6.1 for Windows). Each treatment consisted of 10
plates with five explants each, using each explant
as an experimental unit. To observe the significant
differences in the induction percentages, an
analysis of variance (ANVA) was used and the
comparison between the means was made
according to the Tukey test (p ≤ 0.05).
With respect to the disinfection of rocoto seeds, the
methodology used allowed to obtain 100% free of
contamination.
RESULTS
Figure 1. A. Rocoto leaf explants on day 11 in the callus induction medium. B. The callus increases in size for the second week in
the induction medium.
The Biologist (Lima). Vol. 17, Nº2, jul - dic 2019
Hernández Amasifuen
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5
22
51
70 72
0
10
20
30
40
50
60
70
80
90
2, 4-D (mg·L-1)
Callus inducon seng
0 0.25 0.5 0.75 1
Figure 2. Percentage of callus induction in vitro from rocoto leaves at 21 days.
d
c
b
a a
-1
Figure 3. A. Callus formed from rocoto leaves in basal MS medium with 0.75 mg·L of 2,4-D. B. Callus induced at 21 days with
-1
the 1 mg·L 2,4-D treatment with a translucent and compact appearance.
The Biologist (Lima). Vol. 17, Nº2, jul - dic 2019
Effect of 2,4-dichlorophenoxyacetic on rocoto
332
different responses in the explants, when using
auxins, it will have participation in cell
development and differentiation, affecting in vitro
culture considerably since these endogenous
compounds modify the cellular environment, thus
generating a stress and cellular reorganization that
leads to the formation of a mass of undifferentiated
cells (Fehér et al., 2003).
The greatest number of calluses formed were
obtained from the treatment added with 0.75 and 1
-1
mg·L of 2,4-D, in comparison to other authors that
recommended the addition of a high combination
of cytokinins and auxins, or only the addition of
auxins can favor the callus induction, in addition to
the appearance of callus is related to the type of
hormone used during induction. In the case of the
growth regulator that has more reports of callus
induction is auxin 2,4-D (Larson et al., 2006;
Feeney et al., 2007; Meiners et al., 2007; Shiram et
al., 2008; Hernández & Díaz, 2019).
These results represent an important advance in the
use of biotechnological techniques for the genetic
improvement of rocoto, which opens expectations
for the potential use of callus of this species for the
induction of somatic embryos and isolation of
protoplasts.
A methodology was developed to determine the
effect of 2,4-dichlorophenoxyacetic concentration
on in vitro callus induction from cotyledons of
rocoto cv, Serrano, in which using the MS culture
-1
medium added with 0.75 and 1 mg·L of 2,4-D
achieved the highest callus induction with more
than 70% at 21 days.
The authors thank Angel Abel Hernández Cotrina
and Lastenia Amasifuen Ochavano for providing
access to the cultivation areas and supporting the
collection of samples that were donated to the Plant
Biotechnology laboratory.
The authors declare no conflict of interest.
The method of disinfection of rocoto seeds was
effective when immersed in 70% ethanol and then
with sodium hypochlorite, which is a substance
commonly used in the superficial disinfection of
plant material in the process of introduction in the
in vitro culture for presenting A positive effect in
the elimination of resistant microorganisms, due to
the effects of chlorination (Ramirez et al., 2002), is
effective in most pathogenic bacteria, but with an
unpredictable action against fungi and viruses. The
death of microorganisms is due to the direct
combination of chlorine with the proteins of cell
membranes and enzymes, this because chlorine
destroys organisms by being inactivated by
oxidation of cellular material. Likewise, in the
presence of water gives off nascent oxygen (O )
2
that oxidizes organic matter (Folgueras et al.,
2001). In addition, stirring the explants together
with the disinfectant helps to reduce the surface
tension of the water, thus allowing greater contact
of the explant with the disinfectant solution
(Abdelnour & Escalante, 1994; Hernández et al.,
2019a).
On the other hand, ethanol acts by denaturing
proteins, dissolving lipid layers and as a
dehydrating agent, it is lethal to bacteria, but
irregular to fungi and viruses, it does not act on
spores; when combined with antiseptics of another
group has a greater germicidal action (Folgueras et
al., 2001).
It was possible to induce callus from cotyledons of
rocoto by starting auxin 2,4-D in different
concentrations in the MS culture medium, with
which calluses with translucent and compact
appearance were observed. As a result of this
induction, we have that callus formation is highly
dependent on the type of explants, and on the type
and concentration of phytohormone, in the case of
calluses from leaf segments unlike other explants, a
higher percentage of callus formation by
increasing the concentration of 2,4-D also having it
have an effect on the appearance of the calluses,
also presenting a linear effect (Rodríguez et al.,
2014).
The culture medium supplemented with different
concentrations of growth regulators generates
DISCUSSION
ACKNOWLEDGEMENTS
CONFLICT OF INTEREST
The Biologist (Lima). Vol. 17, Nº2, jul - dic 2019
Hernández Amasifuen
Abdelnour, A. & Escalante, V. 1994. Conceptos
básicos del cultivo de tejidos vegetales.
Centro Agronómico de Investigaciones y
Enseñanza. Turrialba. Costa Rica. 38 pp.
Argüelles, A.; Domínguez, G.; Gózalo, A. & Díaz,
H. 2019. Inducción de germinación in vitro
d e s e m i l l a s d e ro co to ( Ca ps ic um
pubescens). IV Congreso Peruano de
Biotecnología y Bioingeniería. Libro de
Resúmenes: 8 -11 p.
Caballero, B.; Márquez, C. & Alberto, B. 2017.
Efecto de la liofilización sobre las
propiedades funcionales del ají rocoto
(Capsicum pubescens). Revista UDCA
Actualidad & Divulgación Científica, 20:
111-119.
Feeney, M.; Bhagwat, B.; Mitchell, J. & Lane, W.
2007. Shoot regeneration from organogenic
callus of sweet cherry (Prunus avium L.).
Plant Cell, Tissue and Organ Culture, 90:
201–214.
Fehér, A.; Pasternak, T. & Dudits, D. 2003.
Transition of somatic plant cells to an
embryogenic state. Plant Cell, Tissue and
Organ Culture, 74:201-228.
Folgueras, M.; Herrera, L. & Carrazana, D. 2001.
La contaminación microbiana en la
micropropagación in vitro de las raíces y
tubérculos tropicales. Taller Internacional.
pp. 183–185. Villa Clara, Cuba. Instituto de
Investigaciones en Viandas Tropicales.
Guevara, M.; Siles, M. & Bracamonte, O. 2000.
Análisis cariotipico de Capsicum pubescens
R&P (Solanaceae) “rocoto”. Revista
Peruana de Biología, 7: 134 – 141.
Hernández, A. & Díaz, H. 2019. Inducción in vitro
de callo embriogénico a partir del cultivo de
anteras en “papa amarilla” Solanum
goniocalyx Juz. & Bukasov (Solanaceae).
Arnaldoa, 26: 277-286.
Hernández, A.; Pineda, A. & Díaz, H. 2019a.
Efecto de la luz y del ácido giberélico en la
germinación in vitro de Capsicum annuum
L. cv. Papri King. Biotecnología Vegetal,
19: 43-51.
Hernández, A.; Pineda, A. & Noriega, H. 2019b.
Aislamiento e identificación de Fusarium
oxysporum obtenidos de zonas productoras
de “ají paprika” Capsicum annumm L.
333
(Solanaceae) en el distrito de Barranca,
Perú. Arnaldoa, 26: 689-698.
Larson, C.; Gómez, C.; Sánchez, M. & Ríos, D.
2006. Inducción de caulogénesis indirecta
en Eucalyptus globulus. Bosque, 27: 250-
257.
Lucana, C. 2012. Respuesta de 5 especies de
Capsicum spp. A Phytophthora capsici
Leon, bajo condiciones de invernadero, en
los laboratorios de fitopatología de la
UNALM – Lima. Tesis de Ingeniera.
Universidad Nacional de San Antonio Abad
del Cusco. Cusco. Perú.
Meiners, J.; Schwab, M. & Szankowski, I. 2007.
Efficient in vitro regeneration systems for
Vaccinium species. Plant Cell, Tissue and
Organ Culture, 89:169-176.
Murashige, T. & Skoog, F. 1962. A revised medium
for rapid growth and bioassays with tobacco
tissue culture. Physiology Plant, 15: 473-
497.
Orlinska, M. & Nowaczyk, P. 2015. In vitro plant
regeneration of 4 Capsicum spp. genotypes
using different explant types. Turkish
Journal of Biology, 39: 60-68.
Ramírez, M.; Urdaneta, A. & León, S. 2002.
Establecimiento in vitro de explantes
adultos del guanabo (Annona muricata L.)
tratados con hipoclorito de sodio. Revista de
la Facultad de Agronomía – Luz, 19: 1-8.
Rodríguez, M.; Latsague, M.; Chacón, M &
Astorga, P. 2014. Inducción in vitro de
callogénesis y organogénesis indirecta a
partir de explantes de cotiledón, hipocótilo
y hoja en Ugni molinae. Bosque, 35: 111-
118.
S a n a t o m b i , K . & S h a r m a , G . J . 2 0 0 7 .
Micropropagation of Capsicum annuum L.
Notulae Botanicae Horti Agrobotanici Cluj-
Napoca, 35: 57-64.
Sánchez, J. 2015. Relación del color y las
características fisiológicas y fisicoquímicas
del rocoto (Capsicum pubescens). Tesis de
Ingeniera. Universidad Nacional de
Trujillo, Trujillo. Perú.
Sardón, E. 2015. Fortalecimiento de la cadena de
valor del rocoto fresco (Capsicum
pubescens) de la selva central para el
mercado de Lima. Tesis de Maestría.
Universidad Nacional Agraria La Molina,
Lima. Perú.
Shiram, V.; Kumar, V. & Shitole, M. 2008. Indirect
BIBLIOGRAPHIC REFERENCES
The Biologist (Lima). Vol. 17, Nº2, jul - dic 2019
Effect of 2,4-dichlorophenoxyacetic on rocoto
334
Vallejo-Gutiérrez, A.; Mejía-Carranza, J.; García-
Velasco, R. & Ramírez-Gerardo, M. 2019.
Respuesta de genotipos de Capsicum
pubescens al daño ocasionado por el
complejo fúngico de la marchitez. Revista
Mexicana de Fitopatología, 37: 50–70.
organogénesis and plant regeneration in
Helicteres isora L., an important medicinal
plant. In vitro Cellular and Developmental
Biology-Plant, 44:186-193.
Valdez, I. 2017. Caracterización fenotípica de
quince accesiones de germoplasma de
rocoto (Capsicum pubescens Ruiz &
Pavón.) en la estación INIA Santa Rita
Arequipa. Tesis de Ingeniera. Universidad
Nacional de San Agustín, Arequipa. Perú.
Received October 17, 2019.
Accepted December 28, 2019.
The Biologist (Lima). Vol. 17, Nº2, jul - dic 2019
Hernández Amasifuen
