© The Authors, 2022, Published by the Universidad del Zulia*Corresponding author: fcobos@utb.edu.ec
Keywords:
Pasture improvement
Variability
Resistance
Mutation induction
Induced mutagenesis in janeiro grass by ethyl-meta-sulfonate and its effect on spittlebug
control
Mutagénesis inducida en pasto janeiro mediante etil- meta-sulfonato y su efecto sobre el control de
salivazo
Mutagênese induzida em grama de janeiro por etil-meta-sulfonato e seu efeito no controle da
cigarrinha
Juan Gómez Villalva
1
Fernando Cobos Mora
1*
Edwin Hasang Moran
2
Ana Luzmeira Eguiluz de la Barra
3
Israel Cortez Herrera
1
Rev. Fac. Agron. (LUZ). 2022, 39(3): e223938
ISSN 2477-9407
DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v39.n3.04
Animal Production
Associate editor: Dra. Rosa Razz
University of Zulia, Faculty of Agronomy
Bolivarian Republic of Venezuela
1
Universidad Técnica de Babahoyo, Ecuador.
2
Universidad Agraria del Ecuador.
3
Departamento de Fitotecnia, Universidad Nacional Agraria
La Molina, Perú.
Received: 16-11-2021
Accepted: 13-07-2022
Published: 03-08-2022
Abstract
In this research, the median lethal dose (LD
50
) and resistance to spittlebug
(Mahanarva andigena) were determined, using a mutagenizing agent ethyl
meta-sulfonate (EMS) in Janeiro grass (Eriochloa polystachya Kunth) as a
means to generate mutations. The study was carried out at the laboratory
and greenhouse level, using a Complete Random Design (DCA) with ten
treatments and three repetitions, which consisted of 5 doses of EMS (0.00%,
0.25%, 0.50%, 0.75% and 1.00%). for 2 impregnation times (24 and 48
hours). According to the results, the doses of ethyl meta-sulfonate (EMS)
inuenced the setting of the stolons of the janeiro grass, presenting a high
rate of deation in doses higher than 0.50%. Treatments with doses of 0.25%
EMS at 48 hours achieved the best agronomic performance up to 95 days,
where plant height had its best development with use close to that determined
as DL
50
(0.32%). The janeiro grass impregnated with EMS presented slight
damage caused by the spittle.
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2-7 |
Resumen
En esta investigación se determinó la dosis letal media (DL
50
)
y la resistencia a salivazo (Mahanarva andigena), utilizando ethyl
metano-sulfonato (EMS) agente mutagenizante en pasto Janeiro
(Eriochloa polystachya Kunth) como medio para generar mutaciones.
El estudio fue realizado a nivel de laboratorio e invernadero, usando
para ello un Diseño Completo al Azar (DCA) con diez tratamientos y
tres repeticiones, que consistieron en 5 dosis de EMS (0,00 %, 0,25
%, 0,50 %, 0,75 % y 1,00 %) por 2 tiempos de impregnación (24 y
48 horas). De acuerdo a los resultados, las dosis de ethyl metano-
sulfonato (EMS) tuvieron efecto en el prendimiento de los estolones
del pasto janeiro, presentándose un alto índice de deación en dosis
superiores a 0,50 %. Los tratamientos con dosis de 0,25 % de EMS
a 48 horas, lograron el mejor comportamiento agronómico hasta los
95 días, donde la altura de planta tuvo su mejor desarrollo con el
uso cercano a la determinada como DL
50
(0,32 %). El pasto janeiro
impregnado con EMS presentó daños leves causado por el salivazo.
Palabras clave: Mejoramiento de pastos, variabilidad, resistencia,
inducción de mutaciones.
Resumo
Nesta pesquisa, a dose letal mediana (DL
50
) e a resistência à
cigarrinha (Mahanarva andigena) foram determinadas, utilizando-
se um agente mutagenizante etil meta-sulfonato (EMS) em capim-
janeiro (Eriochloa polystachya Kunth) como meio de gerar mutações.
O estudo foi realizado em nível de laboratório e casa de vegetação,
utilizando um Desenho Aleatório Completo (DCA) com dez
tratamentos e três repetições, que consistiu em 5 doses de EMS (0,00
%, 0,25 %, 0,50 %, 0,75 % e 1,00 %) por 2 tempos de impregnação
(24 e 48 horas). De acordo com os resultados, as doses de etil meta-
sulfonato (EMS) tiveram efeito na xação dos estolões do capim-
janeiro, apresentando alto índice de deação em doses superiores
a 0,50%. Os tratamentos com doses de 0,25% EMS às 48 horas
obtiveram o melhor desempenho agronômico até 95 dias, onde a
altura das plantas teve seu melhor desenvolvimento com uso próximo
ao determinado como LD
50
(0,32%). O capim-janeiro impregnado
com EMS apresentou danos leves causados pelo salivazo.
Palavras-chave: Melhoramento genético, variabilidade,
melhoramento, mutações.
Introduction
In the early 1950s, improved cultivars were achieved through
mutagenesis. At the beginning of the 20th century, mutagenesis
techniques were applied to obtain rapidly mutant individuals and
increase variability in plants (Rojas et al., 2016). FAO published in
1994 that 1.800 cultivars were obtained by mutagenesis. Currently,
most of the crops exploited in the world come from mutated cultivars
(González, 2014). For instance, 70% of the wheat (Triticum aestivum
L.) sown in Italy came from mutated crops (González, 2014).
Despite the heavy reliance on chemical mutagenesis, traditional
genetic screens do not readily reveal the underlying mutational
process. This is because geneticists select phenotypes and, as a result,
only a small minority of mutations within a target gene are examined
(Greene et al., 2003).
Corrales et al. (2019) mentioned that the induction of mutations
might be a viable option to create genetic improvement. This is
because the practice is capable of inducing mutability with mutagenic
agents.
The use of mutagenic agents, such as ethyl meta-sulfonate (EMS),
has a fundamental role in the generation of variability (Porch et al.,
2009). One of the most widely used chemical mutagen in breeding
programs is EMS, which could produce tolerance related to phyto
and entomopathogenic agents. This is because it creates many point
mutations in almost all the studied genera. Moreover, the periodicity
of induced mutations is independent of the genome volume (Greene
et al., 2003; Rojas et al., 2016).
In janeiro grass (Eriochloa polystachya Kunth) it is difcult
to nd genetic variability to improve its productive potential and
nutritional quality by conventional methods. This is because of its
largely asexual reproductive system and its short evolutionary period.
Despite of all those drawbacks, janeiro grass could increase forage
production (Nobel, 1999).
The use of EMS in its genetic improvement for the specic case
of janeiro grass has not been reported in the literature. Consequently,
this study aims to determine the mean lethal dose (LD
50
) to induce
mutations with EMS in janeiro grass and evaluate resistance against
spittlebug (Mahanarva andigena).
Materials and methods
Study location
The research, related to the collection and sowing of janeiro
grass, was carried out in the rainy season (January to May) of the
year 2020. It was developed at the laboratory and greenhouse level
at the Faculty of Agricultural Sciences of the Technical University
of Babahoyo, Ecuador, located 7.5 km from the Babahoyo-Montalvo
road, geographically located at 79°32 ́ W longitude and 01°49 ́
S latitude, elevation of 8 m.a.s.l., temperature of 30.4 °C, relative
humidity 65.5% (greenhouse), evaporation 1012.4 mm, heliophany
830.4 hours (Cobos et al., 2021).
Stolons management and experimental procedure
For this study, janeiro grass stolons of 8 cm in length were used,
with a node where they emitted their root, which were rinsed and
disinfected with sterile distilled water (three rinses) (Gómez et al.,
2020).
The methodology established by Pankhurst et al. (2004) was used
for impregnation. The Biotechnology Laboratory at the Faculty of
Agricultural Sciences applied a modied protocol to carry out the
impregnation. The proposed protocol is briey described as follows:
Approximately 2.000 stolons at different EMS concentrations
(0.00%, 0.25%, 0.50%, 0.75%, 1.00%), were used in the present
work. Once the dilutions were prepared, they were poured into bottles
where the plant material was located to avoid splashing. Thereafter,
200 stolons, sterilized with ethanol at 70% (v/v) for 10 min and
H
2
O
2
at 5% (v/v) for 10 min were submerged for 24 hours and 48
hours. After this time, sterile deionized water (v/v) was added to the
glasses with the stolons at room temperature. Finally, the solution
was removed and the stolons treated with distilled water were rinsed
5 times at least 15 minutes each rinse.
The stolons treated with EMS and the untreated controls were
sown in germinating trays located in a greenhouse. This was done
to determine the mean lethal dose (LD
50
). After 30 days, the stolons
were transplanted into definitive covers to evaluate their vigor and
resistance to the spittlebug attack.
The nymphs were collected as foam masses from areas affected
by spittlebug. The transportation was done from the Bucay area to the
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Babahoyo canton in containers with plant material; thus, the nymphs
were kept during the transfer (Castro et al., 2005).
400 plants.treatment
-1
were transplanted from janeiro grass to
the greenhouse and having completed 50 days of physiological
development, they were inoculated with the nymphs of the spittlebug
pest insect, which was done random. Each treatment contained a
number of 5 nymphs. In other words, the study area was exposed to
50 spittlebug nymphs.
Variables evaluated
Percentage of germination and/or seizure. The germination
percentage (take-off) was evaluated at 10, 20 and 30 days. The result
was expressed as a percentage of germinated stolons.
The percentage of seizure was determined by applying the
following equation:
No. of stolons sown - No. dead stolons x 100
% take-up =
No. of stolons sown
Percentage of mortality
The percentage of mortality was determined at 10, 20 and 30
days, considering the stolons that were not alive.
No. of stolons sown - No. living stolons x 100
% mortality =
No. of stolons sown
Growth vigor
It was based on a descriptor (table 1) with a scale established by
CIAT’s standard evaluation system for rice (Rodríguez et al., 2019).
This variable was evaluated after 115 days in the presence of the
spittlebug pest insect.
Table 1. Scale used to determine plant vigor.
Category Scale
Very vigorous plant 1
Vigorous Plant 3
Intermediate or normal plants 5
Plants less vigorous than normal 7
Very weak and small plants 9
Number of spittlebugs per treatment
It was recorded by counting the number of spittlebugs in ten
random plants present in each treatment at 60 days.
Pest severity
The damage in the janeiro grass was determined at 60 days,
observing the damage caused in the plants and the presence of living
individuals, according to Bhattarai (2019). This scale presented in
table 2 is used to determine the severity of the pest in crops.
Table 2. Scale to determine severity.
Intensity of damage Living individuals
No harm 0
Mild 1
Moderate 2
High 4
Strong 6
Very strong >8
Experimental design and treatments
A Complete Random Design (DCA) was used for the statistical
analysis, which consisted of ten treatments and three repetitions (Table
3). The factors under study included 5 doses of EMS (0.00%, 0.25%,
0.50%, 0.75% and 1.00%) and 2 impregnation times (24 and 48
hours), resulting in a total 10 treatments for three repetitions. The data
was subjected to a variance analysis. Moreover, a regression analysis
was performed to nd a relationship between the impregnation dose
and the variables with the statistical package Statgraphics Centurión
XVI. II, with a linear probit model.
Table 3. Study treatments applied in janeiro grass.
Treatments
EMS concentrations
(%)
Hours
T1 0.00% 24
T2 0.25% 24
T3 0.50% 24
T4 0.75% 24
T5 1.00% 24
T6 0.00% 48
T7 0.25% 48
T8 0.50% 48
T9 0.75% 48
T10 1.00% 48
Results and discussion
In the rst phase of germination with different levels of EMS,
mortality (gure 1) presented higher values in EMS concentrations
from 0.6 to 1%. Similar values are reported by Mendoza (2020) in his
research on mutagenesis induction in Eustoma grandiorum cultivars
by using EMS at concentrations of 0.75M and 1M, the death rate was
greater than 60%. On the other hand, Yadav et al. (2016) in LD
50
with
EMS in mustard seeds reports that doses higher than 1% are highly
lethal regardless of genotype and species, and that with a concentration
of 0.50% EMS at three days for the variety RH-749, NRCHB-101 and
Sinapis alba, germinated 33.88 and 25% respectively; while at 6 days
was 62.94 and 57%, respectively.
LD
50
in janeiro grass impregnated at 24 hours
Figure 1 show the regression obtained from the percentage of
mortality against EMS doses. The resulting LD
50
by Probit analysis
was 0.49% of the EMS, in which there was a 50% mortality of janeiro
grass. The most propitious dose was 0.50 at 24 hours of impregnation.
The applied equation was y = -0.797923 + 1.60861 * LD
50
EMS
janeiro, with an R
2
of 58.61 percentage of deviation.
According to the LD
50
results in janeiro grass impregnated at 24
hours, the mortality increases with the EMS doses. Those results are
similar to a research developed by López (2011) who induced genetic
variability by EMS in cultivation in vitro of Cenchrus ciliaris L. This
author reported an LD
50
of 0.5% and mentioned that the germination
percentage decreases as the dose and time increases.
LD
50
in janeiro grass impregnated at 48 hours
Figure 2 shows the linear regression tted to a Probit regression
model, being the equation of the tted model Y = -0.952779 + 2.94161
* B. EMS dose (%) with an R
2
= 79.62. The obtained result indicates
that LD
50
was 0.32% EMS, in which the mortality of the janeiro grass
was 50% with an impregnation time of 48 hours. In a study carried
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4-7 |
Figure 1. LD
50
in janeiro grass impregnated at 24 hours.
Figure 2. Dl
50
in janeiro grass at 48 hours of impregnation.
out by Costa (2017) which impregnated EMS in vitro vegetative
material of artichoke (Romanesco) obtained an LD
50
of 0.37% of
the EMS and observed that at high concentrations the explants
become necrotic and die. Khalil et al. (2018) mentioned that a study
carried out in two varieties of sugarcane (Saccharum ofcinarum)
does not follow the obtained results in janeiro grass. This is because
the LD
50
mutagenic treatment of ROC22 callus was 0.1% EMS for
17 h, while, for callus FN39, was 0.1% EMS for 14 h.
In the present investigation at 24 and 48 hours, the lowest
concentrations of EMS showed higher values. This agrees with
Sharma et al. (2005) who reported a higher mutagenic efcacy at
low concentrations of EMS in beans. According to Konzak et al.
(1965), the efciency at low concentrations of a mutagenic agent
is due to biological damage that increases with increasing dose at a
faster rate than mutations.
Growth vigor
Table 4 displays the values of the evaluated characteristics.
Those values show the growth and development processes of
janeiro grass at 95 days. The data was used to make a comparison
with a descriptive analysis and to determine the vigor of growth.
It can be evidenced that the dose of 0.50% EMS at 24 hours and
0.25% EMS at 48 hours of impregnation stood out statistically in
all variables (height, stem diameter, number of leaves, length and
width of the leaf); presenting better physiological development. On
the other hand, the plants showed growth retardation at a higher
dose of EMS.
The height of the plant varies depending on the doses of EMS.
There were signicant differences between treatments, the higher
the dose the lower the growth. These results agree with what
was expressed by Rojas et al. (2016) on optimal concentration
of EMS in Bean (Phaseolus vulgaris L.) cv. ‘DOR 364’ whose
high doses negatively inuenced the variables related to growth
and development. On the other hand, in coriander cultivation
(Coriandrum sativum L) Kumar and Pandey (2020) mention that
the height of the plant is reduced in high concentrations of EMS.
However, the treatments with a lower concentration of EMS (0.1%
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Table 4. Physiological development at 95 days in janeiro grass by
EMS.
Treatments
Height
(m)
Stem
diameter
(mm)
N° of
sheets
Blade
length
(cm)
Blade
width
(cm)
T7 1.54
a
4.70
a
11.60
a
38.9
a
1.99
a
T3 1.33
b
4.40
abc
9.90
ab
36.9
ab
1.93
a
T8 1.23
bc
4.60
ab
9.70
abc
36.7
ab
1.71
abc
T4 1.21
bc
3.90
bcd
9.80
abc
34.8
abc
1.80
ab
T2 1.18
bc
4.00
abcd
10.20
ab
34.8
abc
1.84
ab
T9 1.06
cd
3.60
de
8.70
bc
34.7
abc
1.49
c
T6 1.06
cd
2.80
abcd
8.80
bc
33.3
bc
1.88
ab
T1 1.05
cd
3.80
cd
8.40
bc
34.5
bc
1.87
ab
T10 0.94
d
3.80
cd
8.70
bc
32.2
c
1.59
bc
T5 0.91
d
4.00
de
7.60
c
32.3
c
1.47
c
Average 1.15 3.96 9.34 34.91 1.76
Signicance ** ** **
** **
CV (%) 11.14 13.50 16.83 8.45 11.52
ns = not important; **: Very signicant (<0,0001)
and 0.3%) had a stimulating effect on plant height compared to the
control. The reduction in the height of janeiro grass could be attributed
to the inactivation of auxins in the plant by increasing exposure to
EMS (Ashok et al., 2009). Mutagens can inhibit an energy supply
system resulting in mitosis inhibition that may be associated with
seedling growth depression (Emrani et al., 2011).
The diameter of the evaluated stem presented signicant
differences (p<0.0001), which shows that it increases as time passes
and the dose of EMS decreases. Mostafa (2015) point out that
in Mahogany (Khaya senegalensis) seedlings impregnated with
dimethyl sulfate (DMS) the stem diameter is reduced from 1.70 cm to
0.70 cm when increasing the doses with respect to the control.
The foliar emission in the janeiro grass is affected by the doses
and hours of impregnation of EMS applied, decreasing the number of
leaves. This is compared with the study carried out by Akhtar (2014)
who evaluated mutagens on the morphological behavior of tomato
(Solanum lycopersicum); where the highest number of leaves was
counted in doses of 8 mM (millimole) EMS (70 leaves) and 16 mM
(61 leaves). As the dose level of EMS increased, number of leaves
decreased from 29 to 20 at 24 mM and 32 mM, respectively.
Regarding the length of the leaf, it was evidenced that there
was high signicance (p=0000.1) obtained a better longitudinal
development in T7 with respect to the control. A study carried out by
Suthakar and Mullainathan (2015) in Sorghum bicolor L. mentioned
that, in the case of leaf length, they obtained maximum development
with EMS doses at 40 mM. In a study carried out by Hasang et al.
(2020) in janeiro grass with gamma rays (60co), it was shown that
some variables were higher with 52 Gy irradiation compared to the
control treatment. What showed a stimulating effect of irradiation.
Table 5 shows the averages of the growth vigor variable for
treatments T3 and T7 reached values of 1.80. Consequently, the plants
are considered as very vigorous since they t with the value 1 of the
CIAT scale that was used to evaluate vegetative vigor. Note that those
values were obtained from a descriptive category, where the doses for
the treatments T3 and T7 were 0.50% EMS at 24 hours and 0.25% of
EMS at 48 hours, respectively.
Table 5. Growth vigor at 115 days and in the presence of spittlebug,
in janeiro grass by EMS.
Treatments Growth vigor Category
T1 3.70 Vigorous plants
T2 3.40 Vigorous plants
T3 1.80 Very vigorous plants
T4 5.00 Intermediate or normal plants
T5 7.80 Plants less vigorous than normal
T6 3.20 Vigorous plants
T7 1.80 Very vigorous plants
T8 5.20 Intermediate or normal plants
T9 8.00
Plants less vigorous than normal and very
weak and small plants
T10 8.40
Plants less vigorous than normal and very
weak and small plants
Number of spittlebugs per treatment at 60 days of inoculation
Table 6 shows the values of the number of spittlebugs at 60 days
of inoculation in each treatment. For which we proceeded to extract
the nymphs from each plant per study, which allowed us to dene
that the treatments T1 and T6 0.00% of EMS at 24 hours and 0.00%
of EMS at 48 hours, respectively, presented a greater population of
spittlebug. Followed by treatments T2 and T3 that showed between
15 and 17 nymphs. Finally, the treatments with the highest dose of
EMS 1.00% at 24 and 48 hours showed a lower number of spittlebugs
with 2.00 nymphs.
Table 6. Spittlebug number at 60 days after inoculation, in janeiro
grass using EMS
Treatments Initial Population Population Final
T1 5 nymphs 19.00 nymphs
T2 5 nymphs 15.00 nymphs
T3 5 nymphs 13.00 nymphs
T4 5 nymphs 4.00 nymphs
T5 5 nymphs 2.00 nymphs
T6 5 nymphs 20.00 nymphs
T7 5 nymphs 17.00 nymphs
T8 5 nymphs 7.00 nymphs
T9 5 nymphs 3.00 nymphs
T10 5 nymphs 2.00 nymphs
Total 50.00 nymphs 102.00 nymphs
Regarding the number of nymphs, the results obtained at 60 days
show that there was a total increase of 102.00 nymphs during the
evaluation. During this period, the spittlebug nymphs went through
the different stages (egg, nymph, adult).
Pest severity per plant
Table 7 shows the average severity of each treatment under study,
for which the number of spittlebugs per plant was used. Using the
severity scale, we can observe that treatments T4 and T5, with doses
of 0.75 and 1.00% of EMS impregnated at 24 hours, and treatments
T9 and T10, with doses of 0.75 and 1.00% of EMS impregnated at 48
hours, they presented values of 0 live individuals, considering those
treatments without damage based on the severity scale used; while
treatments T3 and T8 with doses of 0.50% EMS at 24 and 48 hours,
presented values of 1 living individual and treatments T1, T2 with
doses of 0.00% and 0.25% of EMS at 24 hours and treatments T6 and
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T7 with doses of 0.00 % and 0.25 % of EMS at 48 hours presented
values of 2 live individuals, causing slight and moderate damage,
respectively, such as drying of the foliage, deterioration of its quality
in the control treatments.
Table 7. Severity of the pest (spittlebug) by plants at 60 days after
inoculation, in janeiro grass using EMS.
Treatments
EMS
dose
(%)
Hours of
Impregnation
Nymph presence
per plant / average
Crop
damage
intensity
T1 0.00 24 2 Moderate
T2 0.25 24 2 Moderate
T3 0.50 24 1 Mild
T4 0.75 24 0 No harm
T5 1.00 24 0 No harm
T6 0.00 48 2 Moderate
T7 0.25 48 2 Moderate
T8 0.50 48 1 Mild
T9 0.75 48 0 No harm
T10 1.00 48 0 No harm
The observed spittlebug damage agrees with the description made
by Sotelo et al. (2003). Those entomologists of the CIAT, indicated
that the intense attack of the Cercópids causes total drying of the
foliage, whereas, slight attacks generate a growth retardation, and
reduces the forage production. The damages of this pest have been
registered in humid regions planted with susceptible grasses.
Conclusions
The treatments with doses of 0.25% of EMS at 48 hours, managed
to maintain the best agronomic performance until 95 days, where the
plant height had its best development similarly to the one determined
as LD
50
(0.32%).
The doses of ethyl-metha- sulfonate (EMS) had a direct effect
on the seizure of the stolons of the janeiro grass, presenting a high
deation index in doses higher than 0.50%. Therefore, the best soak
time was 48 hours.
The janeiro grass impregnated with EMS under greenhouse
conditions showed slight damage caused by spittlebug. Thus, it
cannot be stated that there was tolerance in this research work.
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