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Revista Mexicana de Fitopatología
ISSN: 0185-3309
[email protected]
Sociedad Mexicana de Fitopatología, A.C.
México
Alvarez Moya, Carlos; Santerre Lucas, Anne; Zúñiga González, Guillermo; Padilla Camberos,
Eduardo; Feria Velasco, Alfredo
A Model on Agave tequilana Weber for Detection of Damaged DNA from Diseased Cells and Cells
Exposed to Glyphosate
Revista Mexicana de Fitopatología, vol. 19, núm. 1, enero-junio, 2001, pp. 78- 83
Sociedad Mexicana de Fitopatología, A.C.
Texcoco, México
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7 8 / Volumen 19, Número 1, 2001
A Model on Agave tequilana Weber for Detection of Damaged
DNA from Diseased Cells and Cells
Exposed to Glyphosate
Carlos Alvarez-Moya, Anne Santerre-Lucas, Universidad de Guadalajara, CUCBA,
Laboratorio de Genética, Departamento de Biología Celular y Molecular, km 15.5 Carr.
Nogales, Zapopan, Jalisco, México CP 45110; Guillermo Zúñiga-González, Centro de
Investigación Biomédica de Occidente, Laboratorio de Mutagénesis, IMSS, Sierra Morena
800, Guadalajara, Jalisco, México; Eduardo Padilla-Camberos, and Alfredo FeriaVelasco, División de Patología y Biotecnología Ambiental, CIATEJ (SEP-CONACYT),
Av. Normalistas 800, Guadalajara, Jalisco, México. Correspondence to: [email protected]
(Received: February 26, 2001 Accepted: June 1, 2001)
Abstract.
Alvarez-Moya, C., Santerre-Lucas, A., Zúñiga-González, G.,
Padilla-Camberos, E. and Feria-Velasco, A. 2001. A model on
Agave tequilana Weber for detection of damaged DNA from
diseased cells and cells exposed to glyphosate. Revista
Mexicana de Fitopatología 19:78-83.
The comet assay is a valuable tool to assess for genetic
damage induced by chemicals. However, except for one report,
this test has been used in animal cells only. In this study, the
alkaline comet assay was used in order to detect the presence
of lesions in genetic material of Agave tequilana plants. To
do this, a simple methodology was designed to obtain the
nuclei. Three groups of plants were studied: (a) diseased plants
(Agave with wilting) directly taken from crops; (b) healthy
plants obtained by somatic embryogenesis; and (c) healthy
plants exposed separately to a well known mutagen, namely
ethyl methane sulfonate and glyphosate, a herbicide.
Glyphosate is extensively applied to agave crops. All plant
groups, including the diseased plants, showed a statistically
significant difference (p < 0.001) in tail length when compared
to healthy plants. The results indicate that the comet assay
may be used to diagnose genetic damage in any kind of plant
not traditionally used as genotoxicity biomonitors.
Additional keywords: Plant genotoxicity test, embryogenesis,
maleic hidrazide, ethyl methane sulfonate.
Resumen. La prueba del cometa es una excelente herramienta
para evaluar daño genético inducido por sustancias químicas,
sin embargo, salvo por un reporte, ésta ha sido utilizada
solamente en células animales. En este estudio se empleó la
prueba del cometa alcalino con el propósito de detectar la
existencia de lesiones en el material genético de plantas de
Agave tequilana, para ello, se diseño una metodología simple
para la obtención de los núcleos. Tres grupos de plantas
fueron estudiados: (a) plantas enfermas obtenidas
directamente de los cultivos; (b) plantas sanas obtenidas por
embriogénesis somática y (c) plantas sanas expuestas, por
separado, a un mutágeno conocido: etil metanosulfonato y a
un herbicida: glyphosato, el cual, se aplica extensamente en
los cultivos de agave. Todos los grupos de plantas,
incluyendo las enfermas, presentaron una diferencia, respecto
a la longitud de la cola, estadísticamente significativa (p <
0.001) cuando se compararon con las plantas sanas. Los
resultados indican que la prueba del cometa puede ser
utilizada para diagnosticar daño genético en cualquier tipo
de planta no utilizadas tradicionalmente como biomonitores
de genotoxicidad.
Palabras clave adicionales: Pruebas de genotoxicidad en
plantas, embriogénesis, hidrazida málica, etil
metanosulfonato.
To detect genetic damage induced by chemical or physical
agents, a large number of animal testing and bacteria are used
(Ames et al., 1973; Underbrink et al., 1973; Graf et al., 1984;
Heflich, 1991; Galli and Schiestl, 1995). With bioassays, the
genotoxic effect of a specific agent on a test organism is
determined and results may be extrapolated to other
organisms. The design of new assays allowing to detect
genetic damage in any organism where genetic abnormalities
are suspected might be significant. Narendra et al. (1988)
reported a test known as comet assay, used to detect DNA
damage in animals from individual cells. This system uses
cells immersed in an agarose gel. The cells, after being lysed
and the DNA freed are subjected to electrophoresis. The
migration is proportional to both time of electrophoresis and
DNA damage. When chemical or physical agents are
8 0 / Volumen 19, Número 1, 2001
Verschaeve (1996). Glass slides were first covered with a normal
melting point 1% agarose, which was left to solidify and was
then removed. After this, 300 ml of normal melting point 0.6%
agarose were placed on the slide. Afterwards, 100 ml of low
melting point 0.5% agarose were mixed with 10 ml of nuclear
suspension and the mixture was placed over the first layer. A
third layer with 100 ml of low melting point 0.5% agarose was
added to cover the second layer. Slides with nuclei were
immersed in a lysis solution (2.5 mM NaCl, 10 mM Na2EDTA,
10 mM Tris-HCl, 1% sodium lauryl sarcosine, 1% Triton X100, and 10% DMSO, pH 10) for 24 h at 4°C. After lysis, slides
were placed on a horizontal electrophoresis system with a
high pH alkaline buffer (30 mM NaOH, 1 mM Na2EDTA, pH
13) for 3 h at 4°C. Electrophoresis was carried out for 10 min at
200 mA and 9 volts. Once completed, the slides were washed
with distilled water for one min and immediately stained with
0.5 ml of ethidium bromide (20 ml/1ml) for 15 min. Thereafter,
slides were washed twice with distilled water and a glass
cover was placed over the gel for microscopy observation.
Comets were observed under a Reicher fluorescence
microscope with a 515-560 nm excitation filter. Micrographs
of nuclei were taken with a 40X magnification by using a
color ASA 400 film. DNA migration was measured with a
graded ruler under the microscope eyepiece. Measurements
were made from the center of the nucleus to the end of the
tail.
Statistical Analysis. Data were analyzed with a computer
sofware program Co Stat which contains the ANOVA F-test
for variance and the Student-Newman-Keuls t-test. The
significance of differences between control groups with three
test groups was determined by Dunnett´s t statistic at the
0.05 probability (Ma et al., 1994).
RESULTS
Nuclei of Agave tequilana were stained with orcein (Fig. 1a)
and subjected to the comet assay (Fig. 1b). The tail was
readily observable and made measuring the length possible.
Table 1 shows the tail length in micrometers of controls,
glyphosate treated plants and plants diseased with wilting.
Data in each column are significantly higher than expected
from randomization (p < 0.0001). Even if only differences
between the negative and positive (EMS) controls were
expected, results also show strong differences between the
negative control and the group of diseased plants (p < 0.001)
Table 1. Tail length (mm) observed in Agave tequilana nuclei of the groups studied.
Number
Healthy plants
Plants with
Plants treated with
Plants treated
nuclei
(Negative control)
wilting disease ethylmethane sulfonate with glyphosate
1
95
113
150
200
2
90
113
138
200
3
75
113
175
170
4
75
113
175
170
5
75
113
138
170
6
73
113
175
170
7
70
113
175
160
8
73
113
163
160
9
73
125
150
150
10
88
125
125
150
11
88
125
175
150
12
88
125
175
150
13
95
125
150
150
14
97
125
175
150
15
88
125
16
95
125
17
80
138
18
95
138
19
88
138
20
100
138
21
95
22
88
23
88
24
95
25
95
26
95
Mean 86.808 ± 9.282
122.80 ± 10.402
159.93 ± 17.709
164.29 ± 18.127
Data in each column are significantly higher than expected from randomization (p < 0.0001).
8 2 / Volumen 19, Número 1, 2001
applied to plants either cultivated for commercial purposes
or not, which are in contact with pesticides, chemical
contaminants or excessive solar incidence. We do not think
the test would be useful to detect indirectly presence of
biological pathogens because they don’t produce genetic
damage (except virus); the comet assay system will detect
only damaged DNA. Hence, finding the genetic material status
of Agave tequilana crops diseased with wilting, is particularly
significant because of the use of a large amount of herbicides,
in many cases without the proper technical advice. It is
possible that the etiology of agave wilting could be due to
excessive application of pesticides. One of the most widely
used herbicides in agave crops is glyphosate. It is used in
80% of comercial A. tequilana. The literature has been
contradictory about this herbicide; the IPCS (1994) accepts it
is only somewhat toxic and not genotoxic, but other authors
report certain genotoxic (Sram et al., 1998) and carcinogenic
(Moses, 1992) activity. Thus, it was very important to evaluate
the effect of glyphosate directly on Agave tequilana cells
and, at the same time, find out if genetic damage in sick plant
cells is present at all. To achieve this, it was necessary to
develop a simple methodology to separate nuclei. This
worked out properly (Fig. 1), even, if we expected to see only
DNA lesions on cells exposed to EMS, we were surprised to
find genetic damage on cells of diseased plants and plants
exposed to glyphosate. While there are differences statistically
significant between diseased plant cells and cells of plants
exposed to glyphosate (p < 0.001), both groups are also
different from the negative control (p < 0.001). Even if there is
evidence to indicate this, we cannot claim that in diseased
Agave tequilana crops, DNA is damaged due to the
glyphosate effect because other chemical or physical agents
could also have caused it. Because of new agave crops
developed from plantules (asexual reproduction), current
crops are virtually the same as those available a century ago.
This could lead, maybe, to mutations in the Agave tequilana
DNA, causing a greater sensitivity to fungi and bacteria
(Erwinia and Fusarium) naturally occurring in the plant. At
early stage, the mutated plants can´t be differentiated from
the healthy ones, so the growers use them indiscriminately
for propagation. Thus an early test of DNA damage would be
very useful. Moreover, possibly mutated plants resistant to
wilting could be obtained too. Although some authors
attribute the disease to Erwinia (Romero, 1998), it cannot be
ruled out a possible participation of a genetic damage of A.
tequilana in the physiopathology of the disease, which needs
to be further investigated.
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