Cytogenetic Activities of Fungicide on the Root Apicalmeristems of Onion Plant (Allium cepa L.)

 

S. Selvaraju*, M. Vasanth, R. Rajarajan and V. Raghupathy

Department of Plant Biology and Plant Biotechnology, D. G. Vaishnav College (Autonomous), Arumbakkam, Chennai- 600 106, Tamil Nadu, India

*Corresponding Author E-mail: selvarajuvaishnav@gmail.com

 

ABSTRACT:

Higher plants are recognized as excellent genetic models to detect environmental mutagens, and are therefore, frequently used in monitoring studies. When this study, the cytogenetic activities of systemic fungicide (Benomyl) were investigated in the mitotic cell division in onion plant (Allium cepa L.).Onion roots were treated with 1g/L, 2g/L and 3g/L concentrations of benomyl and distilled water as control at 3 hours, 6 hours and 12 hours duration. All the concentrations used, caused several abnormalities in mitotic cell divisions and the Mitotic Index in the onion root tip cells decreased when the concentrations of benomyl solution is increased. Based on our findings, it is reported that benomyl has some negative effects on mitotic divisions in onion root tip cells.

 

KEYWORDS: Benomyl–Allium cepa L.– Root tip – Mitotic Index.

 

 

INTRODUCTION:

In agriculture, plant diseases are controlled primarily by chemicals (pesticides, bactericides, nematicides, etc) P. C. Garciaa et al., 2002.As many as 400 chemicals are being used as pesticides (Grover and Tyagi, 1980). Helsel (1987) estimated that about 17% of applied pesticides are fungicides.

 

Pesticides when used in small amounts have several advantages. However, in high concentrations they act on DNA, plant metabolism and regular cell division (Tripathy et al. 1993). Many genotoxic studies have been carried out to detect the harmful effect of different pesticides have some hazardous effects in addition to their benefits. Their undesirable residues in water, food and in environment may cause health problems.

 

Chromosomal a normalizes induced by some of these compounds were found to be linked with their capacity to induce mutations (Wuu and Grant 1966, Panda and Sharma 1979, Gichner et al., 1982). Chromosomal anomalies produced by pesticides, therefore, have been regarded as reliable evidence of the genotoxicity (Grant 1982, Ma 1982).

 

Fungicides are metabolic inhibitors and their modes of action can be classified into different groups; inhibitors of electron transport chain, inhibitors of enzymes, inhibitors of nucleic acid metabolism, protein synthesis and sterol synthesis (WHO, 1994). When they are used to control fungal diseases by killing the fungus that causes the disease. They are most commonly used against diseases of agricultural crops in many countries of the world. Constant use of these chemicals may result in changing the hereditary constitution of an organism (Wuu and Grant, 1966 and 1967). When some chemicals accumulated within food chain to a toxic level, these chemicals affect directly the public health (Fisun and Rasgele, 2009).

 

There is not much information on their effects on different plants. The few investigated fungicides were found to exert C-mitotic activity and induce chromosomal abnormalities in a number of crop plants (Fiskesjo 1969, Ahmed and Grant 1972, Spasojevic 1974). Some fungicides were also found to induce chromosomal stickiness, bridges and lagging (Bielecki, 1974, Al-Najjar and Soliman, 1980). The interest in the impact of fungicides is mainly related to their toxicity. Like all pesticides, fungicides also affect human health and the environment, hence the need for assessing their effects (Adams and Moss 2008).

 

In context, Dryanowska (1987) and Cantor et al. (1992) showed that the frequency of cancer increases among people who have been exposed directly or indirectly to fungicides. So those should be screened before the use in order to select which are least toxic (Mann, 1977). Generally, toxic effects of environmental pollutants cause genetic damage on plant cells (Kovalchuk et al., 1998, Fisun and Rasgele, 2009).

 

Benomyl was first reported as a fungicide in 1968 and introduced onto the market in 1971 by the U.S Company Du Pont (Tomlin, 1994). It is a systemic, benzimidazole fungicide that is selectively toxic to microorganisms and to invertebrates, especially earthworms. It is used against a wide range of fungal diseases of field crops, fruits, nuts, ornamentals, mushrooms, and grasses. In Turkey, benomyl is used especially in the treatment of Pyricularia oryzae Cav. in rice. Although the field use of pesticides has now become a common practice in rice cultivation.

 

The aim of this study is to determine the influence of benomyl in onion (Allium cepa L.) root tip cells during mitosis.

 

MATERIALS AND METHODS:

Chemistry of Benomyl:

Benomyl (C14H18N4O3) is a colorless crystalline solid (pure compound).It is a broad spectrum benzimidazole carbamate fungicide with molecular weight=290.32 g/mol.(Fig. 1)

 

Fig. I-Benomyl Structure

 

Preparation of onion bulbs:

The plant used as test material was Allium cepa L. The root meristems of Allium cepa consist of diploid (2n= 16) set of chromosomes. Clean and healthy bulbs of onion were chosen for each treatment group. Before starting the experiments, dry scales of bulbs were removed and then the onion bulbs were induced to root by placing them on culture tubes filled with distilled water with the base of the onion touching the surface of the water at room temperature. When the roots reached 1.5 - 2 cm in length, they were treated with different concentrations of fungicide benomyl dissolved with distilled water (1g/L., 2 g/L. and 3g/L.) for 3, 6 and 12 hours. Similarly, distilled water is used as Control.

 

Squash preparation:

For mitotic studies, the root tips of Allium cepa L. were fixed in Acetic acid – Ethyl alcohol1:3 (v/v) mixture for overnight, followed by 5-7 minutes treatment in 45% acetic acid. Then root tips were hydrolyzed in 1N HCl at 60ºC for 5 minutes, followed by staining with 2% Aceto-orcein, following the methods described by Sharma and Sharma (1980). The cover slips were sealed on the slides with clear fingernail polish as suggested by Grant, 1982. After proper fixation and staining, appropriate squash preparations were made for each of the treatment and control.

 

Scoring of slides:

Effects of chemical treatment and control on different slides were observed under light microscopy. The mitotic index (MI) was calculated and different types of chromosomal aberrations were also observed and scored.

 

RESULTS AND DISCUSSION:

Mitotic Index (M. I):

According to Smakakinel et.al. (1996) mitotic index is an acceptable measure of cytotoxicity for all living organisms. Mitotic index and chromosomal aberration analysis of A. cepa root tip assay are used to detect potential genotoxicity of chemical substances (Kumar and Panneerselvam, 2007; Abu and Mba, 2011).Induction of mitotic abnormalities on root tip cells of plants may cause a decrease in mitotic index (Panneerselvam et al., 2012).

 

In the present study, benomyl decreased the mitotic index at all concentrations and at all treatment periods when compared with control. Similar type of result is also found by Fisun and Rasgele (2009) on Allium cepa L. by using fungicide Raxil. The decrease of mitotic index was dose dependent. At all treatment periods, the highest concentration of benomyl decreased mitotic Index more than other used concentrations (Table-I) (Fig-II). Sudhakar et.al (2001) the decrease in mitotic index may be due to inhibition of DNA synthesis at S- phase. Since it decreased the M. I in root tip cells of Allium cepa L. Benomyl can be accepted as a toxic agent in this study.

 

Table- I: Mitotic indices of the root apical meristems of Allium cepa L. treated with Benomyl

Duration (Hours)

Concentration

(g/ L)

Total No. of Cells analyzed (N)

Total No. of divided cells (n)

Mitotic Index (M. I) = n x 100

                                        N

 

3

Control

538

215

39.96

1

522

196

37.54

2

526

181

34.41

3

524

166

31.67

 

6

Control

526

224

42.58

1

531

142

26.74

2

514

112

21.78

3

518

97

18.72

 

12

Control

541

260

48.05

1

512

76

14.84

2

520

58

11.15

3

516

32

06.20

Chromosomal aberration:

Benomyl significantly increased the percentage of aberrated cells at all concentrations and treatment periods in mitotic cell divisions when compared with control. It has been shown by many investigators that several other fungicides induce chromosomal aberrations in different plants (Badr, 1998; Pandy et al., 1994; Armbruster et al., 1991; Badr, 1983; Behera et al., 1982 and Mann, 1977).

 

In our study we observed some abnormalities in meristematic cells of Allium cepa L. The most common aberrations were fragments, bridges, C- Mitosis, stickiness, ring chromosome, disturbed anaphase, metaphase and telophase in cell division (Table- II) (Fig- II). The genotoxic effects were noticed in the form of chromatin bridges, chromatin fragments and ring chromosomes. Ring chromosomes are the result of loss of chromosomes from the telomeric side. Chromatin bridges could happen during the translocation of the unequal chromatid exchange and cause structural chromosome mutation. This type of aberration was also observed in the mitosis of Viciafaba and Allium cepa after treatments with food additives (Gomurgen, 2005 and Turkoglu 2007). Disturbed metaphase, anaphase and telophase might be due to the disturbance of the spindle apparatus. The chromosomal damage produced by chemicals may to their effect on DNA (Grant, 1978).

 

Fig- II: Mitotic Indices of root apical meristems of Allium cepa L. treated with Benomyl at various level of concentration and duration

 

Fig- III Benomyl induced aberrations in root apical meristems of Allium cepa L.

 

Table-II: Benomyl induced chromosomal aberrations in root apical meristems of Allium cepa L.

 

Concentration (g/ L)

Total No. of divided cells (N)

Total No. of aberrant cells (n)

% of aberrant cells =  n X 100

                                         N

 

3

Control

215

0

0

1

196

06

3.06

2

181

15

8.28

3

166

21

12.65

 

6

Control

224

0

0

1

142

32

22.53

2

112

30

26.78

3

97

32

32.98

 

12

Control

260

0

0

1

76

37

48.68

2

58

35

60.34

3

32

24

75.00

 

 

CONCLUSION:

Cytogenetic activities of fungicide (benomyl) were investigated in root meristems of Allium cepa L. Higher concentration and longer duration of treatment is toxic to cells.In our opinion, more detailed studies should be done on different types of the chemicals, which are used as pesticides.

 

The outcome of this study suggests, safety measures to farmers avoid direct contact with high concentration of benomyl contaminated mud while working in the fields or in fields irrigated with benomyl contaminated surface/ ground water and increase public awareness about ill effects of fungicides in water, food and the environment. Meanwhile the use of this fungicide should be under control in agricultural fields.

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Received on 24.10.2014          Accepted on 25.11.2014        

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