A possible alternative to synthetic agricultural chemicals is through the use of allelopathy. Adonis species are rich sources of secondary metabolites. Such allelochemicals offer potential for the development of future pesticides. Allelochemicals influence plant growth and cause morphological alterations. This visible effect could be due to primary effects at cellular or molecular level. Changes in the mitotic activity and disturbances in different phases of mitotic division are accepted as indicators of cytotoxic influence. Mitotic abnormalities and induction of micronuclei in interphase cells are parameters used to determine genotoxicity. The purpose of the current study was to establish the possible allelopathic effect of Adonis vernalis L. water extracts through evaluation of root growth inhibition effect and cytogenetic alterations. Adonis vernalis L. growing wild in Bulgaria was used in the present study. Two types of water extracts were prepared: Hot and Cold Water Extract of A. vernalis (HWЕА and СWЕА). A 72-h root growth inhibition test was provided in order to determine the toxicity level of extracts. EC50 values were determined. For toxicity test, seeds of Triticum aestivum L. cv. GTW were used. Cytotoxic and genotoxic potential of water extracts (EC50) were evaluated using Allium cepa L.-test. The EC50 for HWEA and CWEA was determined 1.83 g/l and 0.78 g/l respectively. Significant influence on mitotic activity values and a marked decrease in percentage of telophase cells were observed after treatment with both extracts. Adonis extracts also induced different mitotic abnormalities in root-tip cells of Allium cepa L. The percent of interphase cells with micronuclei increased significantly only after treatment with HWEA. The results indicated growth inhibitory, cytotoxic and genotoxic effects of crude water extracts of A. vernalis L. These effects demonstrated the presence of water soluble allelochemicals in Adonis aerial parts.
Agrochemicals used in conventional agriculture pose health and environmental risks. Alternative of synthetic agrochemicals is via allelopathy. Allelopathy has been defined as suppression of growth of one plant species by another through release of toxic substances [
Adonis vernalis L. (Ranunculaceae) has been used in traditional medicine for centuries as cardiotonic, diuretic, sedative and vasoconstrictor [
Commonly cited effects of allelopathy include reduced seed germination and seedling growth. Morphological changes in response to allelochemicals could be due to effects on cellular or molecular level. So, establishment of primary action of allelochemicals is of great importance [
The purpose of the current study was to establish the possible allelopathic effect of Adonis vernalis L. water extracts through evaluation of root growth inhibition effect and cytogenetic alterations.
A. vernalis growing wild in the vicinity of Shumen (Jivkovo, Bulgaria) (latitude 43˚43'N; longitude 27˚00'E, altitude 300 m) was used in this study. The aerial parts of plants were collected at the flowering stage. The plant specimens were identified and authenticated by Zh. Nanova (Taxonomist), Faculty of Natural Sciences, Shumen University, Bulgaria. Collected plant materials were dried at room temperature.
Aerial parts of plants cut about 20 cm from the top were used in laboratory tests. Two types of water extracts were prepared: 1) Hot Water Extract of A. vernalis (HWЕА)―the dried stems, leaves and flowers were covered with boiling distilled water, left for 60 min and then allowed to cool to room temperature; 2) Cold Water Extract of A. vernalis (СWЕА)―the dried plant tissue was placed in distilled water and left to stay for 24 h at room temperature.
The test solutions were prepared at concentrations: HWЕА―1, 2, 3, 4 g/l; СWЕА―0.2, 0.4, 0.6, 0.8, 1 g/l. The solutions were prepared before each experiment.
Seeds of Triticum aestivum L. cv. GTW were thoroughly rinsed with tap water and sterile distilled water. After that seeds were placed between two sheets of filter paper and dried at 25˚C.
Thirty seeds were placed on filter paper in each of ten Petri dishes (11 cm in diameter). Five ml of each extract or distilled water as a control were applied to the seeds. The dishes were sealed and incubated at 25˚C ± 1˚C for 72 h. The length of the roots of germinated seeds was measured. The percentage root growth inhibition in relation to the control for each extract was determined. Seeds that did not germinate were not included in the root elongation test. A growth curve was drawn: root length as percent of control (ordinate) against test concen- trations (abscissa). From the growth curve EC values were obtained: EC50 = the effective concentration that decreased root growth about 50% when compared to the negative control group (distilled water, 100%) [
To determine the possible cytotoxic and genotoxic effects EC50 concentrations of extracts were used in Allium cepa-test. Thirty seeds of A. cepa were placed on filter paper in each of three Petri dishes (11 cm in diameter), containing 5 ml of distilled water. The Petri dishes were sealed and incubated at 25˚C ± 1˚C for 72 h. Twenty germinated seeds with equal length of roots (~1 cm) were removed and placed on filter paper in each of another three Petri dishes. Five ml of water extract (at concentration corresponding to EC50 value) were added to two dishes, and incubated at 25˚C ± 1˚C for 3 h. Distilled water was used as a negative control and methyl methanesulfonate (11 mg/l, for 24 h) was used as a positive control. After treatment, the roots were fixed in Clarke’s fixative (95% ethanol: acetic acid glacial, 3:1) for 90 min, hydrolysed in 3 N HCl for 8 min and in 45% acetic acid (CH3COOH) for 30 min at room temperature and stained for 40 min in 2% aceto-orcein. After staining, the terminal root tips (1 - 2 mm) were cut off and squashed in 45% CH3COOH. Each sample consisted of six root meristems. At least 1000 cells of each root meristem were analyzed. The microscopic analysis included estimation of the mitotic indices and aberrant cells. The mitotic index was determined as a ratio between the number of cells in mitosis and the total number of analyzed cells. The index of each phase of mitotic division was calculated as a ratio between the cell number in the respective period and the number of dividing cells. Abnormalities in mitotic cells were evaluated. Interphase cells were analyzed for the presence of micronuclei.
Experimental data were processed by Student’s t-test. In root inhibition test we chose as an experimental unit the root. The calculations were carried out on the assumption that roots used in each treatment made one sample, and each sample was tested against the control sample. In Allium cepa test we chose as an experimental unit the cell, instead of the root. The calculations were carried out on the assumption that all the cells of the six root meristems made one sample, and each sample was tested against the negative control.
A 72-h root growth inhibition test was provided in order to determine the toxicity level of hot (HWEA) and cold (CWEA) extracts. EC50 (value that reduces the control group root length by half) was calculated from the data collected. For toxicity test, seeds of T. aestivum were used. T. аestivum is proved to be appropriate test object in toxicity assays [
The effects of HWEA on root growth of T. aestivum seeds revealed dose dependent increase of the inhibitory effect. The EC50 value for HWEA was determined―1.83 g/l. The cold extract had stronger inhibitory effect in comparison to hot extract. Respectively, the EC50 value for CWEA was about two fold lower than EC50 for HWEA―0.78 g/l.
The root growth inhibition test is proved to be useful tool for detection of the concentrations used for cyto- toxicity and genotoxicity evaluation of different chemical compounds [
We evaluated the possible cytotoxic and genotoxic effects of Adonis water extracts using Allium cepa-test at concentrations, corresponding to determined EC50 values. Treatment time of 3 h was used [
The elongation of the roots depends on different physiological processes, including cell division [
The choice of concentration tested in cytogenic studies is of importance since the chromosome aberration score is possible only if mitotic index value is above 1% [
Adonis water extracts also induced mitotic abnormalities in root-tip cells of Allium cepa (
Abnormal metaphases and anaphases are indicators of microtubule malformation effect of Adonis extracts [
Sample | Cells analyzed (number) | Dividing cells (number) | MI% (±SD) | Prophase PhI% (± SD) | Metaphase PhI% (± SD) | Telophase PhI% (± SD) |
---|---|---|---|---|---|---|
NC | 9013 | 513 | 5.69 ± 0.23 | 26.71 ± 0.44 | 25.54 ± 0.44 | 28.07 ± 0.45 |
HWEA | 8792 | 408 | 4.64 ± 0.21** | 29.17 ± 0.46 | 27.94 ± 0.45 | 21.08 ± 0.41** |
CWEA | 7850 | 367 | 4.68 ± 0.21** | 31.07 ± 0.46 | 24.52 ± 0.43 | 20.16 ± 0.40** |
PC | 7004 | 265 | 3.78 ± 0.19*** | 20.76 ± 0.41* | 31.70 ± 0.47* | 20.76 ± 0.41* |
Sample: NC: negative control (distilled water); HWEA: Hot Water Extract of A. vernalis; CWEA: Cold Water Extract of A. vernalis; PC: positive control (methyl methanesulfonate, 11 mg/l); MI%: Mitotic Index (%), PhI%: Phase Index (%).Data are expressed as means ± SD (standard deviation), *P ≤ 0.05, **Р ≤ 0.01, ***Р ≤ 0.001.
Sample | Abnormalities in mitotic cells | ||||||
---|---|---|---|---|---|---|---|
Number of cells analyzed | Spindle abnormalities in metaphase (%) | Spindle abnormalities in anaphase (%) | Laggard and vagrant chromosomes (%) | Bridges in ana-telophase (%) | Fragments in ana-telophase (%) | Total abnormalities in mitotic cells (% ±SD) | |
NC | 513 | ? | 1.52 | 0.20 | 0.20 | 0.20 | 2.12 ± 0.15 |
HWEA | 408 | 1.96 | 3.19 | 1.20 | 2.45 | 3.19 | 11.99 ± 0.33*** |
CWEA | 367 | 1.36 | 4.63 | 1.09 | 1.09 | 2.45 | 10.62 ± 0.31*** |
PC | 265 | ? | 0.75 | 5.28 | 1.51 | 9.43 | 16.97 ± 0.37*** |
Sample: NC: negative control (distilled water); HWEA: Hot Water Extract of A. vernalis; CWEA: Cold Water Extract of A. vernalis; PC: positive control (methyl methanesulfonate, 11 mg/l). Data are expressed as means ± SD (standard deviation), ***Р ≤ 0.001.
It must be noticed that some aberrant cells are scored in untreated control. Presence of chromosome aberrations in negative control is recorded in other studies as well [
The frequency of micronuclei in interphase cells is presented in
Micronuclei represent extranuclear bodies of chromatin material (
Allelopathic effects could result from the interaction of different secondary metabolites. Crude extracts of A. vernalis are used in traditional medicine. Such extracts possess proved biological activity. The results of the present study also indicated growth inhibitory, cytotoxic and genotoxic effects of crude water extracts. Cytotoxicity and genotoxicity tests were provided using concentrations corresponding to EC50 value: 1.83 g/l for HWEA and 0.78 g/l for CWEA. At concentrations corresponding to EC50 both extracts inhibited cell division at one and the same extent, but HWEA exerted stronger genotoxic effect in comparison with CWEA.
Adonis water extracts decreased root length of T. aestivum, inhibited cell division and induced chromosomal alterations in Allium cepa. These results demonstrated the presence of water soluble allelochemicals in Adonis aerial parts.
Sample | Number of cells analyzed | Micronuclei (number) | Micronuclei, % (mean ± SD) |
---|---|---|---|
NC | 8500 | 12 | 0.14 ± 0.04 |
HWEA | 8384 | 31 | 0.37 ± 0.06** |
CWEA | 7483 | 15 | 0.20 ± 0.05 |
PC | 6739 | 33 | 0.49 ± 0.07*** |
Sample: NC: negative control (distilled water); HWEA: Hot Water Extract of A. vernalis; CWEA: Cold Water Extract of A. vernalis; PC: positive control (methyl methanesulfonate, 11 mg/l). Data are expressed as means ± SD (standard deviation), **Р ≤ 0.01, ***Р ≤ 0.001.
This work was supported by the “European Social Fund 2007-2013, Human Resources Development Opera- tional Programme” under Grant (BG051PO001-3.3.06-0003) and the Bulgarian Ministry of Education and Science under Grant (RD-08-213/10.03.2014).
Asya PenchevaDragoeva,Vanya PetrovaKoleva,Zheni DimitrovaNanova,Borislav PetrovGeorgiev, (2015) Allelopathic Effects of Adonis vernalis L.: Root Growth Inhibition and Cytogenetic Alterations. Journal of Agricultural Chemistry and Environment,04,48-55. doi: 10.4236/jacen.2015.42005