Mitotic index and pollen viability of Helianthus annuus l . , after treatment with Pb ( NO 3 )

Earlier research indicates that the presence of the lead in the soil demonstrates inhibitory influence on mitosis, meiosis, fertility and the complete growth and development of the plant. Thus this experiment aimed to investigate the influence of known concentrations of lead applied as Pb(NO3)2, on the mitotic cycle and fertility of the sunflower (Helianthus annuus L.). In the process, this cytotoxin was added in various proportionally increasing concentrations (40, 80, 160, 320 mg/kg) in two separate growth phases, in elongation phase and in flowering phase. It has been analysed what was the influence of various concentrations of lead over sunflower production of fertile and sterile pollen grains in correlation with the miotic index. Changes of the mitoitic index (MI) of the treated plants were detected, which was the main indicator for proliferation status of the cell population, and proportionally the increase of lead concentration in the soil resulted in decreasing of fertility and increasing of sterility. The obtained results on the influnce of lead on the cytogenic parameters, and the influence on development of this important agricultural crop, indicate the importance of undertaking activities, greater responsibilities and caution of mankind, being the main factor for waste water release and agricultural activities, i.e. inappropriate use of agricultural chemicals.


Introduction
Different chemicals in nature may have negative influence on plants, especially the heavy metals (Påhlsson 1989;Barceló & Poschenrieder 1990;Nagajyot et al. 2010;de Vries 2013).Heavy metals can cause DNA damage and various chromosome abnormalities, and their cancerous effects on animals and humans are connected with their mutagenic activities (Aitken & Krausz 2001;Ercal et al. 2001;Bal & Kasprzak 2002;Hengstler et al. 2003;Gichner et al. 2006).Some metals like cadmium, mercury, lead and arsenic precipitate on the upper horizontal soil profile.In most cases, heavy metals are accumulated on the upper layer of the soil, due to the fact that pedogenic processes of soil formation were not effective long enough after pollution to cause distribution of the metals in the soil profile.Heavy metals are absorbed through plant roots from the soil solution and only a small part through the above ground atmosphere.Generally, there are two aspects of the interaction of plants and heavy metals: heavy metals can have negative influence on plants, or plants can express some of the protection mechanisms, including detoxification (Baker 1981;Kumar et al. 1995;Salt et al. 1995;Brooks 1998;Mejáre & Bülow 2001;Alkorta et al. 2004;Babula et al. 2008;Shah et al. 2010;DalCorso 2012;Ali et al. 2013).Ac-cording to Van Assche and Clijesters (1990), only some of the metals are phytotoxic and those are fractions that enter into the cell metabolism.
It was determined that plant species genotypes show significant differences in pollution tolerance (Antonovics et al. 1971;Gartside & McNeilly 1974;Shaw 1989;Hall 2002).Absorption of lead in most plants is more intensive through their roots, rather than through the above ground parts (Ouzounidou et al. 1995).Accumulation of lead in the roots could be one type of protection of the above ground parts from high concentration of this element from the surrounding environment.Lead is considered to be a general protoplasmic toxin, which is cumulative and affects slowly and with subtlety.Lead contaminated soil causes significant lowering productivity of different plants, and that represents serious problem in agriculture (Mulev 1997).Numerous data point to the damaging effects of the excess concentrations of lead and other heavy metals on the growth and development of a plant organism (Degrassi & Rizzoni 1981;Druskovic 1984;Lefebre & Vernet 1990;Neuman et al. 1995;Cuéllar et al. 1999;Cvetanovska et al. 2005;Vasilevska 2005;Dimeska et al. 2006Dimeska et al. , 2007)).Taking into consideration the frequent toxic influence, this research was aiming to investigate the influence of the excess concentrations of lead on the growth and the development of the sunflower (Helianthus annuus L.), an annual plant and one of the most impotant spieces in Asteraceae family from economical point of view.Primarily, we examined the effects detected on cytogenetic level, i.e. in mitotic division of the root meristem of the sunflower, as well as the fertility of pollen seeds as an indicator for regularity of the meiotic karyokinesis.Aiming to precisely determine the pollution effects, known concentrations of Pb(NO 3 ) 2 are applied on soil.

Material and Methods
Mature commercial sunflower seeds were planted in containers of 10 kg each, and various concentrations of Pb(NO 3 ) 2 were applied on the soil (40, 80, 160, 320 mg/ kg).Simultaneously, three groups were formed: a control group, the group consisting of treated sunflowers in elongation phase and the group consisting of sunflowers treated in flowering phase.
The material for cytological analyses was processed according to Tjio & Levan (1950), as well as the standard "squash" method.Isolated roots were treated with 8hydroxyquinoline (0,002 М), for 24 hours, on 4 0 C temperature, then fixed in a Clarke's solution (absolute ethanolglacial acetic acid in 3:1 ratio), for 48 hours, on 4 0 C temperature.After fixation, they were transferred in 75% ethanol, for 24 hours.Roots were put for hydrolysis in 0,1N HCL, for 15 minutes on 60 0 C.Staining of the chromosomes is performed with leuco-basic fuchsin (Darlington & La Cour 1962) and Gomori's hematoxylin according to Konstantinov et al. (1985).
Mitotic index (MI) is calculated as a ratio of cells in mitosis to the total number of meristem cells, given in percentage.
For pollen analyses flower heads were treated with Clarke's fixative, and then transferred in 70% ethanol.Pollen grains were stained according to the Alexander method (1969), where fertile pollen grains are stained red and sterile green (Petrovic & Vuchenovic 1992).Pollen fertility was expressed in percentage, as a ratio of the number of viable pollen grains to the total number of pollen grains.

Results and discussion
The results obtained in this research indicate that the applied concentrations of lead in the sunflower's soil cause changes of the mitotic index of the plant which is the main indicator of the proliferation status of the cell population.The values of the mitotic index of treated plants show relative decrease compared to control (Tab.Macedonian Journal of Ecology and Environment Various concentrations of Pb(NO 3 ) 2 applied on the sunflower in elongation phase, caused decrease in pollen viability proportional to the concentration increase (Tab.3).In plants treated with Pb(NO 3 ) 2 in their soil in elongation phase, decrease in pollen viability was evident.This change points to the fact that the sunflower, in its growth phase, absorbed part of the applied lead through the root, which caused irregularities in meiosis, resulting in increase of the sterile pollen grains.It is important to indicate that within the frame of fertile seeds' morphological structure, i.e. their appearance, size and form remain unchanged, regardless of the applied lead concentration (Fig. 2).These characteristics are especially important for the fertilization ability and cell selection, emphasized in these processes.This is a result of the great resistance of this crop towards the heavy metals, especially lead, de-pending on the age of the plant (Cvetanovska et al. 2007).There is not significant decrease in pollen viability of plants treated with lead in the flowering phase (Fig. 3).Although soil and plants in the elongation phase were treated with the same concentrations of Pb(NO 3 ) 2 no significant differences were detected.Therefore, there are indications that H. annuus can be considered as a potential phytoremediator of lead, one that manages to protect itself from the excess concentrations and fast translocation of the lead in the upper parts of the plant does not exist (Lefebre & Vernet 1990;Couland et al. 1999).When the application of lead is during the elongation phase, before flowering, which is a period of microsporogenesis, that process remains unimpaired for the mutagen and results in normal cells leading to normal pollen seeds formation (Vasilevska 2005).Mitotic index, acting as main indicator in confirming the effects of the lead, perfectly correlates to the plant condition and its cytogenetics, in various cytotoxic concentrations.In fact, the index constantly gradually decreases as the lead concentrations increase.Differences occur at the mitotic index depending on the phase of development of the sunflower, which has been confirmed in the literature data (Dimeska et al. 2011).With applying of different concentrations in the elongation phase, mitotic index indicated higher values which were different in the flowering phase, which correlates more to the development stage and the nature of this phytoremediator that possesses individual adaptive value mechanisms of protection.

Figure 1 .Figure 2 .
Figure 1.M itotic index of H. annuus sam ples from plants treated w ith P b(NO 3 ) 2 in elongation phase and flowering phase

Figure 3 .
Figure 3. Fertile and sterile pollen seeds of H. annuus, treated w ith 40 m g/ k g Pb(NO 3 ) 2 , concentration I 2

Table 1 .
M itotic index (M I ) of H. annuus root m eristem , treated w ith various concentrations of

Table 2 .
M itotic index (M I ) of H. annuus root m eristem , treated w ith various concentrations of