Effects of heavy metals on male gametes of sweet cherry

Abstract

Fruit set is affected by different environmental, biological, physical and chemical factors. Abnormal conditions in pollination, fertilization and fruit set period can reduce orchard yield. Sweet cherry is one of the most important stone fruits of Iran which was ranked third with 200,000 tons in 2012 in the world by an FAO report. Pollen germination and tube growth are the major factors of fruit set in sweet cherry. In polluted cities, this phenomenon may be affected by heavy metals stresses. In this study, the effects of different concentrations of heavy metals – cadmium, lead, copper and mercury by 0 (control), 50, 100, 150, 200 and 250 ppm) – were studied on pollen germination and tube growth of 10 sweet cherry cultivars in vitro. The results demonstrated that both traits were significantly affected by different levels of heavy metals, cultivars and interaction among them. Pollen germination and tube growth of all cultivars were reduced with increase in metals concentrations. In the concentration of 250 ppm of all heavy metals, pollen germination and tube growth was close to 0 in most cultivars. Cadmium showed the highest toxicity on pollen germination and tube growth and among the cultivars, “Zard-eDaneshkadeh” exhibited highest sensitivity, while “Colt” demonstrated highest tolerance to stress from the studied metals.

Keywords:

• Cherry
• pollen germination
• tube growth
• heavy metals

Introduction

Heavy metals, such as cadmium, copper, lead and mercury, are major environmental pollutants, particularly in areas and cities with high anthropogenic pressure, such as Tehran. Inorganic and organic fertilizers, sewage sludge, irrigation waters, fungicides and pesticides, are the most important sources of heavy metals to agricultural fields. Plants growing in these areas can have altered metabolism, growth reduction, lower biomass production and metal accumulation (Nagajyoti et al. 2010). Some of these heavy metals are bio accumulative; they do not break down in the environment and are not easily metabolized. Some metals, including cadmium, lead and mercury, are strongly poisonous to metal-sensitive enzymes, resulting in growth inhibition and death of organisms (Nagajyoti et al. 2010). Higher absorption of such ions is strongly toxic to metabolic actions (Nagajyoti et al. 2010). For example, seed germination, seedling growth, photosynthesis, plant water status, mineral nutrition, enzymatic activities, pollen germination and pollen tube growth can be affected by toxic heavy metals (Wolters and Martensn 1987).

Researchers have reported the inhibitory effect of heavy metals on pollen germination in some plant species. Generally, dicotyledons are more sensitive to these materials than monocotyledons (Baker 1972; Sawidis and Reiss 1995; Sawidis 1997; Gür and Topdemir 2008; Sabrine et al. 2010; Acharya et al. 2011; Mohsenzadeh et al. 2011). In other studies, some intracellular effects of cadmium, chromium and lead on pollen tubes have been described. These metals caused apex expansion and also affected the pattern of structural polymer distribution along the pollen tube cell wall (Sawidis 2008). Effects of some heavy metals on the ultra-structure of organelles and their distribution in pollen tubes have been reported previously. For example, chromium causes chromatin condensation, mitochondria swelling, cytoplasm vacuolization and perturbed arrangement of EPR cisternae. Lead caused water imbalance, alterations in membrane permeability, disturbs mineral nutrition partial disassembly and longitudinally oriented pollen actin bundles in some plants (Breygina et al. 2012). Some studies have demonstrated that copper was the second most toxic metal with respect to seed germination, root elongation and coleoptiles and hypocotyls growth in Triticum aestivum and Cucumis sativus among mercury, cadmium, cobalt, lead and zinc (Gür and Topdemir 2008). Some studies have shown that high concentration of copper causes chromosome anomalies in plant organs. Mercury interferes with mitochondrial activity and induces oxidative stress by triggering the generation of ROS. This leads to the disruption of bio membrane lipids and cellular metabolism in plants (Kappler and Kristen 1987; Kalbande et al. 2008).

Pollens are considered to be highly sensitive to air pollutants. Pollen germination and tube growth are inhibited by various heavy metals, such as copper, lead, cadmium, cobalt and mercury. Thus, the in vitro culture of pollen can provide a sensitive standard system with which the biological activity of various toxic metals at the cellular level can be investigated (Baker 1972; Gur and Topdemir 2005; Sawidis 2008). In most previous studies, the pollens of various plant species have been utilized to determine the cytotoxic effects of environmental pollutants. This is a simple and inexpensive cultivation which does not require sterile conditions, providing fast germination and growth of the tube. Moreover, the absence of chloroplasts allows the pollen tubes to be used as an appropriate representation for the toxicological review of compounds harmful to animals and humans (Sawidis 2008; Semra et al. 2009; Sharafi 2014a).

Currently, there are some reports regarding the existence of heavy metals effects on ornamental and forest trees pollen (Shkarleto 1972; Cox 1983; Chaney and Strickland 1984; Holub and Ostrolucka 1984; Kappler and Kristen 1987; Andrej 1996; Azmat et al. 2006; Kalbande et al. 2008) but less information has been reported regarding the effect of lead and copper on pollen grains and pollen tubes of fruit trees (Bolat and Pirlak 1999; Sharafi 2014a).

Sharafi (2014a) studied the effects of copper and lead on pollen germination traits in five almond cultivars and reported significant effects of cultivars and different concentrations of copper and lead on pollen germination and tube length.

Munzuroglu and Gur (2000) and Munzuroglu et al. (2003) studied the effects of heavy metals on pollen germination and tube growth of apples (Malus silvestris Miller cv. Golden).

Sweet cherry (Prunus avium L.) is one of the most important stone fruit crops of the Rosaceae family. Most sweet cherry cultivars and genotypes are self-incompatible (FAO 2012). Therefore, pollination, fertilization and commercial production are affected by heavy metals in polluted cities such as Tehran. In this research, effects of different concentrations of cadmium, copper, lead and mercury were studied on pollen germination and tube growth of 10 sweet cherry cultivars in vitro.

Materials and methods

In the spring of 2014, 10 sweet cherry cultivars were selected including “Colt”, “Estella”, “Lapins”, “Napleone”, “Takdaneh”, “Gillas-e-Sefid”, “Sourati-e-Lavasanat”, “Siah-e-Shabestar”, “Siah-e-Mashhad” and “Zard-e-Daneshkadeh”, which are grown in different regions of Tehran. Flower buds in balloon stage were gathered and transmitted to the laboratory in Shahed University. Petals and sepals were separated and anthers were isolated from flower buds and placed in Petri dishes for 72 h to release pollens and then pollens were gathered in distilled glasses. Pollen germination percentage and pollen tube growth of cultivars were tested immediately. Pollens planted in the standard in vitro medium (containing 1% agar, 15% sucrose) were treated with 0 (control), 50, 100, 150, 200 and 250 ppm of cadmium (CdCl₂), copper (CuCl₂) lead (PbCl₂) and mercury (HgCl₂) solutions for 24 h at 24°C and finally pollen tube growth was stopped with the addition of chloroform. Pollen germination percentage (PGP) and pollen tube length (PTL) were measured under light microscope. Seven microscopic areas were counted randomly for evaluation of PGP and PTL. When the pollen tube was as long as its diameter, it was considered to have germinated and measurements of pollen tube length were recorded directly by an ocular micrometer fitted to the eyepiece on microscope based on μm scale. Factorial experiment was performed with two factors: (1) cadmium (Cd), copper (Cu), lead (Pb) and mercury (Hg) at six different concentrations; and (2) sweet cherry cultivars; based on completely randomized design (CRD) with six replications (six Petri dishes). Data were analyzed using SAS software and comparison of means was done with Duncan’s multiple range tests (DMRT).

Results

Analysis of variance (ANOVA) of data demonstrated significant differences for pollen germination percentage and pollen tube length among 10 sweet cherry cultivars in different concentrations of Cd, Cu, Pb and Hg (Table 1). Interaction among cultivars and different concentrations of all metals had significant effect on pollen germination and tube length at p < 0.01% (Table 1).

Table 1. Analysis of variances of pollen germination percentage and pollen tube length in five sweet cherry cultivars.

Source of variation (SOV)	Df	Mean square
		Pollen germination percentage					Pollen tube length (μm)
		Cadmium	Copper	Lead	Mercury		Cadmium	Copper	Lead	Mercury
Cultivar	9	296.2**	17,322.2**	690**	1888.2**		460.5**	9818.8**	1800.1**	260.8**
Heavy metal concentrations	5	426.2**	33,881.0**	4375.3**	3319.8**		480.5**	1056.5**	1806.2**	263.5**
Cultivar*Metal	45	58.5**	16,157.5**	74**	1585.5**		4.8**	10.8**	18.4**	20.56**
Error	180	5.2	16.2	5.9	162.1		0.25	0.55	1.0	0.14
Total	239
CV		8.7	11.9	6.9	13.4		13.9	14.8	12.3	12.5

** Significant at p < 0.01% level.

* Significant at p < 0.05% level and ns non-significant.

Comparison of means among different concentrations of Cd, Cu, Pb and Hg showed that by increasing their concentration, pollen germination and pollen tube length were significantly reduced and closed to zero in all cultivars, in 250 ppm (Table 2). For instance, in controls treated with 0 ppm Cd, mean of PGP was 84.6%, whereas in treatments with 250 ppm, mean of PGP was1%, and PTL mean was 99.1 μm in controls while it was 4 μm in treatments (Table 2). Moreover, in controls treated with 0 ppm of Cu, treatment mean of PGP was 87.9%, while it was 8% in 250 ppm concentration, and PTL mean was 140.6 μm in controls while it was only 2.8 μm in treatments (Table 2). Nevertheless, PTL was affected drastically in comparison with PGP in all studied sweet cherry cultivars (Table 2). Comparison of toxicity effects on PGP and PTL among Cd, Cu, Pb and Hg demonstrated that cadmium had the highest toxicity on pollen germination and tube growth among them. It was shown that toxic effect of Cd, Cu, Pb and Hg was as follows: Cd>Cu>Hg>Pb on PGP and PTL in sweet cherry cultivars (Table 2).

Table 2. Mean comparison of pollen germination percentage and pollen tube length among different concentrations of cadmium, copper, lead and mercury.

Concentration (ppm)		Pollen germination percentage				Pollen tube length (μm)
	Cadmium	Copper	Lead	Mercury	Cadmium	Copper	Lead	Mercury
0 (control)	89.1 ± 0.9^a	89.1 ± 0.9^a	89.1 ± 0.9^a	89.1 ± 0.9^a	192.4 ± 2.1^a	192.4 ± 2.1^a	192.4 ± 2.1^a	192.4 ± 2.1^a
50	52.6 ± 0.6^b	55.7 ± 0.6^b	67.4 ± 0.7^b	53.8 ± 0.6^b	76.6 ± 0.8^b	90.9 ± 1.3^b	130.2 ± 1.4^b	50.2 ± 0.6^b
100	25.9±0.3 ^c	29.0 ± 0.3^c	40.7 ± 0.5^c	29.9 ± 0.3^c	39.0 ± 0.4^c	50.7 ± 0.6^c	70.6 ± 0.8^c	20.9 ± 0.3 ^c
150	10.3 ±0.2^d	12.7 ± 0.2^d	24.3 ± 0.3^d	15.9 ± 0.2^d	30.3 ± 0.4^d	40.8 ± 0.5^d	60.4 ± 0.7^d	11.4 ± 0.2^d
200	5.5±0.1^e	7.2 ± 0.1^e	6.2 ± 0.1^e	7.7 ± 0.1^e	18.7 ± 0.2^e	20.8 ± 0.3^e	30.8 ± 0.4^e	7.4 ± 0.7^e
250	0.1 ±0.1^f	0.8 ± 0.1^f	3.2 ± 0.1^f	2.3 ± 0.1^f	0.3 ± 0.1^f	2.8 ± 0.1^f	9.5 ± 0.1^f	2 ± 0.1^f

Note:

Same letters show no significant difference among genotypes of each column.

Means of pollen germination percentage and pollen tube length in copper treated medium were from 29.1% (Lapins) to 33.0% (Gilas-e-Sefid) and 30.9 μm (Lapins) to 92.2 μm (Takdaneh), respectively (Table 3). Moreover, in cadmium treated medium, PGP and PTL were from 27.1% (Siah-e-Sahbestar) to 38.8% (Lapins) and 23.1 μm (Colt) to 60.0 μm (Sourati-e-Lavasanat), respectively (Table 3). However, in lead treated medium, means of PGP and PTL were from 31.2% (Lapins) to 45.1% (Siah-e-Sahbestar) and 47.1 μm (Colt) to 124.6 μm (Siah-e-Sahbestar), respectively. Finally, PGP and PTL were from 27.3% (Siah-e-Sahbestar) to 33.7% (Colt) and 17.8 μm (Colt) to 47.7 μm (Siah-e-Mashhad) in sweet cherry cultivars, respectively (Table 3).

Table 3. Mean comparison of pollen germination percentage and pollen tube length among 10 sweet cherry cultivars treated with cadmium, copper, lead and mercury.

Cultivar		Pollen germination percentage				Pollen tube length (μm)
	Cadmium	Copper	Lead	Mercury	Cadmium	Copper	Lead	Mercury
“Takdaneh”	31.5 ± 0.4^bc	29.8 ± 0.3^bc	34.3 ± 0.4^c	31.2 ± 0.4^b	34.9b ± 0.4^c	92.2 ± 1.3^a	93.6 ± 1.3^e	30.0 ± 0.4 ^f
“Siah-e-Shabestar”	27.1 ± 0.3^c	29.8 ± 0.3^bc	45.1 ± 0.5^a	27.3 ± 0.3^d	35.3 ± 0.4^c	40.8 ± 0.5^k	124.6 ± 2.1^a	30.5 ± 0.4^e
“Zard-e-Daneshkadeh”	26.8 ± 0.3^c	30.9 ± 0.4^b	30.6 ± 0.4^d	27.7 ± 0.3^d	50.9 ± 0.6^b	30.9 ± 0.4^b	99.8 ± 1.3^d	30.7 ± 0.4^d
“Lapins”	38.8 ± 0.4^a	29.1 ± 0.3^c	31.2 ± 0.4^d	28.6 ± 0.3^c	40.3 ± 0.5^bc	46.1 ± 0.5^h	62.5 ± 0.7^h	20.6 ± 0.3k^e
“Sourati-e-Lavasanat”	31.9 ± 0.4^b	29.9 ± 0.3^c	35.6 ± 0.4^c	29.7 ± 0.3^c	63.0 ± 0.7^a	59.3 ± 0.6^f	68.7 ± 0.7^g	26.0 ± 0.3^g
“Estella”	29.4b ± 0.3^c	31.9 ± 0.4^ab	42.3 ± 0.5^b	30.8 ± 0.4^b	47.8 ± 0.5^bc	87.1 ± 0.9^b	79.1 ± 0.8^f	41.6 ± 0.5^c
“Gilas-e-Sefid”	29.2b ± 0.3^c	33.0 ± 0.4^a	42.0 ± 0.5^b	31.8±0.4^ab	37.6 ± 0.4^bc	70.2 ± 0.8^e	118.0 ± 2.1^b	23.6 ± 0.3^h
“Napleon”	28.7 ± 0.3^bc	29.3 ± 0.3^c	33.6 ± 0.4^d	33.8 ± 0.4^a	59.1 ± 0.6^b	81.4 ± 0.9^c	54.8 ± 0.6^l	44.8 ± 0.5^b
“Colt”	28.8 ± 0.3^bc	32.8 ± 0.4^ab	43.6 ± 0.5^b	33.7 ± 0.4^a	23.1 ± 0.3^d	34.6 ± 0.4^i	47.1 ± 0.5^i	17.8 ± 0.2^i
“Siah-e-Mashhad”	27.6 ± 0.3^c	31.9 ± 0.4^ab	39.1 ± 0.4^c	29.3 ± 0.3^b	31.8 ± 0.4^c	50.6 ± 0.6^g	110.7 ± 2.1^c	47.4 ± 0.5^a

Note:

Same letters show no significant difference among genotypes of each column.

However, cadmium demonstrated the highest toxicity on pollen germination and tube growth among metals and cultivar “Zard-eDaneshkadeh” demonstrated highest sensitivity, while “Colt” showed highest tolerance to studied heavy metals stress among the cultivars (Table 3).

Finally, interaction among cultivars and different concentrations of cadmium, copper, lead and mercury significantly decreased both pollen germination percentage and pollen tube length of all sweet cherry cultivars, whereas both traits were closed to zero in all cultivars, in 250 ppm (Tables 4 and 5). For instance, PGP of “Takdaneh” was from 81.8% in the control to 0.9% in the 250 ppm cadmium treated medium. Also, PTL was from 13.5 μm in the control to 0.7 μm in the 250 ppm concentration (Table 4).

Table 4. Comparison of means for pollen germination percentage and pollen tube length (interaction among five sweet cherry cultivars and different concentrations of cadmium, copper, lead and mercury.

Cultivar		Pollen germination percentage				Pollen tube length (μm)
	Concentrate (ppm)	Cadmium	Copper	Lead	Mercury	Cadmium	Copper	Lead	Mercury
“Takdaneh”	0 (control)	83.0 ± 0.9^a	83.0 ± 0.9^a	83.0 ± 0.9^a	83.0 ± 0.9^a	15.9 ± 0.2 a	15.9 ± 0.2 a	15.9 ± 0.2 a	15.9 ± 0.2 a
	50	56.6 ± 0.6^b	56.5 ± 0.8^b	65.6 ± 0.6^b	52.7 ± 0.7^b	3.2 ± 0.1 ^b	8.1 ± 0.1 ^b	10.9 ± 0.2 ^b	4.1 ± 0.1^b
	100	29.9 ± 0.3^c	29.8 ± 0.3^c	38.9 ± 0.4^c	26.0 ± 0.3^c	3.0 ± 0.1 ^c	4.7 ± 0.1 ^c	6.3 ± 0.1 ^c	2.4 ± 0.1 ^c
	150	13.5 ± 0.4^d	13.4 ± 0.2^d	22.5 ± 0.2^d	9.6 ± 0.2^d	2.9 ± 0.1 ^d	4.0 ± 0.1 ^c	5.3 ± 0.1 ^d	2.0 ± 0.1 ^d
	200	6.6 ± 0.1 ^e	8.8 ± 0.1 ^e	4.2 ± 0.1 ^e	7.4 ± 0.1 ^e	2.8 ± 0.1 ^e	1.7 ± 0.1 ^d	2.2 ± 0.1 ^e	0.9 ± 0.1 ^e
	250	0.9 ± 0.1 ^f	0.0 ± 0.0 ^f	3.0 ± 0.1 ^f	1.9 ± 0.1 ^f	0.7 ± 0.1 ^f	0.0 ± 0.1 ^e	0.7 ± 0.1 ^f	0.2 ± 0.1 ^f
“Siah-e-Shabestar”	0 (control)	82.4 ± 0.9^a	82.4 ± 0.9^a	82.4 ± 0.9^a	82.4 ± 0.9 ^a	23.0 ± 0.3^a	23.0 ± 0.3^a	23.0 ± 0.3^a	23.0 ± 0.3^a
	50	46.8 ± 0.5 ^b	55.1 ± 0.6^b	55.0 ± 0.6^b	48.6 ± 0.5^b	2.0 ± 0.1^b	11.8 ± 0.2^b	15.7 ± 0.2^b	6.0 ± 0.1^b
	100	20.1 ± 0.3 ^c	28.4 ± 0.3 ^c	28.3 ± 0.3 ^c	21.9 ± 0.3^c	1.9 ± 0.1^c	6.8 ± 0.1^c	9.0 ± 0.1^c	3.4 ± 0.1^c
	150	6.7 ± 0.1^d	12.0 ± 0.1^d	11.9 ± 0.1^d	7.7 ± 0.1^d	1.8 ± 0.1 ^d	5.7 ± 0.1^d	7.6 ± 0.1^d	2.9 ± 0.1^d
	200	4.4 ± 0.1 ^e	6.3 ± 0.1^e	4.5 ± 0.1^e	6.8 ± 0.1^e	1.5 ± 0.1 ^e	2.8 ± 0.1^e	3.7 ± 0.1^e	1.4 ± 0.1^e
	250	0.7 ± 0.1 ^f	2.6 ± 0.1^f	2.2 ± 0.1^f	1.7 ± 0.1^f	1.2 ± 0.1^f	0.6 ± 0.1^f	0.9 ± 0.1^f	0.3 ± 0.1^f
“Zard-e-Daneshkadeh”	0 (control)	98.0 ± 1.3^a	98.0 ± 1.3^a	98.0 ± 1.3^a	98.0 ± 1.3^a	17.7 ± 0.2^a	17.7 ± 0.2^a	17.7 ± 0.2^a	17.7 ± 0.2^a
	50	68.1 ± 0.7^b	69.2 ± 0.7^b	72.9 ± 0.8^b	69.4 ± 0.7^b	0.3 ± 0.1^b	9.0 ± 0.1^b	12.1^b	4.6 ± 0.1^b
	100	41.4 ± 0.5^c	42.5 ± 0.5^c	46.2 ± 0.5^c	50.4 ± 0.6^c	0.3 ± 0.1^b	5.2 ± 0.1^c	7.0 ± 0.1^c	2.6 ± 0.1^c
	150	25.0 ± 0.3^d	26.1 ± 0.3^d	29.8 ± 0.3^d	33.4 ± 0.4^d	0.2 ± 0.1^c	4.4 ± 0.1^c	5.9 ± 0.1^d	2.2 ± 0.1^c
	200	6.0 ± 0.1^e	7.0 ± 0.1^e	10.6 ± 0.1^e	5.6 ± 0.1^e	0.2 ± 0.1^c	2.9 ± 0.1^d	3.8 ± 0.1^e	1.5 ± 0.1^d
	250	1.9 ± 0.1^f	2.2 ± 0.1^f	4.8 ± 0.1^f	1.3 ± 0.1^f	0.1 ± 0.1^d	0.8 ± 0.1^e	1.1 ± 0.1^f	0.4 ± 0.1^d
“Lapins”	0 (control)	80.8 ± 0.9^a	80.8 ± 0.9^a	80.8 ± 0.9^a	80.8 ± 0.9^a	14.1 ± 0.2^a	14.1 ± 0.2^a	14.1 ± 0.2^a	14.1 ± 0.2^a
	50	49.3 ± 0.5^b	52.1 ± 0.6^b	57.7 ± 0.7^b	56.8 ± 0.6^b	5.0 ± 0.1^a	7.2 ± 0.1^b	9.6 ± 0.1^b	3.7 ± 0.1^b
	100	22.6 ± 0.3^c	25.4 ± 0.3^c	31.0 ± 0.4^c	30.1 ± 0.4^c	4.9 ± 0.1^b	4.2 ± 0.1^c	5.6 ± 0.1^c	2.1 ± 0.1^c
	150	7.0 ± 0.1^d	9.0 ± 0.1^d	14.6 ± 0.2^d	13.7 ± 0.2^d	4.8 ± 0.1^b	3.5 ± 0.1^c	4.7 ± 0.1^c	1.8 ± 0.1^c
	200	6.9 ± 0.1^d	7.6 ± 0.1^e	4.4 ± 0.1^e	8.4 ± 0.1^e	4.6 ± 0.1^b	2.2 ± 0.1^d	2.9 ± 0.1^d	1.1 ± 0.1^c
	250	1.1 ± 0.1^e	0.0 ± 0.0^f	2.7 ± 0.1^f	2.6 ± 0.1^f	4.5 ± 0.1^ab	0.0 ± 0.0^e	0.6 ± 0.1^e	0.2 ± 0.1^d
“Siah-e-Mashhad”	0 (control)	86.3 ± 0.9^a	86.3 ± 0.9^a	86.3 ± 0.9^a	86.3 ± 0.9^a	21.2 ± 0.3^a	21.2 ± 0.3^a	21.2 ± 0.3^a	21.2 ± 0.3^a
	50	52.6 ± 0.6^b	57.6 ± 0.6^b	61.1 ± 0.7^b	45.7 ± 0.5^b	0.8 ± 0.1^b	10.9 ± 0.2^b	14.5 ± 0.2^b	5.5 ± 0.1^b
	100	25.9 ± 0.3^c	30.9 ± 0.4^c	34.4 ± 0.4^c	19.0 ± 0.2^c	0.6 ± 0.1^b	6.3 ± 0.1^c	8.4 ± 0.1^c	3.2 ± 0.1^c
	150	9.5 ± 0.1^d	14.5 ± 0.2^d	18.0 ± 0.2^d	6.7 ± 0.1^d	0.6 ± 0.1^b	5.3 ± 0.1^c	7.0 ± 0.1^c	2.7 ± 0.1^d
	200	4.3 ± 0.1^d	6.7 ± 0.1^e	5.1 ± 0.1^e	6.5 ± 0.1^d	0.5 ± 0.1b^c	3.1 ± 0.1^d	4.1 ± 0.1^d	1.5 ± 0.1^e
	250	0.8 ± 0.1^e	2.3 ± 0.1^f	3.6 ± 0.1^e	1.5 ± 0.1^e	0.4 ± 0.1b^c	0.8 ± 0.1^e	1.1 ± 0.1^e	0.3 ± 0.1^f

Note:

Same letters show no significant difference among genotypes of each column.

Table 5. Comparison of means for pollen germination percentage and pollen tube length (interaction among five sweet cherry cultivars and different concentrations of cadmium, copper, lead and mercury).

Cultivar		Pollen germination percentage					Pollen tube length (μm)
	Concentrate (ppm)	Cadmium	Copper	Lead	Mercury		Cadmium	Copper	Lead	Mercury
“Estella”	0 (control)	91.1 ± 1.3 ^a	91.1 ± 1.3 ^a	91.1 ± 1.3 ^a	91.1 ± 1.3 ^a		28.3 ± 0.3^a	28.3 ± 0.3^a	28.3 ± 0.3^a	28.3 ± 0.3^a
	50	54.7 ± 0.6^b	51.7 ± 0.6^b	81.1 ± 0.9^b	47.8 ± 0.5^b		13.5 ± 0.2^b	14.5 ± 0.2^b	19.3 ± 0.2^b	7.3 ± 0.1^b
	100	28.0 ± 0.3^c	25.0 ± 0.3^c	54.4 ± 0.6^c	21.1 ± 0.3^c		12.6 ± 0.2^c	8.4 ± 0.1^c	11.1 ± 0.2^c	4.2 ± 0.1^c
	150	11.6 ± 0.2^d	9.1 ± 0.1^d	38.0 ± 0.4^d	9.7 ± 0.1^d		11.7 ± 0.2^d	7.0 ± 0.1^d	9.4 ± 0.1^d	3.6 ± 0.1^d
	200	4.5 ± 0.1^e	5.8 ± 0.1^e	5.6 ± 0.1^e	5.7 ± 0.1^e		10.8 ± 0.2^e	4.3 ± 0.1^e	5.7 ± 0.1^e	2.2 ± 0.1^e
	250	2.0 ± 0.1^f	0.0 ± 0.0^f	1.8 ± 0.1^f	3.9 ± 0.1^f		1.8 ± 0.1^f	0.0 ± 0.0^f	1.0 ± 0.1^f	0.4 ± 0.1^f
“Gilas-e-Sefid”	0 (control)	85.6 ± 0.9^a	85.6 ± 0.9^a	85.6 ± 0.9^a	85.6 ± 0.9^a		24.7 ± 0.3^a	24.7 ± 0.3^a	24.7 ± 0.3^a	24.7 ± 0.3^a
	50	45.7 ± 0.5^b	51.2 ± 0.6^b	70.3 ± 0.8^b	54.6 ± 0.6^b		12.7 ± 0.2^b	4.3 ± 0.1^b	16.9 ± 0.2^b	6.4 ± 0.1^b
	100	19.0 ± 0.2^c	24.5 ± 0.3^c	43.6 ± 0.5^c	27.9 ± 0.3^c		7.3 ± 0.1^c	3.9 ± 0.1^c	9.7 ± 0.1^c	3.7 ± 0.1^c
	150	5.8 ± 0.1^d	8.4 ± 0.1^d	27.2 ± 0.3^d	11.5 ± 0.2^d		6.2 ± 0.1^d	3.6 ± 0.1^c	8.2 ± 0.1^d	3.1 ± 0.1^d
	200	5.3 ± 0.1^e	6.7 ± 0.1^e	7.4 ± 0.1^e	7.9 ± 0.1^e		4.0 ± 0.1^e	2.7 ± 0.1^d	5.4 ± 0.1^e	2.0 ± 0.1^e
	250	1.8 ± 0.1^f	0.2 ± 0.1^f	3.7 ± 0.1^f	2.3 ± 0.1^f		0.2 ± 0.1^f	1.6 ± 0.1^d	1.5 ± 0.1^f	0.3 ± 0.1^f
“Napleone”	0 (control)	92.3 ± 1.3 ^a	92.3 ± 1.3 ^a	92.3 ± 1.3 ^a	92.3 ± 1.3^a		26.5 ± 0.3^a	26.5 ± 0.3^a	26.5 ± 0.3^a	26.5 ± 0.3^a
	50	47.8 ± 0.5^b	54.5 ± 0.6^b	73.1 ± 0.8^b	57.0 ± 0.6^b		6.3 ± 0.1^b	13.6 ± 0.2^b	18.1 ± 0.2^b	6.9 ± 0.1^b
	100	21.1 ± 0.3^c	27.8 ± 0.3^c	46.4 ± 0.5^c	30.3 ± 0.4^c		5.2 ± 0.1^c	7.8 ± 0.1^c	10.4 ± 0.2^c	4.0 ± 0.1^c
	150	7.3 ± 0.1^d	11.4 ± 0.2^d	30.0 ± 0.4^d	13.9 ± 0.2^d		3.2 ± 0.1^d	6.6 ± 0.1^d	8.8 ± 0.1^d	3.3 ± 0.1^d
	200	5.9 ± 0.1^e	7.7 ± 0.1^e	7.3 ± 0.1^e	8.9 ± 0.1^e		2.7 ± 0.1^e	4.3 ± 0.1^e	5.7 ± 0.1^e	2.2 ± 0.1^e
	250	1.4 ± 0.1^f	0.0 ± 0.0^f	1.9 ± 0.1^f	3.1 ± 0.1^f		1.8 ± 0.1^f	0.2 ± 0.1^f	1.3 ± 0.1^f	0.5 ± 0.1^f
“Colt”	0 (control)	92.5 ± 1.3^a	92.5 ± 1.3^a	92.5 ± 1.3^a	92.5 ± 1.3 ^a		10.6 ± 0.2^a	10.6 ± 0.2^a	10.6 ± 0.2^a	10. ± 0.26^a
	50	50.2 ± 0.6^b	56.0 ± 0.6^b	76.9 ± 0.8^b	55.7 ± 0.6^b		9.0 ± 0.1^b	5.4 ± 0.1^b	7.2 ± 0. ^b	2.7 ± 0.1^b
	100	23.5 ± 0.3^c	29.3 ± 0.3^c	50.2 ± 0.6^c	29.0 ± 0.3^c		8.6 ± 0.1^c	3.1 ± 0.1^c	4.2 ± 0.1^c	1.6 ± 0.1^c
	150	7.6 ± 0.1^d	12.9 ± 0.2^d	33.8 ± 0.4^d	12.6 ± 0.2^d		5.1 ± 0.1^d	2.6 ± 0.1^d	3.5 ± 0.1^c	1.3 ± 0.1^c
	200	3.5 ± 0.1^e	8.4 ± 0.1^e	6.0 ± 0.1^e	9.3 ± 0.1^e		2.0 ± 0.1^e	1.6 ± 0.1^e	2.2 ± 0.1^d	0.8 ± 0.1^d
	250	0.2 ± 0.1^f	0.0 ± 0.1^f	3.6 ± 0.1^f	3.5 ± 0.1^f		1.4 ± 0.1^f	0.0 ± 0.0^f	0.6 ± 0.1^e	0.2 ± 0.1^e
“Sourati-e-Lavasanat”	0 (control)	79.5 ± 0.8^a	79.5 ± 0.8^a	79.5 ± 0.8^a	79.5 ± 0.8^a		12.4 ± 0.2^a	12.4 ± 0.2^a	12.4 ± 0.2^a	12.4 ± 0.2^a
	50	54.4 ± 0.6^b	53.8 ± 0.6^b	60.9 ± 0.7^b	50.8 ± 0.6^b		2.6 ± 0.1^b	6.3 ± 0.1^b	8.4 ± 0.1^b	3.2 ± 0.1^b
	100	27.7 ± 0.3^c	27.1 ± 0.3^c	34.2 ± 0.4^c	24.1 ± 0.3^c		1.5 ± 0.1^c	3.7 ± 0.1^c	4.9 ± 0.1^c	1.9 ± 0.1^c
	150	11.3 ± 0.2^d	10.7 ± 0.2^d	17.8 ± 0.2^d	8.9 ± 0.1^d		1.4 ± 0.1^c	3.1 ± 0.1^c	4.1 ± 0.1^c	1.6 ± 0.1^c
	200	6.2 ± 0.1^e	7.9 ± 0.1^e	6.5 ± 0.1^e	7.0 ± 0.1^e		1.1 ± 0.1^c	1.9 ± 0.1^d	2.5 ± 0.1^d	0.9 ± 0.1^d
	250	0.5 ± 0.1^f	0.8 ± 0.1^f	3.1 ± 0.1^f	1.8 ± 0.1^f		0.1 ± 0.1^d	0.3 ± 0.1^e	0.6 ± 0.1^e	0.1 ± 0.1^e

Note:

Same letters show no significant difference among genotypes of each column.

Furthermore, PGP of “Estella” was between the range of 91.1% in control to 2.0% in 250 ppm; also, PTL was between the range of 14.4 μm in control to 1.8 μm in 250 ppm in cadmium treated medium (Table 5). These results are in line with independent effects of sweet cherry cultivars and concentrations of cadmium, copper, lead and mercury (Table 5).

Discussion

Results in this study confirmed that by increasing the concentration of cadmium, copper, lead and mercury, pollen germination percentage and pollen tube length were reduced very significantly and close to zero in all studied sweet cherry cultivars at 250 ppm. In comparison with the control medium, both traits of pollens in all studied cultivars decreased at least three times in 100 ppm. Nevertheless, difference in means of PGP and PTL among the studied cultivars demonstrated higher variety in PGP in comparison with PTL (Tables 2, 3 and 4).

In this study, all cultivars which were treated with Cd showed a decrease in both traits of pollens in comparison with Cu, Pb and Hg, which showed the higher poisonous effect of Cd in sweet cherry. These results may be related to genetic differences of cultivars to heavy metals, as reported by many researchers (Sawidis 2008; Sharafi 2014a).

According to these results, Gür and Topdemir (2005) have germinated quince and plum pollen in a culture medium containing Cd, Cu, Hg and Pb. Their results demonstrated that these heavy metals led to a significant reduction in pollen germination and tube growth. A relationship was found between the concentration of heavy metal salts, and pollen germination and tube growth. However, Cd had the most inhibiting effect on pollen germination and tube growth of plums, and Cu had the least. Mercury exhibited the highest toxicity, whereas pollen germination and tube growth rate was less affected by copper in quince plants. Consequently, all heavy metals examined have negative effect on pollen germination and tube growth in quince and plum plants, but their toxicity levels varied.

Our results were also in line with Gür and Topdemir (2008), who reported that heavy metals led to a significant reduction in pollen germination and tube growth of apricot and cherry. Their results showed a decrease in pollen germination and tube elongation as concentrations of metal increased. Copper had the highest toxic effect on pollen of apricot while lead had the least effect. Cherry pollen germination and tube growth was mostly inhibited by mercury and cadmium, but only weakly by lead.

Sharafi (2014a) studied the effects of Cu and Pb on pollen germination traits in almond cultivars and reported significant differences for PGP and PTL among five almond cultivars in different concentrations of Cu and Pb; however, interaction among cultivars and different concentrations of Cu and Pb had significant effect on PGP and PTL.

Acharya et al. (2011) reported that high levels of Cd, Cr, Zn, Cu, Hg and Pb reduced pollen germination and pollen tube length in Jatropha curcas, but their toxicity levels varied. Thus, pollen germination and pollen tube length were negatively affected at highest concentration of Hg (200 μM). Applications of Cd, Cr, Pb and Hg exhibited more toxic effects in comparison to Zn and Cu. Also, different pollen abnormalities were seen in higher concentrations of metal stress. Stunted pollen tube growth, pollen tube rupture and multiple pollen tubes were observed especially at higher concentrations of Cr, Pb and Hg. Hg at toxic levels inhibits pollen germination, pollen tube growth and seed germination, causing ultra-structural changes. There are also findings indicating that heavy metals cause cytogenetic anomalies in plants, like the inhibition of mitosis division, decrease in the mitotic index (cell division frequency), and chromosomal anomalies (Acharya et al. 2011).

Some researchers have reported that low concentrations of some heavy metals could encourage pollen germination and tube growth (Sawidis and Reiss 1995). Low metal concentrations can motivate enzymatic actions while high concentrations can decrease these processes. This occurrence, called hormesis (from the Greek word horme, impulse), has been described as a stimulatory effect of a sub inhibitory dose of any toxic substance to any organism (Sawidis 2008). Hormesis in pollen germination and growth has been found in some plant species, such as Acer pseudoplatanus and Plantago depresa (Xiong and Peng 2001), but it was not observed in this study.

In our experiments, toxicity effects of Cd, Cu, Hg and Pb on pollen germination was observed at 50 ppm concentrations and it was highly induced at 100 ppm; finally it was close to 0 at 250 ppm in sweet cherry cultivars.

Nevertheless, different effects of heavy metal application have been reported in some fruit trees, for example it was reported that copper has a weak effect on pollen germination and tube growth in quince and plum plants (Gur and Topdemir 2005). These different observations may be attributed to different tolerances of different plants to heavy metal stress (Gür and Topdemir 2005).

Plants growing on metal-contaminated sites need to develop some degree of tolerance to metal toxicity in order to survive. The mechanisms for metal tolerance proposed are: (a) metal sequestration by specially produced organic compounds; (b) compartmentalization in certain cell compartments; (c) metal ion efflux; (d) organic ligand exudation. Inside cells, proteins such as ferritins and metallothioneins, and phytochelatins, participate in excess metal storage and detoxification. When these systems are overloaded, oxidative stress defense mechanisms are activated.

The study of Breygina et al. (2012) has indicated that copper, nickel and mercury are the most toxic elements on pollen germination in tobacco. Tuna et al. (2002) have reported same results in other tobacco cultivars.

Copper has an essential role in many physiological pathways in plants, including photosynthesis, respiration, carbohydrate distribution, and protein metabolism. However, it is toxic to plants at high concentration. Toxicity of copper in plants is higher than that of other heavy metals such as nickel, zinc and chromium. Some studies have demonstrated that copper was the second most toxic metal on seed germination, root elongation and coleoptile and hypocotyls growth in Triticum aestivum and Cucumis sativus among mercury, cadmium, cobalt, lead and zinc (Gür and Topdemir 2008). Some studies have shown that high concentration of copper causes chromosome anomalies in plant organs (Kappler and Kristen 1987; Kalbande et al. 2008).

Lead is a common heavy metal pollutant which is released from loaded gasoline and industrial processes and has not been shown to be essential in plant metabolism. It was shown that Pb stimulates the formation of free radicals and reactive oxygen species which can damage plant cells. Lead inhibits the activity of enzymes at cellular level by reacting with their sulfhydril groups. High Pb concentration also induces oxidative stress by increasing the production of ROS in plants. The primary cause of cell growth inhibition arises from a Pb-induced stimulation of indol-3 acetic acid (IAA) oxidation. Lead is also known to affect photosynthesis by inhibiting activity of carboxylating enzymes. A high level of Pb also causes inhibition of enzyme activities, water imbalance, alterations in membrane permeability and disturbs mineral nutrition. The effect of lead depends on the concentration, type of salts and plant species involved. Though effects are more pronounced at higher concentrations and durations, in some cases, lower concentrations might stimulate metabolic processes (Sharma and Dubey 2005; Nagajyoti et al. 2010).

Lead tolerance in Festuca ovina is an inherited characteristic, evolved by the production of compounds within the plants, specifically for protection against the toxic effects of heavy metals. A small number of genes probably produce the major effects, and modifiers for dominance are present, which are probably affected by the genome as a whole (Wolters and Martensn 1987; Sharma and Dubey 2005; Nagajyoti et al. 2010).

Mercury is strongly phytotoxic to plant cells at high levels. Toxic levels of Hg can induce visible injuries and physiological disorders in plants. For instance, Hg can connect to water channel proteins, thereby inducing leaf stomata to close and physical obstruction of water flow in plants. Furthermore, Hg cations have a high affinity for sulfydryl groups and consequently can disturb almost any function where critical or non-protected proteins are involved. An Hg ion may bind to two sites of a protein molecule without deforming the chain, or it may bind two neighboring chains together or a sufficiently high concentration of Hg may lead to protein precipitation. With organomercurials, the Hg atom still retains a free valence electron so that salts of such compounds can form a monovalent ion (Nagajyoti et al. 2010).

On the other hand, cadmium is not an essential microelement for plant growth, nor does it partake in the progression of cell metabolism, and thus, it is poisonous even at very low concentrations. Cd has no beneficial effects on plants and minimizing its content in biological systems is desirable (Nagajyoti et al. 2010). Scientists have observed that the existence of Cd decreases cell wall plastic extensibility and damages normal cell elongation in growing pollen tubes, causing morphological and structural alterations. Microscopic observations show that Cd acts primarily on cell wall development in germinating and growing pollen tubes. This can be explained by the interface of metal ions with the anionic contents of secretory vesicles and the fact that pollen tube cell walls contain large quantities of pectins and callose, but less cellulose (Sawidis and Reiss 1995; Nagajyoti et al. 2010). This characteristic of pollen tube cell walls may result in a reaction different from that of other plant cells that possess a normal cellulosic cell wall. Consequently, normal growth is inhibited, the cell diameter increases, and walls become thicker. This phenomenon was observed in pollen tubes of all sweet cherry cultivars in this study. A growing pollen tube is a highly active secreting cell, exhibiting oriented exocytosis at the tip of the tube, including vesicle transport and membrane fusion (Nagajyoti et al. 2010).

The most obvious feature of a normally growing pollen tube is the accumulation of vesicles at the tip region, causing the formation of the characteristic vesicle zone. The inhibition or disappearance of this zone that follows the increase in Cd concentration results in a decrease in pollen tube growth. The loosening of the tube wall structure, concomitantly with increase in wall thickness, might result directly from an interference of the Cd with wall polysaccharides or from an influence on the secretary mechanism. The formation of uneven pollen tubes deviating from the normal, straight growth would usually imply changes in the cytoskeletal pattern, and actin filaments are known to be involved in the regular tip growth of pollen tubes (Nagajyoti et al. 2010). Oriented pollen tube growth depends on the presence of cortical microtubules; however, in our experiment, Cd treatment did not seem to affect this system. Thus, it is more likely that the reason for deviated tube growth is not the breakdown of the actin filament system but the inactivation, and by accumulating in the stigmatic secretion, concentrations can be reached that directly impair the germination and growth of pollen tubes (Nagajyoti et al. 2010).

Although most of the available evidence of Cd toxicity to various organisms comes from laboratory experiments, contamination of air, soil, and water by this metal could lead to a similar reaction in vivo as well as other heavy metals (Sharafi 2014a).

Wang et al. (2015) studied the effects of As, Hg, Cd, Cr and Cu on pollen germination and tube growth of Picea wilsonii in vitro. They reported that microscopic evaluation revealed that heavy metals had various degree of toxicity on P. wilsonii pollen tube development. It was shown that Hg and Cd had most toxic effects on decrease in pollen germination, while Cr and Cu showed relatively lower toxicity. Also, pollen tubes showed varying shapes in response to different heavy metal stress. Pollen tubes treated with Cd, Hg and As were usually characterized by irregularly increasing diameters and swelling tips with distinct cytoplasmic vacuolation. On the other hand, except for the slightly increased diameters, no obvious abnormal shape was observed in tubes treated with Cr or Cu. Lyso-Tracker Green staining indicated that only Cd-treated pollen tubes demonstrated numerous vacuole-like acidic organelles, though cytoplasmic vacuolization was also observed in pollen tubes treated with Hg and As. In brief, they indicated that different heavy metals have various effects on Picea wilsonii pollen germination and tube growth, and that in vitro pollen culture might be used as a competent system for biomonitoring of air pollution (Wang et al. 2015).

Conclusion

In this research, it was concluded that cultivar “Colt” with highest pollen germination percentage could be selected for sweet cherry orchard establishment for pollination of commercially growing cultivars in polluted cities such as Tehran. Although the special features of pollen tube walls may result in a different reaction to heavy metals, this could play a decisive role for the whole plant because pollen tubes are part of the plant reproductive system. The air pollutants Cd, Cu, Hg and Pb, when present in elevated concentrations in plants, might contribute to the disappearance of some sensitive cultivars from regions of high pollution by decreasing their reproductive ability and could also influence reproduction in higher plants.

Disclosure statement

No potential conflict of interest was reported by the authors.

Acknowledgments

We would like to acknowledge the research team of Shahed University.