Effects of different types of heavy metals on pollen germination and tubegrowth of the‘Huanghua’pear

Abstract:【Objective】Heavy metal pollution has become one of the serious environment problems alongwith social and economy development. These excessive heavy metals often exhibit negative effects onplant cells or organ growth, such as pollen germination and tube growth. During the pear reproductive peri⁃od, pollen grains are exposed in air and are prone to pollution by some heavy metals. However, the effectsof heavy metals on pear pollen germination and tube growth remain unknown. This present study evaluatesthe properties of Ni, Cd, Pb, Cu and Zn treatments on pear pollen germination and tube growth, respectively.【Methods】Pollen grains from the cultivar‘Huanghua’(Pyrus pyrifolia) are collected from the Zhejiang A & F University Fruit Experimental Yard, preserved by drying in air at ambient temperature (25 °C)for 12 h, and then stored in silica gel at -20 °C. Mature pear pollen grains were cultured in a culture medi⁃um in the dark at 25 °C for 3 h with a constant temperature shaker (100 r·min^-1). The culture medium con⁃tained 0.03% CaCl2，0.01% H3BO3，10% sucrose, 15% polyethylene glycol 4 000 and 30 mmol·L^-1 MES，pH 6.0 (adjusted with Trizma base). Ni2+, Cd2+, Cu2+, Pb2+ and Zn2+ were added to the culture medium before the pollen grains culture. The final treatment concentrations of Ni2+, Cd2+, Cu2+, Pb2+or Zn2+ were 25,50 and 100 μmol·L^-1, respectively. When the germinated tube length was more than the diameter of thepollen grain, this pollen grain was considered as pollen germination. The germination rate is equal to thenumber of germinated pollen/total pollen number; 100 pollen grains (or tubes) were counted in each re⁃peat. Pollen tube length and shape changes of the pear pollen tube tip were captured and analyzed by a microscope with a CCD camera and Image-Pro plus6.0 software, respectively. Data were analyzed with bothExcel and SPSS 22.0 software. The tip-localized reactive oxygen species (ROS) were stained by Nitrobluetetrazolium (NBT; 1 g·L^-1) and 5-(and-6)-chloromethyl-2’, 7’-dichlorodihydrofluor- escein diacetate(CM-H2DCF-DA; 20 μmol·L^-1) for 5 min, respectively. The stained samples were visualized by using aOLYMPUS BX51 microscope equipped with a CCD camera Sensys 1401E (Olympus Optical, Tokyo, Ja⁃pan).【Results】25, 50 or 100 μmol·L-1 Ni2+, Cd2+, Cu2+, Pb2+ and Zn2+ showed inhibition of the‘Huang⁃hua’pear pollen germination rate and tube growth. The inhibition degree increased along with the concentration of the above heavy metal, and exhibited a dose-dependent correlation. The pollen germination rateshowed no statistical significance between 25 and 0 μmol·L^-1 Pb2+ treatments, whereas the 25 μmol·L^-1 Pb2+ treatments significantly (P<0.05) inhibited pollen tube length. Moreover, Ni displayed a similar nega⁃tive manner to Pb treatments. This result indicated that 25 μmol·L^-1 Pb2+ or Ni2+ showed more negative ef⁃ects on tube elongation than pollen germination. In contrast to Pb and Ni, the 25 μmol·L^-1 Cd2+, Zn2+ orCu2+ treatments significantly (P<0.05) inhibited pollen germination and tube growth. At the same concen⁃tration in our present work, Cd showed less negative effects on pollen germination and tube growth thanZn and Cu. Furthermore, Cu treatments showed the highest toxicity on the pear pollen germination rateand tube growth. The pollen germination rate was 10.0% after treatment with 25 μmol·L^-1 Cu2+, which ex⁃tremely significantly (P<0.01) inhibited the pollen germination rate in contrast to the control (52.6%). Theinhibition of the pollen tube average length was consistent with the pollen germination rate after Cu treatments. Among the above five different heavy metals, Pb treatments displayed the least effects on the pol⁃len germination rate and tube growth. Additionally, the pollen tube tip shape is essential for rapid tubegrowth. Features of different heavy metals on the shape of the pear pollen tube tip were investigated afterdifferent heavy metal treatments. Three main forms of swollen tip could be observed in the pear pollentube after different heavy metal treatments. The swollen tip rate was the lowest after Pb treatment, whereasthe Cu treatment showed the highest effect. The swollen tip rate by the Cu treatment was significantly (P<0.05) more than the Pb, Ni, Cd and Zn treatments, respectively. Furthermore, the tip-localized ROS gradient plays an essential role for pear pollen tube growth. Interestingly, the tip-localized ROS gradient wasdisturbed by the above heavy mental treatments, which suggested that the tip-localized ROS gradient disturbance could be involved in the inhibition processes of pear pollen tube growth after heavy metal treatments.【Conclusion】Different concentrations of Ni2+, Cd2+, Cu2+, Pb2+ and Zn2+ showed various negative effects on pear pollen germination and tube growth in this work. This negative degree displayed a dose-dependent manner after one the above five heavy metal treatments. Three main types of swollen pollen tubetip were found after heavy metal treatments, and the rate of swollen pollen tube tip induced by Cu and Pbshowed the highest and the lowest, respectively. Furthermore, pollen tube tip-localized ROS was disrupted by heavy metal treatments. Combined with the pollen germination rate, tube length and tube swollen tiprate analysis after different heavy metal treatments, our results showed that a negative effect trend of differ⁃ent heavy metals was Pb＜Ni＜Cd＜Zn＜Cu on‘Huanghua’pear pollen germination and tube growth.