Materials and Methods:
Specimens of Biscutella laevigata L. were collected from two isolated sites: (1) Jaworzynka Valley in the West Tatra Mts. (mountain population), the control population growing on Humic-Rendzic Leptosols composed of Triasic dolomite, located at 1100 m. a.s.l. (Szarek-Łukaszewska & Niklińska 2002, after Skiba 1983). (2) Over 100-years old zinc-lead mine spoils in Bolesław (calamine population) where calamine flora has evolved. The spoil is located in Upper Silesia, the most industrial region in South Poland, rich in zinc and lead ores (zinc blende and lead-glance with a high content of silver and secondary minerals). Exploitation of ores using open-cast technology ceased at the beginning of the 20th century and the spoil is overgrown with grassland as a result of spontaneous succession. The substrate contains high concentrations of heavy metals, Zn (49700 mg/kg), Pb (2830 mg/kg), Mn (1810 mg/kg), Fe (47800 mg/kg), Cd (154 mg/kg) (Szarek-Łukaszewska & Niklińska 2002).
Flower buds, flowers, fruits (siliculas) at different developmental stages were fixed in situ in FAA (mixture of acetic acid, formaldehyde, ethyl alcohol 70%; 4:6:90 v/v) for 24 hours, stored in 70% ethanol at 4 °C until used.
Pollen viability was assessed with Alexander dye (Alexander 1969).
For paraffin sections, samples/materials were dehydrated in an increasing ethanol series; 100% ethanol was removed by chloroform (in proportions 3:1; 1:1; 1:3; 0:1 with paraffin in 57°C). Then, material was embedded in paraffin blocks and sectioned at 10 μm on a rotary microtome (Adamas Instrumenten BV, HM 340E), sections were stained with Heidenhain’s hematoxylin (FLUKA) combined with alcian blue (FLUKA) and mounted in Entellane (Aldrich).
A clearing technique using methyl salicylate was used for visualization of male and female gametophytes in anthers and ovules, respectively. After dehydration in an ethanol series, material was cleared in a mixture of 100% ethanol and methyl salicylate (Sigma-Aldrich) in proportions 3:1; 1:1; 1:3, in pure methyl salicylate and observed in a drop of methyl salicylate with Nomarski differential interference contrast (DIC) optics (Young et al., 1979, slightly modified).
For detection of metals in flowers and seeds dithizone staining was applied (Seregin and Kozhevnikova 2011).
Immunohistochemical detection of pectin distribution in cell walls of embryo tissues, using primary antibodies for low- and high-esterified homogalacturonan, LM19 and LM20, was preceded according to the protocol of Milewska-Hendel et al. (2017). Slides were mounted in a Fluoromount (Sigma-Aldrich) antifade medium. Negative control was performed for each probe by incubated with a blocking buffer instead of primary antibodies and revealed the absence of any signals. Observations and documentation were carried out using a Nikon Eclipse Ni-U microscope equipped with a Nikon Digital DS-Fi1-U3 camera with corresponding software (Nikon, Tokyo, Japan). Immunohistochemical analyses were performed on at least three samples from analysed species from each studied area. The photos are representative of the obtained results.
A total of 30 randomly taken specimens were analyzed. For female lineage observation over 1300 ovules were studied at various developmental stages: archesporial cells, megasporocytes, megasporogenesis, megagametogenesis, embryogenesis, mature embryos (for all techniques); additionally for male lineage observation 130 flower buds from very small to just before anthesis were analyzed in the following stages: archesporial tissue, microsporocytes, microsporogenesis, microgametogenesis, mature pollen grains; for pollen viability over 4000 mature pollen grains were counted from both sites (5 flowers from each 10 randomly chosen plants). Studies were carried out for two growing seasons. The authors did not observe any strong weather conditions (e.g. drought, floods) that could be an impact on the results.
Statistic chi-square test was used in Microsoft Office Excel 2010 to find significant differences between populations.