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.