Computational protocol: Floral nectar production and carbohydrate composition and the structure of receptacular nectaries in the invasive plant Bunias orientalis L. (Brassicaceae)

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Protocol publication

[…] In 2012, the structure of floral nectaries was studied in nectar-bearing flowers at the 2nd day of anthesis. The distribution of secretory glands in fresh flowers (n = 10) was investigated under an Olympus SZX12 stereoscopic microscope. Then, the nectaries were prepared for investigations by means of light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM).For LM examination, semi-thin sections of nectaries were prepared. Floral material was fixed in 2.5 % glutaraldehyde in phosphate buffer (pH 7.4; 0.1 M) for 12 h at 4 °C, followed by three washes in phosphate buffer. Then, it was treated with 1 % osmium tetraoxide solution at 0 °C for 1.5 h and washed three times in distilled water. After dehydration in a graded ethanol series, the samples were infiltrated in a medium grade LR White acrylic resin (Sigma-Aldrich). Following polymerization at 60 °C, sections were cut by a Reichert Ultracut-S ultramicrotome and a glass knife at a thickness of 0.7–0.9 μm. For general histology, semi-thin sections were stained with 1 % (w/v) aqueous methylene blue-Azur B solution. The presence of insoluble polysaccharides was tested with Periodic acid-Schiff’s (PAS) reagent after blocking free aldehyde groups (O’Brien and McCully ). LM observations were conducted by means of a Nikon Eclipse E200 (Nikon Corp., Tokyo, Japan), and measurements were taken with NIS-Elements Br 2 imaging software (Nikon Corp., Tokyo, Japan).Moreover, other semi-thin sections were stained with auramine O for the presence of cutinized cell walls (Heslop-Harrison ) and examined by means of a Nikon Eclipse 90i equipped with fluorescein isothiocyanate filter (EXP. 465-495, DM 505; BA 515-555). Autofluorescence of chlorophyll in plastids was tested in fresh, hand-cut sections of nectary using a Nikon 90i fluorescence microscope with UV-2B filter. In each case, control sections were used.The material for TEM was fixed as above. Ultra-thin sections (60–70 nm thick) were cut from the embedded material, subsequently stained with uranyl acetate and post-stained in lead citrate (Reynolds ). Then, the sections were examined with an FEI Technai-G2 Spirit Bio TWIN transmission electron microscope at an accelerating voltage of 120 kV. TEM images were taken using a Megaview G2 Olympus Soft Imaging Solution camera.For observations in SEM, flower bases were fixed in 2.5 % glutaraldehyde in phosphate buffer (pH 7.4; 0.1 M) at 4 °C for 12 h. The material was then washed in phosphate buffer and dehydrated in a graded acetone series, respectively. The plant material was subsequently subjected to critical-point drying using liquid CO2, sputter-coated with gold and examined with TESCAN/VEGA LMU SEM (TESCAN, Brno, Czech Republic) at an accelerating voltage of 30 kV. [...] Descriptive statistics were calculated for data on nectar characteristics and nectar tissues measurements and are presented as mean values ± SD (standard deviation). For glucose, fructose and sucrose content in nectar, coefficient of variation (CV) was computed. The differences in nectar amount, nectar concentration and nectar sugar quantity per flower between years of study were subjected to separate one-way ANOVAs. Additionally, the differences in the mean values of nectar carbohydrate composition (glucose and fructose) between individual plants were analysed. When significant differences were detected, post hoc comparison was made by means of the HSD Tukey test. The level of statistical significance required to measure differences between the means for all analyses was P = 0.05. All data analyses were performed using STATISTICA 6.0 (StatSoft Inc., Kraków, Poland) software. […]

Pipeline specifications

Software tools NIS-Elements BR, Statistica
Applications Miscellaneous, cryo-EM
Diseases Lactose Intolerance
Chemicals Fructose, Glucose