2.2. Sample Preparation

Each genotype was sampled in three biological replicates (individual plants). Flower buds 10 mm long (measured from the pedicel insertion point to the tip of the bud) with colored petals and yellow anthers were collected from the first inflorescence of individual plants for a metabolic analysis. These flower buds (two days before opening) contain completely developed and mature pollen. Anthers were isolated from the flower buds of all genotypes at the same time and fixed by liquid nitrogen immediately after they were separated from the floral buds and weighed. Parallel fixation of flower buds at different stages of development (I and II meiotic division, mature pollen grains) was carried out from the same plants for cytological observation and scoring pollen fertility.

Anthers frozen in liquid nitrogen were disrupted in a bead mill (Tissue Lyser LT, beats per second, three times for 2 min). Extraction was provided with 80% methanol. After extraction, the samples were purified from tissue debris by centrifugation for 10 min at 15,000 g. The resulting extract was treated with a vacuum evaporator. The dry sediment was then dissolved in pyridine containing the internal standard nC23 (tricosan), then a silylating agent was added in the proportion BSTFA (N,O-Bis(trimethylsilyl)trifluoroacetamide): TMCS (Trimethylchlorosilane) as 99:1 and the samples were derivatized by incubating them at 90 °C for 20 min.