Chromosome Doubling

Chromosome doubling was performed on C. officinalis ‘Nova,’ ‘Orange Beauty,’ and ‘WUR 1553-7’. Seeds were stored under low humidity in a seed storage room at 4°C before use. Dry seeds (about 1000 seeds per cultivar) were primed in 150 ppm GA₃ for 72 h at room temperature. Per treatment 90 seeds were immersed in different antimitotic agents: 200, 400, 800 ppm colchicine (Duchefa, Netherlands); 20, 40, 80 ppm trifluralin (Sigma-Aldrich, Belgium); 20, 40, 80 ppm oryzalin (Duchefa, Netherlands) and a control treatment using water, during 24 h at room temperature. Stocks of the antimitotic agents were prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Belgium). After treatment the seeds were washed thoroughly with tap water (three times for 5 min). Subsequently for every treatment seeds were germinated on filter paper in Petri dishes. The 90 seeds per treatment were put in three Petri dishes each containing 30 seeds and incubated in the germinator (GC10, Flohr Instruments, Netherlands) with a daylight period of 12 h at 22°C. For the 90 seeds per treatment seed germination was recorded during 14 days. Germinated seeds were transplanted in a plant tray containing a Saniflor peat mixture [(Van Israel, Geraardsbergen, Belgium) 1.5 kg/m³; fertilizer: 12N:14P:24 K including trace elements, pH 5.0-6.5, EC 0.450 mS/cm] and placed in the greenhouse (greenhouse conditions: light 16 h in case of shorter day period, day/night: ventilation temperature: 18/22°C, heating temperature 15/18°C etc.). Both germination rate (%) after 14 days and survival rate (%) 3, 6, and 9 weeks after transplanting were calculated.

The ploidy level of the treated seedlings was determined 3, 6, and 9 weeks after transplant using a Cyflow Space flow cytometer (Partec, Germany) equipped with a UV-LED. Plants that were considered chromosome doubled were reanalyzed 6 months later. The protocol used is based on Otto (1990). Approximately 0.5 cm² of young leaf tissue was chopped in a 500 μL extraction buffer containing 0.1 M citric acid monohydrate and 0.5% Tween-20. Samples were filtered through a 50 μm CellTrics filter to eliminate cell debris. Then 750 μL of staining buffer containing 0.4 M Na₂HPO.12HO, 2 mg/L 4′, 6-diamidino-2-phenyllndole (DAPI), 0.1% polyvinylpyrrolidone (PVP) was added. All histograms were analyzed using FloMax software.