The DNA content was evaluated by FCM according to Carra et al. [50]. A total of 88 plantlets regenerated from somatic embryos were used and compared with the meristem tips of mother plants. The analysis was carried out with the Partec PAS flow cytometer (Partec GmbH, Munster, Germany), equipped with a mercury lamp. Fully expanded and young leaves (0.01 g) were chopped in a glass Petri dish with 1 mL nuclei extraction OTTO buffer 1 [51] and three drops of Tween 20. After 3 min, 1 mL of OTTO buffer 2 [51] was added. In addition, PVP-10 (1%) was added to plant samples to neutralize interference by cell metabolites in the analysis. The solution was filtered through a 30 μm Cell-Trics disposable filter Partec, and 400 µL of staining solution containing 4,6-diamidino-2-phenylindole (DAPI) was added. Routinely, 3000–4000 nuclei for each sample were measured, and histograms of DNA content were generated using the Partec software package (FloMax). Three replicates for each sample were carried out. The fluorescence intensity emitted was normalized by isolated nuclei from Pisum sativum L., optimal DNA reference standard for plant cytometric analyses [52]. This calibration was checked periodically.

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