4.1. Experimental Design

In this experiment, studies were carried out in two separate experiments and were repeated twice. Germination of Lolium perenne, Medicago sativa and Medicago x varia was performed in controlled conditions in a vegetation chamber (Sanyo, Osaka, Japan, Versatile Environmental Test Chamber). Germination was carried out in accordance with constant temperature (25 °C) (day/night) and 8/16 h (day/night) photoperiod light conditions under a cool, white light (110 µM PAR/m²/s). The seeds were first disinfected with a 1% sodium hypochlorite solution for five minutes and then rinsed three times with distilled water. The seeds of Medicago sativa and Medicago x varia were additionally subjected to mechanical scarification (rubbed with fine sandpaper for 1 min.) in order to stimulate germination. Next, the seeds of both species were placed on two layers of filter paper (grammage 65 g/m²) with a moisture content of 60% (moistened with distilled water), and pre-cooling at 8 °C in the dark for 96 h (4 days) was applied as a procedure to break the dormancy of the seeds. For all the germination variants, 50 seeds of each species for the pure-germination conditions and 25 seeds of each species for the mixed-germination conditions (both species in a 1:1 ratio on a 10 cm diameter Petri dish) were used. Petri dishes and filter paper were disinfected in an oven at 120 °C for 3 h. As a precaution to minimize evaporation, the Petri dishes were wrapped with plastic transparent bags. Germination data were collected based on the vigor (germination energy, GE) after 5 days and final counts (germination capacity, GC) after 10 days [35]. The GE was determined considering seeds with a radicle that was 2 mm long, while the GC was calculated as follows [53]: GC = (number of germinated seeds/total number of seeds) × 100. To estimate the morphological parameters of the tested species, 15 normal seedlings were sampled randomly per replication (from each Petri dish) at the end of the study according to the ISTA Rules [35] and Handbook of Seedling Evaluation [54], as type D group A 1-2-3-1. The number and length of the roots and leaves of the seedlings were determined. The distance from the crown to the root tip and leaf tip was measured as the root and leaf length, respectively. The fresh weight as well as the dry weight of the seedlings, after drying in an oven at 80 °C for 24 h, was evaluated. The obtained results were expressed per seedling (mg per plant).