*2.2. Evaluation of LTG and Coleoptile Length*

Seeds from each F2 plant were harvested five weeks after heading and dried in a greenhouse for two weeks. To break dormancy, seeds were stored in a dry oven at 50 ◦C for 72 h. For normal temperature and low-temperature germination tests, 20 and 30 seeds were used with three replicates, respectively. Healthy and normal seeds were carefully selected and used in the germination test. The seeds from each plant were placed into 9-cm Petri dishes onto one layer of filter paper and 5 mL of distilled water added. Dormancy breakage was checked at the normal germination temperature (30 ◦C) for four days. The low-temperature germination test was conducted at 13 ◦C under the dark condition for eight days. Germinated seeds of each plant were counted daily and defined as germination rate. The germination rate (%) was calculated as follows: Germination rate (%) for a given day = (number of germinated seeds until the given day/total number of seeds) × 100. The germination tests were repeated two times under similar conditions. The seeds were considered germinated when coleoptile emerged from the seed (Figure S1).

Coleoptile length measurement was conducted based on previous studies [6,14]. F3 seeds of F2 plants were used to measure coleoptile length. Twenty normal and healthy seeds per plant were placed on one layer of filter paper in 9-cm Petri dishes and 10 mL of distilled water was poured into each Petri dish. The measurements were performed with two replicates using parental lines under normal (30 ◦C) and low-temperature (13 ◦C) conditions. In the F3 generation, coleoptile length was measured under low-temperature conditions (13 ◦C) with one replicate. Distilled water was renewed daily and maintained at 30 ◦C and 13 ◦C conditions for the normal and low-temperature conditions, respectively. Coleoptile length was measured using a ruler. In the normal temperature treatments, measurements were carried out from 1 to 4 days after incubation (DAI) and, in the low-temperature treatments, measurements were carried out from 5 to 10 DAI. For the normal and low-temperature conditions, coleoptile lengths of the grains were measured at room temperature (26 ◦C) and at seed storage temperature (10 ◦C) conditions, respectively, and mean lengths of the coleoptile were used in further analyses.
