Selection of ABA-insensitive and sensitive alfalfa populations
The germinated seeds were isolated and labeled as S0-50 under 50 μM ABA treatment, while the un-germinated seeds were washed with sterile water and regerminated under ABA-free conditions to produce S0-0 plants. The isolated plants were grown further in flower pots (Φ=10 cm, h=15 cm) filled with 200 g soil in a greenhouse with a photoperiod of 14h/10h (day/night) for four months. Other S0-50 plants were planted in the field (Beijing, China, N40.09, E116.22). We harvested seeds from each individual S0-50 plant (S1) respectively, as biological replicates.
The mature seeds of S1 were germinated on wet filter paper containing 50 μM ABA after sterilization. Three days later, we picked out the germinating seeds (S1-50), while the ungerminated seeds (S1-0) were washed with sterile water. Then, S1-50 and S1-0 regenerated on wet filter paper for four days for PEG treatment.