2.3 Experiment under different light intensities and wavelengths for germination
In the implementation of the experiment, seeds of M. zehntneriwere immersed in distilled water for 10 minutes, according to results previously obtained in our laboratory (Magnani and Cardoso, 2022). Later, using a laminar flow cabinet, seeds were subjected to asepsis, aiming at the reduction or even elimination of microorganisms. This was performed in 15 mL Falcon® tubes and seeds were immersed in 70% alcohol (v/v) for one minute and then in a solution containing 30% sodium hypochlorite (2.0-2.5% active chlorine) added with 5 drops of neutral detergent for every 100 mL solution, for 15 minutes under constant stirring, followed by three washes in previously autoclaved deionized water.
In the last rinse, approximately 2 mL deionized water with pH adjusted to 5.8 was kept as a vehicle for the inoculation of seeds on the plates. All seeds, 20 per plate, were sown in clear, smooth, and sterile polystyrene Petri dishes, previously filled with two layers of filter paper saturated with 5 mL sterile deionized water at pH 5.8 per dish.
Subsequently, dishes containing the seeds were arranged under different intensities and wavelengths given by LED lamps and cultivated in a grow room, as follows: blue LED (Phillips Greenpower LED Research Module Blue, ⁓440 nm), red LED ( Phillips Greenpower LED module HF deep red, ⁓660 nm), blue(1)/red(1.5) LED (LabPar, with wavelength peaks at 447 nm, range 420-470 nm - blue and 667 nm, range 625-680 nm – red) and, as a white LED control (Ourolux®, Brazil), with peaks at 440-450 nm (blue), 540-550 nm (green) and 610-620 nm (red).
The photosynthetically photon flux densities (PPFD) were measured using a PPFD Quantum meter, Apogee Instruments®, Model SQ-520 (USA) of each light source and were obtained by placing the dishes in equidistant proportions from the LEDs (Table 1).
The experiment was a completely randomized design, in a 3 x 4 factorial (PPFD x wavelength) with four replications composed of individual Petri dishes containing 20 seeds each.
As a complementary experiment, darkness influence on the germination ofM. zehntneri seeds was tested, with seeds kept protected from light, in a grow room, for three periods of 10, 20, and 30 days of darkness. In this case, seeds under germination conditions were only exposed to a light source after remaining in the respective periods in the dark. For this, the same procedure of preparation and inoculation of seeds was carried out and the experimental design was completely randomized.
Petri dishes from both experiments were sealed with a transparent PVC film and kept in a grow room at 26 ± 1º C, and a photoperiod of 14 hours. Germination was evaluated twice a week; seeds were considered germinated when hypocotyl-radicle protrusion was equal to or greater than 0.1 cm. In the end, the Germination Percentage (G%), Average Germination Speed (AGS), and Germination Speed Index (GSI) were calculated.