2.4 In vitro germination of M. zehntneri under different concentrations of phytoregulators
The main objectives of this experiment were to evaluate the effect of different classes of plant growth regulators on the germination ofM. zehntneri seeds and to develop a methodology for in vitro germination as an alternative to the method of seed germination in Petri dishes.
Procedures before sowing and aiming at seed asepsis were carried out in the same way as described in the previous experiment. In the last rinse, approximately 2 mL deionized water (pH⁓5.8) was maintained so that this solution containing the seeds could be used as a vehicle for inoculation in culture flasks containing 30 mL MS culture medium (Murashige and Skoog, 1962), containing sucrose (20 g L-1), inositol (100 mg L-1), activated charcoal (1 g L-1), and the pH was adjusted to 5.8 before the addition of agar (6.4 g L-1). Flasks containing the culture medium were autoclaved at 120ºC for 25 minutes.
Phytoregulators tested for germination of M. zehntneri seeds and added to the MS culture medium were 6-benzylaminopurine (BAP) 1 mg L-1; gibberellic acid (GA3) 1 mg L-1; and the combination of the two, BAP (1 mg L-1) and GA3 (1 mg L-1) and a control without addition of phytoregulators.
Also, we evaluated the effect of pre-treatment of seeds in a solution containing 100 μL L-1 Ethrel® (240 g/L Ethephon - Bayer®, Brazil) for 24 hours before the experiment and later inoculated in culture media containing the same treatments described above. After inoculation, flasks containing the seeds were kept in a grow room under the same conditions as in the previous experiment, but using only the LED in the blue (1) and red (1.5) wavelengths.
The experiment was conducted in a Completely Randomized Design (CRD), in a 2 (pre-treatment with Ethrel®) x 4 (phytoregulators in the culture medium) factorial. In total, six replications were performed per treatment, each replication consisting of a flask containing 30 mL culture medium, with 10 seeds per flask.
Germination was checked twice a week, and seeds were only considered germinated when embryo protrusion was equal to or greater than 0.1 cm. After 42 days, the germination percentage (%G), the AGS, and GSI were also evaluated.