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.