Effect of humic substances on proliferation activities of Abies cephalonica Loud. embryogenic cell masses: biochemical and cytological approaches

The somatic embryogenesis (SE) of Abies cephalonica is poorly studied and regeneration of plantlets is only sporadic. Early stages of SE might be crucial for the successful completion of the overall process [1,2]. In Norway spruce, cells within the proliferating embryogenic tissue can pass through three consecutive developmental stages, which are characterized by distinct cell aggregates defined as pro-embryogenic mass (PEM) I, PEM II, and PEM III [2]. Humic substances (HS) are supramolecular associations of heterogeneous molecules deriving from microbial degradation of natural organic materials and are ubiquitous in water, soil, and sediments [3]. They exhibit positive effects on some physiological aspects of plant growth and metabolism [4]. We cultivated embryogenic cell masses of A. cephalonica on different proliferation media (control, 1 mg/Petri plate humic or fulvic acids) and, at regular intervals (3, 7, 14 and 21 days), the effects of HS on the proliferation rate, consecutive developmental stages of PEMs, level of ATP and glucose 6-P were evaluated. We show that humic acids had significant effects just on the biochemical parameters evaluated, such as the level of ATP and glucose 6-P, which were increased on days 3, 7 and 14. Fulvic acids were able to enhance significantly the proliferation rate, especially during the early stages, and to affect the proportion of PEMs, in particular enhancing the percentage of PEM III at day 14. Furthermore, fulvic acids induced an increase of cellular ATP concentration, but not of glucose 6-P. These results indicate that HS might play an important role in improving the proliferation of PEM, thus affecting the subsequent maturation process of A. cephalonica.