Modulating ascorbic acid levels to optimize somatic embryogenesis in Picea abies (L.) H. Karst. Insights into oxidative stress and endogenous phytohormones regulation.

Global warming has adversely affected Picea abies (L.) H. Karst. forests in Europe, prompting the need for innovative forest-breeding strategies. Somatic embryogenesis (SE) offers promise but requires protocol refinement. Understanding the molecular mechanisms governing somatic embryo development is essential, as oxidative stress plays a crucial role in SE regulation. Ascorbic acid (ASA), is a vital antioxidant that can potentially control oxidative stress. In the present study, we normalized ASA concentrations in induction and proliferation media to enhance embryogenic tissue (ET) regeneration and proliferation capacity of mature explants. The media were supplemented with ASA at 0 mg l-1, 25 mg l-1, 50 mg l-1, 100 mg l-1, and 200 mg l-1. The accumulation of hydrogen peroxide (H2O2) and endogenous phytohormones, including auxins, cytokinins, brassinosteroids, abscisic acid, and gibberellin, was measured in non-embryonic calli and ET. Subsequently, their impact on ET induction and multiplication was analyzed. Our results demonstrate that application of ASA at concentrations of 25 mg l-1 and 200 mg l-1 led to increased H2O2 levels, potentially inducing oxidative stress while simultaneously reducing the levels of all endohormone groups. Notably, the highest ET induction frequency (approximately 70%) was observed for ASA at 50 mg l-1. These findings will enhance SE induction procedures, particularly in more resistant explants, underscoring the significance of ASA application to culture media.

Cryopreservation of Abies alba × A. numidica and Pinus nigra embryogenic tissues by stepwise dehydration method.

BACKGROUND: Cryopreservation makes it possible to preserve plant biodiversity for thousands of years in ex situ storage. The stepwise dehydration method is a simple and versatile cryopreservation technique based on the vitrification phenomenon. However, the commonly used dimethyl sulfoxide (DMSO) in this cryopreservation technique is considered harmful for plant material, thus alternative methods are needed to be applied. RESULTS: In this study, the possibility of cryopreservation of embryogenic tissues (ETs) of Abies alba x A. numidica and Pinus nigra was investigated. Before freezing, ETs were partially dehydrated in the presence of increasing concentrations of sucrose (from 0.25 to 1.0 M) for 7 days, followed by desiccation of the tissues over silica gel for 2 and 2.5 h, respectively. After these pretreatments, the plant material was frozen in liquid nitrogen (LN; -196 °C). For both coniferous trees the ET survival rate was high and reached 84.4% for A. alba x A. numidica (28 days) and 86.7% for P. nigra (35 days) after recovery of the tissues from liquid nitrogen (LN). The regenerated tissue of A. alba x A. numidica was characterized by more intense growth after storage in LN compared to tissue that had not been cryopreserved (control). The tissue of this tree also undertook relatively rapid growth after thawing from LN. In turn, the ET growth of P. nigra was significantly lower after thawing compared to the other treatment. CONCLUSIONS: The present study demonstrated, that the stepwise dehydration method could be successfully applied to the cryostorage of ETs of both studied trees. To the best of our knowledge, this is the first report on ET cryopreservation based on this method for Abies and Pinus genus representatives, which may be the alternative way for efficient, long-term preservation of germplasm in LN.