Valorisation of a threatened plant species through in vitro culture elicitation of phenolic compounds
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Abstract: Ex situ conservation strategies, such as in vitro culture, have been suggested for the recovery
of threatened plants close to extinction. In vitro culture or micropropagation is a plant-multiplication
technique that allows to obtain a large number of clones in a short period of time and limited space,
which can be used in reintroduction programs and for research purposes (1). This biotechnological tool
also allows increasing the production yield of secondary metabolites, such as phenolic compounds, thus
emerging as an efficient system to obtain high added-value bioactive compounds (2). This approach
allows valorising the culture of threatened plants for their commercial use in different industrial sectors
interested in bioactive compounds, such as the food and pharmaceutical industries. Therefore, the aim
of this research was to study the phenolic compounds elicitation by cytokinins in roots and aerial parts
of the in vitro cultured endangered plant Eryngium viviparum J. Gay (Fam. Apiaceae).
E. viviparum explants were cultured in vitro in a solidified MS medium. A 3-level factorial design was
implemented to study the effect of two cytokinins, 6-benzylaminopurin (BAP) and kinetin (KIN), at 0, 1
and 2 mg L-1, on the elicitation of phenolic compounds. The obtained plant material was lyophilized,
reduced to powder, and submitted to a solid-liquid extraction with ethanol:water (80:20, v/v) to obtain
combined extracts (3). Then, phenolic compounds were identified and quantified by HPLC-DAD-ESI/MS
(3) and the main phenolic compounds with known bioactivities were correlated with both cytokinins.
Seventeen phenolic compounds were identified in all E. viviparum hydroethanolic extracts,
corresponding to 12 phenolic acids and 5 flavonoids. Rosmarinic and chlorogenic acids were the main
bioactive phenolic acids. Regarding flavonoids, tectorigenin-O-glucuronide was identified and quantified
only in the aerial parts. In the roots, the increase of phenolic acids was correlated with the presence of
KIN in the culture media. Concerning the aerial parts, cytokinins did not correlate with phenolic acids,
but KIN correlated with the increase of tectorigenin-O-glucuronide. In addition, the roots had a higher
phenolic composition content than the aerial parts and the combination of both cytokinins promoted a
doubling of the phenolic compounds concentration in some samples. Therefore, this study demonstrated
the efficacy of in vitro culture in adding value to the threatened plant species E. viviparum, as the levels
of valuable secondary metabolites were increased by elicitation, thus being a sustainable approach that
can help save and valorise other endangered plants.
