Natacha Coelho

Thymus lotocephalus wild plants and in vitro cultures produce different profiles of phenolic compounds with antioxidant activity.

We compared the phenolic metabolites and antioxidant activities of Thymus lotocephalus G. López & R. Morales wild plants and in vitro cultures using different extraction solvents. HPLC-DAD analysis allowed the identification and quantification of phenolic (caffeic and rosmarinic) acids and flavones (luteolin and apigenin) in extracts from both sources. The in vitro cultures accumulated large amounts of rosmarinic acid. However, extracts from both sources were able to neutralise free radicals in different test systems (TEAC and ORAC assays), to form complexes with Fe(2+) and to protect mouse brains against Fe(2+)-induced lipid peroxidation. The solvent significantly influenced the phenolic content and antioxidant activity of the extracts, water/ethanol being the most efficient for the extraction of antioxidant phytochemicals. We conclude that in vitro cultures of T. lotocephalus represent a promising alternative for the production of valuable natural antioxidants and an efficient tool for the in vitro biosynthesis of rosmarinic acid, therefore avoiding the need to exploit populations of wild plants.

Morphological, physiological and oxidative stress markers during acclimatization and field transfer of micropropagated Tuberaria major plants

Tuberaria major (Willk.) P. Silva and Rozeira is a critically-endangered rock rose species endemic to Portugal. Because the species needs to be preserved, this study evaluated the morphological and physiological traits of micropropagated T. major plants during acclimatization and field transfer. There were no significant differences between wild and micropropagated plants in the field, although the latter underwent significant changes during acclimatization. Leaf pubescence and leaf mass per area increased during acclimatization whereas the chlorophyll content and chlorophyll/carotenoid ratio declined to eventually match those of wild plants. Stomatal conductance (gs) and transpiration rates (E) also declined substantially during acclimatization, thus preventing uncontrolled wilting. Photosynthetic rate (PN) was initially negative but increased during the later stages of acclimatization. Maximum quantum yield of PSII (Fv/Fm) remained constant at 0.78–0.85, showing that the plants were healthy and unstressed. PSII quantum efficiency (ϕPSII) was initially low but increased during acclimatization along with photosynthetic performance as the energy partitioning in PSII was adjusted. This was balanced by the decline in non-regulated energy dissipation (ϕNO) from an initially high value. Electrolyte leakage and malondialdehyde content remained constant at similar levels in both groups of plants, but H2O2 levels were higher in the field, perhaps indicating the early induction of antioxidant defense systems. The present study shows that T. major has enough phenotypic plasticity to adapt to changing environments and that the procedure described herein can be used for the restoration and preservation of this species.

In vitro propagation of Drosera intermedia in a single step

A simple and efficient protocol for the micropropagation of Drosera intermedia, using cultures initiated from in vitro produced seedlings, is described. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher multiplication rates for ¼ MS (the lowest concentration), but was not affected by the addition of 0.1 mg dm−3 kinetin. In all cases a multiplication percentage above 90 % was recorded. High rooting percentages (up to 100 %) were obtained in multiplication phase on ¼ MS medium without growth regulators. In average 15.8 plantlets per initial shoot was produced after 8 weeks of culture. All plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development.

Estimation of genetic diversity in seedlings of Plantago algarbiensis, an endangered species endemic to the south of Portugal in risk of global extinction

Plantago algarbiensis samp is an endangered species endemic to the Algarve, in southern Portugal. In the present work, the genetic diversity of three populations was assessed by RAPD markers. Samples were amplified using ten primers that generated 145 markers, 80% of which were polymorphic. Tunes population presented the highest polymorphism percentage (73.68%) and Algoz the lowest (67.67%). In the cluster analysis, two major groups were formed, one including individuals from Gambelas and the other clustered together individuals from the other populations. The highest level of genetic diversity, estimated by both Nei’s gene diversity and Shannon’s information measure, was found in Tunes and the lowest in Algoz. From the estimates of Shannon’s index, the proportion of the diversity within populations was 86.12% and that among populations was 13.88%. Similar results were obtained by AMOVA analysis. A correlation was found between geographic distance and genetic differentiation among populations. The Nm values obtained suggest a high level of gene flow among populations, which was inversely proportional to the distance between populations. RAPDs proved to be a useful tool to determine the genetic diversity of P. algarbiensis populations. The data obtained can be used to develop effective conservation strategies to prevent the decline of populations.