Micropropagation of forest reproductive material is becoming an increasingly important tool of climate-smart forest management, whose efficiency is depending on artificial illumination, which in turn can have species-specific effects. To improve the energy-efficiency of micropropagation, light emitting diodes (LED) are becoming more popular; however, they emit light of narrow spectral composition, synergic effects of which can alter plantlet development. Regarding the in vitro cultures of trees, such effects have been scarcely studied. In this study, three clones of silver birch (Betula pendula Roth.) and three clones of hybrid aspen (Populus tremuloides Michx. × Populus tremula L.) from the eastern Baltic region were tested. The responses of leaf and stem anatomy of in vitro cultures to three LED light illumination treatments differing by spectral composition and to illumination by fluorescent tubes were estimated by linear (mixed) models. The studied light treatments had non-interacted effects on stomata density and on the secondary xylem cell wall in the stem of silver birch and in the stomata length, stem radius, and phloem width of hybrid aspen. Furthermore, clone-specific responses to illumination were observed for number of chloroplasts and phloem width of silver birch and for leaf thickness and xylem cell wall thickness of hybrid aspen, implying different mechanisms of shade avoidance. In general, the responses of plantlet anatomy differed according to the width of the light spectrum in case of LED, as well as for fluorescent tubes. Considering the legacy effects of early development of plantlets, adaptability of illumination in terms of spectral composition according to the requirements of genotypes appear highly beneficial for micropropagation of sustainable forest reproductive material.

The aim of the present study was to explore heavy metal tolerance and accumulation potential in Anthyllis vulneraria subsp. maritima plants from coastal sand dunes in controlled conditions. Plants were established from seeds collected in coastal sand dunes and cultivated in substrates in greenhouse conditions. A gradual treatment with CdCl₂, PbOAc, CuSO₄, MnSO₄, and ZnSO₄ was performed until three final concentrations for each metal were reached. The number of leaves, their biomass, and biomass of roots were negatively affected by increasing concentrations of lead (Pb) and manganese (Mn) in substrate, but no negative effect was evident for cadmium (Cd), copper (Cu), and zinc (Zn). Visible effects of metal toxicity were evident for Pb-treated plants (appearance of thinner leaves, yellowing of older leaves), as well as for Mn-treated plants (reduced leaf size, curled leaves, red leaf venation). There was a significant decrease in water content in old leaves at high Pb and increasing Mn concentration, indicating accelerated leaf senescence. Increase in polyphenol oxidase activity in leaves was evident in all the plants treated with heavy metals. In contrast, an increase in peroxidase activity was evident only for plants treated with 50 and 100 mg L⁻¹ Cd, 500 mg L⁻¹ Pb, 200–1000 mg L⁻¹ Mn, and 500 mg L⁻¹ Zn. Metal accumulation potential for Cd and Cu was the highest in the roots, but for Pb, Mn, and Zn, more metal accumulated in old leaves. It can be concluded that A. vulneraria subsp. maritima plants are tolerant to high Cd, Cu, and Zn, but moderately susceptible to Pb and Mn. However, oxidative enzyme activity cannot be unequivocally used as a specific indicator of metal tolerance. In respect to phytoremediation potential, the plants have very good accumulation capacity for Pb, Mn, and Zn.

In plants, phase change from the juvenile stage to maturity involves physiological and anatomical changes, which are initiated and controlled by evolutionary highly conserved microRNAs. This process is of particular significance for the in vitro propagation of woody plant species, as individuals or tissues that have undergone the transition to vegetative maturity are recalcitrant to propagation. Conserved miRNAs differentially expressed between juvenile (including rejuvenated) and mature silver birch tissues were identified using high-throughput sequencing of small RNA libraries. Expression of some miR156 isoforms was high in juvenile tissues and has been previously reported to regulate phase transitions in a range of species. Additional miRNAs, such as miR394 and miR396, that were previously reported to be highly expressed in juvenile woody plant tissues were also differentially expressed in this study. However, expression of miR172, previously reported to be highly expressed in mature tissues, was low in all sample types in this study. The obtained results will provide insight for further investigation of the molecular mechanisms regulating vegetative phase change in silver birch and other perennial woody plant species, by analysing a wider range of genotypes, tissue types and maturation stages. This knowledge can potentially assist in identification of rejuvenated material at an earlier stage than currently possible, increasing the efficiency of silver birch in vitro propagation.

In plants, phase change from the juvenile stage to maturity is a tightly controlled process at the physiological and genetic level, which is controlled by evolutionary highly conserved microRNAs. These phase changes are more pronounced in woody plant species, but the majority of molecular genetic studies on the regulation of this transition has been done in annual model or crop species. This process is of particular significance for the in vitro propagation of woody plant species, as individuals or tissues that have undergone the transition to vegetative maturity are recalcitrant to propagation. Development of effective methodologies for silver birch vegetative propagation are required to increase the efficiency of breeding programs. Conserved miRNAs that were differentially expressed between juvenile and mature silver birch tissues were identified using high-throughput sequencing of small RNA libraries. These differentially expressed miRNAs could potentially be utilized to develop markers indicating the juvenility or maturity of silver birch explants and in vitro cultures. In addition, the obtained results will provide an insight into the molecular mechanisms regulating vegetative phase change in silver birch and other perennial woody plant species.