Micropropagation Protocol and Genetic Stability of the Salix myrtilloides Plants Cultivated In Vitro.

Salix myrtilloides L. is a relict species, threatened with extinction in many European countries. To prevent the loss of the species, tissue culture was established to produce plant material for reintroduction in natural habitats. Micropropagation was chosen as a method to obtain new plants. S. myrtilloides shoots were disinfected with NaOCl, AgNO3, or with a two-step disinfection with NaOCl, and then placed on MS medium supplemented with BA at 1 mg·dm-3 and IBA at 0.1 mg·dm-3. Regenerated shoots were cultivated in presence of BA, KIN, and 2iP to select the treatment with the highest multiplication rate. The obtained plants were acclimatized to ex vitro conditions. Inter-simple sequence repeat (ISSR) and flow cytometric analyses were conducted on in vitro regenerated plants to check their genetic stability. The best disinfection results were obtained when explants were treated with 1.5% NaOCl for 20 min. The highest multiplication rate and good quality plants were noted in the control media, without growth regualtors and in presence of kinetin at 0.5 mg·dm-3. Flow cytometry and ISSR analyses confirmed genetic stability in plantlets, which indicated the possibility to use the in vitro obtained plants for reintroduction.

The Search for Suitable Habitats for Endangered Species at Their Historical Sites-Conditions for the Success of Salix lapponum and Salix myrtilloides Reintroduction.

Restoring endangered plant species to their historical sites is not always possible due to constantly changing habitat conditions. The aim of this study was to test the effects of reintroduction of two relict willow species in eastern Poland. The experiment consisted of planting 48 individuals of Salix lapponum and S. myrtilloides, obtained by micropropagation, at each of the two selected sites and observing their survival after one year. At the same time, selected physicochemical and biocenotic factors of the environment were monitored. About 70% of S. lapponum individuals and 50% of S. myrtilloides plants survived the one-year period. This result can be considered satisfactory and confirms the effectiveness of this means of active protection. The results of measurements of selected abiotic factors of the environment and the observations and ecological analysis of the flora indicated that the habitat conditions of both historical sites have changed, resulting in accelerated succession of vegetation. However, complete habitat degradation did not occur, although the development of a multi-story structure of one of the phytocenoses intensified competition for light and other environmental resources, which narrowed the potential ecological niche of the reintroduced species.

Optimization of Propagation of the Polish Strain of Aldrovanda vesiculosa in Tissue Culture.

Aldrovanda vesiculosa is a rare and critically endangered carnivorous plant species. Its populations have declined worldwide, so there is a need to protect the species from extinction. The research was conducted to establish an effective method of in vitro propagation of the species in order to obtain plants for reintroduction in the wild. The procedures included disinfection, multiplication, and acclimatization of plants. Contamination-free in vitro cultures were established using shoots and turions, which were disinfected with 0.25% sodium hypochlorite. The shoots were first defoliated. The explants regenerated better in liquid 1/5 MS medium than in solidified one. The optimum medium for the multiplication phase contained MS macro- and microelements diluted to 1/10. Plants cultivated in that medium were of good quality, long, and branched. The advantageous effect of medium was also confirmed by the content of photosynthetic pigments in the plant material. The content of chlorophyll a was highest in plants cultivated in 1/5 or 1/10 MS medium. The plants obtained were acclimatized to ex vitro conditions and reintroduced in the wild.

Searching for Optimal Substitute Habitats for Plants by Biological Experiments-A Case Study of the Endangered Species Aldrovanda vesiculosa L. (Droseraceae).

The selection of appropriate locations for the reintroduction of endangered plant species is an important process, because it usually influences the success of the conservation. The aim of this study was to select the optimal substitute habitats for Aldrovanda vesiculosa, taking into account the influence of physical-chemical factors (light intensity, temperature, pH, concentration of dissolved forms of nitrogen and cyanobacterial toxin microcystin-LR) on the efficiency of plant growth. Water analysis and field observations of the habitats of six lakes in Eastern Poland typified as potential substitute habitats for aldrovanda were carried out. The results of the experiments showed that both the concentration and the form in which nitrogen compounds are present in the environment were the factors limiting the growth rate and condition of plants. The second factor that caused the inhibition of aldrovanda growth was microcystin-LR. It was found that the habitat conditions in Lake Brzeziczno were within the ecological tolerance of the species. Particularly important was the low content of mineral compounds and the available forms of nitrogen and phosphorus in the water. Therefore, the probability of development of toxic cyanobacteria, the metabolites of which may affect the growth of A. vesiculosa, is also minimal.

Paphiopedilum insigne Morphological and Physiological Features during In Vitro Rooting and Ex Vitro Acclimatization Depending on the Types of Auxin and Substrate.

To obtain healthy and good quality plants from in vitro cultivation, it is necessary to produce plantlets with well-developed rooting systems because they must undergo acclimatization, a final and a very difficult stage of micropropagation. In the present research, the effect of auxins NAA, IAA and IBA in concentrations of 0.5; 1; 2.5 and 5 mg·dm-3 on the Paphiopediluminsigne in vitro rooting was studied, and it was noted that 1 mg·dm-3 of IAA or IBA enabled the obtaining of a lot of rooted and good quality plantlets. The subsequent influence of the two most advantageous auxins on the acclimatization of plantlets in different substrates (sphagnum moss, sphagnum moss + substrate for orchids, substrate for orchids, substrate for orchids + acid peat) was tested, in the means of morphological features of plants and their physiological parameters, i.e., chlorophyll fluorescence (FV, Fm, Fv/Fm), stress enzyme activity (catalase, ascorbate peroxidase), and water balance. Considering all the tested features, it might be stated that the best results were obtained when explants were rooted in vitro in the presence of 1 mg·dm-3 of IAA and then planted ex vitro in substrate for orchids.

Genetic Stability of the Endangered Species Salix lapponum L. Regenerated In Vitro during the Reintroduction Process.

Salix lapponum L. is a boreal relict species, threatened with extinction in Poland. An 80% decrease in the number of its stands was confirmed in the last half-century, so that to prevent the loss of downy willow, attempts were made to reintroduce this species in natural habitats. Micropropagation was chosen as a first stage of its active conservation. S. lapponum shoots were collected and disinfected with NaOCl, AgNO3, or HgCl2 or with a two-step disinfection with NaOCl and then placed on MS medium with BA 1 mg·dm-3 and IBA 0.1 mg·dm-3. Regenerated shoots were cultivated with addition of BA, KIN, or 2iP, alone or in combination with auxins, to find the highest multiplication rate. Inter-simple sequence repeat (ISSR) analysis and flow cytometric analyses were conducted on in vitro regenerated plants to check their genetic stability. Disinfection was quite difficult and the use of HgCl2 was the most efficient. The highest multiplication rate was obtained in presence of KIN at 0.5 mg·dm-3 + IAA at 0.5 mg·dm-3. The analysis confirmed the genome size stability, which is in agreement with the results obtained by ISSR, revealing no somaclonal variation in plantlets and therefore allowing the use of the obtained plants for reintroduction.

Carex muskingumensis and Osmotic Stress: Identification of Reference Genes for Transcriptional Profiling by RT-qPCR.

Carex muskingumensis is a highly valued perennial ornamental grass cultivated worldwide. However, there is limited genetic data regarding this species. Selection of proper reference genes (RGs) for reverse transcription quantitative PCR (RT-qPCR) data normalization has become an essential step in gene expression analysis. In this study, we aimed to examine expression stability of nine candidate RGs in C. muskingumensis plants, subjected to osmotic stress, generated either by salinity or PEG treatment. The identification of genes exhibiting high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and deltaCt method). The results showed that the combination of two genes would be sufficient for reliable expression data normalization. ADP (ADP-ribosylation factor) and TBP (TATA-box-binding protein) were identified as the most stably expressed under salinity treatment, while eIF4A (eukaryotic initiation factor 4A) and TBP were found to show the highest stability under PEG-induced drought. A set of three genes (ADP, eIF4A and TBP) displayed the highest expression stability across all experimental samples tested in this study. To our best knowledge, this is the first report regarding RGs selection in C. muskingumensis. It will provide valuable starting point information for conducting further analyses in this and related species concerning their responses to water shortage and salinity stress.