Generative processes have been evaluated in six European buttercup species in order to verify the hypothesis that the reproduction efficiency of clonal species is lower than that of nonclonal ones. The study covered common species (Ficaria verna, Ranunculus auricomus, R. bulbosus, R. cassubicus, R. lanuginosus) and the endangered R. illyricus. The following properties have been assessed: pollen viability (staining method), pollen grain germination and the pollen-tube elongation in pistil tissues (fluorescence microscopy), seed formation efficiency, seed viability (tetrazolium test) and germination ability by introducing factors interrupting dormancy (low temperature and gibberellin application). Additionally, the pistil morphology was documented for R. bulbosus, R. illyricus and R. cassubicus using SEM techniques. It was demonstrated that the reproductive efficiency, expressed as the production of viable seeds able to germinate, was significantly higher in the species reproducing sexually (especially in R. lanuginosus) compared to the clonal ones. However, the complexity observed leads to separation of an additional group (cluster) of apomictic species: R. auricomus and R. cassubicus, distinguished by the lowest pollen viability and a low ability of the seeds to germinate. In the vegetatively reproducing R. illyricus, the seed formation efficiency was just 13.2% despite the having highest number of pistils in its flowers. The developed seeds of this species observed in our experiment were viable, but in general effective methods to stimulate their germination have not been proposed yet. Here, the first comparative study concerning the biology of sexual reproduction of R. illyricus is presented in the context of its decreasing distribution in natural habitats.

Pulsatilla turczaninovii is an important medicinal plant, valued for high ornamental value of melliferous flowers. We assessed the efficiency of reproduction under in vitro conditions and the ex situ growth capacity of this important representative of the world flora. The seed germination percentage was assessed, followed by determination of micropropagation rate and rooting efficiency. Then, the possibility of plant development in three consecutive growing seasons was assessed. The in vitro germination percentage was approximately 55%. The highest multiplication coefficient, amounting to 5.17, was obtained on modified MS medium supplemented with 2.5 mg L−1 2iP and 1.0 mg L−1 IAA. Our study provided unique insight on biochemical background of root regeneration in P. turczaninovii. In comparison with standard auxin-supplemented rooting medium, the treatment with 1.0 mg L−1 level of ethylene precursor ACC elevated rooting by about 20%. The total content of soluble sugars was proved to be biomarker of rhizogenesis in the studied species. Their concentration was positively correlated with rooting efficiency, while a level of phenolic was positively correlated with the length of regenerated roots, and their number per rosette. The cultivation of the acclimatized material was successfully carried out and was evaluated over three subsequent years. In the third year of cultivation, the plants entered the stage of generative development and most of them bloomed profusely.

Ranunculus illyricus, a component of xerothermic grasslands, is a declining species and deserves active conservation treatments in many countries preceded by studies on the biology of its reproduction. So far, our knowledge of R. illyricus, a species with two modes of reproduction, has been fragmentary. The purpose of the studies presented here was to describe the annual development cycle of R. illyricus with particular emphasis on the production of underground tuber clusters that serve as vegetative propagation. Based on three-year-long observations in an ex situ collection, the efficiency of vegetative propagation was estimated and compared with the efficiency of generative propagation. It was found that in 3 years the best clones could produce up to 57 progeny clusters followed by flowering specimens in the first season. Meanwhile, the high potential for generative reproduction was suppressed by many limitations including fruit setting, the germination capacity of seeds, seedling survival rate, and additionally, the first flowering plant was observed only in the third year. It seems that the efficiency of vegetative propagation of this species can be higher than the efficiency of generative propagation. Moreover, vegets bloomed in the first year after emergence, whereas the first plant of generative origin was observed to bloom only after 3 years. A large proportion of individuals of vegetative origin can negatively affect the genetic diversity of the population but their survival rate against competing plants is higher. To enhance the existing populations or to create new ones, it would be best to use plants derived from clonal propagation of genets carried out in ex situ conditions.

Aconitum bucovinense Zapał. is the European species of monkshood, endemic to the Eastern and Southern Carpathians. A protocol has been developed for the in vitro regeneration of adventitious shoots by indirect organogenesis from leaf explants. An initiation of cultures carried out on a medium with picloram and kinetin allowed obtaining a callus. More than a 200% FM increase of the callus and at the same time differentiation of adventitious buds were obtained on IBA and BAP supplemented media. Excised buds were used to establish shoot cultures and multiplied after a transfer to nutrient media with an addition of BAP with IBA, IAA or NAA. Almost 70% of rooted shoots were obtained on a 1.5 mg L − 1 IBA and 1.0 mg L − 1 BAP supplemented medium with simultaneous e cient multiplication. An analysis of peroxidase activity revealed its gradual increase in shoots until the appearance of roots. For the rst time, an e cient way to regenerate, multiply and root A. bucovinense shoots has been developed and can be used for ex situ conservation of this species. Key MassageThe system of plant regeneration of Aconitum bucovinense from callus culture was elaborated. The analysis of the peroxidase level showed that its content increases until the rst roots appear.

Streszczenie: Celem przeprowadzonych badań było zbadanie wpływu jonów kadmu na galmanowy ekotyp Alyssum montanum w warunkach kultury in vitro. Zastosowano dodatek 1; 2,5 i 5 µM chlorku kadmu do pożywki namnożeniowej. W trakcie trwania kultury sprawdzano współczynnik namnażania, prowadzono pomiary biometryczne, oceniano świeżą i suchą masę regenerowanych organów oraz kondycję fizjologiczną kultur poprzez pomiar zawartości chlorofili. Badany ekotyp można efektywnie rozmnażać wegetatywnie w obecności jonów kadmu w podłożu hodowlanym, jednak poszczególne stężenia CdCl 2 w różny sposób oddziaływały na oceniane cechy morfometryczne mnożonych mikroroślin. Wyższe wartości pomiarów uzyskano w kulturach prowadzonych na pożywkach wzbogaconych w chlorek kadmu aniżeli na pożywkach kontrolnych bez dodatku CdCl 2 . Najwyższą masę pędów, wynoszącą ponad 11 mg, uzyskano w trakcie kultywacji badanego ekotypu na zmodyfikowanej pożywce WPM wzbogaconej w 0,5 µM CdCl 2 , natomiast największą liczbę pędów na pożywce wzbogaconej w 5 µM tej soli. Słowa kluczowe: smagliczka, kadm, ekotyp galmanowy, kultury in vitro.Summary: The aim of this study was to determine an impact of cadmium ions on calamine ecotype of Alyssum montanum, using in vitro culture. The propagation medium was supplemented with different concentrations of cadmium chloride, that is 0.5 µM; 2.5 µM and 5 µM. During the cultivation of microshoots propagation coefficient, leaf blade surface coefficient, fresh, dry weight and numerous biometric features were evaluated. Studied ecotype can be propagated in vitro on modified WPM medium supplemented with cadmium ions, but individual CdCl 2 concentrations differentially affected evaluated morphometric traits of studied culture. The higher values of some traits were obtained on media supplemented with cadmium chloride in comparison to control ones. The highest shoot mass (above 11 mg) was obtained on modified WPM supplemented with 0,5 µM CdCl 2 , whereas the highest number of regenerated shoots were obtained on the same medium supplemented with 5 µM of the same salt.

Aconitum bucovinense Zapał. is the European species of monkshood, endemic to the Eastern and Southern Carpathians. A protocol has been developed for the in vitro regeneration of adventitious shoots by indirect organogenesis from leaf explants. An initiation of cultures carried out on a medium with picloram and kinetin allowed obtaining a callus. More than a 200% FM increase of the callus and at the same time differentiation of adventitious buds were obtained on IBA and BAP supplemented media. Excised buds were used to establish shoot cultures and multiplied after a transfer to nutrient media with an addition of BAP with IBA, IAA or NAA. Almost 70% of rooted shoots were obtained on a 1.5 mg L− 1 IBA and 1.0 mg L− 1 BAP supplemented medium with simultaneous efficient multiplication. An analysis of peroxidase activity revealed its gradual increase in shoots until the appearance of roots. For the first time, an efficient way to regenerate, multiply and root A. bucovinense shoots has been developed and can be used for ex situ conservation of this species.

Pulsatilla turczaninovii is an important medicinal plant and a material also appreciated by gardeners for its high ornamental value. In this study we assessed the efficiency of reproduction under in vitro conditions of this important representative of the world flora. First, the germination percentage and the following plant development during three growing seasons were assessed, then we tested the possibilities of multiplication by in vitro culture methods. Our study provided new insight on biochemical background of generating adventitious roots in Pulsatilla considered difficult-to-rooting. The germination percentage of studied population reached about 55%. The highest multiplication coefficient of micro-rosettes in tissue culture, amounting to 5.17, was achieved using modified MS medium supplemented with 2.5 mg L-1 2iP and 1 mg L-1 IAA. We proved that in comparison with standard auxin-supplemented rooting medium, the treatment with addition of 1.0 mg L-1 ethylene precursor ACC elevated adventitious rooting by about 20%. The biochemical analyses revealed that total content of soluble sugars was the most impactful biomarker of adventitious rhizogenesis in studied species. Concentration of sugars was positively correlated with rooting efficiency, while the level of phenolic compounds was positively correlated with the root length and their number regenerated per single rosette.