Eryngium maritimum L. is a valuable medicinal species, but since it is protected plant, collection from natural populations is forbidden. Therefore, establishing an efficient system for micropropagation of this species is desirable. To determine the optimal nutritional factors needed for shoot multiplication, root development and secondary metabolites accumulation, different media and plant growth regulators were tested. The highest plant regeneration efficiency (over 96 %), with 4.4 shoots per explant was induced on Murashige and Skoog (MS) medium supplemented with 1.0 mg L⁻¹ benzyladenine (BA) and 0.1 mg L⁻¹ indole-3-acetic acid (IAA). The in vitro-regenerated shoots were rooted (83.3–100 %) and transferred to an experimental plot with 62 % efficiency. Flow cytometric analysis revealed no variation in nuclear DNA content in field- and in vitro-delivered plant material. Ultra high performance liquid chromatography (UHPLC) indicated that multiple shoots and roots from in vitro-regenerated plantlets and adventitious root cultures maintained the production of rosmarinic (RA) and chlorogenic (CGA) acids and triterpenoid saponins found in the rosette leaves and roots of E. maritimum intact plants. UHPLC revealed a 12-fold increase of RA and CGA and 3.2-fold higher accumulation of triterpenoid saponins in roots of in vitro-derived plantlets in comparison to roots from field-grown plants. Adventitious root cultures allowed continuous growth of excised root in liquid media with or without exogenous auxins. The roots grown in liquid medium supplemented with 0.1 mg L⁻¹ IAA showed higher (227-fold) phenolic acids accumulation than those without auxin. Obtained results confirmed that micropropagation is a useful strategy in the protection of endangered species and a renewable source of a high quality plant material for secondary metabolites production.

Background: Polyploid specimens are usually characterized by greater exuberance: they reach larger sizes and/or have a larger number of some organs.Festuca amethystinaL. belongs to the sectionAulaxyper. Based on morphological features, four subspecies ofF. amethystinahave been already identified. On the other hand, it has two cytotypes: diploid and tetraploid. The main aim of our study was to distinguish morphological differences between the cytotypes ofF. amethystina, assuming that its phenotype differs significantly.Methods: The nuclear DNA content was measured by flow cytometry in dry leaves from specimens originating from 13 populations of F. amethystina. Several macrometric and micrometric traits of stems, spikelets and leaf blades were taken into account in the comparative analysis of two cytotypes.Results: In the case of cytotypes, specimens of tetraploids were larger than diploids. The conducted morphometric analysis of leaf cross-sections showed significant differences between the cytotypes.Discussion: The research has confirmed for the first time that in the case ofF. amethystinathe principle of greater exuberance of polyploids is true. Differences between the cytotypes are statistically significant, however, they are not enough to make easy the distinction of cytotypes on the basis of the measurements themselves. Our findings favor the rule known inFestucataxonomy as a whole, i.e. that the ploidy level can be one of the main classification criteria.

Little is known about the stability of plant chimeras after exposure to liquid nitrogen. The aim of this study was to evaluate the effect of cryopreservation on the cytogenetic and genetic stability, as well as vegetative and generative growth of chrysanthemums that are a solid mutant (‘Richmond’) or periclinal chimeras (‘Lady Orange’ and ‘Lady Salmon’). The following encapsulation-dehydration protocol of shoot tip cryopreservation was applied: 0.09 M sucrose concentration and 10 µM abscisic acid during a two-week preculture, followed by a 4-day osmotic dehydration and 3-hour desiccation. Both, the cryopreservation-recovered and control plants were characterized by the same relative DNA content and chromosome number (ploidy level). By applying RAPD and ISSR markers, 18 and 1 and 0 polymorphic loci within cryopreservation-derived ‘Lady Orange’ and ‘Lady Salmon’ and ‘Richmond’ were detected, respectively. The recovered after cryopreservation and control plants had the same colour, diameter and fresh weight of inflorescences, as well as length of ray florets. Cryopreservation also did not affect the flowering time in chrysanthemum. The biochemical assay did not reveal any alternations in the level of anthocyanins and carotenoids in flowers. It was noted, however, that some of the leaves of the cryopreservation-recovered plants were shorter and/or narrower. They had a reduced chlorophyll content, and their internodes were shorter when compared to the untreated control. The inflorescences of ‘Lady Salmon’ opened slower, but faded faster. In conclusion, these results illustrate the practicability of a cryopreservation method that completely protects the chimera chrysanthemum identity.