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The advantages of high dynamic range (HDR) imaging in videodensitometry are presented and discussed on the example of Gentiana extract thin-layer fingerprints visualized under 254 nm. An inexpensive microscope camera, together with security surveillance lens, mounted instead of original camera on videodensitometry chamber, allows to grab HDR images with high tonal range using Python scripts and OpenCL library. HDR imaging preserves linearity in whole tonal range and does not destroy details in the brightest and darkest plate regions, so it can be seen as a good alternative to classical videodensitometry. Moreover, the tonemapping of HDR images can be used to present a plate photograph with enhanced visibility of weak spots and other details. Principal component analysis done on nine classic exposures and HDR image proves that HDR image contains the highest amount of extracted information from the thin-layer chromatographic plate.

Asplenium cuneifolium Viv. is an endangered fern species restricted to European serpentine rocks, which face a risk of being easily damaged by both natural and anthropogenic agents. Establishing a comprehensive system of effective micropropagation and long-term storage of this species is therefore desirable. Freshly collected spores of A. cuneifolium were subjected to direct storage at 5 °C and in liquid nitrogen (LN). The viability of spores stored at 5 °C decreased by 10% after 1 year, whereas storage in LN did not change the initial spore viability even after 3 years. For the initiation of the gametophyte culture, disinfected spores were sown onto half-strength Murashige and Skoog medium (½MS) supplemented with 0.06 M sucrose. Following 6 months, abundant proliferation of secondary gametophytes was achieved. For the cryopreservation of gametophytes, encapsulation-vitrification and encapsulation-dehydration, were compared. Of these two methods, encapsulation-vitrification ensured survival of gametophytes of 64.5–93%, while encapsulation-dehydration guaranteed their 100% viability. The acceleration of syngamy on secondary gametophytes was achieved using a medium with macro- and micronutrients reduced to the 1/8 of the MS free of NH4NO3 and vitamins. The syngamic sporophytes could be multiplied by somatic embryogenesis (SE) induced on etiolated stipe explants in an ½MS medium devoid of any plant growth regulators. Adding 0.3 M of sucrose to the medium almost quadrupled the efficiency of the SE. The genetic stability of gametophytes after cryopreservation, as well as sporophytes obtained from them, was confirmed by flow cytometry, amplified fragment length polymorphism, and inter-simple sequence repeat markers.

Komórki roślinne mają niezwykłą zdolność adaptacji do zmian środowiskowych. Jej przejawem jest tworzenie zarodków wprost z komórek ciała rośliny z pominięciem etapu zapłodnienia. Powstałe tą drogą strukturyrozwijają się w kompletne rośliny, a samproces, dla odróżnienia drogi powstawania i podkreślenia spójności z embriogenezą zygotyczną, jest określany mianem embriogenezy somatycznej (SE). Mimo, iż w2018 roku minęło 60 lat od opisania tego zjawiska po raz pierwszy, to mechanizm przeprogramowania komórki somatycznej w embriogeniczną nadal nie jest w pełni poznany. Jest to krytyczny etap wSE, który może być indukowany przez egzo- i endogenne czynniki oraz stresowe traktowanie. Działanie tych czynników na materiał roślinny wpływa na reorganizację struktury chromatyny, a tym samym na ekspresję genów, co w konsekwencji może uruchamiać program embriogenezy. W niniejszej pracy dokonano przeglądu aktualnej wiedzy na temat tego, jak określana jest tożsamość komórek totipotencjalnych oraz bodźców wymaganych do przeprogramowania rozwoju komórek somatycznych. Podsumowano wiedzę na temat kluczowych molekularnych regulatorów i związanej z nimi sieci powiązań, które kontrolują indukcję SE. Zdefiniowano równieżzagadnienia, które są ważne dla podniesienia zrozumienia mechanizmów leżących u podstaw totipotencji. Na zakończeniepokazano potencjał praktyczny drzemiący w tym procesie i podano przykłady jego wykorzystania.

Plant cells possess the remarkable ability to adapt to environmental changes. It is manifested by formation of embryos directly from the cells of plant body, bypassing the fertilization stage. These embryo structures develop into complete plants. The process itself, to distinguish the path of formation and emphasize consistency with zygotic embryogenesis, is referred to as somatic embryogenesis (SE). Although more than 60 years have passed since the first publication on the phenomenon has been written, the mechanism of reprogramming of a somatic cell into an embryogenic one is still not fully understood. This is a critical step in SE that can be induced by exo- and endogenous factors and stress treatments. The exposition of plant material to these factors affects the reorganization of the chromatin structure and gene expression, which can consequently trigger the program of embryogenesis. The paper reviews current knowledge on how the identity of totipotent cells is determined and the which stimuli are required to reprogram somatic cell development. Knowledge of key molecular regulators and the network of relationships that control the SE induction is summarized. Issues that are important for enhancing the understanding of the mechanisms underlying totipotency are also defined. Finally, the practical potential of SE is demonstrated, and examples of its use are provided.Komórki roślinne mają niezwykłą zdolność adaptacji do zmian środowiskowych. Jej przejawem jest tworzenie zarodków wprost z komórek ciała rośliny z pominięciem etapu zapłodnienia. Powstałe tą drogą strukturyrozwijają się w kompletne rośliny, a samproces, dla odróżnienia drogi powstawania i podkreślenia spójności z embriogenezą zygotyczną, jest określany mianem embriogenezy somatycznej (SE). Mimo, iż w2018 roku minęło 60 lat od opisania tego zjawiska po raz pierwszy, to mechanizm przeprogramowania komórki somatycznej w embriogeniczną nadal nie jest w pełni poznany. Jest to krytyczny etap wSE, który może być indukowany przez egzo- i endogenne czynniki oraz stresowe traktowanie. Działanie tych czynników na materiał roślinny wpływa na reorganizację struktury chromatyny, a tym samym na ekspresję genów, co w konsekwencji może uruchamiać program embriogenezy. W niniejszej pracy dokonano przeglądu aktualnej wiedzy na temat tego, jak określana jest tożsamość komórek totipotencjalnych oraz bodźców wymaganych do przeprogramowania rozwoju komórek somatycznych. Podsumowano wiedzę na temat kluczowych molekularnych regulatorów i związanej z nimi sieci powiązań, które kontrolują indukcję SE. Zdefiniowano równieżzagadnienia, które są ważne dla podniesienia zrozumienia mechanizmów leżących u podstaw totipotencji. Na zakończeniepokazano potencjał praktyczny drzemiący w tym procesie i podano przykłady jego wykorzystania.

Somatic hybridization by protoplast fusion is used in breeding programs to obtain plant material that has inherited valuable traits from two different species, and in order to broaden plant genetic diversity. Somatic hybrids of the genus Gentiana could provide a useful source of new ornamental cultivars and of secondary metabolites. However, in order to evaluate its further usefulness, detailed characterization of the newly created hybrid is essential. Here, genome composition and stability of interspecific gentian somatic hybrids obtained following electrofusion of cell suspension-derived protoplasts of diploid Gentiana kurroo Royle with leaf mesophyll-derived protoplasts of tetraploid G. cruciata L. were characterized using various molecular markers and flow cytometry. AFLP and ISSR analyses revealed that all 21 hybrid plants and 3 lines of hybrid callus were genetically closer to G. cruciata than to G. kurroo. According to the results of chloroplast DNA analysis with the use of CAPS markers, all somatic hybrids inherited chloroplasts from “mesophyll” fusion partner G. cruciata. Flow cytometry revealed that polyploidization occurred, and probably it took place at the early stage of post-fusion culture. In consequence, gradual elimination of nuclear DNA, mixoploidy, and high genetic instability were observed in most hybrid plants and calli during the subsequent 4 years of in vitro culture.

The family Gentianaceae consists of 1736 species, which play an important role in human being existence due to their pharmacological and horticultural values. Many species accumulate bitter iridoid substances used medicinally and in flavorings, while others are cultivated because of beauty of their flowers showing a wide range of colors and patterns. Out of 99 genera belonging to the gentian family, process of somatic embryogenesis (SE) was reported for 5. The first reports, aimed at micropropagation of ornamental cultivars and production of secondary metabolites, concerned Centaurium erythraea Rafn., Eustoma russellianum Grieseb. and Exacum affine Balf. Somatic embryos were induced on different explants cultured in the liquid Murashige and Skoog medium supplemented with auxins and cytokinins. In the 1990s of the last century, significant progress in the exploration of the phenomenon of SE and its biotechnological application was made for the genus Gentiana. The process was induced on various explants and studied at the structural and ultrastructural levels. Regenerated plants were screened for genetic stability using flow cytometry, chromosome counting, and molecular markers. Besides typical indirect SE, the use of leaf fragments enabled to obtain single-cell origin of somatic embryos. On the other hand, proliferation of embryogenic callus in liquid medium resulted in the establishment of long-term embryogenic cell suspension cultures, paving the way not only to study the formation of somatic embryos and the development of regenerants but also to preserve the morphogenic potential of cell aggregates by cryopreservation. Cell suspensions re-established after storage in liquid nitrogen maintained their embryogenic character and allowed to obtain somatic embryo-derived regenerants that were true-to-type at both genetic and epigenetic levels. Another application of SE was related to genetic manipulation purposes. Efficient protocols of plant regeneration from callus-, cell suspension-, or leaf mesophyll-derived protoplasts allowed engaging procedures of somatic hybridization or protoplast electroporation for gentian genome modifications. Also, high embryogenic potential existing in the numerous gentian species enabled successful Agrobacterium-mediated transformation of G. cruciata L. and G. dahurica Fisch.

The morphogenic potential of five Gentiana species of medicinal and ornamental value, i.e.G. cruciata, G. kurroo, G. lutea, G. septemfida, and G. tibetica, was compared using agarose-bead leaf mesophyll protoplast culture. Modified Murashige and Skoog medium containing 2.0 mg l−1 1-naphthaleneacetic acid and 0.1 mg l−1 thidiazuron ensured the highest cell division frequency during both protoplast and protoplast-derived cell culture or callus formation. Indirect plant regeneration mainly via the somatic embryogenesis pathway was achieved for G. kurroo and G. tibetica with the greatest efficiency occurring with MS medium supplemented with 1.0 mg l−1 kinetin, 0.5 mg l−1 gibberellic acid, and 80 mg l−1 adenine sulfate. A considerable percentage of autopolyploid and aneuploid cells was detected in callus lines obtained from protoplasts using flow cytometry. The presence of polyploid cells in calli resulted in the regeneration of 85 % polyploid plants of G. kurroo and 14 % polyploids of G. tibetica. Chromosome counting and stomatal characteristic confirmed the ploidy variation among regenerants.

In spite of the recent description of somatic embryogenesis for ferns, there are a lot of evidences that the morphogenic potential of their cells was the objective of numerous studies. The chapter tries to give the summary of the information of morphogenic potential of both fern generations. The fern body provides a source of various explants, both single-celled and multicellular. Under various plant growth regulator regimes, plant regeneration occurs; however, it is stressed that for some of developmental stages, hormone-free medium is required. For numerous species, the system of sporophyte regeneration was presented and discussed. The attention is paid for callus and cell suspension, and their initiation and culture are maintained as the stage of the great potential which might be explored for fern somatic cell manipulation. Protoplasts are the objective of somatic cell genetic manipulation in plants; however, for ferns the number of papers involving protoplast isolation and their subsequent culture is scant. Independently, the attention is paid for green globular bodies, multicellular structures with unlimited shoot regeneration potential.

Somatic embryogenesis (SE) is a fascinating developmental program that was first described for carrot 60 years ago. In 2015, the first report of SE in the Monilophyta was published for Cyathea delgadii Sternb. Using the data obtained for somatic embryo induction and development in this tree fern, we presented a new model system for studying early events in SE. Here, we summarize what is known of that model system, which requires a hormone-free medium to induce embryogenic competence. Special emphasis is placed on hormonal regulation of somatic-to-embryogenic transition and on the cytomorphological and proteomic changes that occur during early SE. Our research also reveals that SE is able to improve fern productivity to a much greater extent than can current conventional in vitro propagation methods for cryptogamic plants.

The embryogenic cell suspension culture of Gentiana cruciata, cryopreserved by the encapsulation/dehydration method, survived both short- (48 h) and long-term (1.5 years) cryostorage with more than 80% viability. To assess the influence of cryotreatments on the embryogenic potential, a proembryogenic mass was encapsulated and exposed to the following treatments: (1) osmotic dehydration (OD), (2) OD + air desiccation (AD) and (3) OD + AD + cryostorage (LN). The somatic embryogenesis efficiency increased ten times after osmotic dehydration. The AD and LN cryotreatments did not cause any significant alterations in somatic embryo production. We monitored the (epi)genetic stability of 288 regenerants derived from: non-cryotreated, short-term, and long-term cryostored tissue using metAFLP markers and ten primer combinations. Changes in the sequence and DNA methylation levels were studied by subjecting the DNA to digestion with two pairs of isoschisomer restriction enzymes (KpnI/MseI and Acc65I/MseI). Two new AFLP unique DNA fragments at the DNA sequence level, with no differences at the methylation level, were found between regenerants derived from cryopreserved tissue, compared with the non-cryotreated controls. The Acc65I/MseI methylation levels for the three groups of regenerants were not significantly different. Cluster analysis was capable of identifying a number of sub-clusters. Only one of the sub-clusters comprises almost all regenerants derived from non-cryotreated and short-term cryostored tissue. Plantlets derived from long-term cryostored tissue were grouped into separate clusters. The observed AFLP alterations did not appear to be associated with the use of cryopreservation, but were probably related to the process of in vitro culture.

A simple and reliable technique for in vitro multiplication and long-term preservation using liquid nitrogen was developed for gametophytes of Osmunda regalis. The effect of Knop’s medium and various concentrations (1/2, 1/4, 1/8) of mineral salts provided in Murashige and Skoog (MS) basal medium, together in the presence or absence of both ammonium nitrate and a full complement of vitamins on gametophyte proliferation and sporophyte production was determined. Moreover, the effectiveness of gametophyte cryopreservation by vitrification and encapsulation–vitrification techniques was assessed. Maximum gametophyte proliferation (89 %) occurred on the ammonium nitrate- and vitamin-free [(-NH4NO3)(-vit)] MS medium with 1/2 or 1/4 strength mineral salts. The maximum production of sporophytes (30 plantlets per gametophyte clump) required 1/8MS (-NH4NO3)(-vit) medium. The flow cytometric analysis revealed that the sporophytes contained twofold more pg DNA than gametophytes. This confirmed that the sporophytes were obtained by sexual reproduction. The vitrification protocol and PVS2 solution were ineffective for cryopreservation. The greatest survival rate (81.6 %) following cryo-exposure occurred following treatment of encapsulated gametophytes with PVS3 solution for 3 h. This protocol allowed the recovery of gametophyte cultures following 6 weeks after rewarming. Finally, 100 % of sporophytes produced in vitro were successfully acclimated to ex vitro conditions. Application of the in vitro and cryopreservation methods made it possible to improve the number and time of O. regalis sporophyte production. Whole system of micropropagation can be completed in approximately 1 year. The protocols open new avenues for the mass propagation, germplasm conservation and resource management of the species.

For spores, like seeds, the moisture level and storage temperature seem to be crucial factors responsible for their survival. However, in chlorophyllous spores in particular, the problem is not yet fully understood. The aim of this study was to investigate the effect of moisture content (MC; 6.6 and 4.6 %) and storage temperature (15 and −196 °C) on spore germination and gametophyte development in Osmunda regalis. The MC (fresh weight basis) in freshly released spores was 7.5 %, and decreased to 6.6 and 4.6 % during 2 weeks of spore storage at 42 and 18 % relative humidity, respectively. Those spores germinated at 99.9 % within 28 h. The spores with 6.6 and 4.6 % MC were used for the storage at 15 and −196 °C. The deterioration of spores, maintained with 6.6 % MC, proceeds within 1.5 years of storage at 15 °C, starting with month 8. For the spores with 4.6 % MC serious disturbances in germination and capacity to gametophyte development were postponed for more than 6 months. The time required for spore germination increased with the age of the spores, ranging from 28 h in fresh spores to 10 days in spores stored for more than 11 months. Following 7-year-long cryostorage, spore viability remained at 99.9 %, the time taken for germination remained unaltered and gametophyte development was normal. The viability and vigor of spores were directly correlated with their age, the moisture content and storage temperature.

During the last three decades, less than fifteen papers have described the results of scientific investigations in the field of gentian protoplast technology and somatic hybridization. Despite rather limited research already done on this subject, several important goals have been achieved. Protoplast-to-plant systems have been developed either for leading ornamental species or for specific medicinal plants. Two major protoplast sources were evaluated in gentians, namely differentiated leaf mesophyll cells and undifferentiated callus/cell suspensions. Plant regeneration proceeded by the two different pathways of shoot organogenesis or somatic embryogenesis. Some examples of somaclonal variation at the ploidy level were demonstrated within the pool of protoplast-derived regenerants. Totipotency exhibited by gentian protoplasts was exploited to create three different somatic hybrid combinations: intergeneric Swertia mussotii (+) Bupleurum scorzonerifolium , and interspecific Gentiana kurroo (+) G. cruciata and G. cruciata (+) G. tibetica.

Cryopreservation technologies have opened the door to the possibility of long-term storage of valuable biodiverse germplasm of many plant species. However, for successful conservation by cryopreservation , a large amount of work is still needed. This chapter describes the role of plants belonging to the family Gentianaceae in improving our understanding of cryopreservation. Emphasis is on expected problems and benefits originated from enhancing embryogenic capacity by osmotic dehydration stress. In addition, routine application is shown of cryopreservation for long-term conservation of in vitro propagated germplasm of the Gentianaceae.

Key message Somatic embryogenesis has never been reported in ferns. The study showed that it is much easier to evoke the acquisition and expression of embryogenic competence in ferns than in spermatophytes. Abstract We discovered that the tree fern Cyathea del- gadii offers an effective model for the reproducible and rapid formation of somatic embryos on hormone-free medium. Our study provides cyto-morphological evidence for the single cell origin and development of somatic embryos. Somatic embryogenesis (SE) in both primary and secondary explants was induced on half-strength micro- and macro-nutrients Murashige and Skoog medium without the application of exogenous plant growth regulators, in darkness. The early stage of SE was characterized by sequential perpendicular cell divisions of an individual epidermal cell of etiolated stipe explant. These resulted in the formation of a linear pro-embryo. Later their devel- opment resembled that of the zygotic embryo. We defined three morphogenetic stages of fern somatic embryo development: linear, early and late embryonic leaf stage. The transition from somatic embryo to juvenile sporophyte was quick and proceeded without interruption caused by dormancy. Following 9 weeks of culture the efficiency of somatic embryogenesis reached 12-13 embryos per responding explant. Spontaneous formation of somatic embryos and callus production, which improved the effectiveness of the process sevenfold in 10-month-long culture, occurred without subculturing. The tendency for C. delgadii to propagate by SE in vitro makes this species an excellent model for studies relating to asexual embryo- genesis and the endogenous hormonal regulation of that process and opens new avenues of experimentation.

Somaclonal variation, often manifested as the increased ploidy of plants observed following in vitro culture, can be advantageous in ornamental species or those used for secondary metabolite production. Polyploidy occurs especially when plantlets are produced by protoplast and callus cultures. Plants were regenerated from green leaf mesophyll protoplasts of diploid Gentiana decumbens L.f. through somatic embryogenesis. A yield of more than 9 × 105 protoplasts per gram of fresh weight was achieved by incubating fully expanded young leaves in an enzyme mixture containing 1.0% (w/v) cellulase and 0.5% (w/v) macerozyme. Protoplasts, cultured in agarose beads using a modified Murashige and Skoog medium, divided and formed microcalli, with the highest plating efficiency obtained on medium containing 2.0 mg l−1 1-naphthaleneacetic acid and 0.1 mg l−1 thidiazuron. Callus proliferation was also promoted by including thidiazuron in agar-solidified medium, while somatic embryogenesis was induced from microcalli on medium supplemented with 1.0 mg l−1 kinetin, 0.5 mg l−1 gibberellic acid, and 80 mg l−1 adenine sulfate. Flow cytometric analysis and chromosome counting revealed that all regenerants were tetraploid.

Somaclonal variation, often manifested as the increased ploidy of plants observed following in vitro culture, can be advantageous in ornamental species or those used for secondary metabolite production. Polyploidy occurs especially when plantlets are produced by protoplast and callus cultures. Plants were regenerated from green leaf mesophyll protoplasts of diploid Gentiana decumbens L.f. through somatic embryogenesis. A yield of more than 9 × 105 protoplasts per gram of fresh weight was achieved by incubating fully expanded young leaves in an enzyme mixture containing 1.0% (w/v) cellulase and 0.5% (w/v) macerozyme. Protoplasts, cultured in agarose beads using a modified Murashige and Skoog medium, divided and formed microcalli, with the highest plating efficiency obtained on medium containing 2.0 mg l−1 1-naphthaleneacetic acid and 0.1 mg l−1 thidiazuron. Callus proliferation was also promoted by including thidiazuron in agar-solidified medium, while somatic embryogenesis was induced from microcalli on medium supplemented with 1.0 mg l−1 kinetin, 0.5 mg l−1 gibberellic acid, and 80 mg l−1 adenine sulfate. Flow cytometric analysis and chromosome counting revealed that all regenerants were tetraploid.