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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.

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.

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.