Karolina Tomiczak

Autotetraploid plant regeneration by indirect somatic embryogenesis from leaf mesophyll protoplasts of diploid Gentiana decumbens L.f.

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 9u2009×u2009105 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 2.0xa0mgxa0l−1 1-naphthaleneacetic acid and 0.1xa0mgxa0l−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 1.0xa0mgxa0l−1 kinetin, 0.5xa0mgxa0l−1 gibberellic acid, and 80xa0mgxa0l−1 adenine sulfate. Flow cytometric analysis and chromosome counting revealed that all regenerants were tetraploid.

Somatic embryogenesis in ferns: a new experimental system

Key messageSomatic 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.AbstractWe discovered that the tree fern Cyathea delgadii 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 development 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 9xa0weeks 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 embryogenesis and the endogenous hormonal regulation of that process and opens new avenues of experimentation.

Integration of tissue culture and cryopreservation methods for propagation and conservation of the fern Osmunda regalis L.

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 (89xa0%) 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 PVS2xa0solution were ineffective for cryopreservation. The greatest survival rate (81.6xa0%) following cryo-exposure occurred following treatment of encapsulated gametophytes with PVS3 solution for 3xa0h. This protocol allowed the recovery of gametophyte cultures following 6xa0weeks after rewarming. Finally, 100xa0% 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.

The effect of moisture content and temperature on spore aging in Osmunda regalis

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 4.6xa0%) and storage temperature (15 and −196xa0°C) on spore germination and gametophyte development in Osmunda regalis. The MC (fresh weight basis) in freshly released spores was 7.5xa0%, and decreased to 6.6 and 4.6xa0% during 2xa0weeks of spore storage at 42 and 18xa0% relative humidity, respectively. Those spores germinated at 99.9xa0% within 28xa0h. The spores with 6.6 and 4.6xa0% MC were used for the storage at 15 and −196xa0°C. The deterioration of spores, maintained with 6.6xa0% MC, proceeds within 1.5xa0years of storage at 15xa0°C, starting with month 8. For the spores with 4.6xa0% MC serious disturbances in germination and capacity to gametophyte development were postponed for more than 6xa0months. The time required for spore germination increased with the age of the spores, ranging from 28xa0h in fresh spores to 10xa0days in spores stored for more than 11xa0months. Following 7-year-long cryostorage, spore viability remained at 99.9xa0%, 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.

Comparison of the morphogenic potential of five Gentiana species in leaf mesophyll protoplast culture and ploidy stability of regenerated calli and plants

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 2.0xa0mgxa0l−1 1-naphthaleneacetic acid and 0.1xa0mgxa0l−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 1.0xa0mgxa0l−1 kinetin, 0.5xa0mgxa0l−1 gibberellic acid, and 80xa0mgxa0l−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 85xa0% polyploid plants of G. kurroo and 14xa0% polyploids of G. tibetica. Chromosome counting and stomatal characteristic confirmed the ploidy variation among regenerants.

Protoplast fusion in the genus Gentiana: genomic composition and genetic stability of somatic hybrids between Gentiana kurroo Royle and G. cruciata L.

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.

Protoplast Culture and Somatic Cell Hybridization of Gentians

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.

Experimental and Practical Application of Fern Somatic Embryogenesis

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.

Morphogenic Events in Ferns: Single and Multicellular Explants In Vitro

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 and Somatic Embryo Cryopreservation of the Tree-Fern Cyathea Delgadii Sternb.

Cyathea delgadii Sternb. belongs to the Cyathea clade of the genus Cyathea, family Cyatheaceae, also known as Scaly Tree-Ferns. It is one of the many evergreen, non-seasonal tree-fern species from Central and South America, which grow in gallery, montane, cloud and rain forests, as well as in open locations and along paths in Costa Rica, Panama, Venezuela, Columbia, Peru, Ecuador, Bolivia, Argentina, Paraguay, and Brazil.