The Metabolite Content of the Post-Culture Medium of the Tree Fern Cyathea delgadii Sternb. Cell Suspension Cultured in the Presence of 2,4-D and BAP.

The aim of this study was to demonstrate the metabolic profile of post-culture medium as an expression of cell suspension metabolic activity of the tree fern Cyathea delgadii Sternb. The molecular profile of the tree fern's cell culture has been never described, according to our knowledge. The cell suspension was established using ½ MS medium supplemented with various concentrations of 2,4-D and BAP. The optimal concentrations were 2.0 mg·L-1 and 0.2 mg·L-1, respectively. The cell suspension initially showed an organized system of cell division and later unorganized cell proliferation. LC-MS and GC-MS were used to identify the chemical composition of the post-culture medium. The LC-MS analysis results suggested that the color of liquid medium could be due to the presence of flavonoid derivatives, as this group of compounds was represented by eight compounds. After GC-MS analysis based on retention indexes and thanks to mass spectra comparison, 130 natural products were recognized, belonging to various classes of primary and secondary metabolites.

Biotechnology of the Tree Fern Cyathea smithii (J.D. Hooker; Soft Tree Fern, Katote) II Cell Suspension Culture: Focusing on Structure and Physiology in the Presence of 2,4-D and BAP.

The aim of our research was to describe the structure and growth potential of a cell suspension of the tree fern Cyathea smithii. Experiments were performed on an established cell suspension with ½ MS medium supplemented with 9.05 µM 2,4-D + 0.88 µM BAP. In the experiments, attention was paid to the microscopic description of cell suspension, evaluation of cell growth dependent on the initial mass of cells and organic carbon source in the medium, the length of the passage, the content of one selected flavonoid in the post-culture medium, nuclear DNA content, ethylene production, and the antimicrobial value of the extract. For a better understanding of the cell changes that occurred during the culture of the suspension, the following structures of the cell were observed: nucleus, lipid bodies, tannin deposits, starch grains, cell walls, primary lamina, and the filaments of metabolites released into the medium. The nuclear DNA content (acriflavine-Feulgen staining) of cell aggregates distinctly indicated a lack of changes in the sporophytic origin of the cultured cell suspension. The physiological activity of the suspension was found to be high because of kinetics, intensive production of ethylene, and quercetin production. The microbiological studies suggested that the cell suspension possessed a bactericidal character against microaerobic Gram-positive bacteria. A sample of the cell suspension showed bacteriostatic activity against aerobic bacteria.

Somatic Embryogenesis in the Family Gentianaceae and Its Biotechnological Application.

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.

Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb.

Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii.

Protein extraction from Ca-alginate encapsulated plant material for comparative proteomic analysis.

The extensive use of encapsulation material in biotechnology drove the need to develop analytical techniques for this type of material. This study focuses on the specific problems of protein extraction from Ca-alginate encapsulated plant material. Proteomics is one of the fast-developing analysis categories, specifically for stress resistance and developmental changes in plant material. Sample preparation is a critical step in a two-dimensional gel electrophoresis proteome approach and is essential for good results. The aim was to avoid preliminary manipulations and get good quality material for comparative proteome analysis technique 2DE. The phenol extraction method and the complex method with preliminary TCA precipitation, SDS buffer and phenol phase were compared with respect to the efficiency and quality of the resulting 2DE gel. The most appropriate method turned out to be the TCA/phenol method with the phenol fractioning technique adapted to the gentian cell suspension. It resulted in a high protein concentration and good quality sample that could be analyzed using the standard separation procedures of 2DE and spectrometric identification with high efficiency. The work presented here confirms the possibility of obtaining a sufficient protein sample for effective proteomic analysis from a small number of capsules.

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 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.

Somatic embryogenesis in ferns: a new experimental system.

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. We 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 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 embryogenesis and the endogenous hormonal regulation of that process and opens new avenues of experimentation.

Cryopreservation enhances embryogenic capacity of Gentiana cruciata (L.) suspension culture and maintains (epi)genetic uniformity of regenerants.

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.

Cryopreservation strategies for Cyathea australis (R. BR.) domin.

The influence of liquid nitrogen (LN) on the germination of C. australis spores and survival of gametophytes at various stages of development was investigated. Exposure to LN did not change the viability of mature spores (80 percent) but stimulated the germination of immature spores from 1.9 percent to 41 percent. Disinfection before cryopreservation contributed to loss of spore survival. However, some germination capacity was regained (48 percent) if the sterilized spores were enclosed in alginate capsules and subsequently exposed to osmotic desiccation and 5-hour air drying. Development of gametophytes derived from frozen and non-frozen spores was similar. Preculture factors (the period, type and abscisic acid treatment) affected gametophyte viability and growth. A two week preculture on agar significantly increased survival compared to preculture in a liquid medium. Addition of abscisic acid (ABA) to solid or liquid media stimulated explant survival. Highest viability (85 percent) of frozen-thawed gametophytes was achieved by a 2-week preculture in agar with 0.25 M sucrose and 10 muM ABA. Gametophytes developed directly from spores grew and multiplied in vitro at a uniform rate. Young, intensively growing gametophytes and large, proliferating ones survived better (73-80 percent) following cryoexposure than mature, non proliferating gametophytes (50 percent). Less than one quarter of the explant surface was alive in 60-80 percent of the gametophytes that survived cryoexposure.

Cryopreservation by encapsulation of Gentiana spp cell suspensions maintains regrowth, embryogenic competence and DNA content.

A reliable technique for cryopreservation by encapsulation was developed for two suspension cultures of gentian species (Gentiana tibetica and G. cruciata) of different ages and embryogenic potential. The effect of water content, aggregate size and the subculture time on viability was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) test. Regrowth of a proembryogenic mass (PEM) on agar, liquid or agar/liquid media was assayed by measuring the increase in biomass. A water content of 24-30% (fresh weight basis) after 5-6 h dehydration of encapsulated cells of gentians yielded the highest survival (68% for G. tibetica and 83% for G. cruciata) after cryopreservation. Regardless of species, aggregate size and subculture time, the lowest PEM survival was 44%. These parameters did not influence the survival of G. tibetica PEM, but the survival of G. cruciata was higher when the smaller aggregates were cryopreserved on the 5th day of culture. Agar/liquid culture caused the greatest biomass increase. Cryopreservation did not affect the characteristics of suspension cultures and their regrowth after thawing, nor the number and dynamics of somatic embryos formed. Flow cytometry showed that cryopreservation did not change the genome size of the PEMs or regenerants.