Cyathea Delgadii Sternb Research Articles

Temperature and LED lightning affect the regeneration of somatic embryo-derived sporophytes on the internode explants of the tree fern Cyathea delgadii Sternb.

The influence of the temperature and different light qualities emitted by light-emitting diodes (LEDs) and fluorescent lamps (Fl) on the micropropagation of the tree fern Cyathea delgadii was evaluated. The most efficient somatic embryo production was obtained on internode explants at 22 °C. The optimal temperature range for obtaining well-developed plants was 24–28 °C. This stimulated the elongation and development of the first leaf and the formation of the next leaf and roots primordia. Temperatures lower than 24 °C and higher than 28 °C inhibited the formation of young sporophyte organs and delayed their development. The RBUV (35% red, 15% blue, and 50% UV) and B (100% blue) lights and Fl light were beneficial for the sporophyte production on internode explants. However, plants obtained under RBUV light were undeveloped. The white LED light stimulated the number of explants capable of gametophyte production and development. The RB light (70% red and 30% blue) enhanced the number of roots of newly-formed plants. Most of the LED lights tested had a good impact on root elongation compared to Fl light and constant darkness. The R light (100% red) benefits leaf development and elongation. Research shows that temperature and LED lightning play a significant role in the process of morphogenesis in C. delgadii, significantly affecting the embryogenic competence of somatic cells and the development of sporophytes.

Sucrose-induced stress and initial days after explant excision affect the pattern and efficiency of somatic embryogenesis in the tree fern Cyathea delgadii Sternb.

Sucrose-induced stress and initial days after explant excision affect the pattern and efficiency of somatic embryogenesis in the tree fern Cyathea delgadii Sternb.

Cell wall remodeling and callose deposition during the embryogenic transition in the tree fern Cyathea delgadii Sternb.

With the discovery in 2015 of the ability to induce somatic embryos in Cyathea delgadii, learning more about the relationship between the structure of apoplast and cell differentiation has become possible among ferns. In this study, the distribution of arabinogalactan proteins, pectins, extensins, and callose with specific epitopes recognized by monoclonal antibodies was investigated during direct somatic embryogenesis (SE) of C. delgadii. Eight antigens against the arabinogalactan proteins (JIM8, JIM13, LM2), pectins (JIM5, JIM7), extensins (JIM11, JIM12), and callose (anti-1 → 3-β-glucan) were selected. Two types of explants were analyzed, i.e. stipe fragments and internodes, which give rise to embryos of unicellular and multicellular origin, respectively. The study showed that embryogenic transition in C. delgadii is preceded by cell wall remodeling of initial explants. Dynamic changes in JIM13, JIM12, and anti-1 → 3-β-glucan localization were observed. The differences in the distribution of studied epitopes were observed between the cell walls of the epidermis and those located in the other layers of the explant. Moreover, within the somatic embryos, a stronger fluorescence of the examined antibodies was observed, mainly those reacting with arabinogalactan proteins, extensins, and callose. These results also implicated that, with the exception of the earlier appearance of callose in the stipe explants, the uni- and multicellular pathways of somatic embryo differentiation do not differ in the quality of cell wall components. The presented studies document the first time that SE in ferns can be regulated by changes in apoplast structure and they provide a basis for more detailed research.

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.

Enhancing In Vitro Production of the Tree Fern Cyathea delgadii and Modifying Secondary Metabolite Profiles by LED Lighting.

The tree ferns are an important component of tropical forests. In view of this, the enhancement of in vitro production of these plants is needed. Thus, the effect of different light-emitting diodes (LEDs) as well as control fluorescent lamps (Fl) and a 3-week-long period of darkness at the beginning of in vitro culture on micropropagation of the tree fern Cyathea delgadii Sternb. was analysed. Moreover, the photosynthetic pigment content and secondary metabolite profiles were estimated. The period of darkness contributed to a high production of somatic embryo-derived sporophytes and a low production of gametophytes. The formation of new sporophytes was stimulated by RBY (35% red, 15% blue, and 50% yellow) and B (100% blue) lights when the stipe explants or whole young sporophytes were used in the culture, respectively. The elongation of the roots and leaves was stimulated by RBfR light (35% red, 15% blue, and 50% far red), while root production increased under RBY light. The RB (70% red and 30% blue) and B lights stimulated the accumulation of chlorophyll better than Fl light. The most abundant metabolite found in the plant extracts was trans-5-O-caffeoylquinic acid (1.013 µg/mg of dry weight). The extract obtained from plants growing in a greenhouse had the best antioxidant activity.

The Origin of the Cyathea delgadii Sternb. Somatic Embryos Is Determined by the Developmental State of Donor Tissue and Mutual Balance of Selected Metabolites.

Somatic embryogenesis is the formation of a plant embryo from a cell other than the product of gametic fusion. The need to recognize the determinants of somatic cell fate has prompted investigations on how endogenous factors of donor tissues can determine the pattern of somatic embryo origin. The undertaking of this study was enabled by the newly developed experimental system of somatic embryogenesis of the tree fern Cyathea delgadii Sternb., in which the embryos are produced in hormone-free medium. The contents of 89 endogenous compounds (such as sugars, auxins, cytokinins, gibberellins, stress-related hormones, phenolic acids, polyamines, and amino acids) and cytomorphological features were compared between two types of explants giving rise to somatic embryos of unicellular or multicellular origin. We found that a large content of maltose, 1-kestose, abscisic acid, biologically active gibberellins, and phenolic acids was characteristic for single-cell somatic embryo formation pattern. In contrast, high levels of starch, callose, kinetin riboside, arginine, and ethylene promoted their multicellular origin. Networks for visualization of the relations between studied compounds were constructed based on the data obtained from analyses of a Pearson correlation coefficient heatmap. Our findings present for the first time detailed features of donor tissue that can play an important role in the somatic-to-embryogenic transition and the somatic embryo origin.

<strong><em>Cyathea</em> Sm. (Cyatheaceae) on Trindade Island (Brazil): An integrative approach</strong>

Trindade is a Brazilian oceanic island approximately 1,100 km from the American mainland. The Cyathea Sm. plants from Trindade were originally considered an endemic species, Cyathea copelandii Kuhn & Luerssen, but later included in Cyathea delgadii Sternb. Based on molecular data (trnL-trnF, trnG-trnR and rbcL-accD), morphometric analysis of leaf characters, scanning electron microscopy of indusia and spores, and habitat suitability modeling, the present study confirms the status of C. copelandii as a synonym of C. delgadii. Intraspecific molecular variation suggests phylogeographic structuring in C. delgadii and differentiation between the coastal Atlantic Forest domain and the Cerrado domain (savanna) in the interior of Brazil. The Trindade populations are more closely related to the Atlantic Forest populations, indicating a colonization of the island from the latter area.

Explant type and stress treatment determine the uni- and multicellular origin of somatic embryos in the tree fern Cyathea delgadii Sternb.

Cyathea delgadii is the first fern species for which somatic embryogenesis (SE) has recently been described. With this discovery, a new experimental model for exploration of SE was found. In this study, the effect of explant type (stipe and internode), length and diameter of the explant-donor frond, and stress treatment (by sucrose and air desiccation) on somatic embryo origin and SE efficiency was studied. In control culture, somatic embryos originated from single cells of stipe explants, whereas those induced on internodes were of multicellular origin. Although the activation of cell divisions was more abundant and the formation of somatic embryos occurred earlier in a culture of internodes than of stipe explants, the morphogenic capacity of internodes was much smaller. On their surface, the groups of competent cells formed protuberances that give rise to only three somatic embryos per internode. In contrast, almost 92% of stipe explants that were excised from the first frond, measuring 10 mm in length and less than 300 µm in diameter, produced an average of 21 somatic embryos. Stipes treated with sucrose were not able to SE. More than fourfold increase in SE efficiency was obtained on internodes by their treatment with 0.4 M sucrose for 45 min. It was achieved by changing the pathway of somatic embryo differentiation from multicellular to unicellular. These results provide an excellent basis for further research on the mechanism of SE induction associated with a single- and multi-cell proliferation, especially that both types of the embryogenic pathway can occur on the same hormone-free medium.

Effect of TIBA, fluridone and salicylic acid on somatic embryogenesis and endogenous hormone and sugar contents in the tree fern Cyathea delgadii Sternb.

Somatic embryogenesis (SE) in the tree fern Cyathea delgadii was first described in 2015 and since then has been used to exploration of this phenomenon in cryptogamic plants. To deepen the knowledge about the hormonal control of SE, stipe explants were cultured on media supplemented with hormone biosynthesis and transport inhibitors (HBTIs). In the presence of 30 µM 2,3,5-triiodobenzoic acid (TIBA), or 40 µM fluridone or 125 µM salicylic acid (SA), somatic embryo production was totally inhibited. The quantitative analysis of the changes in endogenous hormone and sugar contents was conducted every 2 days within 10-day-long initial culture. The results showed that the concentrations of endogenous indole-3-acetic acid (IAA), abscisic acid (ABA), cytokinins (CKs) and soluble sugars were strongly modified either by TIBA and fluridone. Under their influence, the contents of cytokinins such as c-Z, c-ZR, t-Z, t-ZR, KinR were reduced to barely detectable levels. Treatment with SA results in the changes in endogenous IAA and sugar contents. It also modifies the IAA/CKs ratio; however, excluding the first 2 days of culture, the concentrations of ABA and cytokinins were kept on the control level. All HBTIs significantly increased the kinetin (Kin) content. Our work sheds new light on the relationships between the biosynthetic inhibitors and phytohormones and sugars in the process of early SE. It can be helpful to study the role of hormones in acquisition of embryogenic competence.

The content of endogenous hormones and sugars in the process of early somatic embryogenesis in the tree fern Cyathea delgadii Sternb.

Somatic embryogenesis (SE) of Cyathea delgadii presents a model system for investigating the mechanisms associated with the acquisition of embryogenic competence by single epidermal cells of stipe explants cultured on plant growth regulator-free medium. The present work reveals relationship between endogenous hormone and sugar content in the process of early SE in C. delgadii. By comparing two types of initial explants, i.e. incapable (non-etiolated) and capable (etiolated) of SE, it was established that in etiolated explants, the glucose, fructose, sucrose, and abscisic acid (ABA) contents diminished, but indole-3-acetic acid (IAA) and cytokinins (CKs; i.e. cis/trans zeatin, cis/trans-zeatin riboside, kinetin, kinetin riboside, isopentenyladenosine) contents increased. The ratios between phytohormones revealed that a high concentration of ABA is the main factor inhibiting SE induction. Because of explant excision, a dramatic reduction in concentration of all phytohormones studied was observed, but hormonal balance and sugar content remained almost unchanged. During the 14-day-long culture, the ABA/CKs and ABA/IAA ratios remained constant, whereas the greatest differences were detected for the IAA/CKs and Z-type/iPA cytokinin ratios. Excluding day 6 of culture, cytokinins were found to be the predominant phytohormones over IAA. An almost 12-fold increase in soluble sucrose concentration at day 6 of culture might be the switch to the SE expression phase. Frequent cell divisions leading to somatic embryo formation are clearly associated with increase in trans-zeatin riboside content.

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.

Estrutura populacional e desenvolvimento da fase esporofítica de Cyathea delgadii Sternb. (Cyatheaceae, Monilophyta) no sul do Brasil

Cyathea delgadii Sternb. é uma samambaia arborescente que ocorre em florestas primárias e secundárias da região nordeste até o sul do Brasil. O presente estudo discute a estrutura populacional e o desenvolvimento da fase esporofítica (crescimento do cáudice, produção de frondes, fenologia da produção de esporos) de C. delgadii, em floresta secundária, no Estado do Rio Grande do Sul, Brasil, baseado na observação de 41 plantas, durante 13 meses. A espécie apresentou padrão espacial agregado e uma freqüência expressivamente alta de indivíduos de menor altura, indicando um grande potencial de regeneração da população. O cáudice das plantas cresceu, em média, 4,65 cm ano-1, sendo que as maiores taxas de crescimento foram registradas em plantas mais altas. As taxas de produção de frondes novas (5,75 frondes ano-1) e de senescência (4,92 frondes ano-1) similares evidenciaram uma capacidade de manter um número de frondes estável. Poucas plantas formaram frondes férteis, sendo que a produção e liberação dos esporos ocorreram assincronicamente.

<p><strong>Moss and liverwort epiphytes on trunks of <em>Cyathea delgadii</em> in a fragment of tropical rain forest, São Paulo State, Brazil</strong></p>

This study is a survey of the bryophyte species that occur on the trunks of Cyathea delgadii Sternb. (Cyatheaceae), a native tree fern, encountered in a fragment of Atlantic forest located in the area of the „Parque Estadual das Fontes do Ipiranga (PEFI)“, São Paulo State, Brazil. Specimens of bryophytes were collected from March 2001 to October 2003. We found 35 bryophyte species (12 spp. of mosses and 23 of liverworts). Ceratolejeuenea dentacornuta Steph. is presented as a new record for Brazil. A brief discussion about previous records of bryophyte species growing on trunks of tree ferns in Brazil is also presented.

The survival and establishment potential of spores of Cyathea delgadii Sternb. in soils from Itirapina and Moji Guaçu (SP), Brazil

Germination of spores of Cyathea delgadii was carried out in soils collected in March 1997 (rainy season) at three depths (0-5, 5-10 and 10-15 cm) from the cerrado, open-cerrado, gallery forest and marsh at Moji Guacu and cerradao, cerrado and gallery forest at Pedregulho, in Itirapina, both in the state of Sao Paulo, Brazil. Viability of spores mixed with soil and buried in the cerrado at the Reserva in Moji Guacu were conducted for up to 10 months. The spores germinated in all soils. Germination in the soils was significantly lower than in distilled water. Germination was higher in soils from the gallery forest and cerrado than from open cerrado, cerradao and marsh. The germination was the same in the soil samples from the three depths of gallery forest, cerrado and cerradao. Spores of C. delgadii maintained viability longer when buried in cerrado soil than when dry stored at 4°C. After 10 months germination was 50.5% in buried spores against 3.8% in spores of the same age and harvest, stored at 4°C.

Reserve substances and storage of Cyathea delgadii Sternb. spores

The spores used were collected from a population of trees of Cyathea delgadii Sternb. growing in a gallery forest at the Reserva Biologica e Estacao Experimental de Moji Guacu, Sao Paulo state, Brazil (22°18’S and 47°11’W). The germination of spores of Cyathea delgadii decreases with time when kept in closed bottles under storage at 4°C in darkness. Germination is still very high after storage for one year. Spores stored for three years do not germinate. The results also show a decrease in soluble proteins and an increase in starch after several months storage.

The Effect of Sterilization and Storage Conditions on the Viability of the Spores of Cyathea delgadii

Tests were conducted to evaluate the effects of various sterilization procedures on spores of Cyathea delgadii stored under different conditions with the intent of developing a strat- egy of ex-situ spore conservation of this declining species. Although the percentage of germination after two years of storage was relatively low for all storage and sterilization parameters, dry storage at -12?C resulted in the best survival rate. Elimination of bacterial and fungal contaminants was best when the spores were washed, treated with calcium hypochlorite, incubated, filtered, and washed again before sowing on medium containing Nystatin. The spores of some tree ferns in the Cyatheaceae lose their viability after a few weeks of storage (Page, 1979). The viabilility of Cyathea delgadii Sternb. spores is already decreased after only two months storage at 12?C and low humidity (Randi and Felippe, 1988a) and percent spore germination is signif- icantly decreased after six months of storage under the same conditions (Mar- condes-Ferreira and Felippe, 1984). In general, it is advisable to store fern spores at low temperature and low humidity, sometimes with the use of des- iccators (Dyer, 1979a). More recently, storage in darkness at room temperature and high humidity (imbibed) has been advised for some species (Lindsay et al., 1991). Before storing imbibed spores in the laboratory or before germination of spores stored dry, there is the need to sterilize them. Several chemicals have been used for this purpose (Dyer, 1979b), and either sodium hypochlorite (NaOCl) or calcium hypochlorite (Ca(OCl2)J4H2O) are the most commonly used. Unfortunately, most treatments that kill contaminant fungi and bacteria also reduce fern spore germination. Different concentrations of chemicals and duration of application have been used. Before germination, it is advisable to incubate the spores in darkness for a period of time: the fungi and bacteria start to grow and become more sensitive to the chemicals used for sterilization (Voeller, 1964; Schedlbauer, 1976). The objective of this investigation was to study different sterilization meth- ods and storage conditions in order to develop a procedure for maintaining a source of viable spores of Cyathea delgadii for ex situ conservation, because this species is rapidly disappearing from the Brazilian forests as consequence of commercial exploitation.