The formation of an extracellular matrix surface network (ECMSN), and associated changes in the distribution of arabinogalactan-protein and pectin epitopes, have been studied during somatic embryogenesis (SE) and callogenesis of Trifolium nigrescens Viv. Scanning electron microscopy observations revealed the occurrence of an ECMSN on the surface of cotyledonary-staged somatic embryos as well as on the peripheral, non-regenerating callus cells. The occurrence of six AGP (JIM4, JIM8, JIM13, JIM16, LM2, MAC207) and four pectin (JIM5, JIM7, LM5, LM6) epitopes was analysed during early stages of SE, in cotyledonary-staged somatic embryos and in non-embryogenic callus using monoclonal antibodies. The JIM5 low methyl-esterified homogalacturonan (HG) epitope localized to ECMSN on the callus surface but none of the epitopes studied were found to localize to ECMSN over mature somatic embryos. The LM2 AGP epitope was detected during the development of somatic embryos and was also observed in the cell walls of meristematic cells from which SE was initiated. The pectic epitopes JIM5, JIM7, LM5 and LM6 were temporally regulated during SE. The LM6 arabinan epitope, carried by side chains of rhamnogalacturonan-I (RG-I), was detected predominantly in cells of embryogenic swellings, whilst the LM5 galactan epitope of RG-I was uniformly distributed throughout the ground tissue of cotyledonary-staged embryoids but not detected at the early stages of SE. Differences in the distribution patterns of low and high methyl-esterified HG were detected: low ester HG (JIM5 epitope) was most abundant during the early steps of embryo formation and highly methyl-esterified form of HG (JIM7 epitope) became prevalent during embryoid maturation.

Isolated thylakoids from halophytic Eutrema salsugineum (Thellungiella salsuginea) produces more H2O2 in comparison to glycophytic Arabidopsis thaliana. The first objective of this study was to verify whether this feature is relevant also to the intact chloroplasts and leaves. Enhanced H2O2 levels in chloroplasts and leaves of E. salsugineum were positively verified with several methods (electron microscopy, staining with Amplex Red and with diaminobenzidine). This effect was associated with a decreased ratio of O2.-/H2O2 in E.s in comparison to A.thaliana as detected by electron paramagnetic resonance (EPR) method. As a next step, we tested how this specific ROS signature of halophytic species affect the antioxidant status and down-stream components of ROS signaling. Comparison of enzymatic antioxidants revealed a decreased activity of ascorbate peroxidase (APX), enhanced activity of glutathione peroxidase (GPX), and the presence of thylakoid-bound forms of iron superoxide dismutase (FeSOD) and ascorbate peroxidase (APX) in E.s.. These cues were, however, independent from application of salt stress. The typical H2O2-dependent cellular responses, namely the levels of glucosinolates and stress-related hormones were determined. The total glucosinolate content in E.s water-treated leaves was higher than in A.t. and increased after salinity treatment. Treatment with salinity up-regulated all of tested stress hormones, their precursors and catabolites (abscisic acid, dihydrophaseic acid, phaseic acid, 1-aminocyclopropane-1-carboxylic acid, salicylic acid, jasmonic acid, cis-(+)-12-oxo-phytodienoic acid and jasmonoyl-L-isoleucine) in A.t., whereas in E.s. only a stimulation in ethylene synthesis and abscisic acid catabolism was noted. Obtained results suggest that constitutively enhanced H2O2 generation in chloroplasts of E.s. might be a crucial component of stress-prepardeness of this halophytic species. It shapes a very efficient antioxidant protection (in which glucosinolates might play a specific role) and a fine tuning of hormonal signaling to suppress the cell death program directed by jasmonate pathway.

The aim of this study was to identify and examine the expression pattern of the ortholog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE gene from Trifolium nigrescens (TnSERK) in embryogenic and non-regenerative cultures of immature cotyledonary-stage zygotic embryos (CsZEs). In the presence of 1-naphthaleneacetic acid and N⁶-[2-isopentenyl]-adenine, the CsZE regenerated embryoids directly and in a lengthy culture produced callus which was embryogenic or remained non-regenerative. As revealed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the TnSERK was expressed in both embryogenic and non-regenerative cultures, but the expression level was significantly higher in embryogenic ones. An in situ RNA hybridization assay revealed that the expression of TnSERK preceded the induction of cell division in explants, and then, it was maintained exclusively in actively dividing cells from which embryoids, embryo-like structures (ELSs), callus or tracheary elements were produced. However, the cells involved in different morphogenic events differed in intensity of hybridization signal which was the highest in embryogenic cells. The TnSERK was up-regulated during the development of embryoids, but in cotyledonary embryos, it was preferentially expressed in the regions of the apical meristems. The occurrence of morphological and anatomical abnormalities in embryoid development was preceded by a decline in TnSERK expression, and this coincided with the parenchymatization of the ground tissue in developing ELSs. TnSERK was also down-regulated during the maturation of parenchyma and xylem elements in CsZE and callus. Altogether, these data suggest the involvement of TnSERK in the induction of various developmental programs related to differentiation/transdifferentiation and totipotent state of cell(s).

Microscopy and flow cytometry (FCM) were used to study somatic embryogenesis (SE) from zygotic embryos of Trifolium nigrescens Viv. to determine if there were any relationships between characteristics of somatic embryos (morphology, anatomy, genome size stability) and their regenerability. Embryoids were induced on Murashige and Skoog (MS) medium containing 4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg l⁻¹ N⁶-[2-isopentenyl]-adenine (2iP) either directly from hypocotyls or via an intervening callus, depending on the duration of culture. The morphology of somatic embryos varied from zygotic-like structures to abnormal structures including horn-shaped, polycotyledonary, and fused embryoids. The incidence of abnormalities was higher in callus cultures than in direct regeneration. Horn-shaped embryoids were the most frequent type of abnormal embryos. Only embryoids having zygotic-like morphology regenerated into plantlets. Histological observations revealed that the absence of shoot and root apical meristems along with parenchymatization of embryos were major obstacles to conversion of horn-shaped embryoids. The estimated 2C value for T. nigrescens was 0.9 pg. FCM analysis revealed differences in DNA content between embryoids induced via an intervening callus and those produced directly from explants. Individuals with species-specific as well as increased DNA content were detected among those zygotic-like embryos derived from callus, but all horn-shaped embryoids had increased genome sizes. The observed lack of differences in DNA content between zygotic-like and horn-shaped embryoids, from direct SE, indicated that these phenotypic abnormalities were of physiological origin. The mean DNA content of regenerants was species-specific, suggesting that only diploid embryoids were capable for regeneration into plantlets.