High efficiency protoplast isolation from in vitro cultures and hairy roots of Maesa lanceolata

In vitro cultures of the medicinal plant Maesa lanceolata were established to enable the cultivation of plant material for the production of protoplasts. Callus cultures were initiated using leaves collected from shoot cultures and the root tips from hairy root cultures obtained upon Agrobacterium rhizogenes transformation. For the isolation of protoplast, the different explant material of M. lanceolata was exposed to an enzyme mixture consisting of 1.5% cellulase, 0.5% macerozyme R-10 and 0.5 M mannitol. About 6 x 106 protoplasts g-1 fresh weight were obtained from leaf material and 5 x 105 protoplasts g-1 fresh weight from callus. To obtain high amounts of hairy root protoplasts, the cultures were pretreated with the auxin indole-3-butyric acid (IBA) that stimulated the formation of novel root tips. Using the dissected root tips as starting material, 8 x 105 protoplasts g-1 fresh weight were obtained per preparation. The protoplast isolation method will enable further studies on the transformation and fusion of protoplasts from M. lanceolata

Studies on saponin production in tropical medicinal plants Maesa argentea and Maesa lanceolata

The continuous need for new compounds with important medicinal activities has lead to the identification and characterization of various plant-derived natural products. As a part of this program, we studied the saponin production from two tropical medicinal plants Maesa argentea and M. lanceolata and evaluated several treatments to enhance their saponin production. In this experiment, we present the analyses of saponin production from greenhouse grown plants by means of TLC and HPLC-MS. We observed that the content of saponin from these plants varied depending on organ and physiological age of the plants. In addition, the impact of elicitors on saponin accumulation on in vitro grown plants was analyzed using TLC. The production of saponin was very stable and not affected by treatment with methyl jasmonate, and salicylic acid. In conclusion, Maesa saponins are constitutively produced in plants and the level of these compounds in plants is mainly affected by the developmental or physiological stage

Callose homeostasis at plasmodesmata: molecular regulators and developmental relevance

Plasmodesmata are membrane-lined channels that are located in the plant cell wall and that physically interconnect the cytoplasm and the endoplasmic reticulum (ER) of adjacent cells. Operating as controllable gates, plasmodesmata regulate the symplastic trafficking of micro- and macromolecules, such as endogenous proteins [transcription factors (TFs)] and RNA-based signals (mRNA, siRNA, etc.), hence mediating direct cell-to-cell communication and long distance signaling. Besides this physiological role, plasmodesmata also form gateways through which viral genomes can pass, largely facilitating the pernicious spread of viral infections. Plasmodesmatal trafficking is either passive (e.g., diffusion) or active and responses both to developmental and environmental stimuli. In general, plasmodesmatal conductivity is regulated by the controlled build-up of callose at the plasmodesmatal neck, largely mediated by the antagonistic action of callose synthases (CalSs) and beta-1,3-glucanases. Here, in this theory and hypothesis paper, we outline the importance of callose metabolism in PD SEL control, and highlight the main molecular factors involved. In addition, we also review other proteins that regulate symplastic PD transport, both in a developmental and stress-responsive framework, and discuss on their putative role in the modulation of PD callose turn-over. Finally, we hypothesize on the role of structural sterols in the regulation of (PD) callose deposition and outline putative mechanisms by which this regulation may occur

Hypocotyl adventitious root organogenesis differs from lateral root development

Wound-induced adventitious root (AR) formation is a requirement for plant survival upon root damage inflicted by pathogen attack, but also during the regeneration of plant stem cuttings for clonal propagation of elite plant varieties. Yet, adventitious rooting also takes place without wounding. This happens for example in etiolated Arabidopsis thaliana hypocotyls, in which AR initiate upon de-etiolation or in tomato seedlings, in which AR initiate upon flooding or high water availability. In the hypocotyl AR originate from a cell layer reminiscent to the pericycle in the primary root (PR) and the initiated AR share histological and developmental characteristics with lateral roots (LRs). In contrast to the PR however, the hypocotyl is a determinate structure with an established final number of cells. This points to differences between the induction of hypocotyl AR and LR on the PR, as the latter grows indeterminately. The induction of AR on the hypocotyl takes place in environmental conditions that differ from those that control LR formation. Hence, AR formation depends on differentially regulated gene products. Similarly to AR induction in stem cuttings, the capacity to induce hypocotyl AR is genotype-dependent and the plant growth regulator auxin is a key regulator controlling the rooting response. The hormones cytokinins, ethylene, jasmonic acid, and strigolactones in general reduce the root-inducing capacity. The involvement of this many regulators indicates that a tight control and fine-tuning of the initiation and emergence of AR exists. Recently, several genetic factors, specific to hypocotyl adventitious rooting in A. thaliana, have been uncovered. These factors reveal a dedicated signaling network that drives AR formation in the Arabidopsis hypocotyl. Here we provide an overview of the environmental and genetic factors controlling hypocotyl-born AR and we summarize how AR formation and the regulating factors of this organogenesis are distinct from LR induction

Dynamics of male meiotic recombination frequency during plant development using Fluorescent Tagged Lines in Arabidopsis thaliana

Meiotic homologous recombination plays a central role in creating genetic variability, making it an essential biological process relevant to evolution and crop breeding. In this study, we used pollenspecific fluorescent tagged lines (FTLs) to measure male meiotic recombination frequency during the development of Arabidopsis thaliana. Interestingly, a subset of pollen grains consistently shows loss of fluorescence expression in tested lines. Using nine independent FTL intervals, the spatio-temporal dynamics of male recombination frequency was assessed during plant development, considering both shoot type and plant age as independent parameters. In most genomic intervals assayed, male meiotic recombination frequency is highly consistent during plant development, showing no significant change between different shoot types and during plant aging. However, in some genomic regions, such as I1a and I5a, a small but significant effect of either developmental position or plant age were observed, indicating that the meiotic CO frequency in those intervals varies during plant development. Furthermore, from an overall view of all nine genomic intervals assayed, both primary and tertiary shoots show a similar dynamics of increasing recombination frequency during development, while secondary and lateral shoots remain highly stable. Our results provide new insights in the dynamics of male meiotic recombination frequency during plant development

Cold-induced male meiotic restitution in Arabidopsis thaliana is not mediated by GA-DELLA signaling

Short periods of cold stress induce male meiotic restitution and diploid pollen formation in Arabidopsis thaliana by specifically interfering with male meiotic cytokinesis. Similar alterations in male meiotic cell division and gametophytic ploidy stability occur when gibberellic acid (GA) signaling is perturbed in developing anthers. In this study, we found that exogenous application of GA primarily induces second division restitution (SDR)-type pollen in Arabidopsis, similar to what cold does. Driven by the close similarity in cellular defects, we tested the hypothesis that cold-induced meiotic restitution is mediated by GA-DELLA signaling. Using a combination of chemical, genetic and cytological approaches, however, we found that both exogenously and endogenously altered GA signaling do not affect the cold sensitivity of male meiotic cytokinesis. Moreover, in vivo localization study using a GFP-tagged version of RGA protein revealed that cold does not affect the expression pattern and abundance of DELLA in Arabidopsis anthers at tetrad stage. Expression study found that transcript of RGA appears enhanced in cold-stressed young flower buds. Since our previous work demonstrated that loss of function of DELLA causes irregular male meiotic cytokinesis, we here conclude that cold-induced meiotic restitution is not mediated by DELLA-dependent GA signaling

Developing biostimulants from agro-food and industrial by-products

In modern agriculture, seeking eco-friendly ways to promote plant growth and enhance crop productivity is of priority. Biostimulants are a group of substances from natural origin that contribute to boosting plant yield and nutrient uptake, while reducing the dependency on chemical fertilizers. Developing biostimulants from by-products paves the path to waste recycling and reduction, generating benefits for growers, food industry, registration and distribution companies, as well as consumers. The criteria to select designated by-products for valorizing as biostimulant are: absence of pesticide residue, low cost of collection and storage, sufficient supply and synergy with other valorization paths. Over the years, projects on national and international levels such as NOSHAN, SUNNIVA, and Bio2Bio have been initiated (i) to explore valorization of by-products for food and agriculture industries; (ii) to investigate mode of action of biostimulants from organic waste streams. Several classes of waste-derived biostimulants or raw organic material with biostimulant components were shown to be effective in agriculture and horticulture, including vermicompost, composted urban waste, sewage sludge, protein hydrolysate, and chitin/chitosan derivatives. As the global market for biostimulants continues to rise, it is expected that more research and development will expand the list of biostimulants from by-products. Global nutrient imbalance also requires biostimulant to be developed for targeted market. Here, we review examples of biostimulants derived from agricultural by-products and discuss why agricultural biomass is a particularly valuable source for the development of new agrochemical products

Metabolite profiling of Triterpene Saponins in medicago truncatula hairy roots by liquid chromatography fourier transform Ion Cyclotron resonance mass spectrometry

Triterpenes are one of the largest classes of plant natural products, with an enormous variety in structure and bioactivities. Here, triterpene saponins from hairy roots of the model legume Medicago truncatula were profiled with reversed-phase liquid chromatography coupled to negative-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS). Owing to the accuracy of the FT-ICR MS, reliable molecular formulas of the detected compounds could be predicted, which, together with the generated MS" spectra, allowed the tentative identification of 79 different saponins, of which 61 had not been detected previously in M. truncatula. Upon collision-induced dissociation of saponins that contain a uronic acid residue in the sugar chain, fragment ions resulting from cross-ring cleavages of the uronic acid residues were observed. The identified saponins are glycosides of 10 different sapogenins, of which three were not detected before in M. truncatula. Zanhic acid glycosides, which are prevalent in the aerial parts of M. truncatula, were absent in the hairy root extract's. This metabolite compendium will facilitate future functional genomic studies of triterpene saponin biosynthesis in M. truncptula