Dittrichia viscosa uptake and translocation of the metalloid As is not fully understood and some data are contradictory, but its adaptability to this pollutant is known and is dependent on its genetic variability. D. viscosa is not a hyperaccumulator plant, but it can grow in high-drought conditions while still producing large biomass, even tolerating significant concentrations of As³⁺ and As⁵⁺. In spite of these remarkable characteristics, adaptive modification of performances is not predictable in wild populations. In previous work, we established experimental clonal populations to perform a functional study on the aquaporin NIP1.1. Here, we propose a strategy to select a clonal population of D. viscosa with a defined phenotype related to As tolerance and to reduced NIP1.1 expression levels for phytoremediation applications. From the previous work, we selected four independent clones, two of them belonging to the weak population (W8 and W9) and the other two belonging to the strong population (S1 and S3). The weak and strong populations differ for a different expression ratio root/shoot of DvNip1;1 that brings a different tolerance to As presence. The stress response of the populations, revealed by the CAT enzymatic test, was statistically correlated to the clones, but not to As uptake. Performance of the selected plants on a second unrelated metallic pollutant, Cd, was evaluated, showing that Cd uptake is also independent from the tolerant phenotype. In vitro culture methods using solid media and temporary immersion bioreactors were compared to propose an optimized combined protocol. The procedure yielded propagation of genetically stable tolerant clonal lines with good uptake of As and Cd. The plants, mass-produced with the developed in vitro protocol, were able to maintain their acquired abilities and are potentially able be later applied in phytoremediation or contaminated areas’ re-naturalization.

Viola ucriana Erben & Raimondo is a rare and endangered taxon, endemic to a limited area on Mount Pizzuta in northwestern Sicily, Italy. Its population is significantly threatened by anthropogenic activities, including fires, overgrazing, and habitat alterations. Temporary immersion systems (TISs) have proven effective for large-scale propagation in various protected species, offering potential for ex situ conservation and population reinforcement of V. ucriana. This study aimed to establish a bioreactor-based micropropagation protocol for shoot multiplication and compare the efficacy of a TIS with that of conventional solid culture medium (SCM). Three different plant growth regulators (PGRs) were also compared: 6-benzylaminopurine (BA), zeatin, and meta-topolin-9-riboside (mTR). The starting material originated from seeds collected from mother plants in their natural environment. The best growth outcomes (in terms of shoot multiplication, shoot length, and relative growth rate) were achieved using THE RITA® TIS, with BA (0.2 mg/L) and mTR (0.5 or 0.8 mg/L) outperforming SCM. Anomalous or hyperhydric shoots were observed with all zeatin treatments (especially with 0.8 mg/L) in both the TIS and SCM, suggesting that this cytokinin is unsuitable for V. ucriana biomass production. The rooting phase was significantly improved by transferring propagules onto rockwool cubes fertilized with Hoagland solution. This approach yielded more robust roots in terms of number and length compared to the conventional agar-based medium supplemented with indole-3-butyric acid (IBA). Flow cytometry analysis confirmed the genetic fidelity of the regenerants from the optimal PGR treatments, showing that all plantlets maintained the diploid ploidy level of their maternal plants. Over 90% of the in vitro derived plantlets were successfully acclimatized to greenhouse conditions. This paper represents the first report of V. ucriana biomass multiplication using a RITA^® bioreactor. The stability of the regenerants, confirmed by nuclei quantification via cytofluorimetry, provides guidance in establishing a true-to-type ex situ population, supporting conservation and future reinforcement efforts.

Numerous studies have reported the pharmacological effects exhibited by Dittrichia viscosa, (D. viscosa) including antioxidant, cytotoxic, antiproliferative, and anticancer properties. In our research, our primary objective was to validate a prescreening methodology aimed at identifying the fraction that demonstrates the most potent antiproliferative and anticancer effects. Specifically, we investigated the impact of various extract fractions on the cytoskeleton using a screening method involving transgenic plants. Tumors are inherently heterogeneous, and the components of the cytoskeleton, particularly tubulin, are considered a strategic target for antitumor agents. To take heterogeneity into account, we used different lines of colorectal cancer, specifically one of the most common cancers regardless of gender. In patients with metastasis, the effectiveness of chemotherapy has been limited by severe side effects and by the development of resistance. Additional therapies and antiproliferative molecules are therefore needed. In our study, we used colon-like cell lines characterized by the expression of gastrointestinal differentiation markers (such as the HT-29 cell line) and undifferentiated cell lines showing the positive regulation of epithelial–mesenchymal transition and TGFβ signatures (such as the DLD-1, SW480, and SW620 cell lines). We showed that all three of the D. viscosa extract fractions have an antiproliferative effect but the pre-screening on transgenic plants anticipated that the methanolic fraction may be the most promising, targeting the cytoskeleton specifically and possibly resulting in fewer side effects. Here, we show that the preliminary use of screening in transgenic plants expressing subcellular markers can significantly reduce costs and focus the advanced characterization only on the most promising therapeutic molecules.

Ceratonia siliqua L., or carob, is an evergreen Mediterranean tree valued for its environmental and economic benefits, including soil fertility enhancement and low maintenance requirements. Conventional propagation methods, such as seeds and grafting, have limitations, highlighting the need for large-scale in vitro techniques, including Temporary Immersion Systems (TIS). In this study, we compared two culture systems, solid culture medium (SCM) and TIS RITA ^® , for seedling development. TIS RITA ^® demonstrated superior growth, with higher shoot multiplication rates, longer shoot lengths, and greater fresh weight. It also showed better vigor without callus formation, whereas SCM exhibited some callus at the hypocotyl base. First, we investigated the specificity of zeatin’s effect on carob by comparing it with blueberry shoots. We used the combination of zeatin and a CKX inhibitor to show the specificity of the effect. For carob, 0.2 mg/L zeatin with the CKX inhibitor resulted in the highest shoot induction and fresh weight, while blueberry shoots showed optimal regeneration with 1 mg/L zeatin and the CKX inhibitor. However, the addition of the CKX inhibitor in carob does not show bigger advantages with respect to its absence in terms of shoot regeneration, like the case of the increment of zeatin concentration. As a consequence, other plant growth regulators (PGRs) could be used to substitute this expensive PGR. Shoot multiplication experiments using different PGRs (BAP, m TR, m T and o TR) improved regeneration efficiency and revealed that TIS RITA ^® produced higher shoot numbers and weights compared to SCM. The longest shoots were obtained under specific PGRs concentrations, and morphological differences were observed between the two systems, with TIS RITA ^® generating more elongated shoots. Overall, these findings demonstrate the potential of in vitro techniques, particularly TIS RITA ^® and PGRs application, to improve carob propagation.