Lignans are the main secondary metabolites synthetized by Linum species as plant defense compounds but they are also valuable for human health, in particular, for novel therapeutics. In this work, Linum austriacum in vitro cultures, cells (Cc), adventitious roots (ARc) and hairy roots (HRc) were developed for the production of justicidin B through elicitation with methyl jasmonate (MeJA) and coronatine (COR). The performances of the cultures were evaluated for their stability, total phenols content and antioxidant ability. NMR was used to identify justicidin B and isojusticidin B and HPLC to quantify the production, highlighting ARc and HRc as the highest productive tissues. MeJA and COR treatments induced the synthesis of justicidin B more than three times and the synthesis of other compounds. RNA-sequencing and a de novo assembly of L. austriacum ARc transcriptome was generated to identify the genes activated by MeJA. Furthermore, for the first time, the intracellular localization of justicidin B in ARc was investigated through microscopic analysis. Then, HRc was chosen for small-scale production in a bioreactor. Altogether, our results improve knowledge on justicidin B pathway and cellular localization in L. austriacum for future scale-up processes.

Arundo donax L., the giant reed—being a long-duration, low-cost, non-food energy crop able to grow in marginal lands—has emerged as a potential alternative to produce biomass for both energy production, with low carbon emissions, and industrial bioproducts. In recent years, pioneering efforts have been made to genetically improve this very promising energy crop. This review analyses the recent advances and challenges encountered in using clonal selection, mutagenesis/somaclonal variation and transgenesis/genome editing. Attempts to improve crop yield, in vitro propagation efficiency, salt and heavy metal tolerance by clonal selection were carried out, although limited by the species’ low genetic diversity and availability of mutants. Mutagenesis and somaclonal variation have also been attempted on this species; however, since Arundo donax is polyploid, it is very difficult to induce and select promising mutations. In more recent years, genomics and transcriptomics data are becoming available in Arundo, closing the gap to make possible the genetic manipulation of this energy crop in the near future. The challenge will regard the functional characterization of the genes/sequences generated by genomic sequencing and transcriptomic analysis in a complex polyploid genome.

Plants synthesize specific secondary metabolites for survival, reproduction, environmental resilience, and defense. Among them, lignans are a class of polyphenols with several bioactive properties: chemopreventive, anti-inflammatory, antiviral, and antioxidant. These compounds are often extracted from field-grown plants with very low yields. To overcome these constraints, in vitro tissue cultures provide a tool to optimize large-scale production. Moreover, the use of elicitation to increase secondary metabolite production is gaining importance. The aim of this work was to develop adventitious (ARL) and hairy roots (HRL) from Linum lewisi, a species able to synthesize arylnaphthalene lignans such as justicidin B. The ARL and HRL were obtained for the first time and characterized for their phenol content, antioxidant activity, and the production of justicidin B after treatments with several elicitors and precursor feeding. Through NMR spectroscopy, other four lignans were highlighted and identified in the roots extracts. A pilot-scale bioreactor was adopted to assess the suitability of the developed root cultures for future large-scale production. The ARL and HRL cultures showed a justicidin B production higher than other Linum species cultures described up to now (75.8 mg/L and 82.2 g/L), and the production more than doubled after elicitation with MeJA.

Ocimum basilicum (sweet basil) is an economically important aromatic herb; in Italy, approximately 1000 ha of “Genovese-type” basil are grown annually in greenhouses and open fields and are subjected to Downy Mildew (DM) disease, caused by Peronospora belbahrii, leading to huge crop losses. Mutation of the Susceptibility (S) gene DMR6 (Downy Mildew Resistant 6) has been proven to confer a broad-spectrum resistance to DM. In this work, an effective Genome Editing (GE) approach mediated by CRISPR/Cas9 in O. basilicum ‘Italiko’, the élite cultivar used to produce “Pesto Genovese D.O.P”, was developed. A highly efficient genetic transformation method mediated by A. tumefaciens has been optimized from cotyledonary nodes, obtaining 82.2% of regenerated shoots, 84.6% of which resulted in Cas9+ plants. Eleven T0 lines presented different type of mutations in ObDMR6; 60% were indel frameshift mutations with knock-out of ObDMR6 of ‘FT Italiko’. Analysis of six T1 transgene-free seedlings revealed that the mutations of T0 plants were inherited and segregated. Based on infection trials conducted on T0 plants, clone 22B showed a very low percentage of disease incidence after 14 days post infection. The aromatic profile of all in vitro edited plants was also reported; all of them showed oxygenated monoterpenes as the major fraction.

Sweet basil (Ocimum basilicum L.; Fam. Lamiaceae) is an annual herbaceous plant with a high economic value used in folk medicine, pharmacology, and food production. In Italy, most of the varieties are used to produce the famous “pesto” sauce; however, almost all of them are susceptible to basil downy mildew (BDM) disease, strongly decreasing the growth of the fresh leaves and the survival of the whole plant. Nowadays, CRISPR/Cas9 technology is recognized to be a prominent way to enhance basil genetic breeding. In this work, we present an optimized protocol for in vitro direct regeneration of an elite cultivar, which is the major limiting factor for the transformation of O. basilicum. Regeneration has been obtained from different explants (leaves, cotyledons, cotyledonary nodes); the highest frequency has been obtained from cotyledonary nodes of seedlings germinated on MS medium containing TDZ. This protocol may be used for biotechnological applications as genome editing techniques to obtain basil-downy-mildew-disease-resistant clones.