Maurizio Micheli

Genome editing in autopolyploid plants has the potential to modify all the alleles of a gene of interest at the same time. We attempted to edit the alfalfa glutamate 1-semialdehyde aminotransferase (GSA) gene by CRISPR-Cas9, using a gRNA targeting the second intron and a donor DNA introducing a point mutation for gabaculine resistance. We determined that three GSA copies were present in our lab genotype, and one in three was a previously undetected variant allele differing at the target site, which could not be cut by Cas9. Precise, homology-directed repair was not observed, but five in fourteen transgenic plants carried −1, −3, −4 or −5 deletions at the target site. In three of them, only one of the two standard alleles was mutated. In one event, only the variant allele and a −5 deletion allele were present, demonstrating complete editing of the standard allele. Several rare deletion alleles were detected through deep sequencing, indicating a limited, continued activity of the DSB machinery after in vitro regeneration. No new mutated alleles emerged from sexual reproduction. Interestingly, some clonal progenies from an edited plant presented deletion allele frequencies different from the original plant, likely due to somatic segregation of chimeric mutations. This can possibly be exploited to generate new edited genotypes through clonal reproduction.

The olive tree is one of the most iconic species within the Mediterranean Sea Basin. Countries bordering this sea enjoy a favourable climate that contributes to high-quality agricultural production for numerous horticultural species. The quality of the propagation material is one of the most important factors in determining the value of the final product, regardless of the cultivation-model, climate, and soil characteristics. Therefore, it is crucial to ensure genetic and sanitary certainty of vegetal/propagation material, which can be achieved through nursery productions. These goals are based on efficient propagation systems and methods to obtain high-biological quality plants. Over the last four decades, the application of biotechnology has introduced significant changes in the sector of nursery production. The Authors in this chapter aimed to present through their personal experimental experiences the latest advances in in vitro techniques and technologies that are revolutionizing the field of olive tree nurseries. While some of these methods are currently being employed, others are still undergoing research and development. Experts in this field firmly believe that all these techniques hold great practical value and have immense potential for high-quality nursery production.

Synthetic seed technology, an advanced in vitro propagation method, combines the benefits of micropropagation with the practicality of zygotic seeds, offering an efficient solution for the handling, storage, and transportation of plant propagules. This study represents the first investigation of the role of explant type in blueberry encapsulation. In particular, three non-embryogenic propagules (basal node, median node, and shoot tip) were evaluated in the Brigitta and Duke blueberry cultivars. The artificial endosperm consists of Woody Plant Medium (WPM) macro- and micro-nutrients and Murashige and Skoog vitamins at half concentration, inositol (5 g L⁻¹), Indole-3-butyric acid (IBA) (0.005 mg L⁻¹), zeatin (0.25 mg L⁻¹), and sucrose (50 g L⁻¹). Forty-five days after sowing in in vitro conditions, the encapsulated shoot tips showed better results than basal and median nodes in several parameters, including viability, shoot length, and fresh shoot weight, in both cultivars. In both cultivars, none of the encapsulated propagule types developed roots. These results underscore the advantages of using shoot tips in encapsulation technology for blueberries and provide valuable insights for optimizing encapsulation protocols to improve propagation efficiency.

Nanotechnology offers promising applications in agriculture by enhancing crop growth, yield, and antioxidant defenses. This study is the first to evaluate the effect of biogenic zinc oxide nanoparticles (ZnO-NPs) on the in vitro growth of two blueberry cultivars, Brigitta and Duke. The ZnO-NPs were synthesized biogenically using an extract of Lemna minor L., which is a free-floating aquatic plant, as a capping and modulating agent, and were added to the plant’s growth media at different dosages (0, 2, 6, and 18 mg L−¹). The ZnO-NPs significantly increased the shoot number, fresh biomass, and dry weight in both cultivars without affecting shoot vitality, length, or basal callus formation. Moreover, the increases in carotenoids in both cultivars, as well as chlorophyll and and soluble proteins in the ‘Brigitta’ cultivar, confirm the prompted benefits and possibly evidence genotype-specific metabolic adaptations in response to ZnO-NPs. These results demonstrate that biogenic ZnO-NPs can effectively promote the in vitro growth of blueberry explants, offering improvements in micropropagation efficiency.

In vitro propagation, is becoming the predominant method for blackberry propagation due to its advantages compared to agamic traditional propagation methods. Synthetic seed technology represents a promising approach to further enhance the productivity of in vitro propagation facilitating the exchange of plant materials among laboratories and contributing to germplasm conservation efforts. This study aimed to establish an optimal protocol for the storage and sowing of synthetic blackberry seeds obtained through the encapsulation of clump bases. The synthetic seeds were sown without storage (Control) and after storage periods of 30, 60, and 120 days at 4 °C and 25 °C in the dark, in three different substrates (agarised, perlite, and potting). After forty-five days from sowing viability, regeneration rate, shoot and root numbers and lengths, as well as fresh and dry weights of the plantlets, were assessed. Results indicated that agarised substrate consistently exhibited favourable outcomes, with sustained regeneration rates and robust plantlet development even after prolonged storage at 4 °C. Synthetic seeds sown in perlite and potting substrates demonstrated enhanced regeneration rates following storage at 4 °C for 60 and 120 days. On the contrary, storage at 25 °C resulted in a notable decline in regeneration rate, highlighting its inadequacy for blackberry synthetic seed conservation purposes. These findings underscore the importance of sowing substrate selection and storage temperature in optimizing the storage and sowing protocols for synthetic blackberry seeds.

In vitro propagation, also known as micropropagation, has become the most widely employed method for blackberry propagation, as it overcomes the limitations of the traditional asexual propagation methods (mainly layering and cutting). In this context, synthetic seed technology represents a strategy to enhance the productivity of in vitro propagation and facilitates the exchange of plant materials between laboratories, contributing to germplasm conservation. This study aimed to identify the most suitable vegetative propagule for the encapsulation of blackberry. To this end, uninodal microcuttings (nodes) and the base of clumps were used to produce synthetic seeds for the cultivars Thornfree and Chester. Forty-five days after sowing, viability (percentage of green propagules without browning or necrosis), regeneration (percentage of propagules that sprouted and rooted simultaneously), number of shoots produced, shoot length, number of roots produced, root length, and the fresh and dry weights of the plantlets were measured. The results demonstrated that both considered propagules allowed us to obtain satisfactory regeneration rates. However, plantlets originating from the encapsulated clump’s base had more shoots and roots, resulting in greater fresh and dry weights than the plantlets derived from encapsulated nodes. Therefore, for achieving more robust plantlets and enhancing overall procedural efficiency, we recommend using the base of clumps as a propagule for blackberry encapsulation.

Salinity stress represents an increasing issue for agriculture and has a great negative impact on plant growth and crop production. The selection of genotypes able to tolerate salt stress could be a suitable solution to overcome the problem. In this context, in vitro cultures can represent a tool for identifying the NaCl tolerant genotypes and quickly producing large populations of them. The possibility of exerting selection for tolerance to NaCl by using encapsulation technology was investigated in two genotypes of fig: ‘Houmairi’ and ‘Palazzo’. The effects of five concentrations of NaCl (0, 50, 100, 150 and 200 mM) added to the artificial endosperm were tested on the conversion of synthetic seeds and on the growth of derived shoots/plantlets. Moreover, proline (Pro) and malondialdehyde (MDA), the enzymatic activities of catalase (CAT), guaiacol peroxidase (POD), and EL (Electrolytic Leakage), as well as the chlorophyll content, flavanols, anthocyanins, and Nitrogen Balance Index (NBI) were determined on shoots/plantlet. The obtained results clearly showed that ‘Houmairi’ and ‘Palazzo’ could tolerate salt stress, although a strong difference was found depending on each specific physiological pathway. Indeed, ‘Houmairi’ was revealed to be more tolerant than ‘Palazzo’, with different response mechanisms to salt stress. The use of encapsulated vitro-derived explants proved to be a useful method to validate the selection of genotypes tolerant to salinity stress. Further investigation in the field must validate and confirm the legitimacy of the approach.

In vitro culture, ensuring rapid multiplication and production of plant material under aseptic conditions, represents an excellent tool for ex-situ conservation of tree species biodiversity and can be used for the conservation, among others, of endangered and rare crops. Among the Pyrus communis L. cultivars that have been abandoned over the years due to changed cultivation requirements, but which are still used today in breeding programs, there is the ‘Decana d’inverno’. Pear is generally considered a recalcitrant species for in vitro propagation due to weak multiplication rate, hyperhydricity, and susceptibility to phenolic oxidation. Therefore, the use of natural substances like neem oil (although little explored) represents one of the options to improve the in vitro plant’s tissue culture. In this context, the aim of the present work was to evaluate the effect of adding neem oil (0.1 and 0.5 m L L⁻¹) to the growth substrate in order to optimise the in vitro culture of the ancient pear tree cultivar ‘Decana d’inverno’. The neem oil addition resulted in an increase in the number of shoots produced especially at both concentrations used. On the contrary, an increase in length of proliferated shoots was observed only with the addition of 0.1 mL L⁻¹. The neem oil addition did not affect the explants viability, fresh and dry weights. Therefore, the present study demonstrated for the first time the possibility of using neem oil to optimise the in vitro culture of an ancient pear tree cultivar.

Micropropagation is an in vitro propagation technique, established in the nursery field sector for numerous species, which offers several advantages compared to traditional agamic propagation techniques. In the case of the olive tree, however, despite the advances made through research, it is still little used, due to the recalcitrance to in vitro proliferation and/or rooting of many olive cultivars and the high cost of zeatin, the only cytokinin that makes it possible to achieve a satisfactory proliferation rate in this species. In this context, numerous attempts have been made to identify alternative cytokinin compounds able to improve the proliferation rate of olive tree explants and thus reduce the unitary production cost. In particular, there is a growing interest in the use of natural substances (called in some cases “complex mixtures”), which, when added to the in vitro cultivation substrates, seem to be able to improve proliferation rates. In the present study, neem oil was added to the propagation substrates (partially/totally replacing zeatin) and in the rooting phase for the olive cultivar Moraiolo. In particular, in the proliferation phase, the effect of neem oil (0.1 mL L⁻¹) in substrates containing different zeatin concentrations (0, 1, 2, and 4 mg L⁻¹) was evaluated. For the rooting phase, agarized substrate and soil were used with shoots derived from a standard proliferation substrate (4 mg L⁻¹ zeatin) and from the substrate that gave the best results in the proliferation phase (2 mg L⁻¹ zeatin and 0.1 mL L⁻¹ neem oil). In the proliferation phase, the addition of neem oil in the substrates with low zeatin concentration (1 and 2 mg L⁻¹) induced an increase in the number of adventitious shoots and shoots length. On the contrary, the addition of neem oil in the rooting substrates did not positively influence the rooting phase, but positive results especially in terms of root number and length were observed in explants derived from a neem oil-enriched proliferation substrate compared to the control substrate. Therefore, the present study demonstrated for the first time the positive role of neem oil in the proliferation of olive in vitro with low zeatin concentrations.

It is well known that the products of encapsulation (multifunctional beads and synthetic seeds) can be used as innovative technological tools to integrate micropropagation both for plant germplasm conservation and to simplify the management of propagation materials in nurseries. Nevertheless, the usual concept of encapsulation concerns the use of initial in vitro derived explants. In this study, for the first time, in vivo derived organs of Mentha spicata L., obtained through the excision of fragments (nodes) from stolons of cultivated mother plants, were employed. The artificial endosperm had a tenfold reduced concentration of Murashige and Skoog (MS) substrate, with the addition of sucrose (5 g L⁻¹), 6-benzyl-aminopurine (BAP) (0.1 mg L⁻¹) and 1-naphthalene acetic acid (NAA) (0.01 mg L⁻¹). Moreover, the calcium alginate matrix was enriched with different thiophanate-methyl (TM) concentrations (0, 10, 50, 100 and 200 mg L⁻¹) in order to prevent possible contamination during the conversion in nonsterile conditions. Interesting results were obtained encapsulating every single node of fresh stolon as a bipolar propagule able to develop a whole plantlet (conversion), as the coated seed in other species. The synthetic seeds of spearmint without TM in the artificial endosperm showed a satisfactory ability to convert (56.7%) into plantlets after sowing in soil under nonsterile conditions. TM at 100 and 200 mg L⁻¹ negatively affected the total emergence, which decreased to 30.0 and 33.3%, respectively. In general, in the artificial seeds without TM, higher values for most of the aboveground and belowground plants parameters were recorded compared to naked nodes.