Abstract
Genetic engineering of plants is at the core of environmental sustainability efforts, natural product synthesis of pharmaceuticals, and agricultural crop engineering to meet the needs of a growing population in a chang-ing global climate. Recent progress in genome-editing tool development has revolutionized researcher abili-ties to genetically probe and modify living systems. However, genetic engineering of mature plants and their plastids has remained a challenge owing to the numerous physical barriers that need to be crossed for mature plant genome editing. Nanomaterials and nanobubbles hold great promise to advance our knowledge of –and toolsets for– genome editing, particularly for plant science. The physical barrier presented by the cell wall has limited the ease and throughput with which exogenous bi-omolecules can be delivered to plants. Current techniques suffer from host range limitations, low transfor-mation efficiencies, toxicity, and unavoidable DNA integration into the host genome. The TARGET project will use and develop nanoparticle and nanobubbles platforms (used in the human pharmacology field to deliver drugs) to enable electrostatic grafting of genome engineering biomolecules, which will be leveraged to genet-ically transform mature plants. We would like in this project (i) use and develop nanoparticles which transport across the plant cell mem-branes protein and DNA. (ii) Identify nanoparticles or nanobubbles that are highly efficient for plant cell inter-nalization, and utilize such nanoparticles to deliver DNA, RNA, and Cas9-gRNA RNP to different plant matrices, such as calli, somatic embryos, meristem or protoplasts in a fruit tree species-independent manner.
Results achieved
The project entitled "Development of a DNA editing protocol that does not involve the use of genetic transformation techniques in fruit tree species" is a project in collaboration with research groups of the University of Turin and the Edmund Mach Foundation (FEM). The aim is to develop a new protocol for the delivery of the CRISPR/Cas9 protein without the use of Agrobacterium tumefaciens, in the pear variety 'Conference' (Pyrus communis L.). In this regard, it was decided to test different types of Nanobubbles and Carbon Nanotubes (PEI-NT) which, by incorporating the protein internally or externally, can deliver it inside the cell. To date, this technique is already widely used only on humans, at a medical level, for the transport of drugs in specific therapies. With this PRIN project, therefore, for the first time, we wanted to try to transfer this technique to plants. One of the main adversities of the use of Nanobubbles is the presence of the cell wall in plant cells, which prevents their entry. Due to the technical difficulty in developing an efficient protocol for the incorporation of nanobubbles into plant tissues, it was decided to use the Nicotiana benthamiana species (in vitro model system for its rapid response and well-defined genetic characteristics) in addition to the pear. In particular, in Pyrus communis L. cv 'Conference': the experiments were conducted using leaf tissues and callus cultures and in Nicotiana benthamiana: callus cultures and protoplast cultures were used. Both tobacco (Nicotiana benthamiana) and 'Conference' (Pyrus communis L.) were sub-cultured and maintained in vitro for the production of plant material to be used in experimental trials. Considering that the preliminary approaches did not produce the expected results, it was decided to use a different composition of nanotubes. In addition, this phase focused on solving nanoparticle size issues, with the aim of improving their penetration into the cell membrane. At first, polyethylenimine-functionalized Nanotubes (PEI-NT) engineered with RUBY (reporter system to verify the effective incorporation of nanoparticles) were used to transform young leaves of 'Conference' and Nicotiama benthamiana pretreated with cellulase and ultrasound in order to reduce the barrier effect of the cell walls of plant cells. Since no appreciable results were obtained with the first experimental approaches, other tests were conducted using other nanoparticles such as Mesoporous Silica Nanoparticles (MSN) and PLGA-PEI (Polylactic-co-glycolic acid) nanoparticles to reduce the size of the vectors and improve their stability. These vectors, initially loaded with 10 μg/ml of RUBY or pAVA:GFP (a second reporter system to verify the incorporation of nanoparticles into cultured cells), were tested on three distinct biological matrices: soft tobacco calluses, partially digested pear leaves (using cellulases) and tobacco protoplasts. Confocal fluorescence microscopy analyses of pAVA:GFP provided the best results. The observed fluorescence patterns suggest the possible translocation of the pAVA:GFP plasmid across the cell wall and plasma membrane, followed by its subsequent transcription and transient expression of the GFP reporter gene. As for the "Ruby" system, the experiments did not show any detectable pigment production under all the conditions tested, suggesting insufficient expression levels or the need to increase the concentration of RUBY within NBs. In the future, experiments using the pAVA:GFP system on 'Conference' and other varieties of Pyrus communis should be continued.Dettagli del progetto
Responsabile scientifico: Luca Dondini
Strutture Unibo coinvolte:
Dipartimento di Scienze e Tecnologie Agro-Alimentari
Coordinatore:
Università degli Studi di TORINO(Italy)
Contributo totale Unibo: Euro (EUR) 62.130,00
Durata del progetto in mesi: 28
Data di inizio
12/10/2023
Data di fine:
28/02/2026
