Genome editing applications on grapevine cv. Aglianico for the knockout of susceptibility genes related to fungal diseases

Abstract

Context and purpose of the study. Italy hosts diverse grapevine varieties crucial for viticultural biodiversity. Preserving this biodiversity is essential for maintaining a diversified genetic pool and addressing future challenges such as climate change and emerging plant diseases. Biotechnological approaches, such as genome editing through CRISPR/Cas9 system, have emerged as a significant player in tackling modern viticulture challenge but, until now, its application has been restricted on a few cultivars. Genetic improvement via in vitro delivery of desired constructs requires the regeneration of genome-edited plants. In vitro plant regeneration, a pivotal process in genetic engineering, encounters obstacles, due to factors like genotype and explant-dependent responses. The study focuses on developing an efficient in vitro plant regeneration protocol via somatic embryogenesis to facilitate genome editing of Aglianico, the most important grapevine variety of southern Italy regions.

Material and methods. Stamens and pistils were collected from fruit cuttings at both mature and immature pollen developmental stages. These explants were cultured on three media (MSII, PIV, and NNder) to induce embryogenic callus formation. The embryogenic calli were then transferred to two different media: C1, for long-term propagation, and X6, for embryo formation. Properly developed embryos were transferred to a rooting medium and later to four different media for shoot regeneration. The well-grown shoots were cultured on 3/4 MS medium for micropropagation. Calli propagated in C1 medium were used to isolate protoplasts, providing a platform for the delivery of pDNA and ribonucleoprotein particles (RNPs) to target different susceptibility genes related to fungal diseases.

Results. Preliminary results indicate that the regeneration system employed through the somatic embryogenesis process is optimal for the poorly studied grapevine cv. Aglianico. It successfully produces a high number of plantlets from a small amount of embryogenic calli. From these calli, protoplasts were efficiently isolated, providing an excellent platform for transfection with pDNA and RNPs to target two susceptibility genes: Downy Mildew Resistant 6.1 (DMR6.1) and CERK1 Interacting Protein Phosphatase 1 (CIPP1).