TL;DR
iPB-REG is established as a practical strategy for producing uniform genome-edited fruit trees and provide a valuable platform for DNA-free genetic improvement and functional genomics in clonally propagated perennial crops.
The present study addresses optimization of in-vitro regeneration via direct organogenesis and Agrobacterium-mediated genetic transformation, enabling efficient multiplex CRISPR/Cas9-based genome editing of the phytoene desaturase (PsPDS) gene in pea. Pea (Pisum sativum L.) is an important legume crop valued for food, plant-based protein, vegetable, and green manure. Although genome editing offers a precise and rapid strategy for crop improvement, its application in pea remains challenging due to inherent recalcitrance to in-vitro regeneration and genotype-dependent transformation. The regeneration and Agrobacterium-mediated transformation systems were optimized, and the dicotyledonary node (DCN) was identified as the preferred explant for multiplex CRISPR/Cas9-based genome editing in pea. Among three explant types (embryonic axis, DCN and nodal segment), DCN showed the highest regeneration efficiency, producing 100% shoot bud induction and 39.70 shoots per explant on MS medium augmented with 6-benzylaminopurine (BAP; 6.00 mg/L) and kinetin (1.00 mg/L). Shoot elongation and rooting efficiencies were improved using GA3 (1.00 mg/L), BAP (1.00 mg/L), IAA (0.10 mg/L), and NAA (0.5 mg/L), respectively. Manipulating explant type, Agrobacterium optical density, vacuum infiltration, acetosyringone concentration, infection time, and co-cultivation duration improved the transient transformation efficiency. We noted efficiency from 23.33% to 90.00% in DCN and from 6.66% to 93.33% in embryonic axis explants across 10 pea cultivars. Stable transformed lines generated from the DCN of cultivar Kashi Samridhi were confirmed by GUS staining and PCR. The optimized regeneration and transformation system facilitated targeted editing of phytoene desaturase (PsPDS) in pea, achieving ICE-estimated mutation frequencies of upto 97% in independent lines. The study provides a robust platform for functional genomics and accelerates the deployment of genome-editing technologies for pea improvement.
A complete CRISPR-Cas9-mediated knockout of the BEL5 gene, encoding a transcription factor, is reported, known as one of the key regulators driving tuber formation, and a regulatory role of BEL5 in the timing of tuber onset but, unexpectedly, its dispensability for tuber development in modern cultivated potato is proposed.
Traditional genetic transformation approaches relying on Agrobacterium tumefaciens for the delivery of CRISPR/Cas9 reagents usually provide plants that stably integrate the gene construct in their genome. To meet the EU commission’s proposal for a new legislation on plants obtained by new genomic techniques (NGTs), it is important to develop new protocols that produce transgene-free genome edited plants (NGT category 1). Protoplasts are a promising platform, since delivery of CRISPR/Cas9 reagents as ribonucleoproteins (RNPs) is effective in cells lacking their wall. This allows genetic modifications from a transient application, leaving no traces in the recipient genome apart from the desired targeted mutations. With the aim of implementing transgene-free editing of eggplant (Solanum melongena L.), we adapted and improved a protocol previously established in potato and tomato for the isolation of protoplasts from cotyledonary leaves and subsequent CRISPR/Cas9 reagents delivery. Isolated protoplasts were subjected to in vitro culture and regeneration, and the first shoots were regenerated from calli approximately 4–5 months after isolation. Alongside, two transfection protocols were tested for the delivery of RNPs into eggplant protoplasts, one using polyethylene glycol (PEG) in two concentrations (25% and 40%) and one exploiting two formulations of lipofectamines (Lipofectamine CRISPRMAX™ and Lipofectamine™ 3000), all targeting SmChl_H gene, whose inactivation can cause a chlorotic phenotype. Efficient callus regeneration from transfected protoplasts was obtained and the editing efficiency (calculated as the percentage of edited calli on the total of calli that underwent sequencing) was evaluated. 25% PEG treatment provided the highest editing efficiency, and fully edited biallelic calli were retrieved, showing the expected chlorotic phenotype. Even if the efficiency of in vitro regeneration of plants from calli still needs improvement, edited plants were regenerated from protoplasts, representing the first report of RNP mediated genome editing in eggplant protoplasts.