CRISPR/Cas9-mediated editing of PHYTOENE DESATURASE gene in onion (Allium cepa L.)

Abstract

IntroductionClustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) is a precise genome editing tool used to introduce genetic modifications in a wide range of crop species. Thus far, there is no report of CRISPR/Cas9-mediated genome editing in onions (Allium cepa L.). MethodsIn the present study, we targeted two exons of the gene coding for Phytoene desaturase (AcPDS) in onion cv. Bhima Super. The sgRNA-carrying constructs were co-cultivated with 8-week-old embryogenic calli using an Agrobacterium-mediated transformation protocol and incubated on the media without hygromycin B selection. Results and discussionOut of the total 617 co-cultivated calli, 21 (3.4%) regenerated shoots exhibited three distinct phenotypes: albino, chimeric, and pale green; in comparison to the wild-type non-transformed regenerated shoots. Total chlorophyll content was drastically reduced in albino shoots and significantly decreased in chimeric shoots. Out of the six Cas9 gene PCR-confirmed regenerated shoots, two exhibited the albino phenotype due to insertions/deletions (InDels) and substitution-based mutations in and around the AcPDS target sites. Deep amplicon sequencing revealed a significantly variable InDel frequency between two sgRNAs, ranging from 1.2% to 63.4%, along with a 53.4% substitution frequency. The mutation of the AcPDS gene generated a visually detectable albino phenotype, thus confirming the successful editing of the AcPDS gene. This is the first time a CRISPR/Cas9-mediated genome editing protocol has been successfully established in onion, with the AcPDS gene serving as an example. This study will provide the necessary momentum for researchers to further basic and applied research on onions.

Antioxidants of Non-Enzymatic Nature: Their Function in Higher Plant Cells and the Ways of Boosting Their Biosynthesis

“…The knockout of the PDS gene in many crop species resulted in a phenotype with pigmentation loss. Albino regenerants of cells with edited PDS gene have been obtained for Malus domestica plants, for diploid and octoploid strawberries, yams, and onion [50][51][52][53]. The regenerants of carrot cells with mutations in the region of the DcPDS and DcMYB113-like genes were depigmented [54].…”

CRISPR/Cas9 based genome editing of Phytoene desaturase (PDS) gene in chilli pepper (Capsicum annuum L.)

“… 22 It has been targeted in many CRISPR/Cas mediated genome editing studies as a visible marker to standardize the delivery of CRISPR reagents into plant cells. 23 , 24 , 25 By inhibiting chlorophyll, carotenoid, and gibberellin production, the disruption of PDS results in photobleaching, albino phenotype, and dwarfism. 26 It also allows for simple screening of mutant lines.…”

“… 26 It also allows for simple screening of mutant lines. 27 , 28 Recent studies have shown the establishment of efficient genome editing using the PDS gene through the CRISPR/Cas9 system in several crops, including potato, 24 pea, 29 papaya, 30 coffee, 31 chickpea, 32 hop, 23 onion 25 and squash. 33 Nevertheless, the development of a biolistic-mediated CRISPR/Cas9-reagent delivery system using the PDS gene in chilli pepper has not yet been reported.…”