Demonstration of CRISPR/Cas9-mediated PDS gene editing and Possibility of enhancing Shelf Life of Commercially Important Tomato variety

Author: Sumedha Arora, Prashant Mohanpuria and Salesh Kumar Jindal

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Abstract

CRISPR/Cas9 has been a popular tool for introducing precise mutations at the target site among different crop species to improve several required traits beneficial to farmers and breeders worldwide. Tomato is a model vegetable crop belonging to the family Solanaceae. Here in present study, the phytoene desaturase (PDS) gene from tomato was edited using CRISPR/Cas9 technique. The PDS gene is involved in carotenoid biosynthesis pathway and its editing would lead to albinism in plants. The sgRNA for PDS gene was designed and introduced into tomato system through leaf-disc method leading to precise mutation at target loci. The editing at desired loci was detected in 20 % of the To transgenic tomato plants. Rigorous screening and confirmation are always necessary for detecting true CRISPR edits. For screening of the PDS gene editing, first the T-DNA integration was confirmed through PCR. These plants were further analysed for mutation detection and analysis through Cel-1 assay and Sanger sequencing. About 2 bp deletion was observed in mutated plants at 3-4 bp upstream of PAM site at target loci. The editing of PDS confirmed that the technique can be successfully applied further for editing of Pectate lyase gene in commercially important tomato varieties for enhancing shelf life, which is a complex trait governed by a number of different genes and thus, a real challenge.

Keywords

Phytoene desaturase, tomato, CRISPR/Cas9, Shelf life, Pectate lyase

Conclusion

CRISPR/Cas9 technique has become voguish because of its adaptability, low cost, and versatility. Using modest constructs, we demonstrated the efficiency of this technique in editing PDS gene of tomatoes. To test the efficacy of this technique, the CRISPR edited albino tomato plants with complete or partial loss of PDS gene function were produced. This successful demonstration of the PDS gene of tomatoes has served as a basis for editing another target gene such as PL (for enhancing shelf life) in other tomato commercial varieties. We have explored the PL target gene from different tomato varieties and CRISPR/Cas9 constructs have been prepared using the PL gene.

References

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How to cite this article

Sumedha Arora, Prashant Mohanpuria and Salesh Kumar Jindal (2023). Demonstration of CRISPR/Cas9-mediated PDS gene editing and Possibility of enhancing Shelf Life of Commercially Important Tomato variety. Biological Forum – An International Journal, 15(10): 970-975.