Vegetos
(An International Journal of Plant Research & Biotechnology)
Efficient knockout of TOM1 gene using CRISPR/Cas9 in Indian tomato cultivar ‘Arka Vikas’
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Sandhya Dulam, Jogam Phanikanth, Sheri Vijay, Peddaboina Venkataiah, Allini Venkateswar Rao, Abbagani Sadanandam
Research Articles | Published: 08 January, 2025
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Keywords: Arka Vikas, CRISPR/Cas, Genome editing, n Solanum lycopersicumn , TOM1
Abstract
Tomato is one of the commercial crop grown in all parts of the world. Tobacco Mosaic Virus (TMV) and Tomato mosaic virus (ToMV) cause heavy yield loss in tomato. The tonoplast membrane-associated proteins TOM1 and TOM3 are required for the efficient multiplication of Tobamoviruses. Simultaneous mutations in any one of these genes inhibit the multiplication of Tobamoviruses. We have prepared guide RNA (gRNA) in the exon region of SlTOM1.3 and cloned it into the pHSE401 vector. Polymerase chain reaction (PCR) and sequencing confirmed the plasmid DNA. The confirmed plasmid product (pHSE401-SlTOM1.3) was transferred to Agrobacterium tumefaciens strain LBA4404 for plant transformation. PCR analysis was conducted with non-transformed (WT-wild type) genomic DNA and transformed plants for hptII and Cas9 gene amplification. A total of 16 transformed and wild-type plants performed nucleotide sequencing for mutation analysis in SlTOM1.3 gene. Among 16 plants, mutations were observed in 9 plants. We have observed several insertions and deletions (indels) in target sequence of corresponding gene. A single base pair alteration (either insertion or deletion) in the targeted gene can change the frameshift of a particular gene and result in abnormal protein. So, this CRISPR-Cas9 mediated genome editing protocol developed in tomato will be feasible for developing resistance to several viral pathogens of Tobamovirus genus.
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Author Information
Department of Biotechnology, Kakatiya University, Warangal, India