CRISPR/Cas9-mediated pectate lyase gene editing for enhanced shelf life of tomato (Solanum lycopersicum L.)
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Date
2024
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Punjab Agricultural University
Abstract
Tomato is one of the most consumed vegetable crops worldwide. Tomato fruits are having an inherently short shelf life which is directly related to fruit softening, caused by cell wall modifying enzymes. Among which pectate lyase (PL) controls fruit softening and thus PL gene was targeted using CRISPR/Cas9 to enhance shelf life of tomato variety Punjab Ratta. In silico study of PL genes from Solanaceous crops including tomato was attempted and three sgRNAs were designed from exon-2 of PL gene. The CRISPR/Cas9 constructs containing sgRNA-1 and 2 were used for transformation of tomato. A new in planta transformation protocol was developed with 10.49 % overall transformation efficiency. Floral buds of 3-8 days of developmental stages were used for dipping in Agro suspension which resulted in setting of 150 fruits. The extracted T0 seeds were selected on kanamycin. A total of 3240 T1 generation plants were regenerated from transgenic tomato fruits (T0), out of which 1620 were found kanamycin resistant which were used for DNA isolation and PCR confirmation of T-DNA integration. The 425 bp PL gene target region from 170 transgenic tomato T1 plants was PCR amplified. Upon sequencing eight T1 tomato transgenic plants were found with PL gene edits containing T insertion, T insertions as well as T substitutions, GC substitution by CG as well as TT insertion downstream to PAM site. PL gene expression and pectin content were decreased in fruits of mutated tomato plants but morphological and biochemical characteristics were more or less similar.
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Sumedha (2024). CRISPR/Cas9-mediated pectate lyase gene editing for enhanced shelf life of tomato (Solanum lycopersicum L.) (Unpublished Ph.D. Dissertation). Punjab Agricultural University, Ludhiana, Punjab, India.