Background:
The long-shelf-life is a critical trait for the quality of fleshy fruit, and it is one of the major objectives in breeding programs as it influences fruit marketability for both farmer’s and consumer’s perception. The term ALC was coined from ‘Alco Baca’ Portugal cultivar of tomato from which slow ripening gene alc was discovered in 1960. ALC gene in its heterozygous mutation form was found to be significantly slow down the ripening of tomato with prolonged shelf with acceptable color development and flavor attributes. The present innovation named "CRISPR/Cas9-ALC binary vector construct" was created to generate tomato fruits with long shelf life. Technology Details:
The long-shelf-life is a critical trait for the quality of fleshy fruit, and it is one of the major objectives in breeding programs as it influences fruit marketability for both farmer’s and consumer’s perception. Tomato (Solanum Lycopersicum) has been mostly studied as classic climacteric model species with fleshy fruits to unravel the molecular basis of fruit softening. The term ALC was coined from ‘Alcobaca’ Portugal cultivar of tomato from which slow ripening gene alc was discovered in 1960. ALC gene in its homozygous mutation form was found to be significantly slow down the ripening of tomato with prolonged shelf life but with inferior flavor and poor color development. However, in plants heterozygous for these alleles shown to have a prolonged shelf with acceptable color development and flavor attributes. To generate tomato fruits with long shelf life, CRISPR/Cas9-ALC binary vector construct was created. CRISPR/Cas9 technology was exploited to knockout the ALC gene (Solyc10g006880.2.1). ALC consists of 3 exons and its encoded protein has two domains viz., NAM domain (17-143 amino acid) and NAC domain (11-179 amino acid). Synthetic gRNA was designed to target the second exon of ALC (Figure 1A). It was incorporated into a Cas9 expressing binary vector and confirmed by restriction analysis and sequencing (Figure 1B & C). Figure 1: Overview of ALC gene structure and ALC target gRNA cloning into shuttle vector: A) Schematic of the ALC gene structure consisting of 3 exons with the complete CDS size of 1068 bp. The chosen target guiding sequence (its reverse compliment shaded in blue) for CRISPR/Cas9 modification in exon 2 of ALC is presented, the PAM (its reverse complement) shaded in orange and all the restriction sites needed for cloning are represented. B) Gel picture showing the release of gRNA cassette of 550 bp (Lane 1) and unrestricted gRNA plasmid (Lane 2). C) Chromatogram peaks showing cloning of ALC gRNA sequence.