GE-SP-SP5G-SlER Tomato lines were generated by editing three genes in two different Solanum lycopersicum cultivars, the tomato processing ‘roma’ cultivar M82 and the cherry cultivar Sweet100, using the CRISPR/Cas9 technology.
Aim of the mutations in the target genes SELF-PRUNING (SP), SELF-PRUNING-5G (SP5G) and ERECTA (SlER) was to generate compact and rapid flowering plants suitable for urban farming.
SP5G and SP are regulators of tomato flowering time and shoot architecture. SlER is a regulator of stem length. Triple-knockout in these genes results in condensed shoots, rapid flowering and precocious shoot termination, so-called ‘triple-determinate’ plants.
GE-SP-SP5G-SlER Tomato lines were developed by Agrobacterium tumefaciens-mediated transformation with binary vector pICSL4723. The T-DNA contained the coding sequence for Cas9 and the DNA sequences for target-gene-specific small guide RNAs.
For the M82 cultivar, triple-determinate plants were generated in the background of the natural sp-classic mutation, a missense mutation that has been used in breeding for nearly a century. This mutant background was used as a foundation to mutagenize SP5G and SlER. For the Sweet100 cultivar, all three genes were targeted using CRISPR-Cas9.
For both M82 and Sweet100 multiple SlER mutations were generated. Therefore, several different ‘triple-determinate’ mutants exist with small insertions or deletions in the target genes that resulted in frameshift mutations and eliminated gene function.
Edited plants were backcrossed to unedited parental lines, and progeny containing the desired indels, but confirmed by PCR to lack the T-DNA used to deliver the CRISPR-Cas9 protein, have been identified.
Sources:
USDA-APHIS letter of inquiry 20-149-01_air
Kwon et al. (2020), Rapid customization of Solanaceae fruit crops for urban agriculture, Nature Biotechnology 38:182-188.