Dr. Rameshwar Sharma is a Professor at Repository of Tomato Genomics Resources, Dept. of Plant Sciences, University of Hyderabad, Hyderabad, India.
Research: Understanding of plant development using tomato as model system
– Enhancement of nutraceuticals such as carotenoids and folate in tomato fruits
– Use of forward and reverse genetic tools to understand tomato development
– Harnessing the diversity of important traits in natural accessions of tomato
– Targeted mutagenesis of tomato genes for the trait enhancement
Expanding the horizons of plant trait improvement: NGS-based TILLING and genome editing
The trait enhancement has been the mainstay for the crop improvement right from domestication of crop plants and advent of the agriculture. The judicious selection of traits based on the plants’ phenotype, resilience to survive in adverse climatic conditions, taste, and yield of the produce have been the major targets during domestication. The realization that the traits are governed by the heritable mutations enabled introgressions of the desirable traits to a wider range of cultivars by plant breeders. The ability to enhance the variability in the desired crop species by chemical or physical mutagens gave a big impetus to crop improvement in last century leading to the foundation of mutation breeding. The green revolution that considerably enhanced rice and wheat yield was enabled by introgression of mutations in gibberellic acid synthesis/action. A priori knowledge about the mutagenized gene(s) encoding a particular trait can significantly accelerate the process of mutation breeding. The knowledge about linkage between a trait and its corresponding gene in crop plants has been dramatically enhanced by genome sequencing. The genotype/phenotype linkage permits isolation of gene-specific mutant from a large population of mutagenized plants by reverse genetic tools such as TILLING. We used NGS-based TILLING to identify mutations in an EMS-mutagenized population of tomato consisting of 768 plants. Twenty-five genes belonging to carotenoids and folate metabolism were PCR-amplified and screened to identify potentially beneficial alleles. The NGS-based mutation screening yielded 64 mutations confirmed by Sanger Sequencing. Few of mutant lines were phenotyped in M3-M4 generation and are now under backcrossing. Though the TILLING allows isolation of mutant in the desired gene, during randomness of EMS-mutagenesis the chances to find a suitable allele remains low. In contrast, the gene-editing allows precise targeting of a gene, with an extensive repertoire of variations, leading to loss or gain of function mutations, or even insertion of novel function to a gene. In tomato, we carried out targeted mutagenesis in selected genes using CRISPR/Cas9 system. We targeted the ORF of tomato RIN locus, which is essential for induction of fruit ripening. The CRISPR induced mutants displayed delayed on-vine fruit ripening and reduced ethylene emission. We show that genome editing can be used for trait enhancement in tomato with high efficiency, rare/no off-target mutations, and stable heredity.
Keywords: Tomato, TILLING, Gene-editing, Fruit ripening, Reverse Genetics