We report a highly efficient protocol for the Agrobacterium-mediated genetic transformation of a miniature dwarf tomato (Lycopersicon esculentum), Micro-Tom, a model cultivar for tomato functional genomics. Cotyledon explants of tomato inoculated with Agrobacteriumtumefaciens (Rhizobium radiobacter) C58C1Rif^R harboring the binary vector pIG121Hm generated a mass of chimeric non-transgenic and transgenic adventitious buds. Repeated shoot elongation from the mass of adventitious buds on selection media resulted in the production of multiple transgenic plants that originated from independent transformation events. The transformation efficiency exceeded 40% of the explants. This protocol could become a powerful tool for functional genomics in tomato.

γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that has hypotensive effects. Tomato (Solanum lycopersicum L.) is among the most widely cultivated and consumed vegetables in the world and contains higher levels of GABA than other major crops. Increasing these levels can further enhance the blood pressure-lowering function of tomato fruit. Glutamate decarboxylase (GAD) is a key enzyme in GABA biosynthesis; it has a C-terminal autoinhibitory domain that regulates enzymatic function, and deleting this domain increases GAD activity. The tomato genome has five GAD genes (SlGAD1–5), of which two (SlGAD2 and SlGAD3) are expressed during tomato fruit development. To increase GABA content in tomato, we deleted the autoinhibitory domain of SlGAD2 and SlGAD3 using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 technology. Introducing a stop codon immediately before the autoinhibitory domain increased GABA accumulation by 7 to 15 fold while having variable effects on plant and fruit size and yield. This is the first study describing the application of the CRISPR/Cas9 system to increase GABA content in tomato fruits. Our findings provide a basis for the improvement of other types of crop by CRISPR/Cas9-based genetic modification.

The tomato is an excellent model for studies of plants bearing berry-type fruits and for experimental studies of the Solanaceae family of plants due to its conserved genetic organization. In this study, a comprehensive mutant tomato population was generated in the background of Micro-Tom, a dwarf, rapid-growth variety. In this and previous studies, a family including 8,598 and 6,422 M₂ mutagenized lines was produced by ethylmethane sulfonate (EMS) mutagenesis and γ-ray irradiation, and this study developed and investigated these M₂ plants for alteration of visible phenotypes. A total of 9,183 independent M₂ families comprising 91,830 M₂ plants were inspected for phenotypic alteration, and 1,048 individual mutants were isolated. Subsequently, the observed mutant phenotypes were classified into 15 major categories and 48 subcategories. Overall, 1,819 phenotypic categories were found in 1,048 mutants. Of these mutants, 549 were pleiotropic, whereas 499 were non-pleiotropic. Multiple different mutant alleles per locus were found in the mutant libraries, suggesting that the mutagenized populations were nearly saturated. Additionally, genetic analysis of backcrosses indicated the successful inheritance of the mutations in BC₁F₂ populations, confirming the reproducibility in the morphological phenotyping of the M₂ plants. To integrate and manage the visible phenotypes of mutants and other associated data, we developed the in silico database TOMATOMA, a relational system interfacing modules between mutant line names and phenotypic categories. TOMATOMA is a freely accessible database, and these mutant recourses are available through the TOMATOMA (http://tomatoma.nbrp.jp/index.jsp).

To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1–SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato.

A large amount of γ-aminobutyric acid (GABA) was found to accumulate in tomato (Solanum lycopersicum) fruits before the breaker stage. Shortly thereafter, GABA was rapidly catabolized after the breaker stage. We screened the GABA-rich tomato cultivar ‘DG03-9’ which did not show rapid GABA catabolism after the breaker stage. Although GABA hyperaccumulation and rapid catabolism in fruits is well known, the mechanisms are not clearly understood. In order to clarify these mechanisms, we performed comparative studies of ‘Micro-Tom’ and ‘DG03-9’ fruits for the analysis of gene expression levels, protein levels and enzymatic activity levels of GABA biosynthesis- and catabolism-related enzymes. During GABA accumulation, we found positive correlations among GABA contents and expression levels of SlGAD2 and SlGAD3. Both of these genes encode glutamate decarboxylase (GAD) which is a key enzyme of GABA biosynthesis. During GABA catabolism, we found a strong correlation between GABA contents and enzyme activity of α-ketoglutarate-dependent GABA transaminase (GABA-TK). The contents of glutamate and aspartate, which are synthesized from GABA and glutamate, respectively, increased with elevation of GABA-TK enzymatic activity. GABA-TK is the major GABA transaminase form in animals and appears to be a minor form in plants. In ‘DG03-9’ fruits, GAD enzymatic activity was prolonged until the ripening stage, and GABA-TK activity was significantly low. Taken together, our results suggest that GAD and GABA-TK play crucial roles in GABA accumulation and catabolism, respectively, in tomato fruits.

The Solanaceae family includes several economically important vegetable crops. The tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. Recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. In particular, a miniature cultivar, Micro-Tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the Micro-Tom-background, such as ESTs and mutagenized lines, have been established by an international alliance. To accelerate the progress in tomato genomics, we developed a collection of fully-sequenced 13,227 Micro-Tom full-length cDNAs. By checking redundant sequences, coding sequences, and chimeric sequences, a set of 11,502 non-redundant full-length cDNAs (nrFLcDNAs) was generated. Analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. Classification of functions of proteins predicted from the coding sequences demonstrated that nrFLcDNAs covered a broad range of functions. A comparison of nrFLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. Mapping of the nrFLcDNAs onto currently available tomato genome sequences facilitated prediction of exon-intron structure. Introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the nrFLcDNAs and the tomato genome sequences, the frequency of nucleotide mismatch in exons between Micro-Tom and the genome-sequencing cultivar (Heinz 1706) was estimated to be 0.061%. The collection of Micro-Tom nrFLcDNAs generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. The nrFLcDNA sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional genomics and molecular breeding. Full-length cDNA sequences and their annotations are provided in the database KaFTom http://www.pgb.kazusa.or.jp/kaftom/ via the website of the National Bioresource Project Tomato http://tomato.nbrp.jp .