Constanza Martin-Vásquez

Key Message The identification of the tomato DOR gene that is essential for adventitious organogenesis provides a genetic target for improving regeneration efficiency and root system performance in tissue culture and plant breeding. Abstract Plant rooting and adventitious organogenesis represent major bottlenecks in the application of plant tissue culture techniques. In this study, we characterize the dor mutant (defective in organogenesis and rooting) identified in our collection of tomato T-DNA lines. This mutant exhibits normal callus proliferation but fails to differentiate adventitious buds. Additionally, it displays underdeveloped embryonic roots, and its adventitious roots derived from various explants are also altered. Grafting experiments revealed compromised in vivo development primarily due to its abnormal root system. Upon identifying an allelic mutant in another tomato line, we observed no co-segregation between a T-DNA insert and the phenotype in either of the identified allelic mutants. Through mapping-by-sequencing, we identified Solyc12g098670 as the gene responsible for this mutation, which is homologous to SIGNAL PEPTIDE PEPTIDASE-LIKE (SPPL) genes from Arabidopsis thaliana, particularly SPPL3 and SPPL5. The expression pattern of DOR in tomato is nearly ubiquitous, similar to SPPL3 in Arabidopsis, yet the regeneration and rooting of sppl3 Arabidopsis mutants resemble the wild-type. In both tomato and Solanum pennellii (Correll) D’Arcy, RNAi lines and plants edited by CRISPR/Cas exhibit a dor mutant phenotype. However, by increasing the expression level of DOR, the plants become indistinguishable from the wild-type in terms of in vitro and in vivo development. Overexpression of this gene in the mutant has enabled the regeneration of plants with a wild-type phenotype. These results demonstrate that DOR is the first member of the tomato SPPL gene family known to be associated with root development and adventitious organogenesis.

Byblis, a small genus of carnivorous plants predominantly found in Australia, is characterized by its passive trapping mechanism and unique floral features. The chemical composition of Byblis, including identified phenylethanoid glycosides, particularly acteoside, highlights its pharmacological potential with various biological activities. In vitro culture techniques have been established for propagation, with micropropagation protocols developed for different Byblis species. However, information on genetic transformation, vital for trait modification and enhanced pharmacological interest, remains limited. This study focuses on optimizing micropropagation, adventitious regeneration, and genetic transformation methods for Byblis liniflora. Adventitious regeneration rates were highest in medium with reduced Murashige and Skoog salts (MS/10) and sucrose (3 gL⁻¹) concentrations. Zeatin supplementation (1 mgL⁻¹) further improved regeneration rates and bud development with 100% of regenerated root explants and 8.8 shoots per explant. Liquid MB3 medium supplemented with indole-3-acetic acid (IAA) 5 mgL⁻¹ facilitated efficient rooting and acclimatization. The establishment of an efficient Rhizobium-mediated genetic transformation method yielded transgenic plants expressing green fluorescent protein (GFP). Molecular analysis confirmed transgene integration, marking the first successful genetic transformation in the Byblis genus. These advancements pave the way for exploring gene function and enhancing pharmacological properties, thereby broadening our understanding and utilization of carnivorous plants like Byblis.