RESUMEN
Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be genetically transformed, making it difficult to improve these plants through genetic engineering. In this study, we adapted the recently developed cut-dip-budding (CDB) gene delivery system to transform three previously recalcitrant succulent varieties - the dicotyledonous Kalanchoe blossfeldiana and Crassula arborescens and the monocotyledonous Sansevieria trifasciata. Capitalizing on the robust ability of cut leaves to regenerate shoots, these plants were successfully transformed by directly infecting cut leaf segments with the Agrobacterium rhizogenes strain K599. The transformation efficiencies were approximately 74%, 5% and 3.9%-7.8%, respectively, for K. blossfeldiana and C. arborescens and S. trifasciata. Using this modified CDB method to deliver the CRISPR/Cas9 construct, gene editing efficiency in K. blossfeldiana at the PDS locus was approximately 70%. Our findings suggest that succulents with shoot regeneration ability from cut leaves can be genetically transformed using the CDB method, thus opening up an avenue for genetic engineering of these plants.
RESUMEN
KEY MESSAGE: Promoter activities of RhACS1 and RhACS2 , two rose genes involved in ethylene biosynthesis, are highly sensitive to various abiotic stresses in an organ-specific manner. Our previous studies indicated that two rose (Rosa hybrida) 1-aminocyclopropane-1-carboxylic acid synthase genes, RhACS1 and RhACS2, play a role in dehydration-induced ethylene production and inhibition of cell expansion in rose petals. Here, both RhACS1 and RhACS2 promoters were analyzed using histochemical staining and glucuronidase synthase (GUS) gene reporter activity assays following their introduction into transgenic Arabidopsis thaliana plants. It was found that the promoter activities of both genes were strong throughout the course of development from young seedlings to mature flowering plants in various organs, including hypocotyls, cotyledons, leaves, roots and lateral roots. RhACS1 promoter activity was induced by drought in both rosette leaves and roots of transgenic A. thaliana lines, but was reduced following a re-hydration treatment. In contrast, RhACS2 promoter activity was decreased by drought in rosette leaves, while its response pattern was similar to that of RhACS1 in roots. A mannitol treatment induced the activity of both the RhACS1 and RhACS2 promoters, indicating that both genes are also regulated by osmotic stress. In addition, RhACS2 appeared to be abscisic acid (ABA)-inducible, while RhACS1 was less sensitive to ABA. Finally, four truncated sequences of the RhACS1 promoter were generated and GUS activity assays demonstrated that deleting a 327 bp region between bp 862 and -535 resulted in a substantial decrease of the promoter activity. Taken together, our results suggest that the RhACS1 and RhACS2 promoters respond to abiotic stresses in a developmentally regulated and spatially specific manner.
