Samuel Mortensen, Lauren F Cole, Diana Bernal-Franco, Suphinya Sathitloetsakun, Erin J Cram, Carolyn W T Lee-Parsons
Methods in molecular biology (Clifton, N.J.) 2022Catharanthus roseus produces medicinal terpenoid indole alkaloids, including the critical anti-cancer compounds vinblastine and vincristine in its leaves. Recently, we developed a highly efficient transient expression method relying on Agrobacterium-mediated transformation of seedlings to facilitate rapid and high-throughput studies on the regulation of terpenoid indole alkaloid biosynthesis in C. roseus . We detail our optimized protocol known as efficient Agrobacterium-mediated seedling infiltration method (EASI), including the development of constructs used in EASI and an example experimental design that includes appropriate controls. We applied our EASI method to rapidly screen and evaluate transcriptional activators and repressors and promoter activity. Our EASI method can be used for promoter transactivation studies or transgene overexpression paired with downstream analyses like quantitative PCR or metabolite analysis. Our protocol takes about 16 days from sowing seeds to obtaining the results of the experiment. © 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Samuel Mortensen, Lauren F Cole, Diana Bernal-Franco, Suphinya Sathitloetsakun, Erin J Cram, Carolyn W T Lee-Parsons. EASI Transformation Protocol: An Agrobacterium-Mediated Transient Transformation Protocol for Catharanthus roseus Seedlings. Methods in molecular biology (Clifton, N.J.). 2022;2505:249-262
PMID: 35732950
View Full Text