S01 - Session O1 - Establishing a genome editing platform for banana in Ecuador: challenges and regulation

Authors: Efren Santos *, Eduardo Sánchez, Liliana Villao

New breeding techniques (NBT) comprises different biotechnologies for genetic modification, mainly by editing DNA sequences. In Ecuador, biotechnological crops, which harbour DNA introduced by recombinant DNA technology (e.g. through microparticle bombardment and Agrobacterium tumefaciens insertion), are forbidden by Constitution, unless the Presidency and the Ecuadorian General Assembly approve genetically modified organisms (GMO) following a process of risk assessment. Therefore, application of genetically modified crops for research and commercial purposes have been hindered in Ecuador with few research groups applying the technology. On the other hand, crops that have been modified without the integration of foreign DNA will not be banned, providing an opportunity to implement genome editing techniques for the genetic improvement of crops in Ecuador. The established genetic transformation platform at ESPOL university will be used as a starting point for the standardization and optimization of genome editing, specifically by using CRIPSR/CAS9, starting with banana plants for their genetic improvement for disease resistance. Standardization of the genome editing technique will be performed by employing the luciferase reporter gene system. The established genetic transformation platform will be used to introduce the P35S::luc2::Tnos cassette into banana and luciferase activity will be determined for the standardization of the genome editing protocol in a controlled experiment by knocking-out the luciferase reporter gene by CRISPR/CAS9 in real time. CRISPR/CAS9 editing will be accomplished by genetic transformation of Cas9 and sgRNA in the host. Once the genome editing platform is optimized, genome editing will be performed to knock-out pahogen susceptibility genes identified in transcriptomics studies of susceptible Musa cultivars. Further improvement of the genome editing technique includes the application of the ribonucleoprotein (RNP) complex coupled with biolistic (microparticle bombardment) to generate transgenic-free edited banana plants, which will allow the cultivation of improved plants in Ecuador.