ISHS Acta Horticulturae 1362: XXXI International Horticultural Congress (IHC2022): International Symposium on Breeding and Effective Use of Biotechnology and Molecular Tools in Horticultural Crops
Establishing a genome editing platform of banana in Ecuador: challenges and regulation |
| Authors: | E. Santos, E. Sánchez, L. Villao |
| Keywords: | CRISPR/CAS9, Musa, luciferase, biolistic, Agrobacterium |
| DOI: | 10.17660/ActaHortic.2023.1362.7 |
| Abstract:
New breeding techniques (NBT) comprise different biotechnologies for genetic modification, but mainly by editing DNA sequences. In Ecuador, biotechnological crops, which harbor DNA introduced by recombinant DNA technology (e.g., through microparticle bombardment and Agrobacterium tumefaciens), are forbidden by the constitution, unless the Presidency and the Ecuadorian General Assembly approve the genetically modified organism (GMO), following a process of risk assessment. Therefore, the application of genetically modified crops for research and commercial purposes has 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 improvement of crops. An 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. Banana plants will be used for the standardization of genome editing and 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 cassette P35S::luc2::Tnos 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 the Cas9 and sgRNA in the host. Once the genome editing platform is optimized, genome editing will be performed to knockout pathogen susceptibility genes identified from transcriptomics studies of susceptible Musa cultivars. Further improvement of the genome editing technique includes the application of a ribonucleoprotein (RNP) complex coupled with biolistic (micro-particle bombardment) to generate transgenic-free edited banana plants that will allow the cultivation of improved plants in Ecuador. | |
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