Summary of Tissue Culture Steps for Gene Editing Brachiaria humidicola 16888 Didier Marin & Paul Chavarriaga The Gene Editing Platform April/2023 • Steps for Gene Editing Brachiaria humidicola 16888 from Mature Embryos Naked seeds Whole mature seeds Bleach-sterilized seeds Isolating whole embryo (without cariopsis) Embryogenic Callus Induction. At this stage cells may be gene edited De-novo regenerated gene-edited wole plants will grow on with CRISPR-Cas9/Cpf1 selective media to isolate potential knock-out mutants • Steps for Gene Editing Brachiaria humidicola 16888 from Immature Embryos Donor Plants in Pannicule at Immature seeds Immature Endosperm Greenhouse Anthesis Isolated Immature Immature Embryogenic tissues are gene- De novo Embryo (bottom) Embryogenic tissue editable at this stage regenerated plants dividing on inductive medium The Alliance’s Gene Editing Platform Paul Chavarriaga, Sandra Valdés & Collaborators April, 2023 Colombian Regulation 2018 • ICA resolution #00029299: Regulatory framework. • Gene Edited waxy maize and rice resistant to Xam are conventional lines • New framework will include animals New vareties Proof of concept Climate Change Gene Validation Gene edited, Xam-Resistant rice is a CONVENTIONAL variety in Colombia Bezos Earth Fund supports Gene Editing to make rice a more efficient Carbon storer Trait Gene Phenotype References Yield GN1A -Increase the number of Frontiers | Reassessment of the Four Yield-related grains in the main panicle. Genes Gn1a, DEP1, GS3, and -Increase plant height, IPA1 in Rice Using a CRISPR/Cas9 System panicle size and number of (frontiersin.org) flowers per panicle Photosynthetic OsHXK1 High-photosynthetic Improving the Rice Photosynthetic Efficiency Efficiency efficiency and high yielding and Yield by Editing rice varieties OsHXK1 via CRISPR/Cas9 System - ProQuest Root OsAUX1 Increase Primary Roots https://onlinelibrary.wiley.c om/doi/pdfdirect/10.1111/t Architecture Length pj.12929 Nitrogen CYP75B3 Induce accumulation of WO2022015762 PLANT METABOLITE-MEDIATED Fixation and/or CYP flavones inside the plant INDUCTION OF BIOFILM 75B4 and the exudation of these FORMATION IN SOIL BACTERIA TO INCREASE from the roots into the BIOLOGICAL NITROGEN FIXATION AND PLANT soil, where they activate NITROGEN ASSIMILATION the biofilm synthesis in the (wipo.int) N2-fixing bacteria. Control Edited line #13 Llanura 11, Japonica (165.31.20.3) # filled grains/plant: 1678 # filled grains/plant: 4552 Average of seven plants: 2399 Average of seven plants: 3392.6 Thousand grains weight: 27.9 g Thousand grains weight: 28.3 g Totalgrain weight/plant: 50.2 g Totalgrain weight/plant: 114.2 g # of panicles: 16 # of panicles: 36 Reducing Cd (OsNRAMP5) & Ar (Ls1, Ls2, Ospt8) Absorption • Biofortified lines (Colombia) • High Yielders (Grain number) • Uruguay (Arsenic) • Bangladesh (Cadmium) Gene Editing Cassava Traits Genes Field Tests 2020 to 2023 Waxy Starch GBSS1 >120 lines (3 targets) Waxy Field Tested Haploid Inducers Herbicide Als tolerance (2 targets) Haploid inducers NLD/pL1a/MTL Field Tested (2 genes 2 targets) Xpm R+ SWEET10a & 10b >100 lines (2 genes 4 targets) March 24/2023 Resistance to Xpm wt TRANSGENE-Free Waxy Cassava Lines (pic. 12 July 2022) 1) Waxy 54 x Waxy 8 (14-IX-21) # 2 C-) Negative control TMS 60444 2) Waxy 32 x Waxy 32-S1 (25-X-21): #2 F ) Free 3) Waxy 32 x Waxy 32-S1 (25-X-21): #3 P ) PHSE 401 4) Waxy # 54-S1 (23-IX-21): #2 M ) Marker 5) Waxy # 54-S1 (23-IX-21): #3 +) Waxy 4 #1 6) Waxy # 54-S1 (23-IX-21): #4 +) Waxy 4 #2 +) Waxy 4 #3 Waxy32-S1-P3 Waxy54-S1-P2 Waxy54-S1-P3 Waxy54-S1-P4 Waxy32-S1-P Waxy54 X Waxy8-P2 Selfings & Crosses made in Sep/Oct 2021; In Vitro since May 24/2022 Leverage landrace genomics to engineer thermotolerant cassava Hierarchical whole 30 genome sequencing 20 1 10 and Pangenome 0 Chr 18 Chr 1 (> 400 landraces and Chr 17 Chr 2 Chr 16 Wild Relatives) Chr 3 Chr 15 Natural knockouts Chr 4 Chr 14 mutations Chr 5 Chr 13 Chr 6 Chr 12 Chr 7 Chr 11 Chr 10 Chr 8 Chr 9 4 Rapid validation of candidates with CRISPR 2 Climate modeling 3 Identify Thermotolerant adaptive cassava knockouts 35 30 25 20 15 Cassava landraces Temp. of origin Max temp. Reducing Cd Absorbtion in Theobroma cacao Targeting the Gene TcNRAMP5 WT (Y) Edited line 1 (C) Edited line 2 (C) Edited line 4 (C) Target site sequence from Phytozome (var. Criollo) Acknowledgments: M. Selvaraj The Gene Editing Team R. Escobar Thanks! G. Gallego A. Gkanogiannis H. Ceballos X. Zhang LA. Becerra M. Lorieux B. Szurek P. Díaz-Tatis MF. Alvarez T. Tran Grey Monroe et al J. Tohme Supporters: RTB USDA CNCH HHU/Gates Foundation Ingredion FLAR UC-Davis/FFAR (Left to right; Izquierda a Derecha) Christian Valencia, Paul Chavarriaga, Anibal Penaloza, Maria Hernandez, Orlando Vacca, Arlen Mosquera, Natalia Franco, Gerardo Delgado, Francisco Sanchez, Didier Marín, Sandra Valdés, Jorge Flor.