Development of a protocol for the regeneration of protoplasts from cassava (Manihot esculenta) isolated from friable embryogenic callus. 1,2 Angi M Bueno. 1 Francisco J Sánchez. 1 Magdalena Buriticá. 1 & Paul Chavarriaga. 1) Gene Editing Plataform-The Alliance of Bioversity International & CIAT 2) The National University of Colombia The use of protoplasts in gene editing has revolutionized crop improvement, as these are plant cells lacking a cell wall, facilitating the direct manipulation of their genetic material (Chen et al., 2023). The absence of a cell wall enables the efficient introduction of gene editing tools such as CRISPR/Cas9, allowing for precise modification of target genes (Reed & Bargmann, 2021). This approach is particularly relevant for crops like cassava (Manihot esculenta), a staple food in tropical and subtropical regions that faces significant challenges, including susceptibility to diseases and the presence of toxic compounds in its tissues (Feng et al., 2023; Mukami et al., 2022). The main challenge in the implementation of DNA-free Gene Editing technology lies in the regeneration and development of whole plants from protoplasts (Feng et al., 2020). The embryo development and plant regeneration from protoplasts are key processes, as they determine whether genetic modifications can be incorporated into viable and functional non- transgenic plants. Low efficiency in these processes poses a significant obstacle, limiting experimental success and delaying the application of gene-editing technologies in crop improvement, such as in cassava (Mukami et al., 2022). Therefore, the development of an effective protocol for protoplast regeneration is essential to overcome this obstacle. A well-designed protocol not only optimizes the conditions necessary for successful protoplast regeneration but also enhances the efficiency of the gene-editing process, enabling the faster and more reliable production of modified plants. The following report clearly outlines a developed protocol for regenerating cassava plants of the TMS60444 variety from protoplasts isolated from friable embryogenic callus (CEF). Description of the Experiment The protocol described by Arciniegas, J. (2019) was used for protoplasts isolation and regeneration with some changes. Day 1: Tissue Selection and Enzymolysis 1. Two disks of friable embryogenic callus (CEF) (see Figure 1A) were placed in a small Petri dish (60 x 15 mm) containing 2 ml of Sofiari medium (TM2G) and 2 ml of enzymatic solution (Sofiari et al., 1998). 2. The dish was sealed with plastic wrap and covered with aluminum foil, then incubated in the dark at 28 °C and 30 rpm overnight for 16 hours. Day 2: Isolation 1. The digestion suspension was filtered through a funnel with three layers of Miracloth (22-25 µm) to separate the protoplasts from the debris. The suspension was adjusted to a volume of 5 ml with washing solution W1 in a 5 ml Eppendorf tube. 2. The protoplast suspension was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 3. The pellet was resuspended by inversion with 5 ml of W1 solution. 4. Finally, the protoplast pellet was resuspended in 1-2 ml of Sofiari medium. 5. The protoplast count was performed by placing a 20 μl sample in a Neubauer chamber, which was then observed under a microscope at 40X magnification. 6. The viability of the protoplasts was calculated by adding 20 μl of 0.5% FDA to a 500 μl sample of the protoplast suspension and incubating for 10 minutes in the dark. This was subsequently evaluated under a fluorescence microscope. 7. The protoplasts were cultured in this medium for 13 days in the dark at 28 °C. Note: In the protoplast counting, a cell density of 1 x 10^5 cells/ml was obtained (see Figure 1B), which corresponds to the minimum density required to proceed with cell culture. Day 15: Medium Renewal 1. The Eppendorf tubes were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the pellet was resuspended in 1 ml of Sofiari medium. 2. The tissue was maintained in the same medium for 7 days. Day 22: Inverted Drop Seeding 1. A filter paper was placed in a deep Petri dish and moistened with 3 ml of sterile water. 2. 300-400 μl of the protoplast suspension were taken and 9 drops of equal size were placed inside the lid of the Petri dish (see Figure 1C). 3. Finally, the lid was flipped over and sealed carefully to avoid damaging the drops. 4. The culture continued in the dark at 28 °C for an additional 14 days. Note: During this period, the development of microcalli was observed (see Figure 1D), suggesting that the inverted drop culture method promoted cell division . Day 36: Medium Renewal—Immersion of Drops in Sofiari Medium 1. Six (6) ml of Sofiari medium were added to a deep 60 x 15 mm Petri dish 2. Using a pipette, the drops were carefully removed from the lid and added to the dish. 3. The dishes were sealed with plastic wrap and stored again in the dark at 28°C, where they remained in this medium for an additional 28 days, renovating the medium every 15 days Note: During the liquid culture phase, an increase in both the size and number of calli was observed. The tissue appeared to benefit from the nutrients provided by the medium; the liquid state allowed the calli to absorb nutrients more efficiently. Day 64: Transfer to ME056-2 Medium 1. Mesh squares (2x2 cm) were placed in Petri dishes containing sterile filter paper (Sanchez, et al. 2022). 2. Using a micropipette with a blunt tip, 1-2 ml of tissue were carefully absorbed and deposited onto each mesh to separate the tissue from the medium. 3. Each mesh was transferred to a Petri dish containing solid medium ME056-2, taking care not to dislodge the calli (see Figure 1E) (Sanchez, et al. 2022). 4. Finally, the dish was sealed and stored in an incubator at 28°C, with a 16 h light/8 h dark photoperiod. The tissue remained in this medium for 30 days. Note During culture in ME056-2 medium, the calli began to differentiate into embryos. By 9 days after transfer to the solid medium, globular embryos were observed (see Figure 1F), and by 26 days, cotyledonary embryos had developed (see Figure 1G). These embryos continued their maturation (see Figure 1I). A yellow-orange bacteria was observed growing in the medium and on some tissues, but it did not affect their growth. Day 94: Treatment to Eliminate Bacteria 1. The cotyledonary embryos were transferred individually into 1.5 ml Eppendorf tubes, while the calli were washed together in a 5 ml tube. Each tissue was washed twice with distilled water. 2. They were then quickly washed with a solution of 1 ml/l ppm, and step 1 was repeated. 3. Finally, they were cultured in ME056-2 medium supplemented with 400 μl/L carbenicillin and 2.5 ml/L cefotaxime (stock 1 mg/ml). The cotyledonary embryos were cultured individually in 5 ml Eppendorf tubes containing 2 ml of medium, while the calli were cultured in a Petri dish with 20 ml of medium. They remained in this medium for 3 days (see Figure 1J). 4. After this period, they were transferred again to ME056-2 medium without antibiotics (see Figure 1K). Note: The antibiotic treatment was effective; after three days of culture in the supplemented medium, no further bacterial growth was observed. Continuing the culture in ME056-2 medium allowed the embryos to continue developing, with an increase in cotyledon size observed, and the formation of true leaves began (see Figure 1L). Day 106: Medium Change for Cotyledonary Embryos 1. The cotyledonary embryos were transferred to fresh ME056-2 medium (see Figure 1M). 2. The remaining embryos were left in the same medium to continue them. Figure 1. Regeneration of cassava TMS60444 protoplasts from CEF. A) Friable embryogenic callus (CEF). B) Protoplasts isolated from CEF (40x). C) Protoplast culture in inverted drops. D) Microcalli development after 12 days of culture in inverted drops. E) Transfer to solid ME056-2 medium. F) Monitoring after 9 days of culture in ME056-2 medium. G) Callus development into embryos after 87 days of culture. H) Embryo development after 90 days of culture. I) Growth and maturation of cotyledonary embryos. J) Bacterial treatment in medium supplemented with PPM, carbenicillin and cefotaxime. K) Transfer of embryos to non-supplemented ME056-2 medium. L) Cotyledonary embryos after 106 days of culture. M) Current state of cotyledonary embryos (10/10/24). Conclusions The protocol described for the regeneration of cassava (Manihot esculenta) protoplasts from friable embryogenic callus (CEF) proved to be effective in generating mature cotyledonary embryos, representing a significant advancement in the in vitro regeneration of this crop. The findings highlight the importance of optimizing each phase of the protocol, from the selection of the initial tissue and the elimination of contaminating microorganisms to the use of specific media that promote embryo development. Despite the successes achieved, the protocol still presents areas for improvement, such as increasing regeneration efficiency and optimizing culture conditions to reduce variability in the results. Nevertheless, the successful production of embryos from cassava protoplasts demonstrates the potential of this approach to enhance agronomic traits in the crop through DNA-free Gene Editing, which will significantly contribute to food security and agricultural sustainability in regions where cassava is a staple food. Referencias  Arciniegas Vega, J. P. (2019). Desarrollo de un protocolo para la regeneración de plantas a partir de protoplastos aislados de estructuras embriogénicas organizadas de yuca (Manihot esculenta Crantz). The Alliance internal document.  Chen, K., Chen, J., Pi, X., Huang, L. J., & Li, N. (2023). Isolation, purification, and application of protoplasts and transient expression systems in plants. International Journal of Molecular Sciences, 24(23), 16892. Tomado de: https://www.mdpi.com/1422- 0067/24/23/16892  Feng, W., Fu, H. T., Luo, Y. C., & Huang, J. Q. (2023). Plant Regeneration from Cassava Protoplasts. Tomado de: https://www.intechopen.com/chapters/86089  Feng, W. E. N., Su, W. P., Zheng, H., Yu, B. C., ZHANG, P., & GUO, W. W. (2020). Plant  regeneration via protoplast electrofusion in cassava. Journal of Integrative Agriculture, 19(3), 632-642. Tomado de: https://www.sciencedirect.com/science/article/pii/S2095311919627115 http://www.mdpi.com/1422- http://www.intechopen.com/chapters/86089 http://www.sciencedirect.com/science/article/pii/S2095311919627115  Mukami, A., Juma, B. S., Mweu, C., Ngugi, M., Oduor, R., & Mbinda, W. M. (2022). Plant regeneration from leaf mesophyll derived protoplasts of cassava (Manihot esculenta Crantz). Plos one, 17(12), e0278717. Tomado de: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0278717  Reed, K. M., & Bargmann, B. O. (2021). Protoplast regeneration and its use in new plant breeding technologies. Frontiers in Genome Editing, 3, 734951. Tomado de: https://www.frontiersin.org/journals/genome- editing/articles/10.3389/fgeed.2021.734951/full  Sofiari, E., Raemakers, C. J. J. M., Bergervoet, J. E. M., Jacobsen, E., & Visser, R. G. F. (1998). Plant regeneration from protoplasts isolated from friable embryogenic callus of cassava. Plant Cell Reports, 18, 159-165. Tomado de: https://link.springer.com/article/10.1007/s002990050550  Sánchez, F., Arciniegas, J. P., Brand, A., Vacca, O., Mosquera, A., Medina, A., & Chavarriaga, P. (2022). Metabolic engineering of cassava to improve carotenoids. Carotenoids: Carotenoid and Apocarotenoid Biosynthesis, Metabolic Engineering and Synthetic Biology, 31–60. http://www.frontiersin.org/journals/genome- https://link.springer.com/article/10.1007/s002990050550 Referencias