Abstract
Banana is an important food crop worldwide. However, as banana production is largely affected by pests, diseases, and environmental stresses, there is a need to develop stress-resistant banana varieties. Although modern breeding and conventional genetic engineering techniques exist, they may not provide sufficient precision or speed in introducing desired traits and enhancing banana resilience. Hence, this study focuses on the development of an efficient protoplast isolation and DNA-free CRISPR/Cas9 ribonucleoprotein-mediated protoplast transformation protocols for Berangan cultivar. A total of 1.54 × 107 protoplasts/g FW were isolated from immature male flower buds using an enzymatic mixture of 1% cellulase RS, 1% macerozyme R-10 and 0.15% pectolyase Y-23. Applying 10 min-vacuum infiltrations twice and 0.5 M mannitol significantly increased the number of protoplasts. The isolated protoplasts were transfected with pC-AMBIA1304-GFP and CRISPR/Cas9 ribonucleoprotein complex targeting the stress-related banana Sugar Transport Protein 13 (STP13) using a polyethylene glycol solution. Following 15 minutes of transfection, 76.89% of the treated protoplasts were GFP-positive. DNA sequence analysis confirmed the presence of small deletions (1–3 bp) at the target sites of STP13 with a mutation rate of 4.40–4.90%, indicating that the protocols are suitable for use to modify the genomes of protoplasts of bananas.
Similar content being viewed by others
REFERENCES
Acereto-Escoffié, P., Chi-Manzanero, B., Echeverría-Echeverría, S., Grijalva, R., Kay, A.J., González-Estrada, T., Castaño, E., and Rodríguez-Zapata, L., Agrobacterium-mediated transformation of Musa acuminata cv.“Grand Nain” scalps by vacuum infiltration, Sci. Hortic., 2005, vol. 105, no. 3, pp. 359–371.
Amah, D., van Biljon, A., Brown, A., Perkins-Veazie, P., Swennen, R., and Labuschagne, M., Recent advances in banana (Musa spp.) biofortification to alleviate vitamin A deficiency, Crit. Rev. Food Sci. Nutr., 2019, vol. 59, no. 21, pp. 3498–3510. https://doi.org/10.1080/10408398.2018.1495175
Assani, A., Haicour, R., Wenzel, G., Côte, F., Bakry, F., Foroughi-Wehr, B., Ducreux, G., Aguillar, M.E., and Grapin, A., Plant regeneration from protoplasts of dessert banana cv. Grande Naine (Musa spp., Cavendish sub-group AAA) via somatic embryogenesis, Plant Cell Rep., 2001, vol. 20, no. 6, pp. 482–488. https://doi.org/10.1007/s002990100366
Assani, A., Haicour, R., Wenzel, G., Foroughi-Wehr, B., Bakry, F., Cote, F., Ducreux, G., Ambroise, A., and Grapin, A., Influence of donor material and genotype on protoplast regeneration in banana and plantain cultivars (Musa spp.), Plant Sci., 2002, vol. 162, pp. 355–362.
Belhaj, K., Chaparro-Garcia, A., Kamoun, S., Patron, N.J., and Nekrasov, V., Editing plant genomes with CRISPR/ Cas9, Curr. Opin. Biotechnol., 2015, vol. 32, pp. 76–84. https://doi.org/10.1016/j.copbio.2014.11.007
Bortesi, L., Zhu, C., Zischewski, J., Perez, L., Bassié, L., Nadi, R., Forni, G., Lade, S.B., Soto, E., and Jin, X., Patterns of CRISPR/Cas9 activity in plants, animals and microbes, Plant Biotechnol. J., 2016, vol. 14, no. 12, pp. 2203–2216. https://doi.org/10.1111/pbi.12634
Dellaporta, S.L., Wood, J., and Hicks, J.B., A plant DNA minipreparation: version II, Plant Mol. Biol. Rep., 1983, vol. 1, no. 4, pp. 19–21.
Eriksson, T.R., Protoplast isolation and culture, in Plant Protoplasts, CRC Press, 2018, pp. 1–20.
Evans, D.A. and Bravo, J.E., Plant protoplast isolation and culture, in Plant Protoplasts, Int. Rev. Cytol., 2013, vol. 16, suppl., pp. 33–53.
Haïcour, R., Assani, A., Matsumoto, K., and Guedira, A., Banana protoplasts, in Banana Improvement: Cellular, Molecular Biology, and Induced Mutations. Proceedings of a Meeting Held in Leuven, Belgium, September 24–28, 2001, Science Publishers, Inc., 2001, pp. 111–125.
Huang, H., Wang, Z., Cheng, J., Zhao, W., Li, X., Wang, H., Zhang, Z., Sui, X., An efficient cucumber (Cucumis sativus L.) protoplast isolation and transient expression system, Sci. Hortic., 2013, vol. 150, pp. 206–212.
Karlson, C.K.S., Mohd-Noor, S.N., Nolte, N., and Tan, B.C., CRISPR/dCas9-based systems: mechanisms and applications in plant sciences, Plants, 2021, vol. 10, no. 10, p. 2055.
Kaur, N., Alok, A., Kaur, N., Pandey, P., Awasthi, P., and Tiwari, S., CRISPR/Cas9-mediated efficient editing in phytoene desaturase (PDS) demonstrates precise manipulation in banana cv. Rasthali genome, Funct. Integr. Genome, 2018, vol. 18, no. 1, pp. 89–99. https://doi.org/10.1007/s10142-017-0577-5
Kersey, P.J., Collemare, J., Cockel, C., Das, D., Dulloo, E.M., Kelly, L.J., … and Leitch, I.J., Selecting for useful properties of plants and fungi–Novel approaches, opportunities, and challenges, Plants, People, Planet, 2020, vol. 2, no. 5, pp. 409–420.
Khalid, N. and Tan, B.C., A to Z on banana micropropagation and field practices, in Plant Tissue Culture: Propagation, Conservation and Crop Improvement, Springer, 2016, pp. 101–118.
Khatri, A., Dahot, M.U., Khan, I.A., and Nizamani, G.S., An efficient method of protoplast isolation in banana (Musa spp.), Pak. J. Bot., 2010, vol. 42, no. 2, pp. 1267–1271.
Kim, H., Kim, S.-T., Ryu, J., Kang, B.-C., Kim, J.-S., and Kim, S.-G., CRISPR/Cpf1-mediated DNA-free plant genome editing, Nat. Commun., 2017, vol. 8, no. 1, pp. 1–7.
Kim, S., Kim, D., Cho, S.W., Kim, J., and Kim, J.S., Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins, Genome Res., 2014, vol. 24, no. 6, pp. 1012–1019. https://doi.org/10.1101/gr.171322.113
Lee, W.S., Gudimella, R., Wong, G.R., Tammi, M.T., Khalid, N., and Harikrishna, J.A., Transcripts and microRNAs responding to salt stress in Musa acuminata Colla (AAA Group) cv. Berangan roots, PLoS One, 2015, vol. 10, no. 5, p. e0127526. https://doi.org/10.1371/journal.pone.0127526
Liu, X., Wu, S., Xu, J., Sui, C., and Wei, J., Application of CRISPR/Cas9 in plant biology, Acta Pharm. Sin. B, 2017, vol. 7, no. 3, pp. 292–302. https://doi.org/10.1016/j.apsb.2017.01.002
Londenberg, A., Bartels, F.M., Kqakpo Quaye, J., Boch, J., Ripken, T., and Heinemann, D., Targeted genome editing in potato protoplast via optical delivery of CRISPR/Cas9 ribonucleoproteins, Proc. SPIE, Int. Soc. Opt. Eng., 2020. https://doi.org/10.1117/12.2555288
Malnoy, M., Viola, R., Jung, M.H., Koo, O.J., Kim, S., Kim, J.S., Velasco, R., and Kanchiswamy, C.N., DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins, Front. Plant Sci., 2016, vol. 7, p. 1904. https://doi.org/10.3389/fpls.2016.01904
Matsumoto, K., Crepy, L., Teixeira, J., and Ferreira, F., Isolation and culture of bract protoplasts in banana plants, Natural Resources and the Environment Series, Cassell Tycooly, Ed., Philadelphia, USA, 1988, vol 22, pp. 414–415.
Matsumoto, K., Monte, D.C., Teixeira, J.B., Haicour, R., and Davey, M.R., Banana protoplasts: culture and its applications, Tree For. Sci. Biotech., 2010, vol. 4, no. 1, pp. 32–38.
Murovec, J., Guček, K., Bohanec, B., Avbelj, M., and Jerala, R., DNA-free genome editing of Brassica oleracea and B. rapa protoplasts using CRISPR-Cas9 ribonucleoprotein complexes, Front. Plant Sci., 2018, vol. 9, p. 1594.
Nanasato, Y., Konagaya, K.-I., Okuzaki, A., Tsuda, M., and Tabei, Y., Improvement of Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.) by combination of vacuum infiltration and co-cultivation on filter paper wicks, Plant Biotechnol. Rep., 2013, vol. 7, no. 3, pp. 267–276.
Ntui, V.O., Tripathi, J.N., and Tripathi, L., Robust CRISPR/ Cas9 mediated genome editing tool for banana and plantain (Musa spp.), Curr. Plant Biol., 2019, vol. 21. https://doi.org/10.1016/j.cpb.2019.100128
Panis, B., Vanwauwe, A., and Swennen, R., Plant-regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.), Plant Cell Rep., 1993, vol. 12, nos. 7–8, pp. 403–407. https://doi.org/10.1007/bf00234701
Park, J., Choi, S., Park, S., Yoon, J., Park, A.Y., and Choe, S., DNA-free genome editing via ribonucleoprotein (RNP) delivery of CRISPR/Cas in lettuce, Methods Mol. Biol., 2019, vol. 1917. https://doi.org/10.1007/978-1-4939-8991-1_25
Park, J., Lim, K., Kim, J.-S. and Bae, S., Cas-analyzer: an online tool for assessing genome editing results using NGS data, Bioinformatics, 2017, vol. 33, no. 2, pp. 286–288.
Partovi, R., Farahani, F., Sheidai, M., and Satari, T.N., Isolation of protoplasts banana (Musa acuminate Colla) cvs. Dwarf Cavendish and Valery and research morphological and cytogenetic their plantlets regenerated, Cytologia, 2017, vol. 82, no. 4, pp. 395–401. https://doi.org/10.1508/cytologia.82.395
Razdan, M.K., Introduction to Plant Tissue Culture, New Delhi: Oxford and Ibh Publ., 2003.
Ren, R., Gao, J., Lu, C., Wei, Y., Jin, J., Wong, S.M., Zhu, G., and Yang, F., Highly efficient protoplast isolation and transient expression system for functional characterization of flowering related genes in Cymbidium orchids, Int. J. Mol. Sci., 2020, vol. 21, no. 7. https://doi.org/10.3390/ijms21072264
Rueden, C.T., Schindelin, J., Hiner, M.C., DeZonia, B.E., Walter, A.E., Arena, E.T., and Eliceiri, K.W., ImageJ2: ImageJ for the next generation of scientific image data, BMC Bioinformatics, 2017, vol. 18, no. 1, pp. 1–26.
Sagi, L., Remy, S., Panis, B., Swennen, R., and Volckaert, G., Transient gene expression in electroporated banana (Musa spp., cv. ‘Bluggoe,’ ABB group) protoplasts isolated from regenerable embryogenetic cell suspensions, Plant Cell Rep., 1994, vol. 13, no. 5, pp. 262–266.
Sant’ana, R.R.A., Caprestano, C.A., Nodari, R.O., and Agapito-Tenfen, S.Z., PEG-delivered CRISPR-Cas9 ribonucleoproteins system for gene-editing screening of maize protoplasts, Genes, 2020, vol. 11, no. 9, pp. 1–14. https://doi.org/10.3390/genes11091029
Siddiqui, S., Khatri, A., Khan, I., Nizamani, G., and Khan, R., Improvement of banana (Musa cvs.) through in vitro culture techniques and induced mutations, in In Vitro Mutation Breeding of Bananas and Plantains, IAEA, Ed., Vienna: IAEA, 1995.
Subburaj, S., Chung, S.J., Lee, C., Ryu, S.M., Kim, D.H., Kim, J.S., Bae, S., and Lee, G.J., Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins, Plant Cell Rep., 2016, vol. 35, no. 7, pp. 1535–1544. https://doi.org/10.1007/s00299-016-1937-7
Subramanyam, K., Subramanyam, K., Sailaja, K., Srinivasulu, M., and Lakshmidevi, K., Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration, Plant Cell Rep., 2011, vol. 30, no. 3, pp. 425–436.
Tayi, L., Maku, R.V., Patel, H.K., and Sonti, R.V., Identification of pectin degrading enzymes secreted by Xanthomonas oryzae pv. oryzae and determination of their role in virulence on rice, PLoS One, 2016, vol. 11, no. 12, p. e0166396. https://doi.org/10.1371/journal.pone.0166396
Tripathi, L., Ntui, V.O., and Tripathi, J.N., CRISPR/ Cas9-based genome editing of banana for disease resistance, Curr. Opin. Plant Biol., 2020, vol. 56, pp. 118–126. https://doi.org/10.1016/j.pbi.2020.05.003
Tycko, J., Myer, V.E., and Hsu, P.D., Methods for optimizing CRISPR–Cas9 genome editing specificity, Mol. Cell, 2016, vol. 63, no. 3, pp. 355–370.
Tzean, Y., Lee, M.C., Jan, H.H., Chiu, Y.S., Tu, T.C., Hou, B.H., Chen, H.M., Chou, C.N., and Yeh, H.H., Cucumber mosaic virus-induced gene silencing in banana, Sci. Rep., 2019, vol. 9, no. 1. https://doi.org/10.1038/s41598-019-47962-3
Wall, M.M., Ascorbic acid, vitamin A, and mineral composition of banana (Musa sp.) and papaya (Carica papaya) cultivars grown in Hawaii, J. Food Compost. Anal., 2006, vol. 19, no. 5, pp. 434–445.
Wei, Y.R., Huang, X.L., Li, J., Huang, X., Li, Z., and Li, X.J., Establishment of embryogenic cell suspension culture and plant regeneration of edible banana Musa acuminata cv. Mas (AA), ShengwuGongcheng Xuebao/Chin. J. Biotechnol., 2005, vol. 21, no. 1, pp. 58–65.
Woo, J.W., Kim, J., Kwon, S.I., Corvalan, C., Cho, S.W., Kim, H., Kim, S.G., Kim, S.T., Choe, S., and Kim, J.S., DNA-free genome editing in plants with preassembled CRISPR/Cas9 ribonucleoproteins, Nat. Biotechnol., 2015, vol. 33, no. 11, pp. 1162–1164. https://doi.org/10.1038/nbt.3389
Wu, S., Zhu, H., Liu, J., Yang, Q., Shao, X., Bi, F., Hu, C., Huo, H., Chen, K., and Yi, G., Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana, BMC Plant Biol., 2020, vol. 20, no. 1, p. 425. https://doi.org/10.1186/s12870-020-02609-8
Yoo, S.-D., Cho, Y.-H. and Sheen, J., Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis, Nat. Protoc., 2007, vol. 2, no. 7, p. 1565. https://doi.org/10.1038/nprot.2007.199
Zhang, X.-H., Tee, L.Y., Wang, X.-G., Huang, Q.-S., and Yang, S.-H., Off-target effects in CRISPR/Cas9-mediated genome engineering, Mol. Ther. Nucleic Acids, 2015, vol. 4, p. e264.
Zhu, J., Song, N., Sun, S., Yang, W., Zhao, H., Song, W., and Lai, J., Efficiency and inheritance of targeted mutagenesis in maize using CRISPR-Cas9, J. Genet. Genomics, 2016, vol. 43, no. 1, pp. 25–36.
Funding
This research was supported by the Ministry of Education, Malaysia (Grant no. FRGS/1/2017/STG05/UM/01/2).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
This work does not contain any studies involving human and animal subjects.
CONFLICT OF INTEREST
The authors of this work declare that they have no conflicts of interest.
Additional information
Publisher’s Note.
Pleiades Publishing remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Leh, L.S., Mazumdar, P., Tan, B.C. et al. Establishment of a PEG-Assisted Protoplast Transfection System in Musa acuminata cv. Berangan (AAA) Using a CRISPR/Cas9 Ribonucleoprotein Complex. Biol Bull Russ Acad Sci 50 (Suppl 3), S298–S309 (2023). https://doi.org/10.1134/S1062359023600010
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S1062359023600010