Evaluation and Improvement of Protocols for Ganoderma boninense Protoplast Isolation and Regeneration



  • Anis Farhan Fatimi Ab Wahab FGV Innovation Center (Biotechnology), PT. 23417 Lengkuk Teknologi, 71760 Bandar Enstek, Negeri Sembilan; Department of Biological Science and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
  • Madihah Ahmad Zairun Plant Pathology & Biosecurity Unit, Biology & Sustainability Research Division, 6, Persiaran Institusi, Malaysia Palm Oil Board Bandar, Baru Bangi, 43000 Kajang, Selangor
  • Khairunnisa Hanisah Mohd Daud Department of Biological Science and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
  • Farah Diba Abu Bakar Department of Biological Science and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
  • Izwan Bharudin Department of Biological Science and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
  • Abdul Munir Abdul Murad Department of Biological Science and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia


basal stem rot, a fungal pathogen, protoplast, oil palm


Ganoderma boninense is the causal agent of basal stem rot (BSR) disease of oil palm. The BSR disease reduces oil palm yield by up to 80% of the average oil yield. Attempts to control the disease caused by this fungus in the field showed varying levels of success and cases of infection increased from year to year. Hence, the development of new efficient methods to control the spread of this fungus should be commenced promptly. To ensure a better strategy is created, more thorough research on the method deploy by this fungus to infect the host at the molecular level need to be carried out first. However, the major limitation in endeavoring into the functional analysis of virulence genes related to the pathogenicity of this fungus was hampered by the unavailability of established methods for protoplast isolation with a high regeneration rate to be used in the genetic manipulation analysis. Thus, in this paper, we report an efficient protocol for protoplast isolation and regeneration in G. boninense and successfully used the isolated protoplasts in PEG-mediated transformation analysis. A large quantity of protoplast was obtained using the protocol that utilizes the following parameters: 3 to 4-day-old mycelia, treated with 1% lysing enzyme and 0.02% Driselase, incubated at 30 °C in an osmotic medium containing 0.6 M mannitol at pH 5.8 for 2 h. The highest protoplast yield was in the range of 8.95 × 109 to 3.12 × 1010 cells/mL per 5 g of mycelia used. The regeneration rate ranged from 9.03% to 22.55%, depending on the regeneration media used. By using 5 µg of vector to transform into 1.0 × 107 protoplast/mL, around 3 – 10 mitotically stable putative transformants were successfully obtained and verified via PCR. This protocol will find useful applications in genetic studies to enhance insight into this poorly characterized and understood phytopathogen.


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Ab Wahab, A.F.F., Karim, N.A.A., Ling, J.G., Hasan, N.S., Yong, H.Y., Bharudin, I., Kamaruddin, S., Bakar, F.D.A. & Murad, A.M.A. 2019. Functional characterisation of cellobiohydrolase I (Cbh1) from Trichoderma virens UKM1 expressed in Aspergillus niger. Protein Expression and Purification, 154: 52-61. DOI: https://doi.org/10.1016/j.pep.2018.09.014

Bahari, M.N.A., Sakeh, N.M., Abdullah, S.N.A., Ramli, R.R. & Kadkhodaei, S. 2018. Transciptome profiling at early infection of Elaeis guineensis by Ganoderma boninense provides novel insights on fungal transition from biotrophic to necrotrophic phase. BMC Plant Biology, 18(1): 1-25. DOI: https://doi.org/10.1186/s12870-018-1594-9

Bhadauria, M. & McKee, S.A. 2010. An approach to resource-aware co-scheduling for CMPs. In: Proceedings of the 24th ACM International Conference on Supercomputing, Tsukuba Ibaraki, pp. 189-199. DOI: https://doi.org/10.1145/1810085.1810113

Bharudin, I., Ab Wahab, A.F., Abd Samad, M.A., Xin Yie, N., Zairun, M.A., Abu Bakar, F.D. & Abdul Murad, A.M. 2022. Review update on the life cycle, plan-microbe interaction, genomics, detection and control strategies of the oil palm pathogen Ganoderma boninense. Biology, 11(2): 251. DOI: https://doi.org/10.3390/biology11020251

Bowman, L., Motamed, R., Lee, P., Aleem, K., Berawala, A.S., Hayden, K.L., Bzik, D.J. & Chattopadhyay, D. 2020. A simple and reliable method for determination of optimum pH in coupled enzyme assays. Biotechniques, 68(4): 200-203. DOI: https://doi.org/10.2144/btn-2019-0126

Cheng, Y. & Bélanger, R.R. 2000. Protoplast preparation and regeneration from spores of the biocontrol fungus Pseudozyma flocculosa. FEMS Microbiology Letters, 190(2): 287-291. DOI: https://doi.org/10.1111/j.1574-6968.2000.tb09300.x

Choi, S.H., Kim, B.K., Kim, H.W., Kwak, J.H., Choi, E.C., Kim, Y.C., Yoo, Y.B. & Park, Y.H. 1987. Studies on protoplast formation and regeneration of Ganoderma lucidum. Archives of Pharmacal Research, 10(3): 158-164. DOI: https://doi.org/10.1007/BF02861906

Chong, K.P., Dayou, J., & Alexander, A. 2017. Pathogenic nature of Ganoderma boninense and basal stem rot disease. In: Detection and Control of Ganoderma boninense in Oil Palm Crop. K.P. Chong, J. Dayou and A. Alexander (Eds.). Springer, Cham. pp. 5-12. DOI: https://doi.org/10.1007/978-3-319-54969-9_2

Chou, T.H. & Tzean, S.-S. 2016. Protoplasting, regeneration and transformation of medicinal mushroom Ganoderma multipileum using succinate dehydrogenase mutation gene as a selection marker. Annals of Microbiology, 66(1): 111-120. DOI: https://doi.org/10.1007/s13213-015-1087-0

Chun, S.B., Chin, J.E., Bai, S. & An, G.-H. 1992. Strain improvement of Phaffia rhodozyma by protoplast fusion. FEMS Microbiology Letters, 93(3): 221-226. DOI: https://doi.org/10.1111/j.1574-6968.1992.tb05101.x

Eyini, M., Rajkumar, K. & Balaji, P. 2006. Isolation, regeneration and PEG-induced fusion of protoplasts Pathology, 61(3): 567-578.

Rehman, L., Su, X., Guo, H., Qi, X. & Cheng, H. 2016. Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae. BMC Biotechnology, 16(1): 1-9. DOI: https://doi.org/10.1186/s12896-016-0287-4

Ren, N., Liu, J., Yang, D., Liu, X., Zhou, J. & Peng, Y. 2018. Preparation and regeneration of protoplasts from the ethyl vincamine producing fungus CH1 (Geomyces sp.). Natural Product Communications, 13(2): 145-148. DOI: https://doi.org/10.1177/1934578X1801300209

Rodenburg, S.Y., Terhem, R.B., Veloso, J., Stassen, J.H. & van Kan, J.A. 2018. Functional analysis of mating type genes and transcriptome analysis during fruiting body development of Botrytis cinerea. MBio, 9(1): e01939-17. DOI: https://doi.org/10.1128/mBio.01939-17

Rodriguez, R.J. & Redman, R.S. 1992. Molecular transformation and genome analysis of Colletotrichum species. In: Colletotrichum: Biology, Pathology and Control. J.A. Bailey and M.J. Jegers (Eds.). CAB Int., Wallingford. pp. 47-76

Ruiz-Díez, B. 2002. Strategies for the transformation of filamentous fungi. Journal of Applied Microbiology, 92(2): 189-195. DOI: https://doi.org/10.1046/j.1365-2672.2002.01516.x

Shimizu, M., Nakano, Y., Hirabuchi, A., Yoshino, K., Kobayashi, M., Yamamoto, K., Terauchi, R. & Saitoh, H. 2019. RNA-Seq of in planta-expressed Magnaporthe oryzae genes identifies MoSVP as a highly expressed gene required for pathogenicity at the initial stage of infection. Molecular Plant Pathology, 20(12): 1682-1695. DOI: https://doi.org/10.1111/mpp.12869

Shin, J.H., Han, J.H., Park, H.H., Fu, T. & Kim, K.S. 2019. Optimization of polyethylene glycol-mediated transformation of the pepper anthracnose pathogen Colletotrichum scovillei to develop an applied genomics approach. The Plant Pathology Journal, 35(6): 575-584. DOI: https://doi.org/10.5423/PPJ.OA.06.2019.0171

Siddiqui, Y., Surendran, A., Paterson, R.R.M., Ali, A. & Ahmad, K. 2021. Current strategies and perspectives in detection and control of basal stem rot of oil palm. Saudi Journal of Biological Sciences, 28(5): 2840-2849. DOI: https://doi.org/10.1016/j.sjbs.2021.02.016

Sun, L., Cai, H., Xu, W., Hu, Y., Gao, Y. & Lin, Z. 2001. Efficient transformation of the medicinal mushroom Ganoderma lucidum. Plant Molecular Biology Reporter, 19(4): 383-384. DOI: https://doi.org/10.1007/BF02772841

Wei, Y., Zhou, X., Liu, L., Lu, J., Wang, Z., Yu, G., Hu, L., Lin, J., Sun, X. & Tang, K. 2010. An efficient transformation system of taxol-producing endophytic fungus EFY-21 (Ozonium sp.). African Journal of Biotechnology, 9(12): 1726-1733. DOI: https://doi.org/10.5897/AJB2010.000-3019

Wu, J.D. & Chou, J.C. 2019. Optimization of protoplast preparation and regeneration of a medicinal fungus Antrodia cinnamomea. Mycobiology, 47(4): 483-493. DOI: https://doi.org/10.1080/12298093.2019.1687252

Yu, X., Ji, S.L., He, Y.L., Ren, M.F. & Xu, J.W. 2014. Development of an expression plasmid and its use in genetic manipulation of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher basidiomycetes). International Journal of Medicinal Mushrooms, 16(2): 161-168. DOI: https://doi.org/10.1615/IntJMedMushr.v16.i2.60

Yusoff, A., M. Ashaari, F.H., Abd Samad, M.A., Ab Wahab, A.F.F. & Bharudin, I. 2021. Identification of soil bacteria with antifungus activity towards palm oil pathogen, Ganoderma boninense. Sains Malaysiana, 50(12): 3557-3567. DOI: https://doi.org/10.17576/jsm-2021-5012-08

Zhou, X., Wei, Y., Zhu, H., Wang, Z., Lin, J., Liu, L. & Tang, K. 2008. Protoplast formation, regeneration and transformation from the taxol-producing fungus Ozonium sp. African Journal of Biotechnology, 7(12): 2017-2024. DOI: https://doi.org/10.5897/AJB2008.000-5050



How to Cite

Ab Wahab, A. F. F., Ahmad Zairun, M. ., Mohd Daud, K. H., Abu Bakar, F. D. ., Bharudin, I. ., & Abdul Murad, A. M. (2022). Evaluation and Improvement of Protocols for Ganoderma boninense Protoplast Isolation and Regeneration. Malaysian Applied Biology, 51(5), 43–57. https://doi.org/10.55230/mabjournal.v51i5.2347