Agronomic Performance and Genetic Fidelity of the Selected Elite Cocoa Clones Derived from Somatic Embryogenesis Culture

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Gibson Entuni*
Hollena Nori
Rebicca Edward
Ahmad Kamil bin Mohammad Jaafar


This study was conducted to compare the agronomic performance of four elite cocoa clones (MCBC1, KKM22, KKM4 and PBC230) regenerated from staminode and immature zygotic embryo culture with conventional grafted cocoa clones. From the results, it was found that the KKM4 clone propagated from immature zygotic embryo culture exhibited variations in the fresh pod weight (339.6 g), fresh individual seed weight (4.13 g) and number of flat beans per pod (4 beans) compared with the rest of the regenerated clones. The genetic stability of the somatic embryogenesis cultured clones and the donor clones was then tested using fragment analysis with five SSR primers, i.e. mTcCIR7, mTcCIR18, mTcCIR22, mTcCIR33 and mTcCIR40. Four of these primers identified variations in the allele size and allele addition in KKM4 clone from immature zygotic embryo. Molecular analysis validated that the difference in agronomic performance of the KKM4 clone from immature zygotic embryo culture was due to genetic mutation created during the immature zygotic embryo culture process.

Keywords: agronomic performance; genetic stability; Theobroma cacao; SSR primers; mutation

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[1] Afoakwa, E.O., Kongor, J.E., Takrama, J.F. and Budu, A.S., 2013. Changes in acidification, sugars and mineral composition of cocoa pulp during fermentation of pulp pre-conditioned cocoa (Theobroma cacao) beans. International Food Research Journal, 20, 1215-1222.
[2] Jayasekhar, S. and Ndung’u, I., 2018. Review of economic history of cocoa with special reference to India. Journal of Plantation Crops, 2, 133-138.
[3] Kongor, J.E., Hinneha, M., Walle, D.V., Afoakwa, E.O., Boeckx, P. and Dewettinck, K., 2016. Factors influencing quality variation in cocoa (Theobroma cacao) bean flavour profile - A review. Food Research International, 82, 44-52.
[4] Florez, S.L., Erwin, R.L., Maximova, S.N., Guiltinan, M.J. and Curtis, W.R., 2015. Enhanced somatic embryogenesis in Theobroma cacao using the homologous BABY BOOM transcription factor. BMC Plant Biology, 15,
[5] Li, Z., Traore, A., Maximova, S. and Guiltinan, M.J., 1998. Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using Thidiazuron. In vitro Cellular & Developmental Biology-Plant, 34, 293-299.
[6] Miller, C.R., 2009. An Integrated In Vitro and Greenhouse Orthotropic Clonal Propagation System for Theobroma cacao L. PhD. Penn State University, USA.
[7] Bairu, M.W., Aremu, A.O. and Van Staden, J., 2011. Somaclonal variation in plants: causes and detection methods. Plant Growth Regulation, 2, 147-173.
[8] Ajijah, N., Randriani, E., Rubiyo, A., Sukma, D. and Sudarsono, D., 2015. Field performance of cacao somatic embryogenesis derived plants. Industrial Crop Research Journal, 21, 57-68.
[9] Maximova, S.N., Young, A., Pishak, S. and Guiltinan, M.J., 2008. Field performance of Theobroma cacao L. plants propagated via somatic embryogenesis. In Vitro Cellular & Developmental Biology-Plant, 44, 487-493.
[10] Masseret, B., Gianforcaro, M., Bouquet, J.F., Brulard, E. and Florin, B., 2009. Somatic embryogenesis applied to the creation of a cacao collection. Malaysian Cocoa Journal, 5, 1-10.
[11] Goenaga, R., Guiltinan, M., Maximova, S., Seguine, E. and Irizarry, H., 2015. Yield performance and bean quality traits of cacao propagated by grafting and somatic embryo derived cutting. Horticultural Science, 50, 358-362.
[12] Lopez, R.C.M., Wetten, A.C. and Wilkinson, M.J., 2009. Detection of somaclonal variation during cocoa somatic embryogenesis characterised using cleaved amplified polymorphic sequence and the new freeware Artbio. Molecular Breeding, 25, 501-516.
[13] Ajijah, N., Hartati, R.S., Rubiyo, K., Sukma, D. and Sudarsono, S., 2016. Effective cacao somatic embryo regeneration on Kinetin supplemented DKW medium and somaclonal variation assessment using SSRs markers. Agrivita, 38, 80-92.
[14] Etienne, H. and Bertrand, B., 2001. Trueness-to-type and agronomic characteristics of Coffea arabica trees micropropagated by the embryogenic cell suspension technique. Tree Physiology, 21, 1031-1038.
[15] Lowry, L., 2018. VassarStats: Website for Statistical Computation. [online] Available at:
[16] Everaert, H., Rottiers, H., Pham, P.H.D., Viet Ha, L.T. and Nguyen, T.P.D., 2017. Molecular characterisation of Vietnamese cocoa genotypes (Theobroma cacao L.) using microsatellite markers. Tree Genetics & Genomes, 13,
[17] Johnsiul, L. and Awang, A., 2016. Utilization of molecular markers to detect the authenticity of cocoa clones. International Journal of Agriculture, Forestry and Plantation, 3, 101-114.
[18] Saunders, J.A., Mischke, S., Leamy, E.A. and Hemeida, A.A., 2004. Selection of international molecular standards for DNA fingerprinting of Theobroma cacao. Theoretical and Applied Genetics, 110, 41-47.
[19] Efron, Y., Marfu, J., Faure, M. and Epaina, P., 2003. Screening of segregating cocoa genotypes for resistance to Vascular Streak Dieback under natural conditions in Papua New Guinea. Plant Pathology, 2, 315-319.
[20] Martínez, I.B., Cruz, M.V., Nelson, M.R. and Bertin, P., 2017. Morphological characterization of traditional cacao (Theobroma cacao L.) plants in Cuba. Genetic Resources and Crop Evolution, 64, 73-99.
[21] Dias, L.A.S., 2019. Genetic Improvement of Cacao. [online] Available at:
[22] Loor, R.G., Risterucci, A.M., Courtois, B., Fouet, O., Jeanneau, M. and Rosenquist, E., 2009. Tracing the native ancestors of the modern Theobroma cacao L. population in Ecuador. Tree Genetics & Genomes, 5, 421-33.
[23] Monteiro, W.R., Lopes, U.V. and Clement, D., 2009. Breeding plantation tree crops: Tropical species. London: Springer.
[24] Matthes, M., Singh, R., Cheah, S.C. and Karp, A., 2001. Variation in oil palm (Elaeis guineensis Jacq.) tissue culture derived regenerants revealed by AFLPs with methylation-sensitive enzymes. Theoretical and Applied Genetics, 102, 971-979.
[25] Mgbeze, G.C. and Iserhienrhien, A., 2014. Somaclonal variation associated with oil palm (Elaeis guineensis Jacq.) clonal propagation: A review. African Journal of Biotechnology, 13, 989-997.
[26] Traore, A., and Guiltinan, M., 2006. Effects of carbon source and explants type on somatic embryogenesis of four cacao genotypes. Horticultural Science, 41, 753-758.
[27] Techato, S. and Lim, M., 2000. Improvement of mangosteen micropropagation through meristematic nodular callus formation from in vitro derived leaf explants. Scientia Horticulturae, 86, 291-298.
[28] Kamle, M. and Baek, K.H., 2017. Somatic embryogenesis in guava (Psidium guajava L.): current status and future perspectives. Biotechnology, 7, 203-218.
[29] Roy, B. and Mandal, A.B., 2005. Anther culture response in Indica rice and variations in major agronomic characters among the androclones of a scented cultivar, Karnal local. African Journal of Biotechnology, 4, 235-240.
[30] Lopez, R.C.M., Wetten, A.C. and Wilkinson, M.J., 2009. Progressive erosion of genetic and epigenetic variation in callus derived cocoa (Theobroma cacao) plants. New Phytology, 186, 856-868.
[31] Jayanthi, M. and Mandal, P.K., 2001. Plant regeneration through somatic embryogenesis and RAPD analysis of regenerated plants in Tylophora indica (Burm. F. Merrill). In vitro Cellular & Developmental Biology-Plant, 37, 576-580.
[32] Brito, G., Lopes, T., Loureiro, J., Rodriguez, E. and Santos, C., 2010. Assessment of genetic stability of two micropropagated wild olive species using flow cytometry and microsatellite markers. Trees, 24, 723-732.
[33] Rai, M.K., Phulwaria, M. and Shekhawat, N.S., 2013. Transferability of simple sequence repeat (SSR) markers developed in guava (Psidium guajava L.) to four Myrtaceae species. Molecular Biology Reports, 40(8), 5067-5071.
[34] Bandupriya, H.D.D., Iroshini, W.W.M.A., Perera, S.A.C.N., Vidhanaarachchi, V.R.M., Fernando, S.C. and Santha, E.S., 2017. Genetic fidelity testing using SSR marker assay confirms trueness-to-type of micropropagated coconut (Cocos nucifera L.) plantlets derived from in vitro cultures. The Open Plant Science Journal, 10, 46-54.
[35] Lestari, P., Roostika, I., Nugroho, K., Edison, H.S., Rijzaani, H. and Mastur, I., 2019. Genetic stability of banana plant regenerated from floral axis organogenesis assessed by newly developed SSR markers. Agrivita, 41, 302-315.
[36] Nookaraju, A. and Agrawal, D.C., 2012. Genetic homogeneity of in vitro raised plants of grapevine cv. Crimson seedless revealed by ISSR and microsatellite markers. South African Journal of Botany, 78, 302-316.
[37] Lopes, T., Pinto, G., Loureiro, L., Costa, A. and Santos, C., 2016. Determination of genetic stability in long term somatic embryogenic cultures and derived plantlets of cork oak using microsatellite markers. Tree Physiology, 26, 1145-1152.
[38] Rahman, M. and Rajora, O., 2001. Microsatellite DNA somaclonal variation in micropropagated Trembling aspen (Populus tremuloides). Plant Cell Reports, 20, 531-536.
[39] Pérez, G., Yanez, E., Mbogholi, M., Valle, B., Sagarra, F., Yabor, L., Aragón, C., González, J., Isidrón, M. and Lorenzo, J.C., 2012. New pineapple somaclonal variants: P3R5 and Dwarf. American Journal of Plant Sciences, 3(1), 1-11.
[40] Neelakandan, A. and Wang, K., 2012. Recent progress in the understanding of tissue culture induced genome level changes in plants and potential applications. Plant Cell Reports, 31, 597-620.
[41] Leva, A.R., Petruccelli, R. and Rinaldi, L.M.R., 2012. Somaclonal variation in tissue culture: A case study with olive. In: A. Leva and Rinaldi, L.M.R., eds. Recent Advances in Plant In Vitro Culture. London: Intech, pp. 123-250.
[42] Maximova, S.N., Florez, S., Shen, X., Niemenak, N., Zhang, Y. and Curtis, W., 2014. Genome-wide analysis reveals divergent patterns of gene expression during zygotic and somatic embryo maturation of Theobroma cacao L., the chocolate tree. BMC Plant Biology, 14, 185-198.
[43] Borsani, O., Zhu, J., Verslues, P.E., Sunkar, R. and Zhu, J.K., 2005. Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cells, 123, 1279-1291.