Picloram-Induced Embryogenic Callus Formation and Proliferation from Nucellar Embryo of Mandarin ‘Berastagi’ (Citrus reticulata Blanco)
DOI:
https://doi.org/10.29244/jtcs.13.03.707-719Keywords:
carbohydrate, lactose, picloram, somatic embryogenesis, sucroseAbstract
Mandarin ‘Berastagi’ (Citrus reticulata Blanco) is a key horticultural commodity in North Sumatra, Indonesia, with increasing demand for high quality, genetically uniform planting material. Biotechnological approaches, somatic embryogenesis, hold particularly significant potential enhancing propagation efficiency and crop quality. This study aimed to determine the optimal culture medium and picloram concentration for the induction and proliferation of embryogenic callus from nucellar explants of Mandarin ‘Berastagi’ in vitro. Two experiments were conducted: (1) induction of embryogenic callus from nucellar embryos isolated from young fruits (60–70 days after anthesis) cultured on different basal media supplemented with varying picloram concentrations, and (2) proliferation of embryogenic callus on media containing different carbohydrate concentrations. The Murashige and Wetmore (MW) medium supplemented with 1.5 mg/L picloram resulted in the highest callus induction rate (37.8%) and largest mean callus diameter (17.8 mm). For proliferation, MW medium containing 4% sucrose produced the best response, with an average callus diameter of 14 mm and 96% of explants developing globular stage somatic embryos. These results establish an efficient somatic embryogenesis protocol that can support large-scale propagation and future crop improvement programs in Mandarin ‘Berastagi’.
References
Abdelsalam, A., Chowdhury, K., & Bakry, A. E. (2018). Efficient adventitious morphogenesis from in vitro cultures of the medicinal plant Cymbopogon schoenanthus. Plant Tissue Culture and Biotechnology, 28, 147–160. https://doi.org/10.3329/ptcb.v28i2.39674
Abrahamian, P., & Kantharajah, A. (2011). Effect of vitamins on the in vitro organogenesis of plants. Journal of Plant Sciences, 2, 669–674. https://doi.org/10.4236/ajps.2011.25080
Ahansal, K., Aadel, H., Udupa, S. M., Gaboun, F., Abdelwahd, R., Ibriz, M., & Iraqi, D. (2022). Effect of picloram and 2,4-D on plant regeneration from mature and immature embryos of Moroccan durum wheat varieties. Journal of Plant Biotechnology, 49, 131–138. https://doi.org/10.5010/JPB.2022.49.2.131
Anggreani, N., & Anisah, S. (2021). Analisis kadar glukosa jeruk lokal provinsi Bengkulu menggunakan metode Luff Schoorl. Jurnal Kesehatan Tambusai, 2, 343-347. https://doi.org/10.31004/jkt.v2i4.3192
Ariani, R., Anggraito, Y. U., & Rahayu, E. S. (2016). Respons pembentukan kalus koro benguk (Mucuna pruriens L.) pada berbagai konsentrasi 2,4-D dan BAP. Indonesian Journal of Mathematics and Natural Sciences, 39, 20–28.
Avila-Victor, C. M., Arjona-Suárez, E. D. J., Iracheta-Donjuan, L., Valdez-Carrasco, J. M., Gómez-Merino, F. C., & Robledo-Paz, A. (2023). Callus type, growth regulators, and phytagel on indirect somatic embryogenesis of coffee (Coffea arabica L. Colombia). Plants, 12, 3570. https://doi.org/10.3390/plants12203570
Bahrunnisa, A., Habibah, N. A. (2025). Vanilla callogenesis with the addition of picloram and BAP under dark and light conditions. Life Science, 14, 11-21. https://doi.org/10.15294/unnesjlifesci.v14.i1.22275
Balai Penelitian Jeruk dan Buah Subtropika. (2020). Jeruk nusantara dan sebaran benih. Badan Litbang Pertanian. Baviskar, S. P., & Patil, S. R. (2021). Tissue culture techniques. AkiNik Publications.
Bhardwaj, S., Kumari, M., T., C., Hosamani, M., Paul, M., Moirangthem, A., Dash, A. P., & Verma, S. (2025). Advances in micropropagation and tissue culture for horticultural crops: A review. Plant Cell Biotechnology and Molecular Biology, 26(11–12), 1–10. https://doi.org/10.56557/pcbmb/2025/v26i11-1210039
Catalano, C., Abbate, L., Fatta Del Bosco, S., Motisi, A., Carimi, F., De Michele, R., & Carra, A. (2022). Different cell types affect the transition from juvenile to mature phase in citrus plants regenerated through somatic embryogenesis. Plants, 11, 1811. https://doi.org/10.3390/plants11141811
Chandoliya, R., Sharma, S., Joshi, R., & Sharma, V. (2025). Effect of NAA and picloram on the callus induction in Aloe barbadensis. Research Journal of Biotechnology, 20, 209–212. https://doi.org/10.25303/203rjbt2090212
Gantait, S., & Mahanta, M. (2021). Picloram induced enhanced callus-mediated regeneration, acclimatization, and genetic clonality assessment of Gerbera. Journal of Genetic Engineering and Biotechnology, 19, 152. https://doi.org/10.1186/s43141-021-00269-1
Garcia, C., de Almeida, A-A. F., Costa, M., Britto, D., Valle, R., Royaert, S., and Marelli, J-P. (2019). Abnormalities in somatic embryogenesis caused by 2,4 D: An overview. Plant Cell, Tissue and Organ Culture, 137, 193–212. https://doi.org/10.1007/s11240-019-01569-8
Gerdakaneh, M., & Zohori, M. (2013). The effect of picloram on somatic embryogenesis of different explants of strawberry (Fragaria ananassa). British Biotechnology Journal, 3, 133. https://doi.org/10.9734/BBJ/2013/2370
Indah, P. N., & Ermavitalini, D. (2013). Induksi kalus daun nyamplung (Calophyllum inophyllum Linn.) pada beberapa kombinasi BAP dan 2,4-D. Jurnal Sains dan Seni Pomits, 2, 1–6. https://doi.org/10.12962/j23373520.v2i1.2571
Jin, S. B., Park, S., Park, J., Moon, Y. E., & Lee, D. (2025). Establishment of a regeneration system for ‘Minihyang,’ a domestic citrus variety with high utility as a genetic resource. Plant Breeding, 144, 529-539. https://doi.org/10.1111/pbr.13270
Junairiah, J., Wulandari, D. A., Utami, E. S. W., & Zuraidassanaaz, N. I. (2021). Callus induction and secondary metabolite profile from Elephantopus scaber L. Journal of Tropical Biodiversity and Biotechnology, 6, 59234. https://doi.org/10.22146/jtbb.59234
Kayim, M., & Koc, N. K. (2006). The effects of some carbohydrates on growth and somatic embryogenesis in citrus callus culture. Scientia Horticulturae, 109, 29–34. https://doi.org/10.1016/j.scienta.2006.01.040
Kazmi, S. K., Khan, S., Kabir, N., Mirbahar, A. A., Raziq, M., & Kauser, N. (2015). Embryogenic callus induction, somatic embryogenesis, regeneration, and histological studies of Kinnow mandarin (Citrus reticulata Blanco L.) from nucellar embryo and epicotyl region. Pakistan Journal of Botany, 47, 305–310.
Kosmiatin, M., Purwito, A., Wattimena, G. A., & Mariska, I. (2014). Induksi embriogenesis somatik dari jaringan endosperma jeruk siam (Citrus nobilis Lour.) cv Simadu. Jurnal Agronomi Indonesia, 42, 1–7.
Lathifah, Q., Turista, D., Hermawati, A., & Jannah, R. (2022). Penetapan vitamin C dalam jeruk keprok segar dan jus secara iodometri. Borneo Journal of Medical Laboratory Technology, 4, 297–301. https://doi.org/10.33084/bjmlt.v4i2.3313
Mahadi, I., Syafi’i, Y., & Sari. (2016). Induksi kalus jeruk kasturi (Citrus microcarpa) menggunakan hormon 2,4-D dan BAP dengan metode in vitro. Jurnal Ilmu Pertanian Indonesia, 21, 84–89. https://doi.org/10.18343/jipi.21.2.84
Murugan, T., Awasthi, O. P., Singh, S. K., Chawla, G., Solanke, A. U., Kumar, S., & Jha, G. K. (2023). Molecular and histological validation of modified in ovulo nucellus culture based high-competency direct somatic embryogenesis and amplitude true-to-the-type plantlet recovery in Kinnow mandarin. Frontiers in Plant Science, 14, 1116151. https://doi.org/10.3389/fpls.2023.1116151
Qi, S., Zhao, R., Yan, J., Fan, Y., Huang, C., Li, H., Chen, S., Zhang, T., Kong, L., Zhao, J., & Zhang, J. (2021). Global transcriptome and coexpression network analyses reveal new insights into somatic embryogenesis in hybrid sweetgum (Liquidambar styraciflua × Liquidambar formosana). Frontiers in Plant Science, 12, 751866. https://doi.org/10.3389/fpls.2021.751866
Schuller, A., & Reuther, G. (1993). Response of Abies alba embryonal-suspensor mass to various carbohydrate treatments. Plant Cell Reports, 12, 199–202. https://doi.org/10.1007/BF00237053
Sembiring, J. M. (2024). Analisis faktor faktor yang mempengaruhi produksi dan pendapatan usaha tani jeruk manis (Citrus × sinensis) di Desa Barung Kersap Kecamatan Munte Kabupaten Karo [Doctoral dissertation, Universitas Medan Area]. Universitas Medan Area Digital Repository.
Sharma, P., & Roy, B. (2024). Study on highly efficient direct shoot and root organogenesis from cotyledon and in vitro derived root and leaf of Citrus jambhiri Lush. Indian Journal of Ecology, 51, 162–169. https://doi.org/10.55362/IJE/2024/4210
Sidhu, G. S., Malhotra, P. K., & Rattanpal, H. S. (2023). Optimization of explants, media, plant growth regulators, and carbohydrates on callus induction and plant regeneration in Citrus jambhiri Lush. Journal of Horticultural Sciences, 18, 162–172. https://doi.org/10.24154/jhs.v18i1.2159
Simbolon, S. H. B., Sinulingga, K., & Sitepu, E. R. B. (2025). Sosialisasi Pemanfaatan dan Pengolahan Jeruk Untuk Pengembangan Potensi Desa Sempajaya di Kecamatan Berastagi Kabupaten Karo. Jurnal Pengabdian Masyarakat Bangsa, 3, 4642-4646.
Syahrizal, D., & Puspita, N. A. (2020). Metabolisme dan bioenergetika. Syiah Kuala University Press.
Takamori, L. M., Neto, N. B. M, Vieira, L. G. E., and Ribas, A. F. (2015). Optimization of somatic embryogenesis and in vitro plant regeneration of Urochloa species using picloram. In Vitro Cellular and Developmental Biology Plant, 51, 554 563. https://doi.org/10.1007/s11627-015-9701-1
Tarigan, S. (2017). Analisis kadar vitamin C dalam jeruk (Citrus sp.) lokal dan impor yang beredar di pasar Kota Medan dengan metode volumetri menggunakan 2,6-Diklorofenol indofenol [Magister thesis, Universitas Sumatera Utara]. Universitas Sumatera Utara Digital Repository.
Wahyudhi, A. (2020). Pembentukan poliembrioni pada biji buah jeruk peras (Citrus sinensis L.). Agroscript: Journal of Applied Agricultural Sciences, 2, 49– 55 .
Wahyuni, F. D., Fathoni, A., Sukma, D., Ardie, S. W., Zainuddin, I. M., Qi, B., & Sudarsono, S. (2025). Optimizing somatic embryogenesis in Indonesian cassava genotypes: Effects of 2,4-D or picloram supplementation and explant type. Plant Biotechnology, 25, 1003. https://doi.org/10.5511/plantbiotechnology.25.1003b
Wulansari, A., Purwito, A., Husni, A., & Sudarmonowati, E. (2015). Regeneration ability of embryogenic callus derived from nucellus of siam tangerine and phenotypic variation of its regenerants. In Prosiding Seminar Nasional Masyarakat Biodiversitas Indonesia (vol. 1 no. 1, pp. 97–104). Depok.
Zakiah, Z. & Turnip, M. (2024). Induction of somatic embryogenesis of Pontianak siamese mandarin cotyledon cultures on Murashige Skoog media with the addition of 2,4-D and Kinetin. Jurnal Biologi Tropis, 24, 241–250. https://doi.org/10.29303/jbt.v24i2.6721
Zhang, W., Chen, J., Chen, Q., Wu, H., & Mu, W. (2020). Sugar alcohols derived from lactose: Lactitol, galactitol, and sorbitol. Applied Microbiology and Biotechnology, 104, 9487–9495. https://doi.org/10.1007/s00253-020-10929-w
Downloads
Published
How to Cite
Issue
Section
License
All publications by Journal of Tropical Crop Science is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.



