Effects of Combined 2,4-D and BAP Treatments on the Formation of Leaf-Derived Callus in Pogostemon cablin (Blanco) Benth.

Ira Puspaningtyas, Umi Mukholifah, Maitsa Adzro Fania, Desita Amalia Pratiwi, Reva Firstasa Asmara, Maulida Alfatikhatunnisa, Nurul Jadid

Abstract

Pogostemon cablin (Blanco) Benth., a key source of patchouli oil (2.5 to 5% content), is widely used in the perfume and cosmetic industries but faces propagation challenges due to its time-consuming nature and susceptibility to pathogens. To overcome these limitations, in vitro techniques have been adopted to produce healthy and uniform seedlings efficiently. This study evaluated the effects of varying concentrations of 2,4-D (0 to 1.5 mg l-1) and BAP (0 to 1.5 mg l-1) on the in vitro induction of patchouli calli using axenic leaf explants cultured on Murashige and Skoog (MS) medium for 60 days. Growth parameters assessed included callus emergence time, morphological traits (texture and color), callogenesis percentage, organogenesis, and fresh callus weight. The results indicated that the combination of 2,4-D and BAP significantly influenced callogenesis, organogenesis, and callus morphology. The optimal combination (A0B3: 2,4-D 0 mg l-1 + BAP 1.5 mg l-1) achieved 100% callogenesis and organogenesis, with an average callus fresh weight of 0.25 g and an initiation time of 40.5 days. Calli exhibited a crumbly texture with a yellowish-white color, highlighting the role of auxin and cytokinin interactions in patchouli callus induction. These findings provide a foundation for scaling up in vitro patchouli oil production.

Keywords

auxin; callus; cytokinin; in vitro culture; patchouli

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References

Agarwal, M. (2015). Tissue culture of Momordica charantia L.: A review. Journal of Plant Sciences, 3(1–1), 32–25. https://doi.org/10.11648/j.jps.s.2015030101.14

Anjalani, T. R., Rasmi, S. A., Rahayu, A. E., Ramadhani, M. R. N., Sholihah, M. F., Puspaningtyas, I., ..., & Jadid, N. (2024). Methyl jasmonate stimulates growth and upregulates the expression of Phenylalanine Ammonia-Lyase (PAL) gene in Gynura pseudochina in vitro micropropagation. Biodiversitas Journal of Biological Diversity, 25(5), 1955–1964. https://doi.org/10.13057/biodiv/d250512

Ariani, R., Anggraito, Y. U., & Rahayu, E. S. (2016). Respon pembentukan kalus koro benguk (Mucuna pruriens L.) pada berbagai konsentrasi 2,4-D dan BAP. Indonesian Journal of Mathematics and Natural Sciences, 39(1), 20–28. https://doi.org/10.15294/ijmns.v39i1.7695

Astuti, P., Khairan, K., Marthoenis, M., & Hasballah, K. (2022). Antidepressant-like activity of patchouli oil var. Tapak Tuan (Pogostemon cablin Benth) via elevated dopamine level: A study using Rat Model. Pharmaceuticals, 15(5), 608. https://doi.org/10.3390/ph15050608

Aziz, M. M., Ratnasari, E., & Rahayu, Y. S. (2014). Induksi kalus umbi iles-iles (Amorphophallus muelleri) dengan kombinasi konsentrasi 2,4-D dan BAP secara in vitro. LenteraBio, 3(2), 109–114. Retrieved from https://ejournal.unesa.ac.id/index.php/lenterabio/article/view/8037

Bansal, S., Sharma, M. K., Joshi, P., Malhotra, E. V., Latha, M., & Malik, S. (2024). An efficient direct organogenesis protocol for in vitro clonal propagation of Rubia cordifolia L. Industrial Crops and Products, 208, 117856. https://doi.org/10.1016/j.indcrop.2023.117856

Benjamin, E. D., Ishaku, G. A., Peingurta, F. A., & Afolabi, A. S. (2019). Callus culture for the production of therapeutic compounds. American Journal of Plant Biology, 4(4), 76–84. https://doi.org/10.11648/j.ajpb.20190404.14

Bhat, A. Y., Shahzad, A., Kausar, A., & Rashid, A. (2024). Integrating leaf and root induced shoot regeneration and embryogenesis for the conservation of Atropa acuminata Royle ex Lindl—An endangered Himalayan herb. Plant Cell, Tissue and Organ Culture (PCTOC), 159(3), 73. https://doi.org/10.1007/s11240-024-02937-9

Bojko, M., Kędra, M., Adamska, A., Jakubowska, Z., Tuleja, M., & Myśliwa-Kurdziel, B. (2024). Induction and characteristics of callus cultures of the medicinal plant Tussilago farfara L. Plants, 13(21), 3080. https://doi.org/10.3390/plants13213080

Bondy, S. C. (2023). The hormesis concept: Strengths and shortcomings. Biomolecules, 13(10), 1512. https://doi.org/10.3390/biom13101512

Budisantoso, I., Amalia, N., & Kamsinah, K. (2017). In vitro callus induction from leaf explants of Vanda sp stimulated by 2, 4-D. Biosaintifika: Journal of Biology & Biology Education, 9(3), 492–497. https://doi.org/10.15294/biosaintifika.v9i3.11018

Carsono, N., Juwendah, E., Liberty, L., Sari, S., Damayanti, F., & Rachmadi, M. (2021). Optimize 2, 4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes. Biodiversitas Journal of Biological Diversity, 22(7), 2555–2560. https://doi.org/10.13057/biodiv/d220702

Castro, A. H. F., Braga, K. D. Q., Sousa, F. M. D., Coimbra, M. C., & Chagas, R. C. R. (2016). Callus induction and bioactive phenolic compounds production from Byrsonima verbascifolia (L.) DC. (Malpighiaceae). Revista Ciência Agronômica, 47, 143–151. https://doi.org/10.5935/1806-6690.20160017

Efferth, T. (2019). Biotechnology applications of plant callus cultures. Engineering, 5(1), 50–59. https://doi.org/10.1016/j.eng.2018.11.006

Gurav, S. S., Gurav, N. S., Patil, A. T., & Duragkar, N. J. (2020). Effect of explant source, culture media, and growth regulators on callogenesis and expression of secondary metabolites of Curcuma longa. Journal of Herbs, Spices & Medicinal Plants, 26(2), 172–190. https://doi.org/10.1080/10496475.2019.1689542

Hardjo, P. H., Susanto, D. P. S., Savitri, W. D., & Purwanto, M. G. M. (2019). Shoot multiplication of Pogostemon cablin var. Sidikalang and patchouli oil profile. Nusantara Bioscience, 11(2), 123–127. https://doi.org/10.13057/nusbiosci/n110202

Haring, F., Farid, M., Ridwan, I., Fadhilah, A. N., & Sedayu, N. (2024). Concentration of 2,4-D and BAP combination on callus induction of porang plant (Amorphophallus muelleri Blume). Asian Journal of Plant Sciences, 23(3), 313–320. https://doi.org/10.3923/ajps.2024.313.320

Hemmati, N., Cheniany, M., & Ganjeali, A. (2020). Effect of plant growth regulators and explants on callus induction and study of antioxidant potentials and phenolic metabolites in Salvia tebesana Bunge. Botanica Serbica, 44(2), 163–173. https://doi.org/10.2298/BOTSERB2002163H

Ikeuchi, M., Sugimoto, K., & Iwase, A. (2013). Plant callus: Mechanisms of induction and repression. The Plant Cell, 25(9), 3159–3173. https://doi.org/10.1105/tpc.113.116053

Imelda, M., Wulansari, A., & Poerba, Y. S. (2008). Shoot regeneration from leaf petioles of iles-iles (Amorphophallus muelleri Blume). Biodiversitas Journal of Biological Diversity, 9(3), 173–176. https://doi.org/10.13057/biodiv/d090304

Jadid, N., Anggraeni, S., Ramadani, M. R. N., Arieny, M., & Mas’ud, F. (2024a). In vitro propagation of Indonesian stevia (Stevia rebaudiana) genotype using axenic nodal segments. BMC Research Notes, 17(1), 45. https://doi.org/10.1186/s13104-024-06703-0

Jadid, N., Datus Soleha, I., Esti Rahayu, A., Puspaningtyas, I., Anita Sari, S., Rahmawati, M., Aunurohim, & Hidayati, D. (2024b). Exogenous methyl jasmonate (MeJA) altered phytochemical composition and enhanced the expression of PatAACT gene of in vitro culture-derived patchouli var. Sidikalang (Pogostemon cablin Benth.). Journal of King Saud University - Science, 36(8), 103301. https://doi.org/10.1016/j.jksus.2024.103301

Khairan, K., Bustam, B. M., Yunita, Meilinda, R., & Muna, R. (2021). Pengaruh komposisi 2,4-D dan BAP terhadap pembentukan kalus eksplan pucuk nilam (Pogostemon cablin Benth.) secara in vitro dengan pemotongan horizontal dan vertikal. Biotropic : The Journal of Tropical Biology, 5(1), 9–20. https://doi.org/10.29080/biotropic.2021.5.1.9-20

Kruglova, N. N., & Zinatullina, A. E. (2024). Organogenesis as the realization of the morphogenetic potential of callus cells in in vitro conditions. Russian Journal of Developmental Biology, 55(3), 140–152. https://doi.org/10.1134/S1062360424700127

Litz, R. E., & Jaiswal, V. S. (1991). Micropropagation of tropical and subtropical fruits. Micropropagation (pp. 247–263). Springer Netherlands. https://doi.org/10.1007/978-94-009-2075-0_18

Liu, C., Li, A., Fan, X., Qin, B., & Zhang, L. (2024). Explant browning during callus induction of Juglans mandshurica might be caused by the cooperation of PAL, polyphenol and PPO. South African Journal of Botany, 174, 937–945. https://doi.org/10.1016/j.sajb.2024.09.072

Marković, M., Trifunović-Momčilov, M., Radulović, O., Paunović, D. M., Antonić Reljin, D. D., Uzelac, B., & Subotić, A. (2023). The effects of different auxin–cytokinin combinations on morphogenesis of Fritillaria meleagris using Bulb scale sections in vitro. Horticulturae, 9(8), 910. https://doi.org/10.3390/horticulturae9080910

Marisa, F., Hidayati, L., Sasongko, A. B., & Nuringtyas, T. R. (2021). Callus induction from cotyledon of Gyrinops versteegii (Gilg.) Domke. Jurnal Biologi Tropis, 21(2), 427–433. https://doi.org/10.29303/jbt.v21i2.2629

Muthi’ah, A., Sakya, A. T., Setyawati, A., Samanhudi, & Rahayu, M. (2023). Callus induction of Calotropis gigantea using BAP and 2,4-D in vitro. IOP Conference Series: Earth and Environmental Science, 1177(1), 012021. https://doi.org/10.1088/1755-1315/1177/1/012021

Muzika, N. S., Kamai, T., Williams, L. E., & Kleiman, M. (2024). Characterization of gelling agents in callus inducing media: Physical properties and their effect on callus growth. Physiologia Plantarum, 176(2), e14312. https://doi.org/10.1111/ppl.14312

Nisa, K., Sufardi, S., Rusdi, M., & Indra, I. (2024). Soil quality index and patchouli yields on various cropping systems in Aceh Province, Indonesia: Case study in Aceh Barat Regency. Case Studies in Chemical and Environmental Engineering, 10, 100798. https://doi.org/10.1016/j.cscee.2024.100798

Permadi, N., Akbari, S. I., Prismantoro, D., Indriyani, N. N., Nurzaman, M., Alhasnawi, A. N., ..., & Julaeha, E. (2024). Traditional and next-generation methods for browning control in plant tissue culture: Current insights and future directions. Current Plant Biology, 38, 100339. https://doi.org/10.1016/j.cpb.2024.100339

Rahayu, S., Roostika, I., & Bermawie, N. (2016). The effect of types and concentrations of auxins on callus induction of Centella asiatica. Nusantara Bioscience, 8(2), 283–287. https://doi.org/10.13057/nusbiosci/n080224

Ramadani, M. R. N., & Jadid, N. (2024). A comprehensive review of in vitro precursor feeding strategies for the overproduction of high-value plant secondary metabolites. Arabian Journal of Chemistry, 17(12), 106018. https://doi.org/10.1016/j.arabjc.2024.106018

Rasud, Y., & Bustaman, B. (2020). In vitro callus induction from clove (Syzigium aromaticum L.) leaves on medium containing various auxin concentrations. Jurnal Ilmu Pertanian Indonesia, 25(1), 67–72. https://doi.org/10.18343/jipi.25.1.67

Remakanthan, A., Menon, T. G., & Soniya, E. V. (2014). Somatic embryogenesis in banana (Musa acuminata AAA cv. Grand Naine): Effect of explant and culture conditions. In Vitro Cellular & Developmental Biology - Plant, 50(1), 127–136. https://doi.org/10.1007/s11627-013-9546-4

Rusnak, B., Clark, F. K., Vadde, B. V. L., & Roeder, A. H. (2024). What is a plant cell type in the age of single-cell biology? It’s complicated. Annual Review of Cell and Developmental Biology, 40, 301–328. https://doi.org/10.1146/annurev-cellbio-111323-102412

Rybin, D. A., Sukhova, A. A., Syomin, A. A., Zdobnova, T. A., Berezina, E. V., & Brilkina, A. A. (2024). Characteristics of callus and cell suspension cultures of highbush blueberry (Vaccinium corymbosum L.) cultivated in the presence of different concentrations of 2, 4-D and BAP in a nutrient medium. Plants, 13(23), 3279. https://doi.org/10.3390/plants13233279

Sagai, E., Doodoh, B., & Kojoh, D. (2016). Pengatur zat pengatur tumbuh benzil amino purin (BAP) terhadap induksi dan multiplikasi tunas brokoli Brassica oleraceae L. Var. Italica Plenck. Cocos, 7(6),1–9. https://doi.org/10.35791/cocos.v7i6.13885

Skoog, F., & Miller, C.O. (1957). Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symposia of the Society for Experimental Biology, 11, 118–130. Retrieved from https://scholar.google.co.id/scholar?cites=4322887444565523062&as_sdt=2005&sciodt=0,5&hl=id

Sosnowski, J., Truba, M., & Vasileva, V. (2023). The impact of auxin and cytokinin on the growth and development of selected crops. Agriculture, 13(3), 724. https://doi.org/10.3390/agriculture13030724

Sukamto, L. A. (2011). Effect of physiological age and growth regulators on callus browning of coconut endosperm in vitro culture. Biotropia, 18(1), 31–41. https://doi.org/10.11598/btb.2011.18.1.136

Suminar, E., Sumadi, S., Mubarok, S., Sunarto, T., & Rini, N. S. E. (2017). Percepatan penyediaan benih sumber kedelai unggul secara in vitro. Agrikultura, 28(3). https://doi.org/10.24198/agrikultura.v28i3.15744

Swamy, M. K., & Sinniah, U. R. (2016). Patchouli (Pogostemon cablin Benth.): Botany, agrotechnology and biotechnological aspects. Industrial Crops and Products, 87, 161–176. https://doi.org/10.1016/j.indcrop.2016.04.032

Teoh, S. C., Subramaniam, S., & Chew, B. L. (2023). The effects of 2, 4-dichlorophenoxyacetic acid on the induction of callus from cotyledon and hypocotyl explants of butterfly pea (Clitoria ternatea). Malaysian Applied Biology, 52(1), 61–72. https://doi.org/10.55230/mabjournal.v52i1.2444

Wardani, D. K. (2020). Induksi kalus tanaman nilam (Pogostemon cablin Benth) dengan pemberian konsentrasi auksin jenis 2,4-D (dichlorophenoxyacetic acid) dan picloram. Jurnal Indonesia Sosial Sains, 1(5), 396–401. https://doi.org/10.36418/jiss.v1i5.73

Waryastuti, D. E., Setyobudi, L., & Wardiyati, T. (2017). Pengaruh tingkat konsentrasi 2,4-D dan BAP pada media MS terhadap induksi kalus embriogenik temulawak (Curcuma xanthorrhiza Roxb.). Jurnal Produksi Tanaman, 5(1), 140–149. Retrieved from https://protan.studentjournal.ub.ac.id/index.php/protan/article/view/362/357

Yin, Y., Zhong, R., Li, Y., Guo, B., Li, L., Ma, G., ..., & Zeng, S. (2025). BAP regulates lateral bud outgrowth to promote tillering in Paphiopedilum callosum (Orchidaceae). BMC Plant Biology, 25(1), 241. https://doi.org/10.1186/s12870-025-06256-9

Zarbakhsh, S., Shahsavar, A. R., & Soltani, M. (2024). Optimizing PGRs for in vitro shoot proliferation of pomegranate with bayesian-tuned ensemble stacking regression and NSGA-II: A comparative evaluation of machine learning models. Plant Methods, 20(1), 82. https://doi.org/10.1186/s13007-024-01211-5

Zhai, M., Dai, Q. Q., Zhao, Y., Zhang, S., & Yang, Y. L. (2025). Induction of callus and establishment of suspension culture system in Cassia mimosoides herb. In Vitro Cellular & Developmental Biology-Plant, 61, 229–238. https://doi.org/10.1007/s11627-024-10503-3

DOI: https://doi.org/10.20961/carakatani.v40i2.97331

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