Optimization of Physalis angulata L. callus induction and salinity-induced antioxidant production

Firstian Naufal Sistyananda; Erni Suminar; Anne Nuraini; Muhamad Kadapi; Murgayanti Murgayanti; Syariful Mubarok; Rahmat Budiarto; Eddy Renaldi; Kusumiyati Kusumiyati

doi:10.24198/kultivasi.v25i1.69556

Optimization of Physalis angulata L. callus induction and salinity-induced antioxidant production

Firstian Naufal Sistyananda, Erni Suminar, Anne Nuraini, Muhamad Kadapi, Murgayanti Murgayanti, Syariful Mubarok, Rahmat Budiarto, Eddy Renaldi, Kusumiyati Kusumiyati

Abstract

Groundcherry (Physalis angulata L.) is a plant with many medicinal potentials due to its rich secondary metabolites such as phenolic and flavonoid. However, conventional agriculture practices are still limited, especially in Indonesia. This study was divided into two stages. The first stage was conducted to determine optimal 6-BAP and 2,4-D combination for callus induction, while the second stage was conducted to determine callus phenolic, flavonoid, and antioxidant response to salinity stress. The first stage was arranged in factorial completely randomized design with two factors: 6-BAP (0, 2, and 4 mg/L) and 2,4-D (0, 0.5, 1, and 1.5 mg/L). The second stage was arranged in simple completely randomized design with different NaCl concentration (0, 25, 50, 75, 100 mM) as treatments. The results showed significant interaction (p < 0.05) between 6-BAP and 2,4-D on callus induction. Combination of 2 mg/L 6-BAP and 1 mg/L 2,4-D showed the highest callus formation percentage (46% increase), callus size (60.12% increase), and fresh weight (179.69% increase), and greener compact callus. Application of NaCl as salinity stress at second stage experiment served as an elicitor to enhance callus antioxidant capacity. Salinity level at 100 mM NaCl showed the most accumulation of phenolic content (17.8% increase), flavonoid content (25.17% increase), and antioxidant activities (6.84% IC₅₀decrease). This study demonstrates plant growth regulator optimization with salinity stress elicitation integration as an effective strategy to enhance antioxidant production in P. angulata callus, providing a practical approach for controlled secondary metabolite production.

Keywords

flavonoid; groundcherry; phenolic content; salinity stress; secondary metabolites

Full Text:

PDF

References

Ahmed IM, Nadira UA, Bibi N, Cao F, He X, Zhang G, & Wu F. 2015. Secondary metabolism and antioxidants are involved in the tolerance to drought and salinity, separately and combined, in Tibetan wild barley. Environmental and Experimental Botany, 111: 1–12. https://doi.org/10.1016/j.envexpbot.2014.10.003

Anggraeni D, Ismaini L, Surya MI, Rahmi H, & Saputro NW. 2022. Inisiasi kalus daun Talinum triangulare (Jacq.) Willd pada beberapa kombinasi konsentrasi zat pengatur tumbuh 2,4-dichlorophenoxyatic acid dan benzyl adenine. Jurnal Agrikultura, 33: 276–288.

Aziz MM, Ratnasari E, & Rahayu YS. 2014. Induksi kalus umbi iles-iles (Amorphophallus muelleri) dengan kombinasi konsentrasi 2,4-D dan BAP secara in vitro. Lentera Bio, 3: 109–114. http://ejournal.unesa.ac.id/index.php/lenterabio

Balasundram N, Sundram K, & Samman S. 2006. Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chemistry, 99: 191–203. https://doi.org/10.1016/j.foodchem.2005.07.042

Bozaba TO, & Kuru İS. 2024. The effect of the combined application of elicitors to Salvia virgata Jacq. under salinity stress on physiological and antioxidant defense. BMC Plant Biology, 24: 1–12. https://doi.org/10.1186/s12870-024-05443-4

Carimi F, Zottini M, Formentin E, Terzi M, & Lo Schiavo F. 2003. Cytokinins: New apoptotic inducers in plants. Planta, 216: 413–421. https://doi.org/10.1007/s00425-002-0862-x

Chaidir L, Epi, & Taofik A. 2015. Eksplorasi, identifikasi, dan perbanyakan tanaman ciplukan (Physalis angulata L.) dengan menggunakan metode generatif dan vegetatif. Jurnal Kajian Islam, Sains, Dan Teknologi, 9: 82–103.

Chele KH, Tinte MM, Piater LA, Dubery IA, & Tugizimana F. 2021. Soil salinity, a serious environmental issue and plant responses: A metabolomics perspective. Metabolites, 11: 1–19. https://doi.org/10.3390/metabo11110724

da Silva AL, do Nascimento MN, de Jesus Carneiro V, dos Santos Neto F, da Silva Leite R, & Tanan TT. 2021. Physiological and biochemical indicators of Physalis angulata L. Plants submitted under salinity. Comunicata Scientiae, 12. https://doi.org/10.14295/cs.v12.3450

da Silva Ramos CA, Soares TL, Barroso NS, & Pelacani CR. 2021. Influence of maturity stage on physical and chemical characteristics of fruit and physiological quality of seeds of Physalis angulata L. Scientia Horticulturae, 284. https://doi.org/10.1016/j.scienta.2021.110124

Fadhli H, Ruska SL, Furi M, Suhery, WN, Susanti E, & Nasution MR. 2023. Ciplukan (Physalis angulata L.): Review tanaman liar yang berpotensi sebagai tanaman obat. Jurnal Farmasi Indonesia, 15: 134–141. https://doi.org/10.35617/jfionline.v15i2.144

Fitroh AI, Dwiyani R, Wijaya IKA, & Yuswanti H. 2018. Pengaruh 2,4-D terhadap induksi kalus daun stroberi (Fragaria sp.) dengan media alternatif nutrisi hidroponik AB mix. Agroekoteknologi Tropika, 7: 304–315. https://ojs.unud.ac.id/index.php/JAT304

Golkar P, Alimoradi F, Akbari R, Bazarganipour M, & Peralta-Videa JR. 2025. Exploring the stimulatory effects of graphene oxide-coated zinc oxide nanoparticles on Thymus daenensis Celak. callus under salinity stress: insights into biochemical and morphological changes. Plant Cell, Tissue and Organ Culture, 163. https://doi.org/10.1007/s11240-025-03157-5

Golkar P, Bakhshi G, & Vahabi MR. 2020. Phytochemical, biochemical, and growth changes in response to salinity in callus cultures of Nigella sativa L. In vitro Cellular and Developmental Biology-Plant, 56: 247–258. https://doi.org/10.1007/s11627-020-10058-z

Gozdur K, Szopa A, & Ślesak H. 2024. Effect of salt stress on growth and phenolic compounds production in callus suspension culture of the dioecious species thyrse sorrel (Rumex thyrsiflorus Fingerh.). Plant Cell, Tissue and Organ Culture, 158. https://doi.org/10.1007/s11240-024-02822-5

Gulcin İ. 2025. Antioxidants: a comprehensive review. Archives of Toxicology, 99, 1893–1997. https://doi.org/10.1007/s00204-025-03997-2

Hemerly AS, Ferreira P, de Almeida Engler J, Van Montagu M, Engler G, & Inzé D. 1993. cdc2a expression in Arabidopsis is linked with competence for cell division. The Plant Cell, 5: 1711–1723. https://doi.org/10.1105/tpc.5.12.1711

Hidayat R, Abdullah, & Aminah. 2021. Pemantapan masa kalus kedelai secara in vitro dalam media MS (Murashige dan Skoog) B5 (Gamborg). Jurnal Agrotekmas, 2: 60–70.

Hussen EM, & Endalew SA. (2023). In vitro antioxidant and free-radical scavenging activities of polar leaf extracts of Vernonia amygdalina. BMC Complementary Medicine and Therapies, 23. https://doi.org/10.1186/s12906-023-03923-y

Ighodaro OM, & Akinloye OA. 2018. First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid. Alexandria Journal of Medicine, 54: 287–293. https://doi.org/10.1016/j.ajme.2017.09.001

Indah PN, & Ermavitalini D. 2013. Induksi kalus daun nyamplung (Calophyllum inophyllum Linn.) pada beberapa kombinasi konsentrasi 6-benzylaminopurine (BAP) dan 2,4-dichlorophenoxyacetic acid (2,4-D). Jurnal Sains Dan Seni Pomits, 2: 1–6.

Isah T. 2019. Stress and defense responses in plant secondary metabolites production. Biological Research, 52. https://doi.org/10.1186/s40659-019-0246-3

Javed R, & Gürel E. 2019. Salt stress by NaCl alters the physiology and

biochemistry of tissue culture-grown Stevia rebaudiana Bertoni. Turkish Journal of Agriculture and Forestry, 43: 11–20. https://doi.org/10.3906/tar-1711-71

Kumari A, Kumar V, Ovadia R, & Oren-Shamir M. 2023. Phenylalanine in motion: A tale of an essential molecule with many faces. Biotechnology Advances, 68. https://doi.org/10.1016/j.biotechadv.2023.108246

Li JL, Liu XY, Xie JT, Di YL, Zhu FX. 2015. A comparison of different estimation methods for fungicide EC50 and EC95 values. Journal of Phytopathology, 163: 239-244. https://doi.org/10.1111/jph.12312

Li SM, Zheng HX, Zhang XS, & Sui N. 2021. Cytokinins as central regulators during plant growth and stress response. Plant Cell Reports, 40: 271–282. https://doi.org/10.1007/s00299-020-02612-1

Mao Y, Luo J, & Cai Z. 2025. Biosynthesis and regulatory mechanisms of

plant flavonoids: A review. Plants, 14: 1–30. https://doi.org/10.3390/plants14121847

Mastuti R, & Rosyidah M. 2021. Diversity of bioactive secondary metabolites produced by medicinal plants of Physalis angulata L. (Ciplukan). IOP Conference Series: Earth and Environmental Science, 743. https://doi.org/10.1088/1755-1315/743/1/012081

Mastuti R, Widoretno W, & Harijati N. 2020. Kultur kalus tanaman obat ciplukan (Physalis angulata L.). Biotropika: Journal of Tropical Biology, 8: 26–35.

Munns R, & Tester M. 2008. Mechanisms of salinity tolerance. Annual Review of Plant Biology, 59: 651–681. https://doi.org/10.1146/annurev.arplant.59.032607.092911

Nuranda A, Saleh C, & Yusuf B. 2016. Potensi tumbuhan ciplukan (Physalis angulata Linn.) sebagai antioksidan alami. Jurnal Atomik, 1: 5–9.

Oluwole O, Fernando WB, Lumanlan J, Ademuyiwa O, & Jayasena V. 2022. Role of phenolic acid, tannins, stilbenes, lignans and flavonoids in human health – a review. International Journal of Food Science and Technology, 57: 6326–6335. https://doi.org/10.1111/ijfs.15936

Ortiz A, & Sansinenea E. 2023. Phenylpropanoid derivatives and their role in plants’ health and as antimicrobials. Current Microbiology, 80: 1–13. https://doi.org/10.1007/s00284-023-03502-x

Panche AN, Diwan AD, & Chandra SR. 2016. Flavonoids: An overview. Journal of Nutritional Science, 5. https://doi.org/10.1017/jns.2016.41

Peterson MA, McMaster SA, Riechers DE, Skelton J, & Stahlman PW. 2016. 2,4-D past, present, and future: A review. Weed Technology, 30: 303–345. https://doi.org/10.1614/wt-d-15-00131.1

Pungin A, Lartseva L, Loskutnikova V, Shakhov V, Popova E, Skrypnik L, & Krol O. 2023. Effect of salinity stress on phenolic compounds and antioxidant activity in halophytes Spergularia marina (L.) Griseb. and Glaux maritima L. cultured in vitro. Plants, 12: 1–20. https://doi.org/10.3390/plants12091905

Qari SH, & Tarbiyyah I. 2021. The genetic regulation of secondary metabolic pathways in response to salinity and drought as abiotic stresses. Applied Sciences (Switzerland), 11: 1–18. https://doi.org/10.3390/app11156668

Rasud Y, & Bustaman. 2020. Induksi kalus secara in vitro dari daun cengkeh (Syizigium aromaticum L.) dalam media dengan berbagai konsentrasi auksin. Jurnal Ilmu Pertanian Indonesia, 25: 67–72. https://doi.org/10.18343/jipi.25.1.67

Rybin DA, Sukhova AA, Syomin AA, Zdobnova TA, Berezina EV, & Brilkina AA. 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: 1–18. https://doi.org/10.3390/plants13233279

Samanta A, Das G, & Das SK. 2011. Roles of flavonoids in plants. International Journal of Pharmaceutical Science and Technology, 6: 12-35.

Sari DA, Slameto, & Restanto DP. 2014. Induksi tunas kentang (Solanum tuberosum L.) menggunakan BAP (benzyl amino purine). Berkala Ilmiah Pertanian, 1: 1–4.

Shen N, Wang T, Gan Q, Liu S, Wang L, & Jin B. 2022. Plant flavonoids: Classification, distribution, biosynthesis, and antioxidant activity. Food Chemistry, 383. https://doi.org/10.1016/j.foodchem.2022.132531

Shimamura T, Sumikura Y, Yamazaki T, Tada A, Kashiwagi T, Ishikawa H, Matsui, T., Sugimoto N, Akiyama H, & Ukeda H. 2014. Applicability of the DPPH assay for evaluating the antioxidant capacity of food additives – Inter-laboratory evaluation study –. Analytical Science, 30: 717–721.

Sitorus EN, Hastuti ED, & Setiari N. 2011. Induksi kalus binahong (Basella rubra L.) secara in vitro pada media Murashige & Skoog dengan konsentrasi sukrosa yang berbeda. Berkala Ilmiah Biologi, 13.

Su Y, Liu Y, & Zhang X. 2011. Auxin-cytokinin interaction regulates meristem development. Molecular Plant, 4: 616–625. https://doi.org/10.1093/mp/ssr007

Thorpe T. 2012. History of plant tissue culture. Methods in Molecular Biology, 877: 9–27. https://doi.org/10.1007/978-1-61779-818-4_2

Vargas-Ponce O, Martínez JS, del Pilar Zamora Tavares M, & Mares LEV. 2016. Traditional management of a small-scale crop of Physalis angulata in Western Mexico. Genetic Resources and Crop Evolution, 63: 1383–1395. https://doi.org/10.1007/s10722-015-0326-3

Wartina R, Warnita, & Frizia F. 2011. Pengaruh NAA dan BAP terhadap regenerasi kalus kentang (Solanum tuberosum L.) hasil induksi mutasi ethyl methane sulphonate (EMS). Jurnal Tanaman Hortikultura, 2: 1–9.

Wołosiak R, Drużyńska B, Derewiaka D, Piecyk M, Majewska E, Ciecierska M, Worobiej E, & Pakosz P. 2022. Verification of the conditions for determination of antioxidant activity by ABTS and DPPH assays — A practical approach. Molecules, 27. https://doi.org/10.3390/molecules27010050

Xu L & Wang X. 2025. A Comprehensive Review of Phenolic Compounds in Horticultural Plants. International Journal of Molecular Sciences, 26: 1–25. https://doi.org/10.3390/ijms26125767

Yaowachai W, Luecha P, & Taratima W. 2026. Optimizing salinity and drought stress elicitation to enhance phytochemical accumulation and antioxidant activity in Rhinacanthus nasutus (L.) Kurz calli. International Journal of Agriculture and Biosciences, 15: 679–692. https://doi.org/10.47278/journal.ijab/2025.220

Yari P, Alirezalu A, & Khalili S. 2025. A comparative study of chemical composition, phenolic compound profile and antioxidant activity of wild grown, field and greenhouse cultivated Physalis (P. alkekengi and P. peruviana). Food Production, Processing and Nutrition, 7. https://doi.org/10.1186/s43014-024-00287-9

Yücesan BB, Mohammed A, Arslan M, & Gürel E. 2015. Clonal propagation

and synthetic seed production from nodal segments of Cape gooseberry (Physalis peruviana L.), a tropical fruit plant. Turkish Journal of Agriculture and Forestry, 39: 797–806. https://doi.org/10.3906/tar-1412-86

DOI: https://doi.org/10.24198/kultivasi.v25i1.69556