Callus Induction and Organogenesis of Seven Local Pomegranate Punica protopunica. L Plants Derived from Seeds

Volume 16, Issue 3
Summer 2025
Page 52-59

Document Type : Research Paper

Authors

1 Biology Dept. College of Science, University of Duhok

2 Scientific Research Center, College of science, University of Duhok, Duhok. IRAQ

3 Scientific Research center, College of Science, Duhok University

Abstract
Abstract
This study aimed to establish callus induction and organogenesis to create a reliable micropropagation protocol for the mass production of pomegranates from seeds, which will facilitate future molecular research. In vitro propagation provides a viable method for large-scale production of pomegranate planting material, supporting commercialization efforts. Micropropagation can be achieved through meristem regeneration, adventitious shoot formation, or somatic embryogenesis. Established protocols typically use explants such as leaf segments and cotyledons for callus regeneration.
Seven local cultivars of pomegranate (Punica granatum L.) - Masafik, Melisse, Radisho, Armishte, Sahraban, Halapja, and Dwarf pomegranate - were cultivated in the Kurdistan region of Iraq from October 2020 to February 2023. The purpose was to test their callus induction and organogenesis to establish efficient protocols for mass production and future molecular studies. The results showed that treating the seeds by soaking them in water and a salicylic acid solution separately for 24 hours increased seed germination in some cultivars. It was observed that the leaves responded better than the cotyledons for callus induction. Furthermore, the combination of MS salts plus 1.5 mgl-1 BAP supplemented with 1.5 mgl-1 NAA significantly increased callus induction. In addition, Armishte and Shahraban explants exhibited positive responses for callus induction, followed by Masafik, Radisho, Melisse, Halapja, and Dwarf. The combination of 3.0 mglˉ¹ BAP with 0.5 mgl-1 NAA resulted in a higher percentage of shoot organogenesis and regenerated plantlets derived from callus. Conversely, 2.0 mglˉ¹ BAP + 0.5 mglˉ¹ NAA and 1.0 mgl-1 BAP + 0.5 mglˉ¹ NAA protocols showed lower responses, respectively.

Keywords

Subjects
  • Basu, A. and Penugonda, K. (2009) Pomegranate juice: a heart-healthy fruit juice. Nutrition Reviews, 67(1):49-56. https://doi.org/10.1111/j.1753-4887.2008.00133.x.
  • Khan, N.; Fahad, S. and Naushad, M. (2020). Pomegranates economics and medicinal aspects in the world. SSRN Electronic Journal. DOI:2139/ssrn.3597891
  • Sandoval, C. O.; Fabela-Iiiescas, H.E.; Martinez, E. F.; Ortiz-Rodriguez, M. A.; Cortes, R.C.; Ariza-Ortega, J. A.; Hernandez-Gonzalez, J. C.; Olivo, D.; Miller, H. B. and Cabrera, G. B. (2022) Potential mechanisms of the improvement of glucose homeostasis in type 2 diabetes by pomegranate juice. Antioxidants, 11(3):553. doi: 3390/antiox11030553.
  • Adhami, V. M.; Khan, N. and Mukhtar, H. (2009). Cancer chemoprevention by pomegranate: Laboratory and clinical evidence. Nutr Cancer, 61(6):811-815. https://doi.org/10.1080 /01635580903285064.
  • Syed, D. N.; Chamcheu, J. C.; Adhami, V. M. and Mukhtar, H. (2013). Pomegranate extracts and cancer prevention: molecular and cellular activities. Anticancer Agents Med Chem, 13(8):1149-1161. https://doi.org/10.2174/1871520611313080003.
  • Zarfeshany, A.; Asgary, S. and Javanmard, S. H. (2014). Potent health effects of pomegranate. Advanced Biomedical Research, 3(100): 1-8. doi: 4103/2277-9175.129371
  • Emami Kazemabad, M. J.; Toni, S. A.; Tizro, N.; Dadkhah, P. A.; Amani, H.; Rezayat, S. A.; Sheikh, Z.; Mohammad, M.; Alijanzadeh, D.; Alimohammadi, F.; Shahrokhi, M.; Erabi, G.; Noroozi, M.; Karimi, M. A.; Honari, S. and Deravi, N. (2022). Pharmacotherapeutic potential of pomegranate in age-related neurological disorders. Frontiers in Aging Neuroscience, 14:1-27. DOI:3389/fnagi.2022.955735
  • Guranna P, Hosmani I, Sathyanarayana R, Hegde R, Hipparagi K (2017). Micropropagation in pomegranate (Punica granatum) cv. Bhagwa through indirect organogenesis and assessment of genetic fidelity by RAPD marker. Biotechnology Journal International. 20: 1-8. DOI:10.9734/BJI/2017/38806
  • [9] Omura M, Matsuta N, Moriguchi T and Kazaki I (1987). Adventitious shoot and plantlet formation from cultured pomegranate leaf explants. Sci. 22: 133-134. DOI: https://doi.org/10.21273/HORTSCI.22.1.133
  • Kanwar K, Joseph J and Deepika R (2010). Comparison of in vitro regeneration pathways in Punica granatum Plant cell Tiss. Organ cult. 100: 199-207. DOI:10.1007/s11240-009-9637-4
  • Duncan, D.B. (1955). Multiple ranges and multiple F tests. Biometrics. 11: 1-42. SPSS (2019). Statistical Package for Social Sciences, Ver. 26, User’s guide, IBM publications, USA. https://doi.org/10.2307/3001478
  • Gupta, S.M.; Pandey, P.; Grover, A. and Ahmed, Z. (2011). Breaking seed dormancy in Hip-pophae salicifolia, a high value medicinal plant. Mol. Biol. Plants. 17:403-406. https://doi.org/10.1007/s12298-011-0082-6.
  • Gadapa, M. and Singh, G. (2022). Recent trends in pomegranate propagation: A The Pharma Innovation Journal, 11(5):405-414.https://www.  thepharmajournal.com/.
  • Taheri, M. N.; Gholami, M.; Baninasab, B.; Mobli, M. and Moradi, S. (2014). Pomegranate seed germination and dormancy-breaking techniques. Seed Technology, 36(2):139-149. https://baninasab.iut.ac.ir/pomegranate-seed-germination-and-dormancy-breaking-techniques.
  • Olmez, Z. (2011). Effects of cold stratification and H2SO4 on seed germination of sea buckthorn (Hippophae rhamnoides ). Afr. J. Biotechnol. 10:4586-4590.   http://www.academicjournals.org/AJB
  • Deepika, R. and Kanwar, K. (2010). In vitro regeneration of Punica granatum L. plants from different juvenile explants. Journal of Fruit and Ornamental Plant Research, 18(1):5-22.    Corpus ID: 83669354. http://agro.icm.edu.pl/agro/element/bwmeta1.element.agro-article-c3799408-fd5c-439e-b58e-f7c8c41565b5.
  • Manjitha, K. G. L.; Gunasena, M. D. K. M.; Senarath, W. T. P. S. K. and Samanmalee, L. G. I. (2018). In vitro plant regeneration through organogenesis and somatic embryogenesis of Punica granatum (Lythraceae). International Journal of Scientific & Engineering Research, 9(11):390-395. http://dr.lib.sjp.ac.lk/handle/123456789/8828
  • Bonyanpour, A. and Khosh-Khui, M. (2013). Callus Induction and Plant Regeneration in Punica granatum L. ʻNanaʼ from Leaf Explants. Journal of Central European Agriculture 14(3):928-936. DOI: https://doi.org/10.5513/JCEA01/14.3.1285.
  • Choudhary, R.; Jakhar, M.; Kumar, R.; Jat, H.; Shekhawat, K.; Kumawat, S. and Choudhary, R. N. (2018). In vitro callus induction from different explants in pomegranate (Punica granatum) Journal of Pharmacognosy and Phytochemistry, 7(4): 2656-2659. ·https://www.phytojournal.com/archives/2018/vol7issue4/PartAS/7-3-706-336. pdf.
  • Stimela, T.; Kasili, R. W. and Mamati, E. G. (2019). Pomegranate micropropagation, callogenesis and genetic integrity assessment using simple sequence repeat markers. Journal of Agricultural Science, 11(1): 237-250. https://doi.org/10.5539/jas.v11n1p237
  • Danial, G.H.; Ibrahim, D. A.; Yousef, A. N. and Elyas, S.B. (2019). Rapid protocol of Aloe vera In vitro Iraqi Journal of Agricultural Sciences, 50(5):1377-1382. DOI:10.36103/ijas.v50i5.8041
  • Raspor, M.; Motyka, V.; Kaleri, A.R.; Ninkovic, S.; Tubic, L.; Cingel, A. and Cosic, T. (2021). Integrating the roles for cytokinin and auxin in de novo shoot organogenesis: from hormone uptake to signaling outputs. International Journal of Molecular Science, 22, 8554. https://doi.org/10.3390/ijms22168554.
  • Yousef, A. N.; Danial, G. H.; Ibrahim, D. A. and Barakat, S. E. (2024). Propagation and callus regeneration of potato (Solanum tuberosum) cultvar ,desiree, under salt stress conditions. Science Journal of University of Zakho, 12(3):294-298. https://doi.org/10.25271/sjuoz.2024.12.3.1236
  • Kumar R.; Jakhar, M.L; Verma R. and Jat H.R. (2017).Pomegranate Micropropagation: A Review. Int. J Pure   Biosci2017;5(5):1138- 149. DOI:10.18782/2320-7051.5124
  • Permadi, N.; Akbari, S. I.; Prismantoro, D.; Indriyani, N. N.; Nurzaman, M.; Alhasnawi, A. N.; Doni, F. and 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.
  • Naik, S. K. and Chand, P. K. (2011). Tissue Culture Mediated Biotechnological Intervention in Pomegranate: A Review. Plant Cell Rep. 30: 707–721. https://doi.org/10.1007/s00299-010-0969-7
  • Singh, N. V.; Awachare, C.; Salutgi, U.; Salunkhe, o.; Patil, P. G. and Marathe, R. A. (2022). Micro-propagation and bio-priming in pomegranate imperative for quality planting material. Modern Concepts and Developments in Agronomy, 11(1): 1081-1084. DOI: 31031/MCDA.2022.11.000753
  • Ewes, H. K. M.; Abdel-Raheemb, A. T.; Abu Salhab, A. E. and Rayana, A. O. (2023). Effect of different concentration and combination of some plant growth regulators on Punica granatum anther culture. Current Chemistry Letters, 12: 257-264. DOI: 5267/j.ccl.2023.1.003
  • Pasternak, T. P and Steinmacher, D. (2024), Plant growth regulation in cell and tissue culture in vitro. Plants13(2), 327. https://doi.org/10.3390/plants13020327