Establishment of Punica granatum L. peel cell culture to produce bioactive compounds

  • Rubinovich LiorEmail author
  • Segev Barak
  • Habashi Rida
  • Con Pazit
  • Amir Rachel
Original Article


The pomegranate (Punica granatum L.) fruit harbors remarkable health-beneficial properties. Most of its healthy secondary metabolites are located in their peels. Therefore, pomegranate peels (PPs) can be used to produce high-value healthy compounds. PP cell cultures may also be an attractive alternative source since it can be established throughout the year regardless of seasonal effects. The aim of this study was to establish a novel PP cell culture for future research and biotechnological applications. We first established procedures to sterilize and control oxidative browning of the peel explants. We found that surface sterilizing with 3% sodium hypochlorite for 15 min, frequent sub-culturing of the explants on media containing polyvinylpyrrolidone plus silver nitrate was the optimal treatment for oxidative browning control and callus initiation. We also found that 6-benzylaminopurine (BAP) induced callus initiation, whereas 3-indoleacetic acid (IAA) repressed it. The callus daily growth rate was maintained at 1% regardless of the various growth media examined. Moreover, our results show that although there was a minor effect of the growth media on polyphenol accumulation and antioxidant activity, the exposure to light and high sucrose levels led to higher levels (up to 205 times than the control) of secondary metabolites such as anthocyanins, ellagic acid, punicalagin and gallic acid. In summary, this work laid the foundations for further research that will be needed to study and commercialize PP cell cultures.

Key message

In this present study, we addresses the establishment of pomegranate peel cell cultures for future research and biotechnological applications, such as production of health-beneficial secondary metabolites.


Cell culture Pomegranate peel Punica granatum Secondary metabolites 



The authors wish to thank the Israel Ministry of Economics for funding under the Kamin Fund (No. 53856) and BARD (US-Israel Binational Agricultural Research and Development Fund 364) for funding under Project No. IS-4822-15 R.

Authors contribution

L.R. was the head of this project and was the main writer of this manuscript. B.S., R.H. and P.C. were the lab technicians which carried out most of the tissue culture work. R.A. is the PI and contributed to the planning of the experiments and data analysis.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11240_2019_1609_MOESM1_ESM.jpg (82 kb)
Supplementary material 1 (JPEG 81 kb). Supplemental Fig. A1 Antioxidant capacity of callus and pomegranate fruit skin expressed as Trolox equivalent. Values are means ± SE of four biological replicates (four Petri dishes/fruits, each dish containing nine explants). The experiment was repeated at least three times, with similar results. Significant differences (Tukey-HSD, p < 0.05) are indicated by different letters above the columns
11240_2019_1609_MOESM2_ESM.jpg (40 kb)
Supplementary material 2 (JPEG 39 kb). Supplemental Fig. A2 Anthocyanin localized in the vacuole of pomegranate peel tissue culture cells elicited with sucrose at 5%


  1. Abohatem M, Zouine J, El Hadrami I (2011) Low concentrations of BAP and high rate of subcultures improve the establishment and multiplication of somatic embryos in date palm suspension cultures by limiting oxidative browning associated with high levels of total phenols and peroxidase activities. Sci Hortic (Amst) 130:344–348. CrossRefGoogle Scholar
  2. Abohatem MA, Bakil Y, Baaziz M (2017) Plant regeneration from somatic embryogenic suspension cultures of date palm. Humana Press, New York, pp 203–214Google Scholar
  3. Adams LS, Seeram NP, Aggarwal BB et al (2006) Pomegranate juice, total pomegranate ellagitannins, and punicalagin suppress inflammatory cell signaling in colon cancer cells. J Agric Food Chem 54:980–985. CrossRefGoogle Scholar
  4. Arora J, Goyal S, Ramawat KG (2010) Enhanced stilbene production in cell cultures of Cayratia trifolia through co-treatment with abiotic and biotic elicitors and sucrose. In Vitro Cell Dev Biol Plant 46:430–436. CrossRefGoogle Scholar
  5. Bhat SR, Chandel KPJ (1991) A novel technique to overcome browning in tissue culture. Plant Cell Rep 10:358–361CrossRefGoogle Scholar
  6. Borochov-Neori H, Judeinstein S, Tripler E et al (2009) Seasonal and cultivar variations in antioxidant and sensory quality of pomegranate (Punica granatum L.) fruit. J Food Compos Anal 22:189–195CrossRefGoogle Scholar
  7. Cetin ES (2014) Induction of secondary metabolite production by UV-C radiation in Vitis vinifera L. Öküzgözü callus cultures. Biol Res 47:1–7. Google Scholar
  8. Chastang T, Pozzobon V, Taidi B et al (2018) Resveratrol production by grapevine cells in fed-batch bioreactor: experiments and modelling. Biochem Eng J 131:9–16. CrossRefGoogle Scholar
  9. Chu M, Pedreño MA, Alburquerque N et al (2017) A new strategy to enhance the biosynthesis of trans-resveratrol by overexpressing stilbene synthase gene in elicited Vitis vinifera cell cultures. Plant Physiol Biochem 113:141–148. CrossRefGoogle Scholar
  10. Deepika R, Kanwar K (2010) In vitro regeneration of Punica granatum L. plants from different juvenile explants. J Fruit Ornam Plant Res 18:5–22Google Scholar
  11. Dhama K (2018) The promising pharmacological effects and therapeutic/medicinal applications of Punica granatum L. (Pomegranate) as a functional food in humans and animals. Recent Pat Inflamm Allergy Drug Discov. Google Scholar
  12. Espinosa-Leal CA, Puente-Garza CA, García-Lara S (2018) In vitro plant tissue culture: means for production of biological active compounds. Planta. Google Scholar
  13. Fazal H, Abbasi BH, Ahmad N et al (2016) Sucrose induced osmotic stress and photoperiod regimes enhanced the biomass and production of antioxidant secondary metabolites in shake-flask suspension cultures of Prunella vulgaris L. Plant Cell Tissue Organ Cult 124:573–581. CrossRefGoogle Scholar
  14. Fischer UA, Carle R, Kammerer DR (2011) Identification and quantification of phenolic compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril and differently produced juices by HPLC–DAD–ESI/MSn. Food Chem 127:807–821. CrossRefGoogle Scholar
  15. Gil MI, Tomás-Barberán FA, Hess-Pierce B et al (2000) Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J Agric Food Chem 48:4581–4589. CrossRefGoogle Scholar
  16. Guisti MM, Wrolstad RE (2001) Characterization and measurement of anthocyanins by UV–visible spectroscopy. In: Wrolstad RE, Acree TE, Decker EA et al (eds) Current protocols in food analytical chemistry. Wiley, Hoboken, pp F1.2.1–F1.2.13Google Scholar
  17. Han K-H, Sekikawa M, Shimada K et al (2007) Anthocyanin-rich purple potato flake extract has antioxidant capacity and improves antioxidant potential in rats. Br J Nutr 96:1125. CrossRefGoogle Scholar
  18. He J, Giusti MM (2010) Anthocyanins: natural colorants with health-promoting properties. Annu Rev Food Sci Technol 1:163–187. CrossRefGoogle Scholar
  19. Hiratsuka S, Onodera H, Kawai Y et al (2001) ABA and sugar effects on anthocyanin formation in grape berry cultured in vitro. Sci Hortic (Amst) 90:121–130. CrossRefGoogle Scholar
  20. Howat S, Park B, Oh IS et al (2014) Paclitaxel: biosynthesis, production and future prospects. N Biotechnol 31:242–245. CrossRefGoogle Scholar
  21. Jeandet P, Clément C, Courot E (2014) Resveratrol production at large scale using plant cell suspensions. Eng Life Sci. Google Scholar
  22. Jones AMP, Saxena PK (2013) Inhibition of phenylpropanoid biosynthesis in Artemisia annua L.: a novel approach to reduce oxidative browning in plant tissue culture. PLoS ONE 8:e76802. CrossRefGoogle Scholar
  23. Krishna H, Sairam RK, Singh SK et al (2008) Mango explant browning: effect of ontogenic age, mycorrhization and pre-treatments. Sci Hortic (Amst) 118:132–138. CrossRefGoogle Scholar
  24. Lainé E, David A (1994) Regeneration of plants from leaf explants of micropropagated clonal Eucalyptus grandis. Plant Cell Rep 13:473–476. CrossRefGoogle Scholar
  25. Lansky EP, Newman RA (2007) Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. J Ethnopharmacol 109:177–206. CrossRefGoogle Scholar
  26. Malik S, Cusidó RM, Mirjalili MH et al (2011) Production of the anticancer drug taxol in Taxus baccata suspension cultures: a review. Process Biochem 46:23–34. CrossRefGoogle Scholar
  27. Matsuura HN, Malik S, de Costa F et al (2018) Specialized plant metabolism characteristics and impact on target molecule biotechnological production. Mol Biotechnol 60:169–183. CrossRefGoogle Scholar
  28. McCown HB, Lloyd G (1981) Woody plant medium (WPM)—a mineral nutrient formulation for microculture for woody plant species. Hortic Sci 16:453Google Scholar
  29. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. CrossRefGoogle Scholar
  30. Murkute AA, Patil S (2003) Exudation and browning in tissue culture of pomegranate. Scientia 23:29–31Google Scholar
  31. Murthy HN, Lee EJ, Paek KY (2014) Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell Tissue Organ Cult 118:1–16. CrossRefGoogle Scholar
  32. Naik SK, Chand PK (2011) Tissue culture-mediated biotechnological intervention in pomegranate: a review. Plant Cell Rep 30:707–721. CrossRefGoogle Scholar
  33. Naik SK, Pattnaik S, Chand PK (2000) High frequency axillary shoot proliferation and plant regeneration from cotyledonary nodes of pomegranate (Punica granatum L.). Sci Hortic (Amst) 85:261–270. CrossRefGoogle Scholar
  34. Nasr CB, Ayed N, Metche M (1996) Quantitative determination of the polyphenolic content of pomegranate peel. Z Lebensm Unters Forsch 203:374–378. CrossRefGoogle Scholar
  35. Nataraja K, Neelambika GK (1996) Somatic embryogenesis and plantlet from petal cultures of pomegranate, Punica granatum L. Indian J Exp Biol 34:719–721Google Scholar
  36. Neyrinck AM, Van Hée VF, Bindels LB et al (2013) Polyphenol-rich extract of pomegranate peel alleviates tissue inflammation and hypercholesterolaemia in high-fat diet-induced obese mice: potential implication of the gut microbiota. Br J Nutr 109:802–809. CrossRefGoogle Scholar
  37. Ono NN, Bandaranayake PCG, Tian L (2012) Establishment of pomegranate (Punica granatum) hairy root cultures for genetic interrogation of the hydrolyzable tannin biosynthetic pathway. Planta 236:931–941. CrossRefGoogle Scholar
  38. Patel A, Patil G, Mankad M, Subhash N (2018) Optimization of surface sterilization and manipulation of in vitro conditions for reduced browning in pomegranate (Punica granatum L.) variety Bhagava. Int J Chem Stud 6:23–28Google Scholar
  39. Patil VM (2011) Micropropagation of pomegranate (Punica granatum L.) ‘Bhagava’ cultivar from nodal explant. Afr J Biotechnol 10:18130–18136. Google Scholar
  40. Ramachandra Rao S, Ravishankar GA (2002) Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv 20:101–153. CrossRefGoogle Scholar
  41. Ramirez-Estrada K, Vidal-Limon H, Hidalgo D et al (2016) Elicitation, an effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories. Molecules. Google Scholar
  42. Sae-Lee N, Kerdchoechuen O, Laohakunjit N (2014) Enhancement of phenolics, resveratrol and antioxidant activity by nitrogen enrichment in cell suspension culture of Vitis vinifera. Molecules 19:7901–7912. CrossRefGoogle Scholar
  43. Sandeep C, Suresh CK (2013) Sterilization protocol and control of polyphenol exudation in micropropagation of pomegranate cv. Bhagwa. Biosci Biotechnol Res Asia 10:305–310. CrossRefGoogle Scholar
  44. Schwartz E, Tzulker R, Glazer I et al (2009) Environmental conditions affect the color, taste, and antioxidant capacity of 11 pomegranate accessions’ fruits. J Agric Food Chem 57:9197–9209. CrossRefGoogle Scholar
  45. Seeram NP, Zhang Y, Reed JD et al (2006) Pomegranate phytochemicals. In: Seeram NP, Heber D (eds) Pomegranates: ancient roots to modern medicine. Taylor and Francis Group, New York, pp 3–29Google Scholar
  46. Singh P, Patel RM (2016) Factors affecting in vitro degree of browning and culture establishment of pomegranate. Afr J Plant Sci 10:43–49. CrossRefGoogle Scholar
  47. Teixeira da Silva JA, Rana TS, Narzary D et al (2013) Pomegranate biology and biotechnology: a review. Sci Hortic (Amst) 160:85–107. CrossRefGoogle Scholar
  48. Thanh NT, Murthy HN, Yu KW et al (2005) Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors. Appl Microbiol Biotechnol 67:197–201. CrossRefGoogle Scholar
  49. Tóth K, Haapala T, Hohtola A (1994) Alleviation of browning in oak explants by chemical pretreatments. Biol Plant 36:511–517. CrossRefGoogle Scholar
  50. Tzulker R, Glazer I, Bar-Ilan I et al (2007) Antioxidant activity, polyphenol content, and related compounds in different fruit juices and homogenates prepared from 29 different pomegranate accessions. J Agric Food Chem 55:9559–9570. CrossRefGoogle Scholar
  51. Viuda-Martos M, Fernández-López J, Pérez-Álvarez JA (2010) Pomegranate and its many functional components as related to human health: a review. Compr Rev Food Sci Food Saf 9:635–654. CrossRefGoogle Scholar
  52. Vuong TV, Franco C, Zhang W (2014) Treatment strategies for high resveratrol induction in Vitis vinifera L. cell suspension culture. Biotechnol Rep 1–2:15–21. CrossRefGoogle Scholar
  53. Wilson SA, Keen P, McKee MC et al (2018) Development of an Agrobacterium -mediated transformation method for s Taxus suspension cultures. In Vitro Cell Dev Biol Plant 54:36–44. CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Northern R&DMIGAL – Galilee Research InstituteKiryat ShmonaIsrael
  2. 2.MIGAL – Galilee Research InstituteKiryat ShmonaIsrael
  3. 3.Tel Hai CollegeKiryat ShmonaIsrael

Personalised recommendations