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Vegetos

, Volume 32, Issue 2, pp 181–189 | Cite as

Cloning of mature pomegranate (Punica granatum) cv. Jalore seedless via in vitro shoot production and ex vitro rooting

  • Rachana Modi DineshEmail author
  • Ashok Kumar Patel
  • J. B. Vibha
  • Smita Shekhawat
  • N. S. Shekhawat
Research Articles

Abstract

A novel approach of in vitro shoot amplification and ex vitro rooting for cloning of mature plants of Punica granatum cv. Jalore seedless/soft-seeded has been defined. Surface-sterilized nodal shoots were cultured for axillary meristem activation, bud breaking and shoot amplification. Multiple shoots differentiated by bud breaking from 82.8% of the explants on Murashige and Skoog (Physiol Plant 15:473–497, 1962) MS medium with 13.32 µM 6-benzylaminopurine (BAP). These were amplified by subculturing of fresh shoots and by repeated transfer of mother explants on MS medium + BAP or Kinetin (Kin)/Furfurylaminopurine (FAP) in combination with Indole acetic acid (IAA) or α-naphthalene acetic acid (NAA). MS medium with BAP 2.22 µM + IAA 0.57 µM was found to be most suitable for shoot (14.2 ± 1.03 shoots per culture vessel) multiplication. On half-strength MS medium with 9.84 µM of Indole butyric acid (IBA) and 16.65 µM of activated charcoal (AC), 72.9% of the micro-shoots rooted. Alternatively, base(s) of the micro-shoots treated with 1476 µM of IBA for 300 s. and transplanted on autoclaved soilrite (moistened with one-fourth strength of MS macro-salts) in glass bottles (420 ml; 70 mm diameter × 130 mm height). More than 85% of the IBA-treated shoots rooted ex vitro in a greenhouse. Ex vitro rooting of cloned shoots is a new approach for propagation of pomegranate. The process described is different and superior to all the described tissue culture methods for cloning of pomegranate. This is faster, cost-effective and saves resources enabling acclimatization/hardening with ease while minimizing microbial contamination, thus ensuring quick field transfer of hardened plantlets. This can be applied for mass and clonal propagation of selected genotypes and also for long-term conservation of germplasm of P. granatum.

Keywords

Ex vitro rooting Horticultural fruit plant Micropropagation Lythraceae P. granatum cv. Jalore seedless 

Abbreviations

AC

Activated charcoal

BAP

6-benzylaminopurine

DMRT

Duncan’s multiple range test

IAA

Indole-3-acetic acid

IBA

Indole-3-butyric acid

Kin

Kinetin

MS

Murashige and Skoog (1962)

NAA

α-Naphthalene acetic acid

PFD

Photon flux density

PGRs

Plant growth regulators

RH

Relative humidity

Notes

Acknowledgements

RD acknowledges the University Grants Commission (UGC), Government of India for startup Grant [F-30-16/2014(BSR)]. RD and AKP express their gratitude to the UGC for awarding the Special Assistance Programme (SAP) in the form of Centre of Advanced Study (CAS) to the Department of Botany, Jai Narain Vyas University, Jodhpur [F.5-1/2013(SAP-II)].

Author’s contribution

RMD designed, conducted the experiments and wrote the first draft of the manuscript. AKP and SS assisted in analyzing the data and organized them in tables and figures. VJB looked after the rooting and hardening experiments. NSS conceptualized, supervised the research and prepared the final draft of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they do not have any conflicts of interest.

References

  1. Barathikannan K, Venkatadri B, Khusro A, Al-Dhabi NA, Agastian P, Arasu MV, Choi HS, Kim YC (2016) Chemical analysis of Punica granatum fruit peel and its in vitro and in vivo biological properties. BMC Complement Altern Med 16:264CrossRefGoogle Scholar
  2. Bhansali RR (1990) Somatic embryogenesis and regeneration of plantlets in pomegranate. Ann Bot 66:249–253CrossRefGoogle Scholar
  3. Bonal D, Monteuuis O (1997) Ex vitro survival, rooting and initial development of in vitro rooted vs unrooted microshoots from juvenile and mature Tectona grandis genotypes. Silvae Genet 46:301–306Google Scholar
  4. Bonga JM, von Aderkas P (1992) In vitro culture of trees. Kluwer Academic Publishers, Dordrecht, p 236CrossRefGoogle Scholar
  5. Chandra R, Babu KD (2010) Propagation of pomegranate – A review. Fruit Veg Cereal Biotechnol 4(special issue 2):51–55Google Scholar
  6. Chauhan RD, Kanwar K (2012) Biotechnological advances in pomegranate (Punica granatum L.). In Vitro Cell Dev Biol Plant 48:579–594CrossRefGoogle Scholar
  7. Davarpanah S, Tehranifar A, Abadia J, Val J, Davarynejad G, Aran M, Khorassani R (2018) Foliar calcium fertilization reduces fruit cracking in pomegranate (Punica granatum cv. Ardestani). Sci Hortic 230:86–91CrossRefGoogle Scholar
  8. Debergh PC, Maene LJ (1981) A scheme for commercial propagation of ornamental plants by tissue culture. Sci Hortic 14:335–345CrossRefGoogle Scholar
  9. Deepika R, Kanwar K (2008) Efficient in vitro shoot multiplication and root induction enhanced by rejuvenation of microshoots in Punica granatum cv. Kandhari Kabuli. In: National seminar on physiological and biotechnological intervention in Pomegranate: a review. Plant Cell Rep 30:707–721.Google Scholar
  10. Deepika R, Kanwar K (2010) In vitro regeneration of Punica granatum L. plants from different juvenile explants. J Fruit Ornament Plant Res 18:5–22Google Scholar
  11. Dhinesh KV, Ramasamy D (2016) Pomegranate processing and value addition: review. J Food Process Technol 7:565Google Scholar
  12. Dumas E, Monteuuis O (1995) In vitro rooting of micropropagated shoots from juvenile and mature Pinus pinaster explants: influence of activated charcoal. Plant Cell Tiss Organ Cult 40:231–235CrossRefGoogle Scholar
  13. Duncan DB (1955) Multiple range and multiple F tests. Biometrics 11:1–42CrossRefGoogle Scholar
  14. El-Agamy SZ, Mostafa RAA, Shaaban MM, El-Mahdy MT (2009) In vitro propagation of Manfalouty and Nab El-gamal pomegranate cultivars. Res J Agric Biol Sci 5:1169–1175Google Scholar
  15. Gharib E, Kouhsari SM, Izad M (2018) Punica granatum L. fruit aqueous extract suppresses reactive oxygen species-mediated p53/p65/miR-145 expressions followed by elevated levels of irs-1 in alloxan-diabetic rats. Cell J 19:520–527Google Scholar
  16. Hackett WP (1988) Donor plant maturation and adventitious root formation. In: Davis TD, Haissig BE, Sankhla N (eds) Adventitious root formation in cuttings. Dioscorides Press, Oregon, pp 11–28Google Scholar
  17. Holland D, Habit K, Bar-Yaakov I (2009) Pomegranate: botany horticulture and breeding. Hortic Rev 35:127–191CrossRefGoogle Scholar
  18. Jaidka K, Mehra PN (1986) Morphogenesis in Punica granatum (pomegranate). Can J Bot 64:1644–1653CrossRefGoogle Scholar
  19. Johanningsmeier SD, Harris GK (2011) Pomegranate as a functional food and nutraceutical source. Ann Rev Food Sci Technol 2:181–201CrossRefGoogle Scholar
  20. Kaji BV, Abbasifar A (2017) Transformation of pomegranate (Punica granatum) a difficult—to-transform tree. Biocatal Agric Biotechnol 10:46–52CrossRefGoogle Scholar
  21. Kaji BV, Ershadi A, Tohidfar M (2013) In vitro propagation of two Iranian commercial pomegranates (Punica granatum L.) cvs. ‘Malas Saveh’ and ‘Yusef Khani’. Physiol Mol Biol Plants 19:597–603CrossRefGoogle Scholar
  22. Kaji BV, Ershadi A, Tohidfar M (2014) Agrobactermiu–mediated transformation of pomegranate (Punica granatum L.) ‘Yousef Khani’ using the gus reporter gene. Int J Hortic Sci Technol 1:31–41Google Scholar
  23. Kanupriya C, Radhika V, Ravishankar KV (2013) Mining of miRNAs in pomegranate (Punica granatum L.) by pyrosequencing of part of the genome. J Horti Sci Biotech 88:735–742CrossRefGoogle Scholar
  24. Kanwar K, Thakur K, Ferma V, Sharma RK (2010) Genetic variability of in vitro raised plants of Punica granatum L by RAPDs. Fruit Veg Cereal Sci Biotechnol 4:144–147Google Scholar
  25. Kharchoufi S, Licciardello F, Siracusa L, Muratore G, Hamdi M, Restuccia C (2018) Antimicrobial and antioxidant features of ‘Gabsiʼ pomegranate peel extracts. Ind Crop Prod 111:345–352CrossRefGoogle Scholar
  26. Kothawale SP, Kshirsagar AB, Zahid IH (2015) Response of BA and NAA on shoot proliferation of Punica granatum L. Bionano Frontier 8:18–23Google Scholar
  27. Kotwal GJ (2007) Genetic diversity-independent neutralization of pandemic viruses (e.g. HIV) potentially pandemic (e.g. H5N1 strain of influenza) and carcinogenic (e.g. HBV and HCV) viruses and possible agents of bioterrorism (variola) by enveloped virus neutralizing compounds (EVNCs). Vaccine 26:3055–3058CrossRefGoogle Scholar
  28. Lodha D, Patel AK, Rai MK, Shekhawat NS (2014) In vitro plantlet regeneration and assessment of alkaloid contents from callus cultures of Ephedra foliata (Unth phog), a source of anti-asthmatic drugs. Acta Physiol Plant 36:3071–3079CrossRefGoogle Scholar
  29. Ludwig-Muller J (2000) Indole-3-butyric acid in plant growth and development. Plant Growth Regul 32:219–230CrossRefGoogle Scholar
  30. Mahishi DM, Muralikrishna A, Shivashankar G, Kulkarni RS (1991) Shoot tip culture method for rapid clonal propagation of pomegranate (Punica granatum L.). In: Prakash J, Pierik RLM (eds) Horticulture—new technologies and applications: proceedings of the international seminar on new frontiers in horticulture. Kluwer Academic Publishers, Dordrecht, pp 215–217Google Scholar
  31. Mandal A, Bhatia D, Bishayee A (2017) Anti-inflammatory mechanism involved in pomegranate-mediated prevention of breast cancer: the role of NF-κB and Nrf2 signaling pathways. Nutrients 9:436CrossRefGoogle Scholar
  32. McClelland MT, Smith MAL, Carothers ZB (1990) The effects of in vitro and ex vitro root initiation on subsequent microcutting root quality in three woody plants. Plant Cell Tiss Organ Cult 23:115–123CrossRefGoogle Scholar
  33. McCown BH (1988) Adventitious rooting of tissue cultured plants. In: Davis TD, Haissig BE, Sankhla N (eds) Adventitious root formation in cuttings. Dioscorides press, Oregon, pp 289–302Google Scholar
  34. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  35. Murkute AA, Patil S, Patil BN, Kumari M (2002) Micropropagation in pomegranate, callus induction and differentiation. South Indian Hortic 50:49–55Google Scholar
  36. Murkute AA, Patil S, Singh SK (2004) In vitro regeneration in pomegranate cv. Ganesh from mature trees. Indian J Hortic 61:206–208Google Scholar
  37. Naik SK, Chand PK (2003) Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.). J Plant Phsyiol 160:423–430CrossRefGoogle Scholar
  38. Naik SK, Chand PK (2011) Tissue culture-mediated biotechnological intervention in pomegranate: a review. Plant Cell Rep 30:707–721CrossRefGoogle Scholar
  39. Naik SK, Pattnaik S, Chand PK (1999) In vitro propagation of pomegranate (Punica granatum L. cv. Ganesh) through axillary shoot proliferation from nodal segments of mature tree. Sci Hortic 79:175–183CrossRefGoogle Scholar
  40. Naik SK, Pattnaik S, Chand PK (2000) High frequency axillary shoot proliferation and plant regeneration from cotyledonay nodes of pomegranate (Punica granatum L.). Sci Hortic 82:261–270CrossRefGoogle Scholar
  41. 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
  42. Niu J, Cao D, Li H, Xue H, Chen L, Liu B, Cao S (2018) Quantitative proteomics of pomegranate varieties with contrasting seed hardness during seed development stages. Tree Genet Genomes 14:14CrossRefGoogle Scholar
  43. Omura M, Matsuta N (1990) Suspension culture and plantlet regeneration in dwarf pomegranate (Punica granatum L. var. nana Pers.). Bull Fruit Tree Res 17:19–33Google Scholar
  44. Panwar D, Patel AK, Shekhawat NS (2018) An improvised shoot amplification and ex vitro rooting method for offsite propagation of Tinospora cordifolia (Willd.) Miers: a multi-valued medicinal climber. Ind J Plant Physiol.  https://doi.org/10.1007/s40502-018-0350-3
  45. Patel AK, Agarwal T, Phulwaria M, Kataria V, Shekhawat NS (2014) An efficient in vitro plant regeneration system from leaf of mature plant of Leptadenia reticulata (Jeewanti): a life giving endangered woody climber. Ind Crop Prod 52:499–505CrossRefGoogle Scholar
  46. Patel AK, Lodha D, Ram K, Shekhawat S, Shekhawat NS (2016) Evaluation of physiochemical factors affecting high-frequency plant regeneration of Blyttia spiralis (synonym: Pentatropis spiralis), a threatened climber of medicinal value. Vitro Cell Dev Biol Plant 52:10–19CrossRefGoogle Scholar
  47. Phulwaria M, Rai MK, Patel AK, Kataria V, Shekhawat NS (2013) A genetically stable rooting protocol for propagating a threatened medicinal plant—Celastrus paniculatus. AoB Plants 5:pls054Google Scholar
  48. Phulwaria M, Patel AK, Rathore JS, Ram K, Shekhawat NS (2014) An improved micropropagation and assessment of genetic stability of micropropagated Salvadora oleoides using RAPD and ISSR markers. Acta Physiol Plant 36:1115–1122CrossRefGoogle Scholar
  49. Pijut MP, Beasley RR, Lawson SS, Palla KJ, Stevens EM, Wang Y (2012) In vitro propagation of tropical hardwood tree species: a review. Propagat Ornament Plant 12:25–51Google Scholar
  50. Qin G, Xu C, Ming R, Tang H, Guyot R, Kramer EM, Hu Y, Yi X, Qi Y, Xu X, Gao Z, Pan H, Jian J, Tian Y, Yue Z, Xu Y (2017) The pomegranate (Punica granatum L.) genome and the genomics of punicalagin biosynthesis. Plant J 91:11CrossRefGoogle Scholar
  51. Rahimi HR, Arastoo M, Ostad SN (2011) A comprehensive review of Punica granatum (pomegranate) properties in toxicological, pharmacological, cellular and molecular biology researches. Iran J Pharm Res 11:385–400Google Scholar
  52. Rathore JS, Rathore MS, Shekhawat NS (2005) Micropropagation of Maerua oblongifolia—a liana of arid areas. Phytomorph 55:241–247Google Scholar
  53. Reddy BU, Mullick R, Kumar A, Sudha G, Srinivasan N, Das S (2014) Small molecule inhibitors of HCV replication from Pomegranate. Sci Rep 4:5411CrossRefGoogle Scholar
  54. Sanchez MC, Ballester A, Vieitez AM (1997) Reinvigoration treatments for the micropropagation of mature chestnut trees. Ann For Sci 54:359–370CrossRefGoogle Scholar
  55. Shao TZ, Chen CL, Deng XX (2003) In vitro induction of tetraploid in pomegranate (Punica granatum). Plant Cell, Tissue Organ Cult 75:241–246CrossRefGoogle Scholar
  56. Sharon M, Sinha S (2000) Plant regeneration from cotyledonary node of Punica granatum L. Indian J Plant Physiol 5:344–348Google Scholar
  57. Shaygannia E, Bahmani M, Zamanzad B, Rafieian-Kopaei M (2016) A review study on Punica granatum L. J Evid Based Complementary Altern Med 21:221–227CrossRefGoogle Scholar
  58. Shekhawat NS, Rathore TS, Singh RP, Deora NS, Rao SR (1993) Factors affecting in vitro cloning of Prosopis cineraria. Plant Growth Regul 12:273–280CrossRefGoogle Scholar
  59. Singh SK, Khawale RN (2006) Plantlet regeneration from the nodal segments of pomegranate (Punica granatum L.) cv. Jyoti. In: Kumar A, Roy S (eds) Plant biotechnology and its applications in tissue culture. New Delhi, IK Int Pvt Ltd, pp 107–113Google Scholar
  60. Singh SK, Singh A, Singh NV, Ramajayam D (2010) Pomegranate tissue culture and biotechnology. Fruit. Veg Cereal Sci Biotech 4:35–44Google Scholar
  61. Stover E, Mercure EW (2007) The pomegranate: a new look at the fruit of paradise. HortScience 42:1088–1092CrossRefGoogle Scholar
  62. Teixeira da Silva JA, Rana TS, Narzary D, Verma N, Meshram DT, Ranade SA (2013) Pomegranate biology and biotechnology: a review. Sci Hortic 160:85–107CrossRefGoogle Scholar
  63. The Plant List (2018) http://www.theplantlist.org/tpl1.1/record/kew-2536844. Accessed 5 Feb 2018
  64. Thomas TD (2008) The role of activated charcoal in plant tissue culture. Biotechnol Adv 26:618–631CrossRefGoogle Scholar
  65. Verma N, Mohanty A, Lal A (2010) Pomegranate genetic resources and germplasm conservation: a review. Fruit. Veg Cereal Sci Biotech 4:120–125Google Scholar
  66. Wu S, Tian L (2017) Diverse phytochemicals and bioactivities in the ancient fruit and modern functional food pomegranate (Punica granatum). Molecules 22:1606CrossRefGoogle Scholar
  67. Xue H, Cao S, Li H, Zhang J, Niu J, Chen L, Zhang F, Zhao D (2017) De novo transcriptome assembly and quantification reveal differentially expressed genes between soft-seed and hard-seed pomegranate (Punica granatum L.). PLoS One 12:e0178809Google Scholar
  68. Yang Y, Xiu J, Zhang L, Qin C, Liu J (2012) Antiviral activity of punicalagin toward human enterovirus 71 in vitro and in vivo. Phytomedicine 20:67–70CrossRefGoogle Scholar
  69. Yuan Z, Fang Y, Zhang T, Fei Z, Han F, Liu C, Liu M, Xiao W, Zhang W, Wu S, Zhang M, Ju Y, Xu H, Dai H, Liu Y, Chen Y, Wang L, Zhou J, Guan D, Yan M, Xia Y, Huang X, Liu D, Wei H, Zheng H (2017) The pomegranate (Punica granatum L.) genome provides insights into fruit quality and ovule developmental biology. Plant Biotechnol J.  https://doi.org/10.1111/pbi.12875

Copyright information

© Society for Plant Research 2019

Authors and Affiliations

  • Rachana Modi Dinesh
    • 1
    Email author
  • Ashok Kumar Patel
    • 1
  • J. B. Vibha
    • 1
  • Smita Shekhawat
    • 1
  • N. S. Shekhawat
    • 1
  1. 1.Biotechnology Unit, Department of Botany, UGC–Centre of Advanced Study (CAS)Jai Narain Vyas UniversityJodhpurIndia

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