Exogenous polyamines enhance somatic embryogenesis and Agrobacterium tumefaciens-mediated transformation efficiency in sugarcane (Saccharum spp. hybrid)

  • Dorairaj Sathish
  • Jeevaraj Theboral
  • Venkatachalam Vasudevan
  • Gadamchetty Pavan
  • Chandrasekaran Ajithan
  • Chinnaswamy Appunu
  • Markandan ManickavasagamEmail author
Embryogenesis/Somatic Embryogenesis


The influence of exogenous polyamines (PAs) on somatic embryogenesis from immature leaf roll explants and Agrobacterium tumefaciens-mediated transformation of embryogenic callus of Saccharum spp. (sugarcane) ‘Co 86032’ was examined. Immature leaf roll-derived embryogenic callus was obtained on Murashige and Skoog with Gamborg B5 vitamins (MSB5) medium containing 3 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D). Various concentrations of PAs along with 2 mg L−1 2,4-D and 0.5 mg L−1 kinetin (Kin) were tested for somatic embryo induction. A total of 106 somatic embryos per 250 mg of callus (96.3% responsive explants) were obtained on medium supplemented with 20 mg L−1 putrescine (PUT) and 92.0% of the somatic embryos matured and produced 98 shoots per 250 mg of callus. Somatic embryo induction and maturation was increased more than two- and threefold, respectively, on PUT-supplemented medium compared to control cultures. Histomorphological analyses of various developmental stages verified somatic embryogenesis from immature leaf roll explants. The rooted plantlets were successfully hardened and exhibited normal growth. The efficiency of A. tumefaciens-mediated transformation of embryogenic callus using various concentrations of PAs in the infection, co-cultivation, and regeneration media was also assessed. Putrescine at 20 mg L−1 showed the highest regeneration (54.4%) and transformation (35.8%) efficiencies, which were more than twofold higher than the control treatment. These results demonstrate that exogenously supplied PAs improve plant regeneration using somatic embryogenesis and A. tumefaciens-mediated transformation of embryogenic callus of sugarcane ‘Co 86032’.


Agrobacterium tumefaciens-mediated transformation Histomorphology Polyamines Somatic embryogenesis Sugarcane 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. Ahloowalia BS, Maretzki A (1983) Plant regeneration via somatic embryogenesis in sugarcane. Plant Cell Rep 2:21–25PubMedPubMedCentralGoogle Scholar
  2. Ahmadi B, Shariatpanahi ME, Ojaghkandi MA, Heydari AA (2014) Improved microspore embryogenesis induction and plantlet regeneration using putrescine, cefotaxime and vancomycin in Brassica napus L. Plant Cell Tissue Organ Cult 118:497–505CrossRefGoogle Scholar
  3. Altman A, Nadel BL, Falash Z, Levin N (1990) Somatic embryogenesis in celery, induction, control and changes in polyamines and proteins. In: Nijkamp HJJ, Vander Plas LHW, Van Aartrijk J (eds) Progress in plant cell culture biology. Kluwer Academic Publisher, Dordrecht, pp 454–459CrossRefGoogle Scholar
  4. An F, Zhao Q, Ji Y, Li W, Jiang Z, Yu X, Zhang C, Han Y, He W, Liu Y, Zhang S, Ecker JR, Guo H (2010) Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis. Plant Cell 22:2384–2401PubMedPubMedCentralCrossRefGoogle Scholar
  5. Apelbaum A, Burgoon AC, Anderson JD, Lieberman M (1981) Polyamines inhibit biosynthesis of ethylene in higher plant tissue and fruit protoplasts. Plant Physiol 68:453–456. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Arencibia AD, Carmona ER, Tellez P, Chan MT, Yu SM, Trujillo LE, Oramas P (1998) An efficient protocol for sugarcane (Saccharum spp. L.) transformation of sugarcane mediated by Agrobacterium tumefaciens. Transgenic Res 7:1–10CrossRefGoogle Scholar
  7. Arruda P (2011) Perspective of the sugarcane industry in Brazil. Trop Plant Biol 4:3–8CrossRefGoogle Scholar
  8. Arun M, Chinnathambi A, Subramanyam K, Karthik S, Sivanandhan G, Theboral J, Alharbi SA, Kim CK, Ganapathi A (2016) Involvement of exogenous polyamines enhances regeneration and Agrobacterium-mediated genetic transformation in half-seeds of soybean. 3 Biotech 6(148):148. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Aydin M, Pour AH, Haliloğlu K, Tosun M (2016) Effect of polyamines on somatic embryogenesis via mature embryo in wheat. Turk J Biol 40:1178–1184CrossRefGoogle Scholar
  10. Bai B, Su YH, Yuan J, Zhang XS (2013) Induction of somatic embryos in Arabidopsis requires local YUCCA expression mediated by the down-regulation of ethylene biosynthesis. Mol Plant 6:1247–1260PubMedCrossRefGoogle Scholar
  11. Bajaj S, Rajam MV (1995) Efficient plant regeneration from long-term callus cultures of rice by spermidine. Plant Cell Rep 14:717–720PubMedCrossRefGoogle Scholar
  12. Balzon TA, Luis ZG, Scherwinski-Pereira JE (2013) New approaches to improve the efficiency of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) from mature zygotic embryos. In Vitro Cell Dev Biol Plant 49:41–50CrossRefGoogle Scholar
  13. Batista DS, Dias LLC, Macedo AF, do Rego MM, do Rego ER, Floh ELS, Finger FL, Otoni WC (2013) Suppression of ethylene levels promotes morphogenesis in pepper (Capsicum annuum L.). In Vitro Cell Dev Biol Plant 49:759–764CrossRefGoogle Scholar
  14. Belide S, Hac L, Singh SP, Green AG, Wood CC (2011) Agrobacterium-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting. Plant Methods 7:12. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Bertoldi D, Tassoni A, Martinelli L, Bagni N (2004) Polyamines and somatic embryogenesis in two Vitis vinifera cultivars. Physiol Plant 120:657–666PubMedCrossRefGoogle Scholar
  16. Bleecker AB, Kende H (2000) Ethylene: a gaseous signal molecule in plants. Annu Rev Cell Dev Biol 16:1–18PubMedCrossRefGoogle Scholar
  17. Bonneau L, Beranger-Novat N, Monin J, Martin-Tanguy J (1995) Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis. Plant Growth Regul 16:5–10CrossRefGoogle Scholar
  18. Brisibe EA, Miyake H, Taniguchi T, Maeda E (1994) Regulation of somatic embryogenesis in long term callus cultures of sugarcane (Saccharum officinarum L.). New Phytol 126:301–307CrossRefGoogle Scholar
  19. Butterfield MK, D’Hont AD, Berding N (2001) The sugarcane genome: a synthesis of current understanding, and lessons for breeding and biotechnology. Proc S Afr Sugar Technol Assoc 75:1–5Google Scholar
  20. Chiancone B, Tassoni A, Bagni N, Germanà MA (2006) Effect of polyamines on in vitro anther culture of Citrus clementina Hort. ex Tan. Plant Cell Tissue Organ Cult 87:145–153CrossRefGoogle Scholar
  21. Couee I, Hummel I, Sulmon C, Gouesbet G, Amrani AE (2004) Involvement of polyamines in root development. Plant Cell Tissue Organ Cult 76:1–10CrossRefGoogle Scholar
  22. Cvikrová M, Binarová P, Cenklová V, Eder J, Machácková I (1999) Reinitiation of cell division and polyamine and monoamine levels in alfalfa explants during somatic embryogenesis. Physiol Plant 105:330–337CrossRefGoogle Scholar
  23. Dam A, Paul S, Bandyopadhyay TK (2010) Direct somatic embryogenesis and plant regeneration from leaf explants of Limonium sinensis (Girard) Kuntze. Sci Hortic 126:253–260CrossRefGoogle Scholar
  24. De-la-Peña C, Galaz-Avalos RM, Loyola-vargas VM (2008) Possible role of light and polyamines in the onset of somatic embryogenesis of Coffea canephora. Mol Biotechnol 39:215–224PubMedCrossRefGoogle Scholar
  25. Desai NS, Suprasanna P, Bapat VA (2004) Simple and reproducible protocol for direct somatic embryogenesis from cultured immature inflorescence segments of sugarcane (Saccharum spp.). Curr Sci 87:764–768Google Scholar
  26. Fienberg AA, Choi JH, Lubich WP, Sung ZR (1984) Developmental regulation of polyamine metabolism in growth and differentiation of carrot culture. Planta 162:532–539PubMedCrossRefGoogle Scholar
  27. Galston AW (1983) Polyamines as modulators of plant development. Bioscience 33:382–388CrossRefGoogle Scholar
  28. Gamborg OL, Miller RA, Ojiama K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158CrossRefGoogle Scholar
  29. Guiderdoni E, Demarly Y (1988) Histology of somatic embryogenesis in cultured leaf segments of sugarcane plantlets. Plant Cell Tissue Organ Cult 14:71–88CrossRefGoogle Scholar
  30. Hema BP, Murthy HN (2008) Improvement of in vitro androgenesis in niger using amino acids and polyamines. Biol Plant 52:121–125CrossRefGoogle Scholar
  31. Ho WJ, Vasil IK (1983a) Somatic embryogenesis in sugarcane (Saccharum officinarum L.) I. The morphology and physiology of callus formation and the ontogeny of somatic embryos. Protoplasma 118:169–180CrossRefGoogle Scholar
  32. Ho WJ, Vasil IK (1983b) Somatic embryogenesis in sugarcane (Saccharum officinarum L.): growth and plant regeneration from embryogenic cell suspension cultures. Ann Bot 51:719–726CrossRefGoogle Scholar
  33. Janno N, Grivet L, Segiun M, Paulet F, Domaingue R, Rao PS, Dookun A, D’Hont A, Glaszmann JC (1999) Molecular investigation of the genetic base of sugarcane cultivars. Theor Appl Genet 99:171–184CrossRefGoogle Scholar
  34. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Report 5:387–405CrossRefGoogle Scholar
  35. Kakkar RK, Nagar PK, Ahuja PS, Rai VK (2000) Polyamines and plant morphogenesis. Biol Plant 43:1–11CrossRefGoogle Scholar
  36. Kanchanapoom K, Domyoas P (1999) The origin and development of embryoids in oil palm (Elaeis guineensis Jacq.) embryo culture. Sci Asia 25:193–200CrossRefGoogle Scholar
  37. Karami O, Aghavaisi B, Pour AM (2009) Molecular aspects of somatic-to-embryogenic transition in plants. J Chem Biol 2:177–190PubMedPubMedCentralCrossRefGoogle Scholar
  38. Karami O, Saidi A (2010) The molecular basis for stress-induced acquisition of somatic embryogenesis. Mol Biol Rep 37:2493–2507PubMedCrossRefPubMedCentralGoogle Scholar
  39. Kevers C, Gaspar T, Dommes J (2002) The beneficial role of different auxins and polyamines at successive stages of somatic embryo formation and development of Panax ginseng in vitro. Plant Cell Tissue Organ Cult 70:181–188CrossRefGoogle Scholar
  40. Kevers C, Nathalie LG, Monteiro M, Dommes J, Gasper T (2000) Somatic embryogenesis of Panax ginseng in liquid cultures: a role for polyamines and their metabolic pathways. Plant Growth Regul 31:209–214Google Scholar
  41. Khanna HK, Daggard GE (2003) Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium. Plant Cell Rep 21:429–436PubMedCrossRefGoogle Scholar
  42. Kumar SV, Rajam MV (2005) Polyamines enhance Agrobacterium tumefaciens vir gene induction and T-DNA transfer. Plant Sci 168:475–480CrossRefGoogle Scholar
  43. Kumria R, Rajam MV (2002) Ornithine decarboxylase transgene in tobacco affects polyamines, in vitro morphogenesis and response to salt stress. J Plant Physiol 159:983–990CrossRefGoogle Scholar
  44. Lakshmanan P (2006) Somatic embryogenesis in sugarcane. In Vitro Cell Dev Biol Plant 42:201–205CrossRefGoogle Scholar
  45. Liu MC (1993) Factors affecting induction, somatic embryogenesis and plant regeneration of callus from cultured immature inflorescences of sugarcane. J Plant Physiol 141:714–720CrossRefGoogle Scholar
  46. Malgorzata DG (2001) Direct somatic embryogenesis as a rapid and efficient system for in vitro regeneration of Arabidopsis thaliana. Plant Cell Tissue Organ Cult 64:39–46CrossRefGoogle Scholar
  47. Minocha R, Minocha SC, Long S (2004) Polyamines and their biosynthetic enzymes during somatic embryo development in red spruce (Picea rubens Sarg.). In Vitro Cell Dev Biol Plant 40:572–580CrossRefGoogle Scholar
  48. Minocha R, Smith DR, Reeves C, Steele KD, Minocha SC (1999) Polyamine levels during the development of zygotic and somatic embryos of Pinus radiata. Physiol Plant 105:155–164CrossRefGoogle Scholar
  49. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  50. Nakagawa R, Kurushima M, Matsui M, Nakamura R, Kubo T, Funada R (2011) Polyamines promote the development of embryonal- suspensor masses and the formation of somatic embryos in Picea glehnii. In Vitro Cell Dev Biol Plant 47:480–487CrossRefGoogle Scholar
  51. Nonaka S, Yuhashi K, Takada K, Sugaware M, Minamisawa K, Ezura H (2008) Ethylene production in plants during transformation suppresses vir gene expression in Agrobacterium tumefaciens. New Phytol 178:647–656PubMedCrossRefGoogle Scholar
  52. Rajesh MK, Radha E, Anitha K, Parthasarathy VA (2003) Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines. Plant Cell Tissue Organ Cult 75:41–47CrossRefGoogle Scholar
  53. Rajesh MK, Radha E, Sajini KK, Anitha K (2014a) Polyamine-induced somatic embryogenesis and plantlet regeneration in vitro from plumular explants of dwarf cultivars of coconut (Cocos nucifera). Indian J Agric Sci 84:527–530Google Scholar
  54. Rajesh M, Sivanandhan G, Jeyaraj M, Selvaraj N, Ganapathi A (2014b) An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle. Protoplasma 251:1231–1243PubMedCrossRefGoogle Scholar
  55. Redha A, Suleman P (2011) Effects of exogenous application of polyamines on wheat anther cultures. Plant Cell Tissue Organ Cult 105:345–353CrossRefGoogle Scholar
  56. Reis RS, Vale EM, Heringer AS, Santa-Catarina C, Silveira V (2016) Putrescine induces somatic embryo development and proteomic changes in embryogenic callus of sugarcane. J Proteome 130:170–179CrossRefGoogle Scholar
  57. Robie CA, Minocha SC (1989) Polyamines and somatic embryogenesis in carrot. I. The effects of difluoromethylornithine and difluoromethylarginine. Plant Sci 65:45–54CrossRefGoogle Scholar
  58. Roustan JP, Chraibi KM, Latche A, Fallot J (1993) Relationship between ethylene and polyamine synthesis in plant regeneration. In: Pech JC, Latche A, Balague C (eds) Cellular and molecular aspects of the plant hormone ethylene. Kluwer Acad. Publ, Dordrecht, pp 365–366CrossRefGoogle Scholar
  59. Roustan JP, Latche A, Fallot J (1989) Stimulation of Daucus carota somatic embryogenesis by inhibitors of ethylene biosynthesis: cobalt and nickel. Plant Cell Rep 8:182–185PubMedCrossRefGoogle Scholar
  60. Roustan JP, Latche A, Fallot J (1992) Influence of ethylene on the incorporation of 3,4-[14C] methionine into polyamines in Daucus carota cells during somatic embryogenesis. Plant Physiol Biochem 30:201–205Google Scholar
  61. Sakhanokho HF, Peggy OA, May OL, Peng WC (2005) Putrescine enhances somatic embryogenesis and plant regeneration in upland cotton. Plant Cell Tissue Organ Cult 81:91–95CrossRefGoogle Scholar
  62. Santa-Catarina C, Silveira V, Scherer GFE, Floh EIS (2007) Polyamines and nitric oxide induce morphogenetic evolution in somatic embryogenesis of Ocotea catharinensis. Plant Cell Tissue Organ Cult 90:93–101CrossRefGoogle Scholar
  63. Santanen A, Simola LK (1992) Changes in polyamine metabolism during somatic embryogenesis in Picea abies. J Plant Physiol 140:475–480CrossRefGoogle Scholar
  64. Sathish D, Vasudevan V, Theboral J, Elayaraja D, Appunu C, Siva R, Manickavasagam M (2018) Efficient direct plant regeneration from immature leaf roll explants of sugarcane (Saccharum officinarum L.) using polyamines and assessment of genetic fidelity by SCoT markers. In Vitro Cell Dev Biol Plant 54:399–412CrossRefGoogle Scholar
  65. Scherwinski-Pereira JE, Guedes RS, Silva RA, Fermino PCP, Luis ZG, Freitas EO (2012) Somatic embryogenesis and plant regeneration in açaí palm (Euterpe oleracea). Plant Cell Tissue Organ Cult 109:501–508CrossRefGoogle Scholar
  66. Shah AH, Rashid N, Haider MS, Saleem F, Tahir M, Iqbal J (2009) An efficient, short and cost-effective regeneration system for transformation studies of sugarcane (Saccharum officinarum L.). Pak J Bot 41:609–614Google Scholar
  67. Shu S, Yuan LY, Guo SR, Sun J, Liu CJ (2012) Effects of exogenous spermidine on photosynthesis, xanthophyll cycle and endogenous polyamines in cucumber seedlings exposed to salinity. Afr J Biotechnol 11:6064–6074Google Scholar
  68. Silva TER, Cidade LC, Alvim FC, Cascardo JCM, Costa MGC (2009) Studies on genetic transformation of Theobroma cacao L.: evaluation of different polyamines and antibiotics on somatic embryogenesis and the efficiency of uidA gene transfer by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 99:287–298CrossRefGoogle Scholar
  69. Silveira V, Vita AM, Macedo AF, Dias MFR, Floh EIS, Santa-Catarina C (2013) Morphological and polyamine content changes in embryogenic and non-embryogenic callus of sugarcane. Plant Cell Tissue Organ Cult 114:351–364CrossRefGoogle Scholar
  70. Steiner N, Santa-Catarina C, Silveira V, Floh EIS, Guerra MP (2007) Polyamine effects on growth and endogenous hormones levels in Araucaria angustifolia embryogenic cultures. Plant Cell Tissue Organ Cult 89:55–62CrossRefGoogle Scholar
  71. Tang W, Newton RJ, Outhavong V (2004) Exogenously added polyamines recover browning tissues into normal callus cultures and improve plant regeneration in pine. Physiol Plant 122:386–395CrossRefGoogle Scholar
  72. Taparia Y, Gallo M, Altpeter F (2012) Comparison of direct and indirect embryogenesis protocols, biolistic gene transfer and selection parameters for efficient genetic transformation of sugarcane. Plant Cell Tissue Organ Cult 111:131–141CrossRefGoogle Scholar
  73. Thiruvengadam M, Rekha KT, Jayabalan N, Praveen N, Kim EH, Chung IM (2013) Effect of exogenous polyamines enhances somatic embryogenesis via suspension cultures of spine guard (Momordica dioica Roxb. ex. Wild). Aust J Crop Sci 7:446–453Google Scholar
  74. Tiburcio AF, Kaur-Sawhney R, Ingersoll RB, Galston AW (1985) Correlation between polyamines and pyrrolidine alkaloids in developing tobacco callus. Plant Physiol 78:323–326PubMedPubMedCentralCrossRefGoogle Scholar
  75. Vasudevan V, Subramanyam K, Elayaraja D, Karthik S, Vasudevan A, Manickavasagam M (2017) Assessment of the efficacy of amino acids and polyamines on regeneration of watermelon (Citrullus lanatus Thunb.) and analysis of genetic fidelity of regenerated plants by SCoT and RAPD markers. Plant Cell Tissue Organ Cult 130:681–687CrossRefGoogle Scholar
  76. Wang X, Ikeguchi Y, McCloskey DE, Nelson P, Pegg AE (2004) Spermine synthesis is required for normal viability, growth, and fertility in the mouse. J Biol Chem 279:51370–51375PubMedCrossRefPubMedCentralGoogle Scholar
  77. Wu XB, Wang J, Liu JH, Deng XX (2009) Involvement of polyamine biosynthesis in somatic embryogenesis of Valencia sweet orange (Citrus sinensis) induced by glycerol. J Plant Physiol 166:52–62PubMedCrossRefPubMedCentralGoogle Scholar
  78. Zhang RH, Li J, Guo SR, Tezuka T (2009) Effects of exogenous putrescine on gas-exchange characteristics and chlorophyll fluorescence of NaCl-stressed cucumber seedlings. Photosynth Res 100:155–162PubMedCrossRefPubMedCentralGoogle Scholar
  79. Zhu C, Chen Z (2005) Role of polyamines in adventitious shoot morphogenesis from cotyledons of cucumber in vitro. Plant Cell Tissue Organ Cult 81:45–53CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  • Dorairaj Sathish
    • 1
  • Jeevaraj Theboral
    • 2
  • Venkatachalam Vasudevan
    • 1
  • Gadamchetty Pavan
    • 1
  • Chandrasekaran Ajithan
    • 1
  • Chinnaswamy Appunu
    • 3
  • Markandan Manickavasagam
    • 1
    Email author
  1. 1.Department of BiotechnologyBharathidasan UniversityTiruchirappalliIndia
  2. 2.Department of BiotechnologyBishop Heber CollegeTiruchirappalliIndia
  3. 3.Division of Crop ImprovementIndian Council of Agricultural Research-Sugarcane Breeding InstituteCoimbatoreIndia

Personalised recommendations