Reduction in hyperhydricity and improvement in in vitro propagation of commercial hard fibre and medicinal glycoside yielding Agave sisalana Perr. ex Engelm by NaCl and polyethylene glycol

  • Tukaram D. NikamEmail author
  • Ketki V. Mulye
  • Mahadev R. Chambhare
  • Harichandra A. Nikule
  • Mahendra L. Ahire
Original Article


Agave sisalana is hapaxanthic monocotyledonous plant yielding a hard fibre of commercial value. It is also a source of medicinally important glycosides such as hecogenin and tigogenin. Sisal plantations can play a significant role in reforestation of hilly barren land. Unfortunately, natural propagation is not sufficient to fulfill the demands; besides, hyperhydricity is a severe problem in the in vitro propagation method of A. sisalana. The present study for the first time demonstrates that, the hyperhydricity problem can be solved by inclusion of osmotic stress inducing agents like sodium chloride (NaCl) and polyethylene glycol (PEG 6000) (0.0, 0.1, 0.2, 0.3, 0.4, or 0.5% w/v) in MS medium. The response of hyperhydric shoots, normal shoots and hyperhydric reverted shoots with the treatments was analyzed for water content, chlorophyll content, osmolyte accumulation and oxidative damage. The principal component analysis showed the significant positive and negative variations in the net photosynthesis, stomata conductance, internal CO2, transpiration, and water use efficiency between normal, hyperhydric and hyperhydric reverted shoots among all the time points of day and night period. Besides, detailed anatomical and ultra-structural observations were made to discern the changes that occurred in epidermis, mesophyll cells, vascular bundles, and stomata. About 85 and 58% of hyperhydric shoots were reverted on medium fortified with 0.2% NaCl and 0.1% PEG, respectively. All reverted shoots were normal and survived when transferred to the field conditions. The improved protocol with the treatment of NaCl for recovery of hyperhydric shoots was not cost intensive and without any harmful effects on shoots and plantlets.

Key message

First report on change in physiology and recovery of hyperhydricity with NaCl and PEG in shoots and somatic embryos of Agave sisalana which allows additional 38% plantlets available for plantation.


Asparagaceae CAM plant Hardening Reforestation Osmotic stress Sisal Shoot regeneration Somatic embryo 



The authors are grateful to University Grant Commission (UGC) for financial support for the Project F. No. 37-468/2009 (MS) (SR).

Author Contributions

TDN and MLA conceived the experiments. KVM, MRC, and HAN performed the experiments. TDN, KVM, MLA, and MRC analyzed the data and wrote the manuscript. All authors have reviewed the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11240_2019_1603_MOESM1_ESM.doc (5.5 mb)
Supplementary material 1 (DOC 5632 kb)


  1. Angiosperm Phylogeny Group (APG III) (2009) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants. Bot J Linn Soc 161:105–121CrossRefGoogle Scholar
  2. Ardelean M, Cachita-Cosma D, Ardelean A, Ladasiu C, Mihali VC (2014) The effect of heat stress on hyperhydricity and guaiacol peroxidase activity (Gpox) at the Foliar Lamina of Sedum telephium L. Ssp. Maximum (L.) Krock Vitroplantlets. An Stiintifice Univ Al. I. Cuza din Iasi 60(2):21–31Google Scholar
  3. Arizaga S, Ezcurra E (2002) Propagation mechanisms in Agave macroacantha (Agavaceae), a tropical arid-land succulent rosette. Am J Bot 89(4):632–641CrossRefGoogle Scholar
  4. Badr-Elden AM, Nower AA, Ibrahim IA, Ebrahim MK, Elaziem TMA (2012) Minimizing the hyperhydricity associated with in vitro growth and development of watermelon by modifying the culture conditions. Afr J Biotech 11(35):8705–8717Google Scholar
  5. Binh LT, Muoi LT, Oanh HTK, Thang TD, Phong DT (1990) Rapid propagation of Agave by in vitro tissue culture. Plant Cell Tiss Org Cult 23(1):67–70CrossRefGoogle Scholar
  6. Castillo FJ (1996) Antioxidative protection in the inducible CAM plant Sedum album L. following the imposition of severe water stress and recovery. Oecologia 107(4):469–477CrossRefGoogle Scholar
  7. Chakrabarty D, Park SY, Ali MB, Shin KS, Paek KY (2006) Hyperhydricity in apple: ultrastructural and physiological aspects. Tree Physiol 26:377–388CrossRefGoogle Scholar
  8. Debnath M, Mukeshwar P, Sharma R, Thakur GS, Lal P (2010) Biotechnological intervention of Agave sisalana: a unique fibre yielding plant with medicinal property. J Med Plants Res 4(3):177–187Google Scholar
  9. FAO W (2012) IFAD. The state of food insecurity in the world 65Google Scholar
  10. Fauguel CM, Vega TA, Nestares G, Zorzoli R, Picardi LA (2008) Anatomy of normal, and hyperhydric sunflower shoots regenerated in vitro. Helia 31(48):17–26CrossRefGoogle Scholar
  11. Fontes MA, Otoni WC, Carolino SMB, Brommonschenkel SH, Fontes EPB, Fari M, Louro RP (1999) Hyperhydricity in pepper plants regenerated in vitro: involvement of BiP (binding protein) and ultrastructural aspects. Plant Cell Rep 19(1):81–87CrossRefGoogle Scholar
  12. Franck T, Gaspar T, Kevers C, Penel C, Dommes J, Hausman JF (2001) Are hyperhydric shoots of Prunus avium L. energy deficient? Plant Sci 106(6):1145–1151CrossRefGoogle Scholar
  13. Gao H, Xia X, An L, Xin X, Liang Y (2017) Reversion of hyperhydricity in pink (Dianthus chinensis L.) plantlets by AgNO3 and its associated mechanism during in vitro culture. Plant Sci 254:1–11CrossRefGoogle Scholar
  14. Gao H, Li J, Ji H, An L, Xia X (2018) Hyperhydricity-induced ultrastructural and physiological changes in blueberry (Vaccinium spp.). Plant Cell Tiss Org Cult 133:65–76CrossRefGoogle Scholar
  15. Ghuge SA, Rai AN, Suprasanna P (2010) Comparative effects of NaCl, PEG and mannitol iso-osmotic stress on solute accumulation and antioxidant enzyme system in potato (Solanum tuberosum L.). Plant Stress—Glob Sci Books 4(1):50–55Google Scholar
  16. Gonzalez JTC, Dillon AJP, Perez-Perez AR, Fontana R, Bergmann CP (2015) Enzymatic surface modification of Sisal fibres (Agave sisalana) by Penicillium echinulatum cellulases. Fibres Polym 16(10):2112–2120CrossRefGoogle Scholar
  17. Hazra SK, Das S, Das AK (2002) Sisal plant regeneration via organogenesis. Plant Cell Tiss Org Cult 70:235–240CrossRefGoogle Scholar
  18. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125(1):189–198CrossRefGoogle Scholar
  19. Ivanova M, Van Staden J (2011) Influence of gelling agent and cytokinins on the control of hyperhydricity in Aloe polyphylla. Plant Cell Tiss Org Cult 104:13–21CrossRefGoogle Scholar
  20. Kacem NS, Delporte F, Muhovski Y, Djekoun A, Watillon B (2017) In vitro screening of durum wheat against water-stress mediated through polyethylene glycol. J Genet Eng Biotechnol 15:239–247CrossRefGoogle Scholar
  21. Kundu DK, Sarkar S, Saha AR, Jha AK, Behera MS (2018) Application of micro-irrigation and micronutrients to improve fibre yield and water use efficiency in sisal (Agave sisalana Perr. Ex Engelm.). Int J Curr Microbiol App Sci 7(10):2101–2108CrossRefGoogle Scholar
  22. Lee SB, Jung SJ, Go YS, Kim HU, Kim JK, Cho HJ, Suh MC (2009) Two Arabidopsis 3-ketoacyl CoA synthase genes, KCS20 and KCS2/DAISY, are functionally redundant in cuticular wax and root suberin biosynthesis, but differentially controlled by osmotic stress. Plant J 60:462–475CrossRefGoogle Scholar
  23. Lichtenthaler HK, Buschmann C (2001) Chlorophylls and carotenoids: measurement and characterization by UV-Vis spectroscopy. In: Wrolstad RE, Acree TE, An H, Decker EA, Penner MH, Reid DS, Schwartz SJ, Shoemaker CF, Sporns P (eds) Current protocols in food analytical chemistry (CPFA). Wiley, New York, pp F4.3.1–F4.3.8Google Scholar
  24. McLaughlin SP, Schuck SM (1991) Fiber properties of several species of Agavaceae from the southwestern United States and northern Mexico. Econ Bot 45:480–486CrossRefGoogle Scholar
  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  26. Mwaniki AM, Kisangau DP, Musimba NLR (2017) Socio-economic factors affecting Sisal cultivation and adoption in Kiomo Division, Kitui County. Int J Agron Agri R 11(3):97–103Google Scholar
  27. Nikam TD (1997) High frequency shoot regeneration in Agave sisalana. Plant Cell Tiss Org Cult 51:225–228CrossRefGoogle Scholar
  28. Nikam TD, Bansude GM, Kumar KA (2003) Somatic embryogenesis in Sisal (Agave sisalana Perr. ex. Engelm). Plant Cell Rep 22:188–194CrossRefGoogle Scholar
  29. Olmos E, Hellıin E (1998) Ultrastructural differences of hyperhydric and normal leaves from regenerated carnation plants. Sci Hort 75:91–101CrossRefGoogle Scholar
  30. Parent C, Capelli N, Berger A, Crevecoeur M, Dat JF (2008) An overview of plant responses to soil waterlogging. Plant Stress 2(1):20–27Google Scholar
  31. Picoli EAT, Paiva EAS, Xavier A, Aguiar RM, Carolino SMB, Fari MG, Otoni WC (2008) Ultrastructural and biochemical aspects of normal and hyperhydric Eucalypt. Int J Hort Sci 14:61–69Google Scholar
  32. Piqueras A, Cortina M, Serna MD, Casas JL (2002) Polyamines and hyperhydricity in micropropagated carnation plants. Plant Sci 162:671–678CrossRefGoogle Scholar
  33. Pompelli MF, Martins SCV, Celin EF, Ventrella MC, DaMatta FM (2010) What is the influence of ordinary epidermal cells and stomata on the leaf plasticity of coffee plants grown under full-sun and shady conditions? Braz J Biol 70:1083–1088CrossRefGoogle Scholar
  34. Portillo L, Santacruz-Ruvalcaba F (2006) Factibilidad de uso de un nuevos sistema de inmersion temporal (Orbitabion®) para embriogenesis somatica de Agave tequilana Weber cultivar azul. Bol Nakari 17:43–48Google Scholar
  35. Reyes-Zambrano SJ, Lecona-Guzman CA, Barredo-Pool FA, Calderon JDA, Abud-Archila M, Rincon-Rosales R, Gutierrez-Miceli FA (2016) Plant growth regulators optimization for maximize shoots number in Agave americana L. by indirect organogenesis. Gayana Bot 73(1):124–131CrossRefGoogle Scholar
  36. Rodriguez-Garay B (2016) In: Loyola-Vargas V, Ochoa-Alejo N (eds) Book chapter- somatic embryogenesis: fundamental aspects and applications. Springer International Publishing Switzerland, pp 267–282Google Scholar
  37. Rossetto M, Dixon KW, Bunn E (1992) Aeration: a simple method to control vitrification and improve in vitro culture of rare Australian plants. In Vitro Cell Dev Biol Plant 28:192–196CrossRefGoogle Scholar
  38. Saher S, Piqueras A, Hellin E, Olmos E (2004) Hyperhydricity in micropropagated carnation shoots: the role of oxidative stress. Physiol Plant 120(1):152–161CrossRefGoogle Scholar
  39. Santacruz-Ruvalcaba F, Portillo L (2009) Thin cell suspension layer as a new methodology for somatic embryogenesis in Agave tequilana Weber cultivar azul. Ind Crop Prod 29:609–614CrossRefGoogle Scholar
  40. Santos J, Vieira I, Braz-Filho R, Branco A (2015) Chemicals from Agave sisalana biomass: isolation and identification. Int J Mol Sci 16(4):8761–8771CrossRefGoogle Scholar
  41. Sarkar S, Jha AK (2017) Research for sisal (Agave sp.) fibre production in India. Int J Curr Res 9(11):61136–61146Google Scholar
  42. Sarkar S, Jha AK, Majumdar B, Saha AR (2018) Influence of drip irrigation, manure and fertilizers on production of planting materials in sisal (Agave sisalana Perr. Ex Engelm.). Int J Curr Microbiol Appl Sci 7(9):1934–1941CrossRefGoogle Scholar
  43. Shelke DB, Pandey M, Nikalje GC, Zaware BN, Suprasanna P, Nikam TD (2017) Salt responsive physiological, photosynthetic and biochemical attributes at early seedling stage for screening soybean genotypes. Plant Physiol Biochem 118:519–528CrossRefGoogle Scholar
  44. Steinmacher DA, Guerra MP, Saare-Surminski K, Lieberei R (2011) A temporary immersion system improves in vitro regeneration of peach palm through secondary somatic embryogenesis. Ann Bot 108:1463–1475CrossRefGoogle Scholar
  45. Sullivan CY (1972) Mechanisms of heat and drought resistance in grain sorghum and methods of measurement. In: Rao NGP, House LR (eds) Sorghum in Seventies. Oxford & IBH Pub. Co., New Delhi, pp 247–264Google Scholar
  46. Taiz L, Zeiger E (2010) Plant physiology. Sinauer Associates Inc., Sunderland, MAGoogle Scholar
  47. Tewari D, Tripathi YC, Anjum N (2014) Agave sisalana: a plant with high chemical diversity and medicinal importance. World J Pharm Res 3(8):238–249Google Scholar
  48. Van den Dries N, Gianni S, Czerednik A, Krens FA, de Klerk GJM (2013) Flooding of the apoplast is a key factor in the development of hyperhydricity. J Exp Bot 64:5221–5230CrossRefGoogle Scholar
  49. Watanabe S, Kojima K, Ide Y, Sasaki S (2000) Effects of saline and osmotic stress on proline and sugar accumulation in Populus euphratica in vitro. Plant Cell Tiss Org Cult 63:199–206CrossRefGoogle Scholar
  50. Weber B, Bowker M, Zhang Y, Belnap J (2016) Natural recovery of biological soil crusts after disturbance. In: Weber B, Budel B, Belnap J (eds) Biological soil crusts: an organizing principle in drylands, ecological studies (analysis and synthesis), vol 226. Springer International Publishing Switzerland, Cham, pp 479–498Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Tukaram D. Nikam
    • 1
    Email author
  • Ketki V. Mulye
    • 1
  • Mahadev R. Chambhare
    • 1
  • Harichandra A. Nikule
    • 1
  • Mahendra L. Ahire
    • 2
  1. 1.Department of BotanySavitribai Phule Pune UniversityPuneIndia
  2. 2.Department of BotanyYashavantrao Chavan InstituteSataraIndia

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