Acta Biologica Hungarica

, Volume 65, Issue 3, pp 355–367 | Cite as

Synthetic Seed Production and Physio-Biochemical Studies in Cassia Angustifolia Vahl. — a Medicinal Plant

  • N. A. W. Bukhari
  • Iram SiddiqueEmail author
  • K. Perveen
  • I. Siddiqui
  • M. S. Alwahibi


Synthetic seed technology is an alternative to traditional micropropagation for production and delivery of cloned plantlets. Synthetic seeds were produced by encapsulating nodal segments of C. angustifolia in calcium alginate gel. 3% (w/v) sodium alginate and 100 mM CaCl2 ∙ 2H2O were found most suitable for encapsulation of nodal segments. Synthetic seeds cultured on half strength Murashige and Skoog medium supplemented with thidiazuron (5.0 μM) + indole-3-acetic acid (1.0 μM) produced maximum number of shoots (10.9 ± 0.78) after 8 weeks of culture exhibiting (78%) in vitro conversion response. Encapsulated nodal segments demonstrated successful regeneration after different period (1–6 weeks) of cold storage at 4 °C. The synthetic seeds stored at 4 °C for a period of 4 weeks resulted in maximum conversion frequency (93%) after 8 weeks when placed back to regeneration medium. The isolated shoots when cultured on half strength Murashige and Skoog medium supplemented with 1.0 μM indole-3-butyric acid (IBA), produced healthy roots and plantlets with well-developed shoot and roots were successfully hardened off in plastic pots containing sterile soilrite inside the growth chamber and gradually transferred to greenhouse where they grew well with 85% survival rate. Growth performance of 2 months old in vitro-raised plant was compared with in vivo seedlings of the same age. Changes in the content of photosynthetic pigments, net photosynthetic rate (PN), superoxide dismutase and catalase activity in C. angustifolia indicated the adaptation of micropropagated plants to ex vitro conditions.


Antioxidant enzymes encapsulation rooting synthetic seeds Thidiazuron 


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  1. 1.
    Aebi, H. (1984) Catalase in vitro methods. Enzymol. 105, 121–126.CrossRefGoogle Scholar
  2. 2.
    Agrawal, V., Sardar, P. R. (2003) In vitro organogenesis and histomorphological investigations in Senna (Cassia angustifolia) a medicinally valuable shrub. Physiol. Mol. Biol. Plants. 91, 131–140.Google Scholar
  3. 3.
    Agrawal, V., Sardar, P. R. (2006) In vitro propagation of Cassia angustifolia (Vahl.) through leaflet and cotyledon derived calli. Biol Plant. 1, 118–122.CrossRefGoogle Scholar
  4. 4.
    Agrawal, V., Sardar, P. R. (2007) In vitro regeneration through somatic embryogenesis and organogenesis using cotyledons of Cassia angustifolia Vahl. In Vitro Cell. Dev. Biol. Plants. 43, 585–592.CrossRefGoogle Scholar
  5. 5.
    Ahmad, N., Anis, M. (2010) Direct plant regeneration from encapsulated nodal segments of Vitex negundo. Biol. Plant. 54, 748–752.CrossRefGoogle Scholar
  6. 6.
    Ali, M. B., Hahn, E. J., Paek, K. Y. (2005) Effects of light intensities on antioxidant enzymes and malondialdehyde content during short term acclimatization on micropropagated Phalaenopsis plantlet. Environ. Exp. Bot. 54, 109–120.CrossRefGoogle Scholar
  7. 7.
    Anonymous (1992) The wealth of India: A dictionary of Indian raw materials and industrial products. Vol 3, CSIR, New Delhi, pp. 354–363.Google Scholar
  8. 8.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Annals Biochem. 72, 248–254.CrossRefGoogle Scholar
  9. 9.
    Chai, T. T., Fadzillah, N. M., Kusnan, M., Mahmood, M. (2005) Water stress induced oxidative damage and antioxidant responses in micropropagated banana plantlets. Biol. Plant. 49, 153–156.CrossRefGoogle Scholar
  10. 10.
    Danso, K. E., Ford-Llyod, B. V. (2003) Encapsulation of nodal cuttings and shoot tips for storage and exchange of cassava germplasm. Plant Cell Rep. 21, 718–725.PubMedGoogle Scholar
  11. 11.
    Desjardins, Y. (1995) Photosynthesis in vitro–On the factors regulating CO2 assimilation in micropropagation systems. Acta Hort. 393, 45–62.CrossRefGoogle Scholar
  12. 12.
    Dhinsa, P. S., Plumb-Dhinsa, P., Thorpe, T. A. (1981) Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32, 93–101.CrossRefGoogle Scholar
  13. 13.
    Estrada-Luna, A. A., Davies Jr., F. T., Egilla, J. N. (2001) Physiological changes and growth of micropropagated Chile ancho pepper plantlets during acclimatization and post acclimatization. Plant Cell Tissue Organ Cult. 66, 17–24.CrossRefGoogle Scholar
  14. 14.
    Faisal, M., Ahmad, N., Anis, M. (2006) In vitro plant regeneration from alginate encapsulated microcuttings of Rauvolfia tetraphylla L. Amer. Eurass. J. Agric. Environ. Sci. 1, 1–6.Google Scholar
  15. 15.
    Faisal, M., Anis, M. (2007) Regeneration of plants from alginate encapsulated shoots of Tylophora indica (Burm. F.) Merrill, an endangered medicinal plant. J. Hortic. Sci. Biotechnol. 82, 351–354.CrossRefGoogle Scholar
  16. 16.
    Faisal, M., Anis, M. (2009) Changes in photosynthetic activity, pigment composition, electrolyte leakage, lipid peroxidation, and antioxidant enzymes during ex vitro establishment of micropropagated Rauvolfia tetraphylla plantlets. Plant Cell Tiss. Org. Cult. 99, 125–132.CrossRefGoogle Scholar
  17. 17.
    Faisal, M., Anis, M. (2010) Effect of light irradiations on photosynthetic machinery and antioxidative enzymes during ex vitro acclimatization of Tylophora indica plantlets. J. Plant Interaction 5, 21–27.CrossRefGoogle Scholar
  18. 18.
    Foyer, C. H., Lelandais, M., Kunert, K. J. (1994) Photooxidative stress in plants. Physiol. Plant. 92, 696–717.CrossRefGoogle Scholar
  19. 19.
    Franz, G. (1993) The senna drug and its chemistry. Pharmocology 47, 2–6.Google Scholar
  20. 20.
    Inze, D., Van Montagu, M. (1995) Oxidative stress in plants. Curr. Opinion Biotech. 6, 153–158.CrossRefGoogle Scholar
  21. 21.
    Jana, S., Shekhawat, G. S. (2012) In vitro regeneration of Anethum graveolens, antioxidative enzymes during organogenesis and RAPD analysis for clonal fidelity. Biol. Plant. 56, 9–14.CrossRefGoogle Scholar
  22. 22.
    Kavyashree, R., Gayatri, M. C., Revanasiddaiah, H. M. (2006) Propagation of mulberry variety–S54 by synseeds of axillary bud. Plant Cell Tissue Organ Cult. 84, 245–249.CrossRefGoogle Scholar
  23. 23.
    Mckinney, G. (1941) Absorption of light by chlorophyll solution. J. Biol. Chem. 140, 315–322.Google Scholar
  24. 24.
    McLachlan, S., Zalik, S. (1963) Plastid structure, chlorophyll concentration and free amino acid composition of chlorophyll mutant by barley. Canadian J. Bot. 41, 1053–1062.CrossRefGoogle Scholar
  25. 25.
    Mitrovic, A., Janosevic, D., Budimir, S., Pristov, J. B. (2012) Changes in antioxidative enzymes activities during Tacitus bellus direct shoot organogenesis. Biol. Plant. 56, 357–361.CrossRefGoogle Scholar
  26. 26.
    Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497.CrossRefGoogle Scholar
  27. 27.
    Pence, V. C. (1999) The application of biotechnology for the conservation of endangered plants. In: Benson, E. E. (ed.) Plant conservation biotechnology, Vol. 15. Taylor and Francis, London, pp. 227–241.Google Scholar
  28. 28.
    Pospisilova, J., Haisel, D., Synkova, H., Catsky, J., Wilhelmova, N., Plzakova, S., Prochazkova, D., Sramek, F. (2000) Photosynthetic pigments and gas exchange during ex vitro acclimation of tobacco plants as affected by CO2 supply and abscisic acid. Plant Cell Tiss. Org. Cult. 61, 125–133.CrossRefGoogle Scholar
  29. 29.
    Santamaria, J. M., Davies, W. J., Atkinson, C. J. (1993) Stomata from micropropagated Delphinium plants responded to ABA, CO2, water potential and light but fail to close fully. J. Exp. Bot. 44, 99–107.CrossRefGoogle Scholar
  30. 30.
    Scandalios, J. G. (1993) Oxygen stress and superoxide dismutase. Plant Physiol. 101, 7–12.CrossRefGoogle Scholar
  31. 31.
    Sgherri, C. L. M., Navari-Izzo, F. (1995) Sunflower seedlings subjected to increasing water deficit stress: oxidative stress and defense mechanisms. Physiol. Plant. 93, 25–30.CrossRefGoogle Scholar
  32. 32.
    Siddique, I., Anis, M. (2006) Thidiazuron induced high frequency shoot bud formation and plant regeneration from cotyledonary node explants of Capsicum annuum L. Indian J. Biotechnol. 5, 303–308.Google Scholar
  33. 33.
    Siddique, I., Anis, M. (2007) In vitro shoot multiplication and plantlet regeneration from nodal explants of Cassia angustifolia (Vahl.): a medicinal plant. Acta Physiol. Plant. 29, 233–238.CrossRefGoogle Scholar
  34. 34.
    Siddique, I., Anis, M. (2007) Rapid micropropagation of Ocimum basilicum using shoot tip explants pre-cultured in thidiazuron supplemented liquid medium. Biol. Plant. 51, 787–790.CrossRefGoogle Scholar
  35. 35.
    Siddique, I., Anis, M. (2009) Morphogenic response of the alginate encapsulated nodal segment and antioxidative enzymes analysis during acclimatization of Ocimum basilicum L. J Crop Sci. Biotechnol. 12, 229–234.CrossRefGoogle Scholar
  36. 36.
    Siddique, I., Anis, M., Aref, I. M. (2010) In vitro adventitious shoot regeneration via indirect organogenesis from petiole explants of Cassia angustifolia Vahl. a potential medicinal plant. Appl. Biochem. Biotechnol. 162, 2067–2074.CrossRefGoogle Scholar
  37. 37.
    Siddique, I., Bukhari, N. A., Perveen, K., Siddiqui, I., Anis, M. (2013) Pre-culturing of nodal explants in thidiazuron supplemented liquid medium improves in vitro shoot multiplication of Cassia angustifolia. Acta Biol. Hung. 64, 377–384.CrossRefGoogle Scholar
  38. 38.
    Siddique, I., Javed, S. B., Al-Othman, M. R., Anis, M. (2013) Stimulation of in vitro organogenesis from epicotyl explants and successive micropropagation round in Cassia angustifolia Vahl.: an important source of sennosides. Agroforestry Syst. 87, 583–590.CrossRefGoogle Scholar
  39. 39.
    Van Huylenbroeck, J. M., Debergh, P. C. (1996) Impact of sugar concentration in vitro on photosynthesis and carbon metabolism during ex vitro acclimatization of Spathiphyllum plantlets. Physiol. Plant. 96, 298–304.CrossRefGoogle Scholar
  40. 40.
    Van Huylenbroeck, J. M., Van Laere, I. M. B., Piqueras, A., Debergh, P. C., Bueno, P. (1998) Time course of catalase and superoxide dismutase during acclimatization and growth of micropropagated Calathea and Spathiphyllum plants. Plant Growth Regul. 26, 7–14.CrossRefGoogle Scholar

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© Akadémiai Kiadó, Budapest 2014

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  • N. A. W. Bukhari
    • 1
  • Iram Siddique
    • 1
    Email author
  • K. Perveen
    • 1
  • I. Siddiqui
    • 1
  • M. S. Alwahibi
    • 1
  1. 1.Department of Botany and Microbiology, College of Science, Female Centre for Scientific and Medical CollegesKing Saud UniversityRiyadhSaudi Arabia

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