Abstract
Seeds of neem were collected from different parts of India and analyzed for their azadirachtin content by High Performance Liquid Chromatography (HPLC). In order to assess the effects of genotypic and geographical variation on azadirachtin content in cell cultures, callus development was attempted in the seeds containing high and low concentration of azadirachtin. The concentration of azadirachtin in callus cultures was significantly affected by the explant source. Seed kernels with higher azadirachtin content produced higher azadiractin content in callus cultures and lower azadirachtin content was seen in callus cultures produced from seed kernels with low azadiractin content. The protocol for development of elite stock culture ofAzadirachta indica was established with the objective of selecting a high azadirachtin-producing cell line. The highest azadirachtin-producing cell line was selected and the effects of different media and illumination conditions on growth and azadirachtin production were studied in shake flask suspension culture. Detailed batch growth kinetics was also established. These studies provided elite starter culture and associated protocols for cultivation ofA. indica plant cell culture in the bioreactor.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Govindchari, T. R. (1992) Chemistry and biological investigation onAzadirachta indica (the neem tree).Curr. Sci. 63: 117–121.
Koul, O. M., B. Isman, and C. M. Ketkar (1990) Properties and uses of neem,Azadirachta indica.Can. J. Bot. 68: 1–11.
Jacbson, M. (1988) Focus on phytochemical pesticides. pp. 101–140. In: M. Jacbson (ed.).The neem Tree. CRC Press, Boca Raton Fla, USA.
Schmutterer, H. (1990) Properties and potential of natural pesticides from the neem tree,Azadirachta indica.Ann. Rev. Entomol. 35: 271–297.
Mordue, A. J. and A. Blackwell (1993) Azadirachtin: An update.J. Insect Physiol. 39: 903–924.
Benge, M. D. (1989) Cultivation and propagation of the neem tree. pp. 1–18. In: M. Jacbson (ed.).Focus on Phytochemical Pesticides, The neem Tree. CRC Press, Boca Raton, USA.
Ermel, K. (1995) Azadirachtin content of neem seed kernels from different regions of the world. pp. 222–230. In: M. Schmutterer (ed.).The neem Tree: Source of Unique Natural Products for Integrated Pest Management, Medicine, Industry and Other Purposes. VCH, Weinheim, Germany.
Kumar, J. and B. S. Parmar (1997) Neem oil content and its key chemical constituents in relation to the agroclimatic factors and regions of India.Pestic. Res. J. 9: 216–225.
Prakash, G., S. S. Bhojwani, and A. K. Srivastava (2002) Production of azadirachtin from plant tissue culture: State of the art and future prospects.Biotechnol. Bioprocess Eng. 7: 185–193.
Alfermann, A. W. and M. Petersen (1995) Natural product formation from plant cell biotechnology.Plant Cell Tissue Org. Cult. 43: 199–205.
Fowler, M. W. and A. H. Scragg (1998) Natural Products from higher plants and plant cell culture. pp. 165–177. In: M. S. S. Pais, F. Marituna, and J. M. Novais (eds.).Plant Cell Biotechnology NATO ASI Series. Berlin: Springer Verlag, Germany.
Constable, F., W. G. W. Kurz, and J. P. Kutney (1982) Variation in cell cultures of perwinkle,Catharanthus roseus. pp. 301–314. In: A Fujiwara and M. Maruzen (eds.).Plant Tissue Culture. Tokyo, Japan.
Evans, D. A., W. R. Sharp, and H. P. Medina-Filho (1984) Somaclonal and gametoclonal variation.Am. J. Bot. 71: 759–774.
Sidhu, O. P. and H. M. Behl (1996) Seasonal variation in azadirachtin in seeds ofAzadirachta indica.Curr. Sci. 70: 1084–1085.
Sidhu, O. P., V. Kumar, and H. M. Behl (2003) Variability in neem (Azadirachta indica) with respect to azadirachtin content.J. Agric. Food Chem. 51: 910–915.
Chattopadhyay, S., S. Farkya, A. K. Srivastava, S. S. Bhojwani, and V. S. Bisaria (2002) Bioprocess Considerations for production of secondary metabolites by plant cell suspension cultures.Biotechnol. Bioprocess Eng. 7: 138–149.
Zenk, M. K., H. Shagi (El), H. Arens, J. Stockgt, E. J. W. Weiter, and B. Dens (1977) Formation of indole alkaloids serpantine and ajmalicine in cell cultures ofCatharanthus roseus. pp. 27–44. In: W. Barz, E. Reinhard, and M. K. Zenk (eds.).Plant Tissue Culture and Its Biotechnological Application. Berlin, Germany: Springer-Verlag.
Murashige, T. and F. Skoog (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture.Physiol. Plant 15: 473–497.
Bhojwani, S. S. and M. K. Rajdan (1996) Procuction of secondary metabolites. pp. 537–562. In:Plant Tissue Culture. Theory and Practice. Elsevier, NY, USA.
Miller, G. M. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar.Anal. Chem. 31: 426–428.
Kaushik, N. (2002) Prospecting chemical diversity of neem (Azadirachta indica) trees growing in India for value additionProceedings of 3rd World neem Conference, India.
Wewetzer, A. (1998) Callus culture ofAzadirachta indica and their potential for the production of azadirachtin.Phytoparasitica 26: 47–52.
Smollny, T., H. Wichers, S. Kalenberg, A. Shahsavar, M. Petersen, and A. W. Alfermann (1998) Accumulation of prodophyllotoxin and related lignans in cell suspension culture ofLinum album.Phytochem. 48: 975–979.
Fujita, Y., S. Takahasi, and Y. Yamada (1985) Selection of cell lines with high productivity of shikonin derivatives by protoplast culture ofLithospermum erythorhizon cells.Agric. Biol. Chem. 49: 1755–1759.
Aiken, M. M. and M. M. Yeoman (1986) A rapid screening technique for the selection of high yielding capsaicin cell lines ofCapasicum frutescence Mill. In: P. Morris (ed.).Secondary Metabolism in Plant Cell Cultures. Cambridge University Press, London, UK.
Hara, Y., T. Laugel, T. Morimoto, T. J. Hiratsuka, T. Yoshioka, Y. Fujita, and Y. Yamada (1989) Flow cytometric analysis of cellular berberin contents in high- and low producing cell lines ofCoptis japonica obtained by repeated selection.Planta Med. 55: 151–154.
Adamse, P. (1990) Selection of high yielding cell linesTagets using flow cytometry. pp. 726–731. In: H. J. Nijkamp (ed.).Progress in Plant Cellular and Molecular Biology. Kluwer, Dordecht.
Songstad, D. D., K. L. Giles, J. Park, D. Novakovski, E. Epp, and L. Friesen (1990) Use of nurse cultures to select forPapaver somniferum cell lines capable of enhanced sanguinarine production.J. Plant Physiol. 136: 236–239.
Sundaram, K. M. S. and J. Curry (1996) Effect of UV light absorbers on the photostablization of azadirachtin, a neem based biopesticide.Chemosphere 32: 649–659.
Mathur, A., Y. N. Shukla, M. Pal, P. S. Ahuja, and Uniyal G. C. (1994) Saponin production in callus and cell suspension cultures ofPanax quinquefolium.Phytochem. 35: 1221–1225.
Raval, K., S. Hellwig, G. Prakash, A. R. Plasencia, A. K. Srivastava, and J. Buchs (2003) Necessity of a two-stage process for the production of azadirachtin-related limonoids in suspension culture ofAzadirachta indica.J. Biosci. Bioeng. 96: 16–22.
Allan, E. J., J. P. Easwara, and A. Wood (1994) The production of azadirachtin byin vitro tissue culture of neem,Azadirachta indica.Pestri. Sci. 42: 147–152.
Veersham, C., M. R. Kumar, D. Sowjanya, C. K. Kokate, and S. S. Apte (1998) Production of azadirachtin from callus cultures ofAzadirachta indica.Fitoterapia 69: 423–424.
Balaji, K. (2001)Studies on Tissue Culture of Azadrachta indica (neem). Ph.D. Thesis. Kakatiya University. Warangal, AP, India.
Kearney, M. L., E. J. Allan, J. E. Hooker, and A. J. Mordue (1994) Antifeedant effects ofin vitro culture extract of neem tree,Azadirachta indica against the deser locust (Shistocerca gregaria).Plant Cell Tiss. Org. Cult. 37: 67–71.
Kuruvilla, T., P. Komaraiah, and S. V. Ramakrishna (1999) Enhanced secretion of azadirachtin by permeabilized margosa (Azadirachta indica) cells.Ind. J. Exp. Bot. 37: 89–91.
Balaji, K., C. Veeresham, K. Srisilam, and C. Kokate (2003) Azadirachtin, a novel biopesticide from cell cultures ofAzadirachta indica.J. Plant Biotechnol. 5: 121–129.
Allan, E. J., J. P. Eeswara, A. Jarvis, A. J. Mordue (Juntz), E. Morgan, and T. Stucbbury (2002) Induction of hairy root culture ofAzadirachta indica A. Juss and their production of azadirachtin and other important insect bioactive metabolites.Plant Cell Rep. 21: 374–379.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Prakash, G., Emmannuel, C.J.S.K. & Srivastava, A.K. Variability of azadirachtin inAzarirachta indica (neem) and batch kinetics studies of cell suspension culture. Biotechnol. Bioprocess Eng. 10, 198–204 (2005). https://doi.org/10.1007/BF02932013
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02932013