Cultivos de células en suspensión de Azadirachta indica para la producción de un bioinsecticida
Cell suspension culture of Azadirachta indica for production of a bioinsecticide
F. Orozco–Sánchez1 y M. Rodríguez–Monroy2*
1 Universidad Nacional de Colombia — Sede Medellín. Autopista Norte x Calle 65, Bloque 15. Medellín, Colombia.
2 Centro de Desarrollo de Productos Bióticos. Instituto Politécnico Nacional. Apdo. Postal 24. Yautepec, Morelos. 62731, México. * Autor para la correspondencia: E–mail: mrmonroy@ipn.mx
]]>Recibido 14 de Noviembre 2006
Aceptado 17 de Julio 2007
Resumen
Azadirachta indica es una planta importante para la obtención de bioinsecticidas y la azadiractina (Aza) es el principal compuesto presente en las semillas que presenta esta actividad biológica. Sin embargo, existen limitaciones agroclimáticas para la producción de las semillas y el cultivo de células vegetales in vitro surge como una tecnología alternativa y promisoria para la producción de Aza. En este trabajo, se presenta el estado del conocimiento del desarrollo del cultivo de células de A. indica y se analizan las estrategias usadas para mejorar el crecimiento y la producción de Aza. Se destacan los estudios del uso de agentes permeabilizantes de la membrana celular para liberar la Aza intracelular, la adición de precursores biosintéticos, los efectos del régimen de luz, la temperatura y la formulación de medios. A nivel de biorreactores, el tanque agitado se destaca como la configuración más reportada y los estudios abordan la evaluación de impulsores y la posibilidad de operación en un régimen por lote alimentado. Las productividades calculadas de los reportes, muestran una transferencia de los cultivos de matraces a biorreactor satisfactoria tecnológicamente y permiten vislumbrar el escalamiento futuro de los cultivos de células de A. indica para la producción de un bioinsecticida.
Palabras clave: Azadirachta indica, Azadiractina, biorreactor, bioinsecticida.
Abstract
Seeds of Azadirachta indica are an important source for production of bioinsecticides, being Azadirachtin (Aza) the main compound. However, agricultural and climate adversity limit the seed production, thereby plant cell culture in vitro is an alternative technology for Aza production. In this work, the state of the knowledge of A. indica cell culture is presented and the strategies for improving growth and production of Aza are analyzed. The use of cell membrane permeabilizing agents to liberate intracellular Aza, of biosynthetic precursors, light, temperature, and the effect of media culture are revised. The stirred tank bioreactor has been the most used for cell culture and the studies have been centered on impellers evaluation and the possibility of using the fed–batch regime. Calculated productivities show that the bioreactor is technologically appropriated for cultivation of A. indica cell cultures and bioinsecticide production.
]]> Keywords: Azadirachta indica, Azadirachtin, bioreactor, bioinsecticide.
DESCARGAR ARTÍCULO EN FORMATO PDF
Agradecimientos
Los autores agradecen a CONACYT (P43861) y a la SIP – IPN (20060039) por su apoyo financiero. F. Orozco S. agradece a la Secretaría de Relaciones Exteriores del Gobierno de México por su beca para estudios de doctorado.
Referencias
Abraham, T.; Devaki, N.; Kuruvilla, T. and Joseph, J. (2005). Production of peroxidase from plant cell and callus cultures. International Application published under the Patent Cooperation Treaty. Patent No. WO 2005/116196, PCT/IB2005/001421. [ Links ]
]]>Allan E. J.; Eeswara J. P.; Jarvis A. P.; Mordue, A. J.; Morgan, E. D. and Stuchbury, T. (2002). Induction of hairy root cultures of Azadirachta indica A. Juss and their production of azadirachtine and other important insect bioactive metabolites. Plant Cell Reports 21, 374–379. [ Links ]
Allan, E. J; Eeswara, J. P.; Johnson, J; Mordue, A. J.; Morgan E. D. and Stuchbury, T. (1994). The production of azadirachtin by in vitro tissue culture of neem, Azadirachta indica. Pesticide Science 42, 147–152. [ Links ]
Ambrosino, P.; Fresa, R.; Fogliano, V.; Monti, S. and Ritieni, A. (1999). Extraction of azadirachtin A from neem seed kernels by supercritical fluid and its evaluation by HPLC and LC/MS. Journal of Agriculture and Food Chemistry 47, 5252–5256. [ Links ]
Balaji, K.; Veeresham, C.; Srisilam, K. and Kokate C. (2003). Azadirachtin, a novel biopesticide from cell cultures of Azadirachta indica. Journal of Plant Biotechnology 5(2), 121–129. [ Links ]
Bandyopadhyay, U.; Biswas, K.; Chatterjee, R.; Bandyopadhyay, D.; Chattopadhyay, I.; Kumar, Ch.; Chakraborty, T.; Bhattacharya, K. and Banerjee, R. (2002). Gastrotective effect of neem Azadirachta indica bark extract: possible involvement of H+–K+–ATPase inhibition and scavenging of hydroxyl radical. Life Science 71 , 2845–2865. [ Links ]
]]>Brechelt, A. y Fernández C. L. (1995). El nim, un árbol para la agricultura y el medio ambiente. Experiencias en la República Dominicana. Fundación Agricultura y Medio Ambiente. Amigo del hogar, San Cristobal, República Dominicana. 133 p. [ Links ]
Capataz, J. (2005). Efecto de elicitores abióticos sobre la producción de metabolitos secundarios en suspensiones celulares de Azadirachta indica y su efecto sobre Spodoptera sp. Tesis de Maestría Biotecnología, U. Nacional de Colombia. Colombia. 67 p. [ Links ]
Dai, J.; Yaylayan, V.; Vijaya G. S. and Pare, J. (1999). Extraction and colorimetric determination of azadirachtin–related limonoids in neem seed kernel. Journal of Agriculture and Food Chemistry 47, 3738–3742. [ Links ]
David, E.; Jarvis, A. and Jonesa, G. (2000). Ratio of products formed on photo–oxidation of the neem triterpenoids nimbin and salannin. Arkivok 1(3), 312–319 [ Links ]
Dureja, P., Johnson, S. (2000). Photodegradation of azadirachtin–A: A neem–based pesticide. Current Science 79(12) 1700 – 1703. [ Links ]
Gajalakshmi, S. and Abbasi S. A. (2004). Neem leaves as a source of fertilizer–cum–pesticide vermicompost. Bioresource Technology 92, 291–296. [ Links ]
Gautam, V.; Nanda, K. and Gupta, S. (1993). Development of shoots and roots in anther–derived callus of Azadirachta indica A.Juss – a medicinal tree. Plant Cell Tissue and Organ Culture 34, 13 – 18. [ Links ]
Hollowach – Keller, L. P; Birman, I. and Patterson D. R. (1997). Method for producing azadirachtin by cell culture of Azadirachta indica. United States Patent No. 5,693,423. [ Links ]
Huang, Z.; Shi, P.; Dai, J. and Du, J. (2004). Protein metabolism in Spodoptera litura (F.) is influenced by the botanical insecticide azadirachtin. Pesticide Biochemistry and Physiology 80, 85–93 [ Links ]
Kearney, M.L.; Allan, E; Hooker J. E. and Mordue, J. (1994). Antifeedant effects of in vitro culture extracts of neem tree, Azadirachta indica against the desert locus (Schitocerca gregaria (Fosskal)) Plant Cell Tissue and Organ Culture 37, 67 – 71. [ Links ]
Kintzios, S. E. (2006). Territorial plant derived anticancer agents and plant species used in anticancer research. Critic Reviews in Plant Physiology 25, 79–113. [ Links ]
]]>Kumar, J. and Parmar B. S. (1999). Stabilization of azadirachtin A in neem formulations: effect of some solid carriers, neem oil, and stabilizers. Journal of Agriculture and Food Chemistry 47, 1735–1739. [ Links ]
Kuruvilla, T.; Kkomaraiah, P. and Ramkrishna, S.V. (1999). Enhanced secretion of azadirachtin by permeabilized margosa (Azadirachta indica) cells. Indian Journal of Experimental Biology 37, 89–91. [ Links ]
Lonard, D. M.; Bhasharan, G. and Dahm, R. H. (1996). Control of prothoracic gland activity by juvenile hormone in fourth instar Manduca sexta larvae. Journal of Insect Physiology 42 (3), 205–213. [ Links ]
Mordue, A. J. and Blackwell, A. (1993). Azadirachtin: an update. Journal of Insect Physiology 39 (11), 903–924. [ Links ]
Muñoz–Cruz, W.; Vanegas–Monterrosa, O. A.; Guzman–Rojas, A. A.; Capataz–Tafur, J.; Hoyos–Sánchez, R. A. y Orozco–Sánchez, F. (2006). Estimación de variables de operación de un biorreactor con células de Azadichta indica A. Juss. Revista de la Facultad Nacional de Agronomía, Medellín 59(2), 3467–3478. [ Links ]
]]>Murashige, T. and Skoog. F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiology 15, 473–497. [ Links ]
Naina, N. S; Gupta P. K. and Mascarenhas A. F. (1989). Genetic transformation and regeneration of transgenic neem (Azadirachta indica) plants using Agrobacterium tumefaciens. Current Science 58, 184 – 187. [ Links ]
National Research Council. (1992). Neem: A tree for solving global problems. Ed. National Academic Press. Washington D.C. 141 p. [ Links ]
Neem Foundation, (2006). Growing Neem. Disponible en internet: http://www.neemfoundation.org/contact.htm. Consultado el 15 de abril de 2006. [ Links ]
Parmar, B.S; Balraj; Varshney, M and Shah, D.O. (2004) . Neem oil microemulsion without cosurfactants or alcohols and a process to form the same. United States Patent 6,703,034. [ Links ]
]]>Prakash, G; Bhojwani, S and Srivastava, A. (2002). Production of azadirachtin from plant tissue culture: state of the art and future prospects. Biotechnology and Bioprocess Engineering 7, 185–193. [ Links ]
Prakash, G.; Emmanuel, C.J. and Srivastava, A. K. (2005) . Variability or azadirachtin in Azadirachta indica (neem) and batch kinetics studies of cell suspension culture. Biotechnology and Bioprocess Engineering 10, 198 – 204. [ Links ]
Prakash, G and Srivastava, A.K. (2005a). Production of azadirachtin (biopesticide) by plant cell cultivation of Azadirachta indica in bubble column reactor. Indian Chem. Eng. Congress, Chemcon 2005. New Delhi, 14– 17 Dec. [ Links ]
Prakash, G. and Srivastava, A. K. (2005b). Statistical media optimization for cell growth and azadirachtin production in Azadirachta indica (A. Juss) suspension cultures. Process Biochemistry 40, 3795–3800. [ Links ]
Prakash, G. and Srivastava, A. K. (2006). Modeling of azadirachtin production by Azadirachta indica and its use for feed forward optimization studies. Biochemical Engineering Journal 29, 62 – 88. [ Links ]
]]>Prakash, G. and Srivastava, A. K. (2007). Azadirachtin production in stirred tank reactors by Azadirachta indica suspension culture. Process Biochemistry 42(1), 93–97. [ Links ]
Raval, K.; Hellwig, S.; Prakash, G.; Ramos–Plasencia, A.; Srivastava, A. and Buchs, J. (2003). Necessity of a two–stage process for the production of azadirachtin–related limonoids in suspension cultures of Azadirachta indica. Journal of Bioscience and Bioengineering 96 (1), 16–22. [ Links ]
Raveendra, M; Acharya, L. D. and Udupa, N. (2004). Evaluation of antiplaque activity of Azadirachta indica leaf extract gel – a 6 week clinical study. Journal of Ethnopharmacology 90, 99–103. [ Links ]
Rodríguez–Monroy, M. y Galindo, E. (2003). Las células vegetales ¿frágiles para crecer en biorreactores? BioTecnología 8 (2), 6 – 17. [ Links ]
Royal Botanic Gardens. (2006). Neem. Plant Cultures, exploring plant and people. Royal Garden Botanics, Kew. Disponible en internet: http://www.plantcultures.org.uk/plants/neem_landing.html. Consultado el 15 de abril de 2006. [ Links ]
]]>Sajc, L. Grubisic, D. and Vunjak–Novakovic, G. (2000). Bioreactors for plant engineering: an outlook for further research. Biochemical Engineering Journal 4, 89–99 [ Links ]
Saxena, R. C. (2002). Management of insect pests with neem: Global perspective. In: 2002 Neem Proceedings. Neem Foundation, India. [ Links ]
Schaaf, O. (2000). Rapid and sensitive analysis of azadirachtin and related triterpenoids from neem (Azadirachta indica) by high performance liquid cromatography atmospheric pressure chemical ionization mass spectrometry. Journal of Chromatography A, 886: 89–97. [ Links ]
Sidhu, O. P.; Kumar, V. and Behl H. M. (2003). Variability in neem (Azadirachta indica) with respect to azadirachtin content. Journal of Agriculture and Food Chemistry 51, 910–915. [ Links ]
Stoney, C. (1997). Azadirachta indica – neem, a versatile tree for the tropics and subtropics. Forest, Farm, and Community Tree Network (FACT Net). Arkansa, USA. Disponible en internet: http://food–security.info/food–security.info/Winrock%20Archive/neem.htm. Consultado el 18 de abril de 2006. [ Links ]
Sundaram, K. M. S. and Curry, J. (1996). Effect of some UV light absorbers on the photostabilization of azadirachtin, a neem–based biopesticide. Chemosphere 32 (4), 649-659. [ Links ]
Surf – IP. (2006). Search Patents for Neem. Surf – IP, A Project of the Intellectual Property Office of Singapore. Singapore Government. Disponible en internet: http ://www.surfip.gov.sg/. Consultado el 20 de abril de 2006. [ Links ]
Trejo–Tapia, G.; Sepúlveda–Jiménez, G.; Trejo–Espino, JL.; García–Rojas, C.; de la Torre–Martínez, M.; Rodríguez–Monroy, M. and Ramos–Valdivia, A. 2007. Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst. Biotechnology and Bioengineering 98(1), 230–238. [ Links ]
Veeresham C.; Kumar M. R.; Sowjanya, D.; Kokate, C. K and Apte, S. S. (1998). Production of azadirachtin from callus cultures of Azadirachta indica. Fitoterapia 69, 423–424. [ Links ]
Wewetzer, A. (1998). Callus cultures of Azadirachta indica and their potential for the production of azadirachtin. Phytoparasitica 26 (1),47–52. [ Links ]
]]>