The effect of different hormone combinations on direct and indirect somatic embryogenesis in Agave americana

Document Type : Original Article


1 Department of Genetics and Plant Breeding, Khuzestan Science and research Branch, Islamic Azad University, Ahvaz, Iran

2 Department of Genetics and Plant Breeding, Ahvaz Branch, Islamic Azad University, Ahvaz, Iran



Direct and indirect somatic embryogenesis in Agave Americana through basal parts of micropropagated shoots were considered in this study. For micropropagation, 2 factors (BA and NAA) were utilized in different concentrations. A factorial experiment based on a completely randomized design (CRD) was accomplished with four replicates in a modified Murashige and Skoog medium (MSB). BA in 3 mg/L along with NAA in 0.1 mg/L was the best treatment for multiplication. Callus and embryo induction were evaluated using different auxins in a CRD experiment with three replicates in MS medium supplemented with L2 vitamins. Picloram 2.5 and 2 mg/L and also Dicamba 1 mg/L induced 66.7%, 60%, and 53.3% of explants to somatic embryo, respectively. Also, 2,4-D 2 mg/L had the highest impact on callus induction (95.6%). Embryogenesis was studied using Dicamba and Picloram in the same culture medium in a factorial experiment based on CRD with three replicates. The results of orthogonal polynomial analaysisindicated that high concentrations of Dicamba from 0 to 1.5 mg/L had a significant effect on somatic embryo induction. However, higher concentrations had the opposite effect on somatic embryo numbers. Germination of somatic embryos was performed in MSB medium without growth regulators.


Beyl, C. A. and G. C. Sharma. 1983. 'Picloram induced somatic embryogenesis in Gasteria and Haworthia'. Plant Cell Tiss Org cult. 2:123–132.
Canhoto, J. M., S. C. Rama and G. S. Cruz. 2006. Somatic embryogenesis and plant regeneration in Carob (Ceratonia siliqua L.). In Vitro Cell Dev. Biol. Plant, 42(6)514-519.
Chalupa, V. 1990. 'Plant regeneration by somatic embryogenesis from cultured immature embryos of oak (Querem robur L.) and linden (Tilia cordata Mill.)'. Plant Cell Rep.  9(7):398-401.
Depnath, M., M. Pandey, R.  Sharma, G. S.Thakur  and P. Lal. 2009. 'Biotechnological intervention of Agave sisalana: A unique fiber yielding plant with medicinal property'. J. Med. Plants Res. 4(3) 177-187.
Fehér, A. 2008. 'The initiation phase of somatic embryogenesis: What we know what we don´t'. Acta Biol Szeged. 52:53–56.
Fehér, A. T. P. Pasternak and D. Dudits. 2003. 'Transition of somatic plant cells to an embryogenic state'. Plant Cell Tiss Org cult. 74:201–228.
Fehér, A. 2005.Why somatic plant cells start to form embryos? In: Mujib A, Samaj J (eds) Somatic embryogenesis. Springer-Verlag, Berlin Heidelberg, 85–101.
Fucikovsky, L. 2002. Diseases of some tropical and subtropical plants caused by bacteria, phytoplasmas and spiroplasmas. Universidad de Guadalajara y Colegio de Postgraduados.:175 pp.
George,  F. Edwin, Hall, A. Michael and  De Klerk, Geert-Jan. 2008. Plant Propagation by Tissue Culture.  3rd Ed, 175–204.
George, E. 1993. Plant propagation by tissue culture. 2nd ed., London: Exegetics Ltd.; 574 pp.
Groenewald, E. G., D. C. J. Wessels and A. Koeleman. 1977. 'Callus formation and subsequent plant regeneration from seed tissue of an Agave species (Agavaceae)'. J. Pflanzenphysiol. 81: 369–373.
Henry, Y., P.Vain and J. D. Buyser. 1994. 'Genetic analysis of in vitro plant tissue culture responses and regeneration capacities'. Euphytica ,79:45–58.
Jain, S. M. and P. K. Gupta. 2005.Protocole for somatic embryogenesis in woody plants, 505-515.
Jiménez, V. 2005.' Review: Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis'. Plant Growth Regu.l 47:91–110.
Kulus, D. 2014. 'Micropropagation of selected Agave species'. Interdisci-plinary Journal,4 ,  75-84.
Martínez-Palacios, A., M. P. Ortega-Larrocea, V. M.  Chávez and R. Bye. 2003. 'Somatic embryogenesis and organogenesis of Agave victoriareginae: considerations for its conservation'. Plant Cell Tiss Orgcult. 74:135–142.
Monja-Mio, K. M. and M. L. Robert. 2013. 'Direct somatic embryogenesis of Agave fourcroydes Lem. through thin cell layer culture'. In Vitro Cell. Dev. Biol.—Plant. 49:541–549.
Murashige, T. and F. Skoog. 1962. 'A revised medium for rapid growth and bioassays with tobacco tissue cultures'. Physiol. Plant. 15:473- 479.
Nikam T, G. Bansude and A. Kumar. 2003. 'Somatic embryogenesis in sisal (Agave sisalana Perr. Ex. Engelm)'. Plant Cell Rep 22:188-194.
Oliva, V. F. G., A. C.Castro, I. Herrera,  M. A. R. Mendiola  and F. A. G. Miceli. 2014. 'Response surface methodology of glutamine, asparagine and 2,4-Dichlorophenoxyacetic acid for Agave americana L. embryo number and their optimization in a RITA® automatic bioreactor system'. Int J Biotechnol Wellness Ind.3: 88-94.
Phillips, G. C. and G. B. Collins. 1979. 'In vitro tissue culture of selected legumes and plant regeneration from callus cultures of red clover'. Crop Sci. 19:59-64.
Portillo, L., F. Santacruz-Ruvalcaba, A. Gutiérrez-Mora and B. Rodríguez- Garay. 2007.  'Somatic embryogenesis in Agave tequilana Weber'. In Vitro Cell Dev Biol Plant .43:569–575.
Powers, D. E. and R. A. Backhaus. 1989. ' In vitro propagation of Agave arizonica Gentry and Weber'. Plant Cell Tiss Orgcult. 16: 57-60.
Robert, M. L., J. L. Herrera-Herrera, E. Castillo, G. Ojeda and M. Herrera-Alamillo. 2005. 'An efficient method for the micropropagation of Agave species'. Plant Cell Cult. Protocols. 318:165-178.
Robert, M.L., J. L., Herrera, F. Contreras and K. N. Scorer. 1987. 'In vitro propagation of Agave fourcroydes Lem. (Henequén)'. Plant Cell Tissue Organ Cult. 8:37-48.
Robert, M. L., J. L. Herrera-Herrera, M. A Herrera-Alamillo, A. Quijano and U. Balám. 2004. Manual for the in vitro culture of Agaves. Technical paper Nº 38. United Nations Industrial Development Organization, Vienna, Common Fund for Commodities.
Robert, M. L., J. L. Herrera, J. L. Chan and F. Contreras. 1992.'Micropropagation of Agave spp'. In: Bajaj YPS (eds), Biotechnology in Agriculture and Forestry, vol 19. High-tech and micro propagation III.II.9. Springer Berlin pp 306–329.
Rodríguez-Garay, B., A. Gutiérrez-Mora and B. Acosta-Dueñas. 1996.'Somatic embryogenesis of Agave victoria-reginae Moore'. Plant Cell, Tissue Organ Cult. 46:85–87.
Tejavathi, D. H.,  M. D. Rajanna,  R. Sowmya and K. Gayathramma. 2007. I'nduction of somatic embryos from cultures of Agave vera-cruz Mill'. In Vitro Cell Dev Biol Plant. 43:423–428.
Terzi, M. and F. Lo Schiavo. 1990.  'Developmental mutants in carrot'. pp. 391-397.  In: Nijkamp H.I.J., Van Der Plas I.H.W. & Van Aartrijk J. (eds.) (1990): Progress in Plant Cellular andMolecular Biology. Proc.
Zaffari, G. R., L. E. P. Peres and G. B.  Kerbauy. 1998. 'Endogenous levels of cytokinins, indolacetic acid, abscisic acid, and pigments in variegated somaclones of micropropagated banana leaves'. J. Plant Growth Regul. 17:59–61.