Improvement of maize (Zea mays L.) anther culture embryogenesis and direct regeneration by differentplant growth regulators and gelling agent

Document Type : Original Article


1 Department of Plant Biology, Faculty of Basic Sciences, Behbahan Khatam Alanbia University of Technology, Behbahan, Iran

2 Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khoram Abad, Iran

3 Department ofBiotechnology and Plant Breeding, Faculty of Agriculture, Ramin University of Agriculture and Natural Resources, Khouzestan, Iran


Androgenesis via anther culture or microspore culture is one of the current methods for producing haploid and double haploid plants in maize. To use of this method in maize breeding program it should be able to regenerate enough plantlets. Anthers culture usually is carried out indirectly via callus induction and regeneration on at least two different media. In this study a responsive genotype, ETH-M82, using a new single culture medium was used for embryogenesis and regeneration. We tested different growth regulators (2,4-D; kinetin; NAA & IAA) in modified YP medium. After6-week, direct regeneration on some treatments was observed. The highest frequency of direct formation of plantlets (in 100 anthers) occurred on medium supplemented with 2mgl-1 IAA and 2mgl-1 kinetin (4%).Best results with an average of 3.1plantlets in replication were obtained with the medium solidified with agar, while in difcobactoagar only 1.4 of plantlets in replication was produced. This experiment suggested that agar and plant growth regulators in the medium were beneficial for producing embryo and plantlet from maize anthers.


  1. Guha, S., Maheshwari, S.C., In vitro production of embryos from anthers of Datura. Nature,1964, vol. 204, pp. 497.
  2. Mohammadi, P.P., Moieni, A., Javaran, M.J., Colchicine induced embryogenesis and doubled haploid production in maize (Zea mays L.) anther culture. Iranian J Biotech, 2007, vol. 5(3), pp. 140–146.
  3. Kahrizi, D., Mahmoodi,S.,  BakhshiKhaniki, G.R.,  Mirzaei,M., Effect of genotype on androgenesis in barley (Hordeumvulgare L.). Biharean Biologist, 2011, vol. 5(2), pp. 132–134.
  4. Kahrizi, D., Moieni, A., Bozorgipour, R., Effect of genotype and carbohydrate source on androgenesis of hexaploid wheat (Triticumaestivum L.). Seed Plant, 2000, vol. 16, pp. 41–51.
  5. Jahne, A., Lorz, H., Cereal microspore culture. Plant Sci, 1995, vol. 109, pp. 1–12.
  6. Ateeq, B., Farah, A.M., Ali, M.N., Ahmad, W., Clastogenicity of pentachlorophenol 2,4-D and butachlor evaluated by Allium root tip test. Mutat Re., 2001, vol. 514, pp. 105–113.
  7. Kahrizi, D., Study of androgenesis and spontaneous chromosome doubling in barley (Hordeumvulgare L.) advanced line using isolated microspore culture. InterJ Plant Breed, 2009, vol.2(3), pp. 111–114.
  8. Kahrizi, D., Mohammadi, R., Study of androgenesis and spontaneous chromosome doubling in barley (Hordeumvulgare L.) genotypes using isolated microspore culture. Acta Agronomica Hung, 2009, vol. 57(2), pp. 155–164.
  9. Rashid, A., Induction of haploid plant/cell.In: Rashid, A. (Ed.), Cell physiology and genetics of higher plants. CRC Press, 1988, vol 1. pp. 119–158.
  10. Rakoczy-Trojanowska, M., Smiech, M., Malpszy, S., The influence of genotype and medium on rye (Secalecereale L.) anther culture. Plant Cell Tissue Org Cult, 1997, vol. 48, pp. 15–21.
  11. Ferrie, A.M.R., Palmer, C.E., Keller, W.A.,Haploid embryogenesis. In: Thorpe TA. (Ed.), In Vitro Embryogenesis in Plants. Kluwer Academic Publishers,1995, pp. 309–344.
  12. Dolgykh, Y.I., Establishment of callus cultures and regeneration of maize plants. In: Bajaj YPS, (Ed.), Biotechnology in Agriculture and Forestry, Maize, Springer Verlag, 1994, Vol. 25, pp. 197–209.
  13. Bronsema, F.B.F., Van Oostveen, W.J.F., Prinsen, E., Van Lammeren, A.A.M., Distribution of [14C] dichlorophenoxyacetic acid in cultured zygotic embryos of Zea mays L. JPlant Growth Regul, 1998, vol. 17, pp. 81–88.
  14. Fisher, C., Neuhaus, G., Influence of auxin on the establishment of bilateral symmetry in monocots. Plant J, 1996, vol. 9, pp. 659–669.
  15. Hoekstra, S., van Bergen, S., van Brouwershaven, I.R., Schilperoort, R.A.,Heidekamp, F.,The interaction of 2,4-D application and mannitol pre-treatment in anther and microspore culture of HordeumvulgareL cv. Igri. J Plant Physiol, 1996, vol. 148, pp. 696–700.
  16. Dodds, J.H., Roberts, L.W., Experiments in Plant Tissue Culture. In: Experiments in Plant Tissue Culture. Cambridge Universtiy Press, New York, vol. 1985, pp.156–171.
  17. Hadeler, B.,Scholz, S.,Reski, R.,Gelrite and agar differently influence cytokinin-sensitivity of a moss. J Plant Physiol,1995, vol. 146, pp. 369–371.
  18. Nestakova, M., Havrlentova, M., Farago, J., Effect of gelling agents on in vitro multiplication of two ornamental plants. Biologica Bratislava, 2000, vol. 55(4), 409–411.
  19. Henderson, W.E., Kinnersley, A.M., Corn starch as an alternative gelling agentfor plant tissue culture. Plant Cell Tiss Org Cult, 1988, vol. 15, pp. 17–22.
  20. Sahay, S., The use of psyllium (isubgol) as an alternative gelling agent for microbial culture media. World J Microb Biot, 1999, vol. 15, pp.733–735.
  21. Atanassov, A.,Zagorska, N.,Boyadjiev, P.,Djilianov, D.,In vitro production of haploid plants. World J Microb Biot., 1995, vol. 11, pp. 400–408.
  22. Veramendi,J.,Villafranca, M.J., Sota, V., Mingo-Castel, A.M., Gelrite as an alternative to agar for micropropagation and microtuberization ofSolanumtuberosumL. cv. Baraka. In Vitro Cell Dev Plant, 1997, vol. 33, pp. 195–199.
  23. Chevreau, E., Mourgues, F., Neveu, M., Chevalier, M., Effect of gelling agents and antibiotics on adventitious bud regeneration from in vitro leaves of pear. In Vitro Cell Dev Plant, 1997, vol. 33, pp. 173–179.
  24. Genovesi, A.D., Collins, G.B., In vitro production of haploid plants of corn via anther culture. Crop Sci, 1981, vol. 22, pp. 1137–1144.
  25. Feher, A., Pasternak, T.,Otvos, K.,Miskolczi, P.,Dudits, D., Induction of embryogenic competence in somatic plantcells: a review. Biol Bratislava, 2002, vol. 57,pp.5–12.
  26. Feher, A., Pasternak, T.P., Dudits, D., Transition ofsomatic plant cells to an embryogenic state. Plant Cell Tiss Org Cult., 2003, vol. 74, pp. 201–228.
  27. Gaj, M.D., Factors influencing somatic embryogenesisinduction and plant regeneration with particular reference toArabidopsis thaliana L. Heynh. J Plant Growth Regul, 2004, vol. 43, pp.27–47.
  28. Buter, B., In vitro haploid production in maize. In: Jain SM, Sopory SK,Veilleux RE, (Eds.), In vitro Haploid Production in Higher Plants. Kluwer Academic Publishers 1997, Vol. 4,pp. 37–71.
  29. Jimenez, V.M.,Bangerth, F., Hormonal status of maize explants and of the embryogenic and non-embryogenic callus cultures derived from them as related to morphogenesis in vitro. Plant Sci, 2001, vol.160, pp. 247–257.
  30. Johansson, L.B., Effects of activated charcoal, cold treatment and elevated CO2 concentration on embryogenesis in anther cultures. In: de Gruyter W. (ed) Genetic manipulation in plant breeding, New York, USA, 1986,pp. 257–264.
  31. Buah, J.N., Kawamitsu, Y., Sato, S., Murayama,S.,Effects of different types and concentrations of gelling agents on the physical and chemical properties of media and the growth of banana (Musa spp.) in vitro. Plant Prod Sci., 1999, vol. 2, pp. 138–145.
  32. Huang, L., Chi, D.,Pivotal roles of picloram and gelrite in banana callus culture. Environ Exp. Bot, 1988, vol. 28, pp. 249–258.
  33. Naim, B.J., Furneaux, R.H., Stevenson, T.T., Identification of an agar constituent responsible for hydric control in micropropagation of radiatapine. Plant Cell Tiss Org Cult, 1995. Vol. 43, pp. 1–11.
  34. Stoltz, L.P., Agar restriction of the growth of excised mature iris embryos. J Am Soc Hort Sci, 1971, vol. 96, pp. 618–684.
  35. Romberger, J.A., Tabor, C.A., The Piceaabies shoot apical meristem in culture. Agar and autoclaving effects. Am J Bot,1971, vo. 58, pp. 131–140.