Abstract
The role of nitrogen (N) supplied as inorganic ammonium (NH4 +) and nitrate (NO3 −), and organic glutamine on shoot regeneration, incidence of hyperhydricity and production of good quality shoots in Aloe polyphylla explants was investigated. The omission of total N from the culture medium resulted in low proliferation and hindered shoot growth. Ammonium as the sole source of N depressed shoot regeneration and growth and escalated the frequency of hyperhydricity to ca. 50%. When NO3 − was used as the sole N source, shoots of good quality were produced and hyperhydricity was completely eliminated. Overall, the MS N combination was superior to any single N source for proliferation and growth of shoots, suggesting a synergistic effect between NH4 + and NO3 − on shoot regeneration. Moreover, the relative amounts of NH4 + and NO3 − also influenced the parameters tested. The highest regeneration was obtained with NH4 +:NO3 − ratios (mM) of 20:40, 30:30 and 40:20. Decreasing the ratio of NH4 +:NO3 − lowered the incidence of hyperhydricity. This study also demonstrates the potential of glutamine as the sole source of N, since its application resulted in the production of good quality shoots and almost no hyperhydricity.
Similar content being viewed by others
Abbreviations
- ANOVA:
-
Analysis of variance
- HS:
-
Hyperhydric shoots
- IBA:
-
Indole-3-butyric acid
- MS:
-
Murashige and Skoog (1962) medium
- N:
-
Nitrogen
- NS:
-
Normal shoots
References
Abrie AL, Van Staden J (2001) Micropropagation of the endangered Aloe polyphylla. Plant Growth Regul 33:19–23
Avila A de L, Pereyra SM, Argüello JA (1998) Nitrogen concentration and proportion of NH4 +-N affect potato cultivar response in solid and liquid media. HortScience 33:336–338
Brand MH (1993) Agar and ammonium nitrate influence hyperhydricity, tissue nitrate and total nitrogen content of serviceberry (Amelanchier arborea) shoots in vitro. Plant Cell Tiss Organ Cult 35:203–209
Chauvin JE, Salesses G (1988) Advances in chestnut micropropagation (Castanea sp.). Acta Hortic 227:340–345
Cousson A, Tran Thanh Van K (1993) Influence of ionic composition of the culture medium on de novo flower formation in tobacco thin cell layers. Can J Bot 71:506–511
Daguin F, Letouzé R (1986) Ammonium-induced vitrification in cultured tissues. Physiol Plant 66:94–98
Debergh P, Harbaoui Y, Lemeur R (1981) Mass propagation of globe artichoke (Cynara scolymus): evaluation of different hypotheses to overcome vitrification with special reference to water potential. Physiol Plant 53:181–187
Debergh P, Aitken-Christie J, Cohen D, Grout B, von Arnold S, Zimmerman R, Ziv M (1992) Reconsideration of the term ‘vitrification’ as used in micropropagation. Plant Cell Tiss Organ Cult 30:135–140
Evans NE (1993) A preliminary study on the effects of nitrogen supply on the growth in vitro of nine potato genotypes (Solanum spp). J Exp Bot 44:837–841
Gaspar T (1991) Vitrification in micropropagation. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, volume 17. High-tech and micropropagation I. Springer, Berlin, pp 116–126
George EF (1993) Plant propagation by tissue culture. Part 1: the technology, 2nd edn. Exegetics Ltd, England, pp 654–670
Grimes HD, Hodges TK (1990) The inorganic NO3 −:NH4 + ratio influences plant regeneration and auxin sensitivity in primary callus derived from immature embryos of indica rice (Oryza sativa L.). J Plant Physiol 136:362–367
Han BH, Paek KY, Choi JK (1991) Micropropagation of Gypsophila paniculata using shoot tip culture in vitro. J Korean Soc Hortic Sci 32:394–400
Ivanova M (2009) Regulation of hyperhydricity in Aloe polyphylla propagated in vitro. PhD Thesis, University of KwaZulu-Natal
Ivanova M, Van Staden J (2008) Effect of ammonium ions and cytokinins on hyperhydricity and multiplication rate of in vitro regenerated shoots of Aloe polyphylla. Plant Cell Tiss Organ Cult 92:227–231
Ivanova M, Novák O, Strnad M, Van Staden J (2006) Endogenous cytokinins in shoots of Aloe polyphylla cultured in vitro in relation to hyperhydricity, exogenous cytokinins and gelling agents. Plant Growth Regul 50:219–230
Kevers C, Franck T, Strasser RJ, Dommes J, Gaspar T (2004) Hyperhydricity of micropropagated shoots: a typically stress-induced change of physiological state. Plant Cell Tiss Organ Cult 77:181–191
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Nagakubo T, Nagasawa A, Ohkawa H (1993) Micropropagation of garlic through in vitro bulblet formation. Plant Cell Tiss Organ Cult 32:175–183
Niedz RP, Evens TJ (2006) A solution to the problem of ion confounding in experimental biology. Nat Methods 3:417
Nowak B, Miczynski K, Hudy L (2007) The effect of total inorganic nitrogen and the balance between its ionic forms on adventitious bud formation and callus growth of ‘Wegierka Zwykla’ plum (Prunus domestica L.). Acta Physiol Plant 29:479–484
Pâques M (1991) Vitrification and micropropagation: causes, remedies and prospects. Acta Hortic 289:283–290
Ramage CM, Williams RR (2002) Inorganic nitrogen requirements during shoot organogenesis in tobacco leaf discs. J Exp Bot 53:1437–1443
Riffaud JL, Cornu D (1981) Utilisation de la culture in vitro pour la multiplication de merisiers adultes (Prunus avium L.) sélectionnés en forêt. Agronomie 1:633–640
Sathyanarayana BN, Blake J (1994) The effect of nitrogen sources and initial pH of the media with or without buffer on in vitro rooting of jackfruit (Artocarpus heterophyllus Lam). In: Lumsden PJ, Nicholas JR, Davies WJ (eds) Physiology growth and development of plants in culture. Kluwer, Dordrecht, pp 77–82
Tsai C-J, Saunders JW (1999) Evaluation of sole nitrogen sources for shoot and leaf disc cultures of sugarbeet. Plant Cell Tiss Organ Cult 59:47–56
Tsay H-S (1998) Effects of medium composition at different recultures on vitrification of carnation (Dianthus caryophyllus) in vitro shoot proliferation. Acta Hortic 461:243–249
Vasudevan A, Selvaraj N, Ganapathi A, Kasthurirengan S, Anbazhagan VR, Manickavasagam M (2004) Glutamine: a suitable nitrogen source for enhanced shoot multiplication in Cucumis sativus L. Biol Plant 48:125–128
Vieitez AM, Ballester A, San-José MC, Vieitez E (1985) Anatomical and chemical studies of vitrified shoots of chestnut regenerated in vitro. Physiol Plant 65:177–184
Walch-Liu P, Neumann G, Bangerth F, Engels C (2000) Rapid effects of nitrogen form on leaf morphogenesis in tobacco. J Exp Bot 51:227–237
Williams RR (1993) Mineral nutrition in vitro—a mechanistic approach. Aust J Bot 41:237–251
Woodward AJ, Bennett IJ, Pusswonge S (2006) The effect of nitrogen source and concentration, medium pH and buffering on in vitro shoot growth and rooting in Eucalyptus marginata. Sci Hortic 110:208–213
Ziv M (1991) Vitrification: morphological and physiological disorders of in vitro plants. In: Debergh PC, Zimmerman RH (eds) Micropropagation: technology and application. Kluwer, Dordrecht, pp 45–69
Acknowledgments
The National Research Foundation, Pretoria, South Africa is thanked for financial assistance.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ivanova, M., Van Staden, J. Nitrogen source, concentration, and NH4 +:NO3 − ratio influence shoot regeneration and hyperhydricity in tissue cultured Aloe polyphylla . Plant Cell Tiss Organ Cult 99, 167–174 (2009). https://doi.org/10.1007/s11240-009-9589-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-009-9589-8