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Plant Cell, Tissue and Organ Culture (2005) 82: 57–66

Ó Springer 2005

Optimization of plantain (Musa AAB) micropropagation by temporary immersion system
S. Roels1,*, M. Escalona3, I. Cejas3, C. Noceda2, R. Rodriguez2, M.J. Canal2, J. Sandoval4 & P. Debergh1

Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Gent, Belgium; 2Departamento de Biologı´a Organismos y Sistemas, University of Oviedo, Catedra´tico Rodrigo Urı´a s/n, 33071 Oviedo, Spain; 3 Laboratory for Plant Cell and Tissue Culture, University of Ciego de Avila, Carretera Ciego - Moron km 9, 69450 Ciego de Avila, Cuba; 4 Corporacion Bananero Nacional, P.O. Box 390, 7210 Guapiles, Costa Rica (*requests for offprints; Fax: +32-9-264-6225; E-mail:

Received 11 June 2004; accepted in revised form 24 November 2004

Key words: in vitro culture, meta-topolin, multiplication rate, Musaceae, temporary immersion

Abstract The positive and reliable effect of temporary immersion systems on in vitro shoot proliferation was already proved for different plant genera and it is now presented as an alternative for plantain micropropagation.Some culture parameters affecting the efficiency of the twin flasks system or temporary immersion bioreactor (Escalona et al., 1999) were investigated. Three different cytokinins (benzyladenine, thidiazuron and meta-topolin) were added to the culture medium and meta-topolin at a concentration of 4.4 lM was proved to be the most efficient. Successive subcultures (28 days per subculture) were performed onmedium supplemented with meta-topolin, revealing a decrease in multiplication after the 6th subculture. Multiplication rate was not changed within the ranges of immersion times (4, 12 or 22 min) and frequencies (every 3, 5 or 7 h) tested. The size of the bioreactor (250, 1,000, 5,000 or 10,000 ml) and the volume of medium per inoculum (10, 20 or 30 ml) were also evaluated and appeared to have aninfluence on the multiplication. A proportion of 25–100 ml of headspace per inoculum and 30 ml of medium per inoculum resulted in a multiplication rate >13 in 28 days. Abbreviations: BA – N6-benzyladenine; MET – meta-topolin; SP medium – standard proliferation medium; TDZ – thidiazuron; TIB – temporary immersion bioreactor

Introduction Bananas and plantains (Musa spp.) are among the most importantfood crops in the world, with a production approximating 102 million tons per year (FAO, 2002); one third is plantain. However, expansion of plantain production is limited by shortage of plant material. The transmission of harmful insects, nematodes, viruses and black Sigatoka disease by field-grown suckers has

prompted interest in the use of aseptic culture techniques (in vitro). High productioncosts generally limit the commercial use of in vitro micropropagation. Using liquid media is considered to be the ideal solution for automation and reducing production costs. However, the use of liquid media can be responsible for other problems such as asphyxia, hyperhydricity and the need for more complex equipment (Etienne and Berthouly, 2002). Several

58 methods have been proposed toavoid these problems, one being the twin flasks system or temporary immersion bioreactor (TIB), which allows temporary immersion of the explants (Escalona et al., 1999). Alvard et al. (1993) made a comparison between five different liquid medium culture methods and gelled culture medium for multiplication of the banana ‘Grande Naine’. The highest multiplication rate (>5 in 20 days) was observed forexplants subjected to temporary immersion, and the highest accumulation of dry matter was obtained in aerated liquid medium and temporary immersion. In plantain (AAB group) the growth of axillary buds in vivo is inhibited by a high degree of apical dominance. Conversely, many well-developed suckers are often observed on banana (AAA group). Ortiz and Vuylsteke (1994) have shown that plantain-banana...
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