Development of embryogenic cell suspensions from shoot meristematic tissue in bananas and plantains (Musa spp.)
Introduction
Bananas and plantains are monocotyledonous, perennial herbs, cultivated in nearly 120 countries of the humid and sub humid tropical regions. In the developing world, Musa species are one of the major food sources [1]. Bananas and plantains are, however, prone to many pests and diseases such as fungi, viruses, bacteria, insects, and nematodes [2]. Due to sterility of most important banana and plantain varieties, genetic improvement through conventional breeding is seriously hampered [3], [4]. Hence, genetic engineering of bananas and plantains is needed. The choice of candidate tissues for genetic engineering in banana is restricted to meristematic tissue and embryogenic cell suspensions. The low transformation frequency of explants isolated from multiple meristem cultures indicates that this tissue is not suitable for genetic transformation [5]. In contrast, transformation of embryogenic cell suspensions (ECS) of bananas and plantains is very efficient [5], [6], [7]. ECS are also the material of choice for regenerable protoplast production [8], [9], [10] and mutation breeding [11], [12]. In bananas and plantains, four procedures exist for the development of embryogenic cell suspensions. They differ mainly in the source of the explant: zygotic embryos [13], [14], rhizome slices, and leaf sheaths [15], immature (fe)male flowers [16], [17], [18], and multiple meristem cultures [19], [20]. In this paper, we focus on multiple meristem cultures as the starting material from which an approximate 3 mm top layer (i.e. scalp) is excised and cultured on embryogenesis induction medium. The procedure to develop embryogenic cell suspensions derived from shoot meristematic tissue is henceforth referred to as scalp-method. The main advantage of this procedure is that no field access is required, unlike with most of the other methods. As such, virus-indexed plant material can be used and there is no seasonal dependence of the embryogenic response. In this paper, we demonstrate on a large-scale the application of the scalp-method for the establishment of embryogenic cell suspensions in a large range of banana and plantain varieties. Also we provide tools to monitor quality control during the entire process.
Section snippets
Materials and methods
The following subsequent in vitro phases are involved in the scalp-methodology: preparation of multiple meristem cultures, embryogenesis induction, suspension initiation and maintenance, and plant regeneration (Table 1).
Results
The different in vitro phases involved in the scalp-methodology, the resulting plant material and the duration of the different steps are indicated in Table 1.
Discussion
This paper presents for the first time both a qualitative and quantitative account of the establishment of Musa embryogenic cell suspensions derived from shoot meristematic tissue. It involved large-scale experiments with 24,375 explants of 18 varieties classified into 5 genome types. As such, we were able to demonstrate that the scalp-method is applicable to a wide range of banana and plantain varieties. Unlike most dicotyledoneous plants [25] and seed setting monocots [26], we found that the
Acknowledgements
This work was possible due to the financial support of DGIC (Directorate-General for International Cooperation), Belgium through a grant to IITA (International Institute of Tropical Agriculture) and INIBAP (International Network for the Improvement of Banana and Plantain). This research falls within the strategy of ProMusa. We thank F. Côte and R. Domergue of CIRAD (Centre de Coopération Internationale en Recherce Agronomique pour le Développement) for the fruitful discussions on somatic
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