Hannelore Strosse

Establishment of embryogenic cell suspensions and plant regeneration of the dessert banana 'Williams' (Musa AAA group)

Summary ‘Williams’ is one of the most important dessert banana cultivars in the World. Improvement through genetic engineering depends on the development of embryogenic cell suspension (ECS) cultures. Yellow, compact meristematic globules and yellow-whiteish, friable embryogenic calli were induced from scalps (i.e., the top layer of highly proliferating meristem cultures). About 10% of induced explants gave rise to embryogenic calli. Embryogenic calli were used subsequently to establish ECS cultures. After 3 months of selection and sub-culturing, homogeneous ECS cultures with a high regeneration capacity were obtained. More than 80% of the cell culture consisted of embryogenic cell aggregates. Regenerable somatic embryos emerged about 3 weeks after the ECS was plated onto regeneration medium. Their weight increased 4.8- to 13-fold after 8 weeks of culture. The number of somatic embryos regenerated per ml settled cell volume (SCV) of embryogenic cells (i.e., somatic embryo recovery frequency) ranged between 0.92 – 2.17 105, depending on incubation and regeneration conditions. The conversion frequency from somatic embryo to plant varied between 42.5 – 55.5%, and was independent of regeneration conditions.

Potential of flow cytometry for monitoring genetic stability of banana embryogenic cell suspension cultures

Cell suspensions are the material of choice for rapid multiplication and for genetic engineering strategies such as in vitro mutagenesis and genetic transformation. Effective use of cell suspension cultures relies on the knowledge of several key parameters, which include genetic stability, kinetics of the cell cycle and a mode of plant regeneration. Here we report on the use of DNA flow cytometry for quality monitoring of banana cell suspension cultures. The method facilitates detection of ploidy changes and the occurrence of aneuploidy, which result in somaclonal variation of cell-suspension-derived plants. Flow cytometry could also be used to analyse the cell cycle kinetics by calculating the ratio of cells in the G2 and G1 phase of the cell cycle. This is important to determine the most appropriate moment for mutagenic treatment or for genetic transformation but also as an indicator on the proportion of cycling cells. In addition, the unicellular origin of somatic embryos was verified by treating embryogenic cell suspensions with colchicine and by determining the ploidy of regenerated plants by flow cytometric analysis. None of the plants regenerated from colchicine-treated embryogenic cell suspensions were mixoploid (chimeric). The application of flow cytometry will be discussed in relation to (a) the monitoring of genetic instability in DNA content of cell suspensions (b) the analysis of cell cycle and (c) the origin of somatic embryos of bananas and plantains.