An Improved Protocol for Microcallus Production and Whole Plant Regeneration from Recalcitrant Banana Protoplasts (Musa spp.)

Abstract

One important limitation for routine production of somatic hybrids in banana (Musa spp.) is the difficulty in protoplast regeneration. To facilitate protoplast regeneration in banana, the crucial step of microcallus production was optimised for the following parameters: nurse culture medium, duration of microcalli on nurse culture, differing nurse cells, and filter composition. A comparative study between two nurse cell media, Ma2 and PCM, significantly affected the number of microcalli produced, which was 90 × 103 per Petri dish on Ma2 with 0.5 μM zeatin and 9.0 μM 2,4 D, and 30 × 103 per Petri dish on PCM. Moreover, continuous production of microcalli was achieved on Ma2 and the frequency of embryogenic cell aggregates was higher among microcalli on Ma2-medium. However, no cell division was observed in protoplasts cultured on Ma2 in which nurse cells were maintained for 2 weeks suggesting a requirement of effective presence of nurse cells for cell division of banana protoplasts. Use of a filter in conjugation with nurse cells resulted in greater than 7-fold increase in the number of microcalli. Flow cytometry analysis of 124 protoplast-derived plants showed the presence of hexaploid plants (mother plant is triploid) at the frequency of 4%. Together, these data are indicative of the complex factors involved in the regulation of plant cell division and growth. Each individual aspect must be optimised for efficient protocol development.