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, Ma 2 and PCM, significantly affected the number of microcalli produced, which was 90 Â 10 3 per Petri dish on Ma 2 with 0.5 lM zeatin and 9.0 lM 2,4 D, and 30 Â 10 3 per Petri dish on PCM. Moreover, continuous production of microcalli was achieved on Ma 2 and the frequency of embryogenic cell aggregates was higher among microcalli on Ma 2 -medium. However, no cell division was observed in protoplasts cultured on Ma 2 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.

Date Palm Cell and Protoplast Culture

“…Recently nurse culture technique has been successfully used to address the regeneration issues in recalcitrant protoplasts (Assani et al 2006;Horita et al 2002;Sun et al 2004). The number of microcalli we obtained was close to those obtained in earlier study in banana (Assani et al 2001).…”

“…The most frequently employed purification approach comprises filtration and centrifugation (Gamborg et al 1981). For purification of date palm protoplasts, the following protocol, described in Assani et al (2006), was employed. After enzymatic maceration, the digested mixture was filtered through 100/25-mm metallic mesh combination to remove debris.…”

“…Non-metabolizable osmoticum like sorbitol, manitol, inositol and sucrose, alone or as a mixture, have been employed for several plant protoplast systems (Evans and Bravo 1983;Kao and Michayluk 1975). Also solutions of mineral salts, such as KCl and CaCl 2 have been used as osmoticum (Assani et al 2006;Chabane et al 2007).…”

Date Palm Status and Perspective in Algeria

“…The product of maceration is fi ltered using two sieves (100/25 µm). Purifi cation of protoplasts is carried out according to the protocol described by Assani et al ( 2006 ) by two centrifugations at 65 g for 5 min each in a washing solution consisting of 204 mM KCl and 67 mM CaCl 2 . The protoplasts are then cultured in a liquid medium of the same composition as the callus.…”

Oligosaccharides induce changes in protein patterns of regenerating spruce protoplasts

“…GGMOs are able to influence developmental processes of spruce embryos [8], division and differentiation of cells [9], and viability and regeneration capacity of spruce protoplasts [10]. The success of protoplast regeneration is determined by cells viability and ability to synthesize new cell walls and to divide, but the rapidity of cell wall formation is influenced by many exo-and endogenous factors [11][12][13][14][15]. As galactoglucomannan oligosaccharides are components of primary and secondary cell walls of conifers, it was of interest to study the effect of GGMOs, as biologically active molecules, on spruce protoplasts.…”