Myo-inositol increases the plating efficiency of protoplast derived from cotyledon of cabbage (Brassica oleracea var. capitata)

Abstract

This study describes the effect of myo-inositol on sustained cell division and plant regeneration from cotyledon-derived protoplast of cabbage (Brassica oleracea var. capitata). Freshly isolated protoplasts were cultured in modified Murashige and Skoog (MS) medium removed ammonia ions and containing <TEX>$0.4\;mg\;l^{-1}$</TEX> thiamine HCl, <TEX>$100\;m</TEX><TEX>g\;l^{-1}$</TEX> myo-inositol, <TEX>$2\;mgl^{-1}$</TEX> 2,4-D, <TEX>$0.5\;mgl^{-1}$</TEX> BA, <TEX>$30\;gl^{-1}$</TEX> sucrose and several concentrations of myo-inositol (2, 4, 6, 8, 10% (w/v)) as an osmotic stabilizer. After 3 weeks of culture in the dark at <TEX>$25^{\circ}C$</TEX>, the plating efficiency of cabbage protoplasts reached to <TEX>$22.5{\pm}2.9%$</TEX> when cultured in modified MS medium supplemented with <TEX>$2\;mgl^{-1}$</TEX> 2,4-D, <TEX>$0.5\;mgl^{-1}$</TEX> BA, <TEX>$30\;gl^{-1}$</TEX> sucrose and 8% (w/v) of myo-inositol at a density of <TEX>$2{\times}10^5$</TEX> protoplasts/ml. Rapidly growing cell colonies after 3 weeks of culture were transferred to the same culture medium removed osmoticum. To induce shoot regeneration from calluses, calluses with about 2 mm in diameter were transferred to the MS medium containing <TEX>$2\;mgl^{-1}$</TEX> BA and <TEX>$0.5\;mgl^{-1}$</TEX> NAA. After further three weeks of incubation onto the medium in the light, green shoots were formed on the surface of calluses at a frequency of 30%. Upon transfer to half-strength MS basal medium, roots were formed onto the bottom of regenerated shoots without auxin treatments. These regenerated plantlets were successfully acclimatized to soil transfer, grown to normal mature plants. The cabbage protoplast culture system established in this study could be applied for production of somatic hybrids or cybrids by asymmetric protoplast fusion and mass proliferation of elite somatic clones of cabbage.