Establishment of Embryogenic Suspension Cultures of Wheat by Continuous Callus Selection

Abstract

Embryogenic callus, derived from immature embryos of an Australian wheat (Triticum aestivum L. cv. Hartog) was used to establish liquid cultures which consisted of cell clumps (2-8 mm in diameter); these have been maintained for 20 months and still retain their embryogenic potential. This was achieved by continuous selection of embryogenic clumps at subculture. Incubation of callus in medium containing low concentrations of 2,4-D facilitated the selection of embryogenic cell clumps. After more than 1 year in liquid medium, 37% of the cell clumps formed embryoids and green plants on differentiation medium. The addition of silver nitrate to the subculture medium, which contained 5 μM 2,4-D, enhanced the differentiation frequency of the callus. A study of embryogenic calli from different wheat cultivars showed genotype was important for the retention of embryogenic potential in liquid medium. Fine suspension cultures, consisting of rapidly growing small aggregates of 2-200 cells, were derived from liquid cultures of cv. Hartog. Cytological examination showed that the cells of the fine suspension cultures had the normal wheat chromosome complement (2n = 42). Following transfer to differentiation medium, the fine suspension cultures formed soft as well as solid callus, from which embryoids and green plants have been obtained. Protoplasts isolated from the fine suspension cultures formed green plants with morphologically normal shoots and roots.