Fine Suspension Cultures Research Articles

Establishment of Fine Suspension Cultures of Triticum tauschii ([Coss.] Schmal.) which remain Embryogenic for Several Years

Immature embryos of 10 accessions of Triticum tauschii, the D genome donor of Triticum aestivum, were used to produce embryogenic callus for the initiation of suspension cultures. For the long-term maintenance of embryogenicity of these suspensions, it was necessary to use different media for the initiation, establishment and maintenance phases. The initiation phase required media supplemented with L-proline, L-asparagine and L-glutamine, together with Dicamba at 12 mg L −1 and maltose. In the establishment phase, it was essential to reduce the concentration of Dicamba to 6 mg L −1 for the rapid production of fine suspension cultures. Finally, the long-term maintenance of a capacity for regeneration depended on the inclusion of 1.1 mg L −1 2,4-dichlorophenoxyacetic acid and 30 g L −1 sucrose in the medium. By the use of these procedures, long-term embryogenic fine suspension cultures were established from two accessions, while non-embryogenic fine suspension cultures were produced from five accessions. Over 90% of plants regenerated from fine suspensions of 1-year-old embryogenic cultures were fertile, and embryogenic suspension cultures retained their regeneration capacity for more than 3 years.

Plant regeneration from protoplasts of Triticum aestivum L. cv. Nakasoushu

A fast-growing, small, granular, embryogenic callus was selected from primary calli induced from the Japanese wheat cultivar Nakasoushu and the Australian wheat cultivar Bodallin. Regenerable and fine suspension cultures were induced three to six months after liquid culture was initiated and were characterized by dense cytoplasm and active division. These suspension cultures routinely provided high yields of protoplasts with about 90% viability when incubated in a modified KMP (Kao and Michayluk, 1975) medium containing 1 mg l-1 2,4-D (2,4-dichlorophenoxyacetic acid), and 1 mg l-1 zeatin. Nakasoushu and Bodallin protoplasts divided at frequencies of 8.6% and 11.1%, respectively, in agarose-solidified media. When Nakasoushu protoplasts were cultured with effective nurse cells of sorghum and wheat, protoplast division increased to 16.9% and 12.6%, respectively. Plating efficiencies varied from 0.03% to 2.5%. After subculture, protocolonies yielded embryogenic calli and somatic embryos, from which green plants were eventually regenerated. Whole plants obtained from Nakasoushu protoplasts were fertile, demonstrating the first report of Japanese cultivars in wheat protoplast cultures.

Establishment of Embryogenic Suspension Cultures of Wheat by Continuous Callus Selection

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.

A method for the rapid production of fine plant cell suspension cultures

A method has been devised which allows the rapid production of fine suspension cultures of small aggregate size from suspension cultures of large average aggregate size, such as those of Capsicum frutescens. The method, which uses a Waring blender for aseptic homogenisation of cultures, has also been shown to be effective in rapidly producing suspension cultures from callus cultures. The suspension cultures so produced are particularly useful for immobilisation, such as in porous polyurethane foam matrices.

The toxicity of monoterpenes to plant cell cultures

The effects of exogenous monoterpenes on cellular viability and growth of fine suspension cultures of Pelargonium fragrans have been investigated in order to establish whether product toxicity might play a role in determining the level of monoterpene accumulation in morphologically undifferentiated plant cell cultures. Producing lines of P. fragrans accumulate monoterpenes in the medium at concentrations which we now demonstrate to be toxic to fine suspension cultures. The problems associated with end-product toxicity must be solved before the potential of plant tissue culture techniques for the commercial production of monoterpenes be fully realised.

Growth Cycle of Suspension Cultures of Lycopersicon esculentum and L. peruvianum

Summary Rapidly growing, fine suspension cultures were established from hypocotyl pieces of Lycopersicon esculentum cv. Lukullus and from cotyledons of L. peruvianum. They are characterized with respect to their growth pattern, nutrient consumption as well as uptake and incorporation of precursors of nucleic acid and protein synthesis. The doubling time of these tomato suspension cultures in a modified Murashige-Skoog medium is ~ 24 h. Analyses of the macronutrient consumption during the growth cycle (carbohydrate, nitrate, ammonia, phosphate) revealed a phosphate-limited growth with exponential increase of cell number and dry weight during 3–5 d. Normally 3% sucrose is used as carbon source; but glucose is suitable as well. In L. esculentum suspensions sucrose is rapidly hydrolysed by a cell wall bound acid invertase, and the liberated glucose and fructose are consumed consecutively. Long-term cultures from both tomato species exhibit very low morphogenetic potential. With L. peruvianum green, heterogeneous suspensions can be obtained after prolonged passages on cytokinin-free media containing indole acetic acid as the sole growth hormone.