142 M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 Electrostimulation experiments attracted considerable interest during the 1990s,but this simple approach has received less attention in recent years.Clearly,this is an area that deserves research effort in the future,particularly for protoplast systems that currently remain recalcitrant in culture 6.2.Supplementation of culture media with surfactants,antibiotics,and polyamines A novel approach for enhancing the mitotic division of plant protoplast-derived cells involves supplementation of the culture medium with nonionic surfactants,especially the commercially available copolymer compounds known as Pluronics.One such surfactant, Pluronic F-68,a polyoxyethylene-polyoxypropylene copolymer,is used extensively as a nontoxic,low-cost cell-protecting agent during the culture of both animal and plant cells (Lowe et al.,2001).For example,in experiments with plant protoplasts,supplementation of culture medium with 0.1%(wt/vol)Pluronic F-68 increased the plating efficiency of protoplasts of Solanum dulcamara by 26%over control (Kumar et al.,1992).The beneficial effects of medium supplementation with Pluronic F-68 have also been assessed in cells of several other plant species,including those following recovery from cryopreservation.Lowe et al.(2001)suggested that Pluronic F-68 may exert a stimulatory effect on cell growth and differentiation by promoting the uptake of nutrients, growth regulators,and oxygen. Some antibiotics stimulate the division of protoplast-derived cells.For example,the cephalosporin antibiotic,cefotaxime,promoted mitotic division and cell colony formation of protoplasts isolated from seedling leaves of the woody plant passionfruit (Passiflora edulis)when added to the culture medium at 250 ug ml(d'Utra Vaz et al., 1993).Ciprofloxacin has also been shown to stimulate division in protoplasts isolated from callus of Allium longicuspis (Fellner,1995),with first cell divisions being observed after 2-6 days of culture.In contrast,protoplasts cultured without this antibiotic required at least 10 days in culture before entering division.In both cases,the mode of action of antibiotics is unclear,although cefotaxime may be metabolised to growth regulator-like compounds. Polyamines influence plant cell morphogenesis by regulating DNA replication, transcription,translation,cell division,and differentiation,and are regarded as a new class of plant growth regulators and a reserve of carbon and nitrogen in cultured tissues (Kakkar et al.,2000).Thus,polyamines stimulated DNA synthesis and mitotic activity in oat protoplasts,with arginine also stimulating division in protoplasts of almond.In other reports,leaf protoplasts of cytoplasmic male sterile and male fertile diploid sugarbeet synthesised new cell walls and underwent sustained division to form callus in the presence of the diamine putrescine (Jazdzewska et al.,2000),with this research group having demonstrated previously that spermine promoted division of the same protoplast system (Majewska-Sawka et al.,1997).As expected,inhibition of the conversion of putrescine to spermidine by difluoromethyl arginine,difluoromethyl ornithine,and cyclohexylamine reduced cell division by 30%in protoplasts of the legume Vigna aconitifolia.Since the existing literature is limited,it will be timely to clarify the validity of polyamines as media supplements in stimulating division and morphogenesis of protoplast-derived cells and tissues in a range of species.Electrostimulation experiments attracted considerable interest during the 1990s, but this simple approach has received less attention in recent years. Clearly, this is an area that deserves research effort in the future, particularly for protoplast systems that currently remain recalcitrant in culture. 6.2. Supplementation of culture media with surfactants, antibiotics, and polyamines A novel approach for enhancing the mitotic division of plant protoplast-derived cells involves supplementation of the culture medium with nonionic surfactants, especially the commercially available copolymer compounds known as Pluronics. One such surfactant, PluronicR F-68, a polyoxyethylene–polyoxypropylene copolymer, is used extensively as a nontoxic, low-cost cell-protecting agent during the culture of both animal and plant cells (Lowe et al., 2001). For example, in experiments with plant protoplasts, supplementation of culture medium with 0.1% (wt/vol) PluronicR F-68 increased the plating efficiency of protoplasts of Solanum dulcamara by 26% over control (Kumar et al., 1992). The beneficial effects of medium supplementation with PluronicR F-68 have also been assessed in cells of several other plant species, including those following recovery from cryopreservation. Lowe et al. (2001) suggested that PluronicR F-68 may exert a stimulatory effect on cell growth and differentiation by promoting the uptake of nutrients, growth regulators, and oxygen. Some antibiotics stimulate the division of protoplast-derived cells. For example, the cephalosporin antibiotic, cefotaxime, promoted mitotic division and cell colony formation of protoplasts isolated from seedling leaves of the woody plant passionfruit (Passiflora edulis) when added to the culture medium at 250 Ag ml1 (d’Utra Vaz et al., 1993). Ciprofloxacin has also been shown to stimulate division in protoplasts isolated from callus of Allium longicuspis (Fellner, 1995), with first cell divisions being observed after 2–6 days of culture. In contrast, protoplasts cultured without this antibiotic required at least 10 days in culture before entering division. In both cases, the mode of action of antibiotics is unclear, although cefotaxime may be metabolised to growth regulator-like compounds. Polyamines influence plant cell morphogenesis by regulating DNA replication, transcription, translation, cell division, and differentiation, and are regarded as a new class of plant growth regulators and a reserve of carbon and nitrogen in cultured tissues (Kakkar et al., 2000). Thus, polyamines stimulated DNA synthesis and mitotic activity in oat protoplasts, with arginine also stimulating division in protoplasts of almond. In other reports, leaf protoplasts of cytoplasmic male sterile and male fertile diploid sugarbeet synthesised new cell walls and underwent sustained division to form callus in the presence of the diamine putrescine (Jazdzewska et al., 2000), with this research group having demonstrated previously that spermine promoted division of the same protoplast system (Majewska-Sawka et al., 1997). As expected, inhibition of the conversion of putrescine to spermidine by difluoromethyl arginine, difluoromethyl ornithine, and cyclohexylamine reduced cell division by 30% in protoplasts of the legume Vigna aconitifolia. Since the existing literature is limited, it will be timely to clarify the validity of polyamines as media supplements in stimulating division and morphogenesis of protoplast-derived cells and tissues in a range of species. 142 M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171