M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 145 Reference has been made to the presence of glass rods in the medium,stimulating division of cassava leaf protoplasts.Growth of these protoplasts was increased further by oxygenated perfluorodecalin between the liquid and underlying semi-solid phases of the medium,generating a triple-layered system (Anthony et al.,1995b). Oxygenated perfluorodecalin also promoted a fivefold increase in the plating efficiency of protoplasts from cell suspensions of the Japonica rice cv.Taipei 309. Importantly,this stimulation was sustained throughout culture,giving a 12%increase in shoot regeneration from protoplast-derived tissues.Crucially,there were no detrimental long-term effects of PFCs on regenerated plants,since the latter were fertile and morphologically similar to seed-derived individuals (Wardrop et al.,1996). Oxygenated PFC also promoted the plating efficiency of protoplasts of passionfruit (Passiflora edulis and P giberti)by up to 62%.Electrofused protoplasts of the two species had a plating efficiency more than 60%of that of electrofused protoplasts cultured without PFC (Wardrop et al.,1997a).As in rice,electrofused protoplasts exhibited a twofold increase in shoot regeneration following culture with oxygen- gassed PFC. 6.6.2.Hemoglobin solution Hb in legumes (leghemoglobin)is associated with nitrogen-fixing rhizobia within nodules,where it facilitates respiratory oxygen flow by the bacteria at concentrations low enough not to inactivate nitrogenase(Hunt et al.,2001).Hb is also present in dicotyledons and monocotyledons that do not fix nitrogen (Arredondo-Peter et al.,1997;Sowa et al., 1999),although its function in these plants is unclear.Each molecule of Hb may bind four molecules of oxygen,but chemical modifications are required prior to Hb being used as a gas carrier in experimental systems (Lowe et al.,2003).Such modifications include cross- linking of the alpha-alpha,beta-beta,and alpha-beta chains to decrease the oxygen affinity of Hb (Jones et al.,1996),polymerisation of Hb molecules with glutaraldehyde (Nishide et al.,1997),or conjugation to macromolecules such as polyethylene glycol (Alayash,2004).Bovine Hb is preferable to that extracted from human blood (Lowe et al., 2003)since oxygen release depends on chloride ions,rather than the anionic organic phosphate,2,3-diphosphoglycerate,with the former being abundant in plant cells.In general,Hb has received little attention as an artificial oxygen carrier in animal,microbial, and plant culture systems. Medium supplementation with the commercial Hb solution,ErythrogenM(Biorelease, Salem,USA),at 1:50(vol/vol)increased the plating efficiency of P hybrida protoplasts by more than 60%(Anthony et al.,1997b).Interestingly,inclusion of the surfactant Pluronic F-68 at 0.01%(wt/vol)in the Erythrogen-containing medium further stimulated the protoplast plating efficiency to 92%over the control.In extending these studies to cereals,Azhakanandam et al.(1997)suspended protoplasts of the rice cv.Taipei 309 in liquid medium at 0.5x 10 ml-and spread 200 ul aliquots of the suspension on the surface of cellulose nitrate filters.The latter were placed on 5 ml aliquots of agarose- solidified medium containing nurse cells of L.multiflorum in Petri dishes.Addition of Erythrogen at 1:50(vol/vol)to the aqueous medium containing the rice protoplasts stimulated their division.As in earlier studies with oxygenated PFC(Wardrop et al.,1996), shoot regeneration from rice protoplasts cultured in Hb-supplemented medium wasReference has been made to the presence of glass rods in the medium, stimulating division of cassava leaf protoplasts. Growth of these protoplasts was increased further by oxygenated perfluorodecalin between the liquid and underlying semi-solid phases of the medium, generating a triple-layered system (Anthony et al., 1995b). Oxygenated perfluorodecalin also promoted a fivefold increase in the plating efficiency of protoplasts from cell suspensions of the Japonica rice cv. Taipei 309. Importantly, this stimulation was sustained throughout culture, giving a 12% increase in shoot regeneration from protoplast-derived tissues. Crucially, there were no detrimental long-term effects of PFCs on regenerated plants, since the latter were fertile and morphologically similar to seed-derived individuals (Wardrop et al., 1996). Oxygenated PFC also promoted the plating efficiency of protoplasts of passionfruit (Passiflora edulis and P. giberti) by up to 62%. Electrofused protoplasts of the two species had a plating efficiency more than 60% of that of electrofused protoplasts cultured without PFC (Wardrop et al., 1997a). As in rice, electrofused protoplasts exhibited a twofold increase in shoot regeneration following culture with oxygen￾gassed PFC. 6.6.2. Hemoglobin solution Hb in legumes (leghemoglobin) is associated with nitrogen-fixing rhizobia within nodules, where it facilitates respiratory oxygen flow by the bacteria at concentrations low enough not to inactivate nitrogenase (Hunt et al., 2001). Hb is also present in dicotyledons and monocotyledons that do not fix nitrogen (Arredondo-Peter et al., 1997; Sowa et al., 1999), although its function in these plants is unclear. Each molecule of Hb may bind four molecules of oxygen, but chemical modifications are required prior to Hb being used as a gas carrier in experimental systems (Lowe et al., 2003). Such modifications include cross￾linking of the alpha–alpha, beta–beta, and alpha–beta chains to decrease the oxygen affinity of Hb (Jones et al., 1996), polymerisation of Hb molecules with glutaraldehyde (Nishide et al., 1997), or conjugation to macromolecules such as polyethylene glycol (Alayash, 2004). Bovine Hb is preferable to that extracted from human blood (Lowe et al., 2003) since oxygen release depends on chloride ions, rather than the anionic organic phosphate, 2,3-diphosphoglycerate, with the former being abundant in plant cells. In general, Hb has received little attention as an artificial oxygen carrier in animal, microbial, and plant culture systems. Medium supplementation with the commercial Hb solution, Erythrogenk (Biorelease, Salem, USA), at 1:50 (vol/vol) increased the plating efficiency of P. hybrida protoplasts by more than 60% (Anthony et al., 1997b). Interestingly, inclusion of the surfactant PluronicR F-68 at 0.01% (wt/vol) in the Erythrogenk-containing medium further stimulated the protoplast plating efficiency to 92% over the control. In extending these studies to cereals, Azhakanandam et al. (1997) suspended protoplasts of the rice cv. Taipei 309 in liquid medium at 0.5
106 ml1 and spread 200 Al aliquots of the suspension on the surface of cellulose nitrate filters. The latter were placed on 5 ml aliquots of agarose￾solidified medium containing nurse cells of L. multiflorum in Petri dishes. Addition of Erythrogenk at 1:50 (vol/vol) to the aqueous medium containing the rice protoplasts stimulated their division. As in earlier studies with oxygenated PFC (Wardrop et al., 1996), shoot regeneration from rice protoplasts cultured in Hb-supplemented medium was M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171 145