M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 137 similar structurally to animal steroidal hormones (Oh and Clouse,1998),can promote division of protoplast-derived cells.Interestingly,Oh et al.(2003)provided evidence that cyclophilin immunophilins may play a role in actively growing protoplast-derived cells and intact plants,particularly during early flower development.There is considerable scope for further investigations into the physiological role(s)of these ubiquitous proteins whose function in plants remains obscure.May and Sink (1995)and Pasterak et al. (2000)reviewed the hormonal combinations and concentrations reported for isolated protoplasts.Sucrose and glucose are the regular choices of carbon sources in most media, although a change in the carbon source from sucrose to maltose promoted shoot regeneration for protoplast-derived cells of cereals (Jain et al.,1995). 4.2.Experimental systems for the culture of isolated protoplasts There have been several approaches developed for protoplast culture,all of which are based on liquid or semi-solid media,or their combination (Evans and Bravo,1983; Eriksson,1985).The ready availability of sterile plasticware has facilitated protoplast isolation and culture.Dispensing protoplast suspensions into Petri dishes is the most simple option,since the medium can be easily replaced to gradually reduce its osmolarity. thereby maintaining protoplast growth.Droplets of suspension of ca.100-150 ul in volume are useful when limited numbers of protoplasts are available (Kao et al.,1970), while droplet array techniques (Potrykus et al.,1979)have facilitated assessments of growth regulator combinations. Isolated protoplasts can withstand the rigours of being embedded in semi-solid media, with agar being first used as the gelling agent (Nagata and Takebe,1971).Protoplasts remain separated in the semi-solid medium,with the latter supporting wall regeneration and promoting mitotic division.The superiority of agarose compared to agar as a gelling agent may be related to its neutrality.Semi-solid media containing suspended protoplasts can be dispensed as a layer or droplets,with the latter usually up to 250 ul in volume in Petri dishes.Dissecting the layer of medium into sectors,with subsequent bathing of the sectors or droplets in liquid medium of the same composition,promotes protoplast growth. Stepwise reduction of the osmotic pressure is readily achieved by changing the bathing medium.Alginate has also been used to semi-solidify media,with gelling being induced by exposure to calcium ions.Crucially,protoplast-derived colonies may be released by depolymerising the alginate by treatment with sodium citrate to remove the calcium ions. Suspending protoplasts in a thin layer of liquid over semi-solid medium was found to stimulate cell colony formation,particularly when a filter paper was included at the liquid/ semi-solid interface (dos Santos et al.,1980).The filter paper can be replaced with a bacterial membrane filter (pore size 0.2 um)to produce a similar effect as,for example,in rice (Jain et al.,1995),or with a cellophane layer,as for protoplasts of the moss,P patens (Schween et al.,2003).Nylon mesh has also been used as a support for protoplasts in both liquid (Russell and McCown,1986)and semi-solid culture systems (Dovzhenko et al., 2003).Removal of the filter paper,bacterial filter,cellophane,or nylon mesh facilitates transfer of protoplast-derived cells to new medium. A novel approach to combine the isolation of protoplasts with their introduction into culture involved the suspension of cells in strontium alginate gel followed by dropping ofsimilar structurally to animal steroidal hormones (Oh and Clouse, 1998), can promote division of protoplast-derived cells. Interestingly, Oh et al. (2003) provided evidence that cyclophilin immunophilins may play a role in actively growing protoplast-derived cells and intact plants, particularly during early flower development. There is considerable scope for further investigations into the physiological role(s) of these ubiquitous proteins whose function in plants remains obscure. May and Sink (1995) and Pasternak et al. (2000) reviewed the hormonal combinations and concentrations reported for isolated protoplasts. Sucrose and glucose are the regular choices of carbon sources in most media, although a change in the carbon source from sucrose to maltose promoted shoot regeneration for protoplast-derived cells of cereals (Jain et al., 1995). 4.2. Experimental systems for the culture of isolated protoplasts There have been several approaches developed for protoplast culture, all of which are based on liquid or semi-solid media, or their combination (Evans and Bravo, 1983; Eriksson, 1985). The ready availability of sterile plasticware has facilitated protoplast isolation and culture. Dispensing protoplast suspensions into Petri dishes is the most simple option, since the medium can be easily replaced to gradually reduce its osmolarity, thereby maintaining protoplast growth. Droplets of suspension of ca. 100–150 Al in volume are useful when limited numbers of protoplasts are available (Kao et al., 1970), while droplet array techniques (Potrykus et al., 1979) have facilitated assessments of growth regulator combinations. Isolated protoplasts can withstand the rigours of being embedded in semi-solid media, with agar being first used as the gelling agent (Nagata and Takebe, 1971). Protoplasts remain separated in the semi-solid medium, with the latter supporting wall regeneration and promoting mitotic division. The superiority of agarose compared to agar as a gelling agent may be related to its neutrality. Semi-solid media containing suspended protoplasts can be dispensed as a layer or droplets, with the latter usually up to 250 Al in volume in Petri dishes. Dissecting the layer of medium into sectors, with subsequent bathing of the sectors or droplets in liquid medium of the same composition, promotes protoplast growth. Stepwise reduction of the osmotic pressure is readily achieved by changing the bathing medium. Alginate has also been used to semi-solidify media, with gelling being induced by exposure to calcium ions. Crucially, protoplast-derived colonies may be released by depolymerising the alginate by treatment with sodium citrate to remove the calcium ions. Suspending protoplasts in a thin layer of liquid over semi-solid medium was found to stimulate cell colony formation, particularly when a filter paper was included at the liquid/ semi-solid interface (dos Santos et al., 1980). The filter paper can be replaced with a bacterial membrane filter (pore size 0.2 Am) to produce a similar effect as, for example, in rice (Jain et al., 1995), or with a cellophane layer, as for protoplasts of the moss, P. patens (Schween et al., 2003). Nylon mesh has also been used as a support for protoplasts in both liquid (Russell and McCown, 1986) and semi-solid culture systems (Dovzhenko et al., 2003). Removal of the filter paper, bacterial filter, cellophane, or nylon mesh facilitates transfer of protoplast-derived cells to new medium. A novel approach to combine the isolation of protoplasts with their introduction into culture involved the suspension of cells in strontium alginate gel followed by dropping of M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171 137