140 M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 exploitation in genetic manipulation,physiological investigations,and plant-pathogen interactions As already mentioned,guard cell protoplasts of sugarbeet are totipotent (Hall et al., 1996).However,the availability of more than one shoot regeneration pathway is useful when protoplasts of a specific crop are the target for genetic manipulation involving recombinant DNA.In this context,Dovzhenko and Koop (2003)developed an efficient procedure to release totipotent protoplasts from friable hypocotyl-derived callus of sugarbeet,providing an altemative system to guard cells.It is interesting to note that in studies with sugarbeet mesophyll protoplasts,Majewska-Sawka and Munster (2003) suggested that the recalcitrance to regeneration of this system may be related to newly synthesised cell wall components containing large quantities of pectins,arabinogalactan proteins,and callose. Protoplasts of ornamental plants have also received attention.For example,Sugimoto and Lidbetter(2003)released protoplasts from cotyledons of Goodenia scaevolina,whilst Nassour and Dorion(2002)and Nassour et al.(2003)demonstrated the totipotency of mesophyll protoplasts from micropropagated plants of Pelargonium xhortorum 'Alain.' The concentrations of ammonium nitrate and calcium chloride in the culture medium were crucial.since division decreased as the ammonium and calcium ions increased to 5.15 and 15 mM,respectively.Protoplast yield and viability were greater from shoots cultured in jars than in test tubes,probably because of the greater headspace and relative humidity in jars. Rose (Rosa hybrida)is a woody plant that is generally regarded as recalcitrant at the protoplast level.Therefore,it is interesting that Kim et al.(2003)succeeded in isolating totipotent protoplasts from 2-week-old embryogenic cell suspensions of the cv.Sumpath. Myoinositol in half-strength MS-based medium was necessary for their sustained division, with Gelrite at 0.4%(wt/vol)being used to semi-solidify the medium.Regenerated plants had a chromosome complement identical to the source material(2n=3x=21),confirming genomic stability in regenerants.Success has also been achieved in culturing protoplasts from monocotyledonous omamentals.Nakano et al.(2003)regenerated diploid and tetraploid plants from callus-derived protoplasts of Agapanthus praecox ssp.orientalis "Royal Purple Select,'with protoplasts being plated in medium semi-solidified with Gellan gum.Protoplast-derived tissues produced somatic embryos in the absence of growth regulators,or adventitious shoots with 1.0 mg I benzyladenine.Lily is also a high-value ornamental plant.Consequently,the protoplast-to-plant system for the oriental hybrid lily cvs.Casablanca,Siberia,and Acapulco represents a major advance in exploiting biotechnological approaches for the improvement of this and probably related species (Horita et al.,2003). Whilst most emphasis has focused on protoplasts of higher plants,recent reports have described the culture of moss protoplasts.In this respect,Schween et al.(2003) regenerated auxotrophic mutants of P patens with semi-solid medium supplemented with ammonium tartrate,enhancing protoplast survival.In other studies,the development of protoplasts to protonemata was investigated in wild-type and mutants of Ceratodon purpureus,a novel feature being that wild-type protonemata exhibited negative geotrophism when cultured in the dark (Wagner and Sack,1998).More recently,some interesting personal reflections on developments in protoplast technologies have been presented by Cocking(2000)and Gamborg(2002).exploitation in genetic manipulation, physiological investigations, and plant–pathogen interactions. As already mentioned, guard cell protoplasts of sugarbeet are totipotent (Hall et al., 1996). However, the availability of more than one shoot regeneration pathway is useful when protoplasts of a specific crop are the target for genetic manipulation involving recombinant DNA. In this context, Dovzhenko and Koop (2003) developed an efficient procedure to release totipotent protoplasts from friable hypocotyl-derived callus of sugarbeet, providing an alternative system to guard cells. It is interesting to note that in studies with sugarbeet mesophyll protoplasts, Majewska-Sawka and Munster (2003) suggested that the recalcitrance to regeneration of this system may be related to newly synthesised cell wall components containing large quantities of pectins, arabinogalactan proteins, and callose. Protoplasts of ornamental plants have also received attention. For example, Sugimoto and Lidbetter (2003) released protoplasts from cotyledons of Goodenia scaevolina, whilst Nassour and Dorion (2002) and Nassour et al. (2003) demonstrated the totipotency of mesophyll protoplasts from micropropagated plants of Pelargonium
hortorum dAlain.T The concentrations of ammonium nitrate and calcium chloride in the culture medium were crucial, since division decreased as the ammonium and calcium ions increased to 5.15 and 15 mM, respectively. Protoplast yield and viability were greater from shoots cultured in jars than in test tubes, probably because of the greater headspace and relative humidity in jars. Rose (Rosa hybrida) is a woody plant that is generally regarded as recalcitrant at the protoplast level. Therefore, it is interesting that Kim et al. (2003) succeeded in isolating totipotent protoplasts from 2-week-old embryogenic cell suspensions of the cv. Sumpath. Myoinositol in half-strength MS-based medium was necessary for their sustained division, with Gelrite at 0.4% (wt/vol) being used to semi-solidify the medium. Regenerated plants had a chromosome complement identical to the source material (2n=3x=21), confirming genomic stability in regenerants. Success has also been achieved in culturing protoplasts from monocotyledonous ornamentals. Nakano et al. (2003) regenerated diploid and tetraploid plants from callus-derived protoplasts of Agapanthus praecox ssp. orientalis dRoyal Purple Select,T with protoplasts being plated in medium semi-solidified with Gellan gum. Protoplast-derived tissues produced somatic embryos in the absence of growth regulators, or adventitious shoots with 1.0 mg l1 benzyladenine. Lily is also a high-value ornamental plant. Consequently, the protoplast-to-plant system for the oriental hybrid lily cvs. Casablanca, Siberia, and Acapulco represents a major advance in exploiting biotechnological approaches for the improvement of this and probably related species (Horita et al., 2003). Whilst most emphasis has focused on protoplasts of higher plants, recent reports have described the culture of moss protoplasts. In this respect, Schween et al. (2003) regenerated auxotrophic mutants of P. patens with semi-solid medium supplemented with ammonium tartrate, enhancing protoplast survival. In other studies, the development of protoplasts to protonemata was investigated in wild-type and mutants of Ceratodon purpureus, a novel feature being that wild-type protonemata exhibited negative geotrophism when cultured in the dark (Wagner and Sack, 1998). More recently, some interesting personal reflections on developments in protoplast technologies have been presented by Cocking (2000) and Gamborg (2002). 140 M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171