M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 139 have been somewhat limited in recent years.Tylicki et al.(2002,2003)used an immunodetection approach to monitor changes in the tubulin cytoskeleton during protoplast culture and plant regeneration in Solanum lycopersicoides.Mononuclear, polynuclear,homogeneous,and anuclear(enucleate)protoplasts were obtained following enzymatic isolation,but only mononuclear protoplasts rearranged their cortical micro- tubules,reestablished their radial and perinuclear tubulin cytoskeletons,and entered division.In the same year,Sasamoto et al.(2003)observed unusual elongate fibres in protoplasts from leaves of Betula platyphylla and embryogenic cells of Larix leptolepis, with calcium and magnesium ions,respectively,having most significant effects on such structures in protoplasts of these genera.The fibres fluoresced with Calcofluor White and Aniline Blue,indicating the presence of cell wall components,including callose(beta-1,3- glucan).More recently,the cellular and molecular investigations of Morse et al.(2004). with protoplasts of 4.thaliana,suggested the presence of a novel class of receptors binding vertebrate atrial natriuretic peptides,which may have a role in plant growth.These findings indicate a possible link between animal hormones and plant growth that warrants further investigation. Several workers have published details of protoplast-to-plant systems,together with the novel use of the green fluorescent protein (GFP)gene from the jellyfish,Aeguorea victoria,as a marker system to identify protoplasts with the potential for somatic embryogenesis,as in protoplasts isolated from leaves of Nicotiana plumbaginifolia transformed previously with a GFP construct(Chesnokov et al.,2002).The importance of chromatin decondensation during dedifferentiation of tobacco leaf protoplasts following their introduction into culture was emphasised by Zhao et al.(2001),who also highlighted possible parallels with differentiation processes in animal systems.These authors speculated that there may be commonality in the contribution that such processes make to cellular development in higher eukaryotes.Protoplasts of red cabbage were mentioned earlier(Chen et al.,2004).A simple protocol for regenerating plants from leaf protoplasts of cabbage,cauliflower,and broccoli,employing Kao-type medium,was developed by Kirti et al.(2001).In addition to studies with protoplasts of crop brassicas,protoplasts isolated from cultured leaf explants of the wild crucifer,Rorippa indica,produced calli, which regenerated plants with the expected diploid chromosome number of 32(Mandal and Sikdar,2003). Other cultural assessments have focused on plants of medicinal value,such as Artemisia judaica and Echinops spinosissima (Pan et al.,2003).Protoplasts of Artemisia developed in medium semi-solidified with 0.6%(wt/vol)SeaPrep agarose;those of Echinops were cultured in MS-based medium with B5 vitamins and semi-solidified with sodium alginate.Whilst embryogenic cell suspensions have been the preferred source tissue for protoplast isolation in cereals,such as rice and rye (Ma et al,2003),it is interesting to note that success was finally achieved,after many years of research,in regenerating plants from leaf protoplasts of the cereal sorghum (Sairam et al.,1999). However,other workers have not commented on the robustness of this system,or the applicability of the protocol to other cereals.Additional advances with monocotyledons include an efficient method for plant regeneration from protoplasts of 6-year-old callus of garlic (Allium sativum;Hasegawa et al.,2002).Davey et al.(2003)have discussed the current status of protoplasts from grain and forage legumes with respect to their culture,have been somewhat limited in recent years. Tylicki et al. (2002, 2003) used an immunodetection approach to monitor changes in the tubulin cytoskeleton during protoplast culture and plant regeneration in Solanum lycopersicoides. Mononuclear, polynuclear, homogeneous, and anuclear (enucleate) protoplasts were obtained following enzymatic isolation, but only mononuclear protoplasts rearranged their cortical micro￾tubules, reestablished their radial and perinuclear tubulin cytoskeletons, and entered division. In the same year, Sasamoto et al. (2003) observed unusual elongate fibres in protoplasts from leaves of Betula platyphylla and embryogenic cells of Larix leptolepis, with calcium and magnesium ions, respectively, having most significant effects on such structures in protoplasts of these genera. The fibres fluoresced with Calcofluor White and Aniline Blue, indicating the presence of cell wall components, including callose (beta-1,3- glucan). More recently, the cellular and molecular investigations of Morse et al. (2004), with protoplasts of A. thaliana, suggested the presence of a novel class of receptors binding vertebrate atrial natriuretic peptides, which may have a role in plant growth. These findings indicate a possible link between animal hormones and plant growth that warrants further investigation. Several workers have published details of protoplast-to-plant systems, together with the novel use of the green fluorescent protein (GFP) gene from the jellyfish, Aequorea victoria, as a marker system to identify protoplasts with the potential for somatic embryogenesis, as in protoplasts isolated from leaves of Nicotiana plumbaginifolia transformed previously with a GFP construct (Chesnokov et al., 2002). The importance of chromatin decondensation during dedifferentiation of tobacco leaf protoplasts following their introduction into culture was emphasised by Zhao et al. (2001), who also highlighted possible parallels with differentiation processes in animal systems. These authors speculated that there may be commonality in the contribution that such processes make to cellular development in higher eukaryotes. Protoplasts of red cabbage were mentioned earlier (Chen et al., 2004). A simple protocol for regenerating plants from leaf protoplasts of cabbage, cauliflower, and broccoli, employing Kao-type medium, was developed by Kirti et al. (2001). In addition to studies with protoplasts of crop brassicas, protoplasts isolated from cultured leaf explants of the wild crucifer, Rorippa indica, produced calli, which regenerated plants with the expected diploid chromosome number of 32 (Mandal and Sikdar, 2003). Other cultural assessments have focused on plants of medicinal value, such as Artemisia judaica and Echinops spinosissima (Pan et al., 2003). Protoplasts of Artemisia developed in medium semi-solidified with 0.6% (wt/vol) SeaPrep agarose; those of Echinops were cultured in MS-based medium with B5 vitamins and semi-solidified with sodium alginate. Whilst embryogenic cell suspensions have been the preferred source tissue for protoplast isolation in cereals, such as rice and rye (Ma et al., 2003), it is interesting to note that success was finally achieved, after many years of research, in regenerating plants from leaf protoplasts of the cereal sorghum (Sairam et al., 1999). However, other workers have not commented on the robustness of this system, or the applicability of the protocol to other cereals. Additional advances with monocotyledons include an efficient method for plant regeneration from protoplasts of 6-year-old callus of garlic (Allium sativum; Hasegawa et al., 2002). Davey et al. (2003) have discussed the current status of protoplasts from grain and forage legumes with respect to their culture, M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171 139