Available online at www.sciencedirect.com BIOTECHNOLOGY ADVANCES ELSEVIER Biotechnology Advances 23(2005)131-171 www.elsevier.com/locate/biotechadv Research review paper Plant protoplasts:status and biotechnological perspectives Michael R.Daveya.*,Paul Anthony", J.Brian PowerKenneth C.Lowe Plant Sciences Division,School of Biosciences,University of Nottingham.Sutton Bonington Campus. Loughborough LE12 5RD.UK School of Biology.University of Nottingham.University Park.Nottingham NG7 2RD.UK Received 10 July 2004;received in revised form 13 September 2004;accepted 23 September 2004 Available online 30 December 2004 Abstract Plant protoplasts("naked"cells)provide a unique single cell system to underpin several aspects of modern biotechnology.Major advances in genomics,proteomics,and metabolomics have stimulated renewed interest in these osmotically fragile wall-less cells.Reliable procedures are available to isolate and culture protoplasts from a range of plants,including both monocotyledonous and dicotyledonous crops.Several parameters,particularly the source tissue,culture medium,and environmental factors,influence the ability of protoplasts and protoplast-derived cells to express their totipotency and to develop into fertile plants.Importantly,novel approaches to maximise the efficiency of protoplast-to-plant systems include techniques already well established for animal and microbial cells,such as electrostimulation and exposure of protoplasts to surfactants and respiratory gas carriers,especially perfluorochemicals and hemoglobin.However,despite at least four decades of concerted effort and technology transfer between laboratories worldwide,many species still remain recalcitrant in culture.Nevertheless,isolated protoplasts are unique to a range of experimental procedures.In the context of plant genetic manipulation,somatic hybridisation by protoplast fusion enables nuclear and cytoplasmic genomes to be combined,fully or partially,at the interspecific and intergeneric levels to circumvent naturally occurring sexual incompatibility barriers.Uptake of isolated DNA into protoplasts provides the basis for transient and stable nuclear transformation,and also organelle transformation to generate transplastomic plants.Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function,cell structure,synthesis of Corresponding author.Tel:+44 115 951 3507:fax:+44 115 951 6334. E-mail address:mike.davey@nottingham.ac.uk (M.R.Davey). 0734-9750/S-see front matter 2004 Elsevier Inc.All rights reserved doi:10.1016f.biotechadv.2004.09.008
Research review paper Plant protoplasts: status and biotechnological perspectives Michael R. Daveya,*, Paul Anthonya , J. Brian Powera , Kenneth C. Loweb a Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK b School of Biology, University of Nottingham, University Park, Nottingham NG7 2RD, UK Received 10 July 2004; received in revised form 13 September 2004; accepted 23 September 2004 Available online 30 December 2004 Abstract Plant protoplasts (bnakedQ cells) provide a unique single cell system to underpin several aspects of modern biotechnology. Major advances in genomics, proteomics, and metabolomics have stimulated renewed interest in these osmotically fragile wall-less cells. Reliable procedures are available to isolate and culture protoplasts from a range of plants, including both monocotyledonous and dicotyledonous crops. Several parameters, particularly the source tissue, culture medium, and environmental factors, influence the ability of protoplasts and protoplast-derived cells to express their totipotency and to develop into fertile plants. Importantly, novel approaches to maximise the efficiency of protoplast-to-plant systems include techniques already well established for animal and microbial cells, such as electrostimulation and exposure of protoplasts to surfactants and respiratory gas carriers, especially perfluorochemicals and hemoglobin. However, despite at least four decades of concerted effort and technology transfer between laboratories worldwide, many species still remain recalcitrant in culture. Nevertheless, isolated protoplasts are unique to a range of experimental procedures. In the context of plant genetic manipulation, somatic hybridisation by protoplast fusion enables nuclear and cytoplasmic genomes to be combined, fully or partially, at the interspecific and intergeneric levels to circumvent naturally occurring sexual incompatibility barriers. Uptake of isolated DNA into protoplasts provides the basis for transient and stable nuclear transformation, and also organelle transformation to generate transplastomic plants. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, synthesis of 0734-9750/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2004.09.008 * Corresponding author. Tel.: +44 115 951 3507; fax: +44 115 951 6334. E-mail address: mike.davey@nottingham.ac.uk (M.R. Davey). Biotechnology Advances 23 (2005) 131 – 171 www.elsevier.com/locate/biotechadv
132 M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 pharmaceutical products,and toxicological assessments.This review focuses upon the most recent developments in protoplast-based technologies. 2004 Elsevier Inc.All rights reserved. Keywords:Genetic manipulation:Molecular farming;Nuclear and organelle transformation;Physiological investigations;Protoplast-to-plant systems;Somatic hybridisation Contents 1. 133 2. Source material for protoplast isolation.。...·.,。··.·,··.·,····· 133 3. Procedures for protoplast isolation.......,..·.......。.·.·.... 134 3.l.Stress during protoplast isolation,...···················· 136 4. Culture techniques for isolated plant protoplasts.···.·······.······· 136 4.1. Culture media。.·······:······+ 136 4.2. Experimental systems for the culture of isolated protoplasts.......... 137 4.3.Plating density and protoplast growth in culture....·....·.·..·. 138 5.Totipotent protoplast systems.... 138 6.Innovative approaches for protoplast culture 141 6.1.Electrical stimulation of protoplasts... 141 6.2.Supplementation of culture media with surfactants,antibiotics,and polyamines 142 63. Manipulation of respiratory gases during protoplast culture.... 。。。 143 6.4. Physical procedures to stimulate gaseous exchange.··,,·,··,····· 143 6.5. Gassing of protoplast cultures.······· 143 6.6. Artificial oxygen carriers:perfluorocarbon liquids(PFCs)and hemoglobin(Hb)solutions······················· 144 6.6.l.Perfluorocarbon liquids.....:。...·...,·。·。.·.·. 144 6.6.2.Hemoglobin solution..,..,,·,··.··,·········· 145 7. Exploitation of protoplast--to-plant technologies···················· 146 7.1. Somatic hybridisation to generate novel plants,,··············· 146 7.1.1.Citrus... 147 7.1.2. 149 7.1.3. Potato and other members of the Solanaceae............. 150 7.1.4. 151 7.1.5. Ornamental plants.. 151 7.1.6.Miscellaneous crop plants 151 7.1.7. Other applications of protoplast fusion........ 152 7.2. Transformation of protoplasts......·.··.·.····· 153 7.2.1.Transformation by DNA uptake into the nucleus of isolated protoplasts 153 7.2.2. Organelle transformation......... 155 7.2.3. Transformation for expression of recombinant proteins 156 7.3.Somaclonal variation.....,...,..,。..··,····+·… 156 8. Miscellaneous studies with isolated protoplasts..··················· 157 9. Conclusions ·*+·…*4·+·++“中小·小*”·4中··”+*+”“ 160 References·················· 160
pharmaceutical products, and toxicological assessments. This review focuses upon the most recent developments in protoplast-based technologies. D 2004 Elsevier Inc. All rights reserved. Keywords: Genetic manipulation; Molecular farming; Nuclear and organelle transformation; Physiological investigations; Protoplast-to-plant systems; Somatic hybridisation Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 2. Source material for protoplast isolation. . . . . . . . . . . . . . . . . . . . . . . . . 133 3. Procedures for protoplast isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 3.1. Stress during protoplast isolation . . . . . . . . . . . . . . . . . . . . . . . . 136 4. Culture techniques for isolated plant protoplasts . . . . . . . . . . . . . . . . . . . . 136 4.1. Culture media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 4.2. Experimental systems for the culture of isolated protoplasts . . . . . . . . . . 137 4.3. Plating density and protoplast growth in culture . . . . . . . . . . . . . . . . 138 5. Totipotent protoplast systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 6. Innovative approaches for protoplast culture . . . . . . . . . . . . . . . . . . . . . . 141 6.1. Electrical stimulation of protoplasts . . . . . . . . . . . . . . . . . . . . . . . 141 6.2. Supplementation of culture media with surfactants, antibiotics, and polyamines 142 6.3. Manipulation of respiratory gases during protoplast culture . . . . . . . . . . . 143 6.4. Physical procedures to stimulate gaseous exchange . . . . . . . . . . . . . . . 143 6.5. Gassing of protoplast cultures . . . . . . . . . . . . . . . . . . . . . . . . . . 143 6.6. Artificial oxygen carriers: perfluorocarbon liquids (PFCs) and hemoglobin (Hb) solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 6.6.1. Perfluorocarbon liquids. . . . . . . . . . . . . . . . . . . . . . . . . 144 6.6.2. Hemoglobin solution . . . . . . . . . . . . . . . . . . . . . . . . . . 145 7. Exploitation of protoplast-to-plant technologies . . . . . . . . . . . . . . . . . . . . 146 7.1. Somatic hybridisation to generate novel plants . . . . . . . . . . . . . . . . . 146 7.1.1. Citrus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 7.1.2. Brassica. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 7.1.3. Potato and other members of the Solanaceae. . . . . . . . . . . . . . 150 7.1.4. Cereals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 7.1.5. Ornamental plants . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 7.1.6. Miscellaneous crop plants . . . . . . . . . . . . . . . . . . . . . . . 151 7.1.7. Other applications of protoplast fusion . . . . . . . . . . . . . . . . . 152 7.2. Transformation of protoplasts . . . . . . . . . . . . . . . . . . . . . . . . . . 153 7.2.1. Transformation by DNA uptake into the nucleus of isolated protoplasts 153 7.2.2. Organelle transformation . . . . . . . . . . . . . . . . . . . . . . . . 155 7.2.3. Transformation for expression of recombinant proteins (dmolecular farmingT) . . . . . . . . . . . . . . . . . . . . . . . . . . 156 7.3. Somaclonal variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 8. Miscellaneous studies with isolated protoplasts. . . . . . . . . . . . . . . . . . . . . 157 9. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 132 M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171
M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 133 1.Introduction Three decades have passed since the Centre National de la Recherche Scientifique, Versailles,hosted the symposium 'Protoplastes et Fusion de Cellules Somatiques Vegetals,'the proceedings of which were published the following year (Ephrussi et al., 1973).Ten years later,the Sixth International Protoplast Symposium was held in Basel (Potrykus et al.,1983).Both conferences focussed on the isolation,culture,fusion,and transformation of protoplasts,with several of the papers presented at these symposia now seen retrospectively as classic publications.The 1980s witnessed many protoplast-based articles,particularly those reporting novel protoplast-to-plant systems for genetic manipulation.During the 1990s,protoplast-based technologies for gene transfer were overshadowed by Agrobacterium and Biolistics -mediated gene delivery to plants. However,public antagonism (especially in Europe)to recombinant DNA technologies renewed interest in exploiting protoplasts in somatic hybridisation,cybridisation, protoclonal variation studies,proteomics,and metabolomics. Cells of primary plant tissues possess cellulosic walls with a pectin-rich matrix,the middle lamella,joining adjacent cells.The living cytoplasm of each cell,bounded by the plasma membrane,constitutes the protoplast.Normally,intimate contact is maintained between the plasma membrane and the wall,since this membrane is involved in wall synthesis.However,in hypertonic solutions,the plasma membranes of cells contract from their walls.Subsequent removal of the latter structures releases large populations of spherical,osmotically fragile protoplasts ('naked'cells),where the plasma membrane is the only barrier between the cytoplasm and its immediate external environment. Protoplast isolation is now routine from a wide range of species;viable protoplasts are potentially totipotent.Therefore,when given the correct chemical and physical stimuli, each protoplast is capable,theoretically,of regenerating a new wall and undergoing repeated mitotic division to produce daughter cells from which fertile plants may be regenerated via the tissue culture process.Protoplast-to-plant systems are available for many species,with an extensive literature relating to their exploitation.It is noteworthy that the basic procedures for protoplast isolation have undergone little change since first reported.However,remarkable progress has been made in the number of species for which protoplast-to-plant systems exist.Furthermore,the later decades of the 20th century witnessed dramatic developments in the genetic manipulation of plants through protoplast fusion and transformation.This review focuses primarily upon more recent and innovative activities involving isolated plant protoplasts. 2.Source material for protoplast isolation The physiological status of the source tissue influences the release of viable protoplasts.Furthermore,seasonal variation,which affects the reproducibility of protoplast isolation from glasshouse-grown plants,can be effectively eliminated using in vitro grown (axenic)shoots,seedlings,and embryogenic cell suspensions.Never- theless,Keskitalo (2001)reported that protoplast isolation from cultured shoots of
1. Introduction Three decades have passed since the Centre National de la Recherche Scientifique, Versailles, hosted the symposium dProtoplastes et Fusion de Cellules Somatiques Ve´ge´tals,T the proceedings of which were published the following year (Ephrussi et al., 1973). Ten years later, the Sixth International Protoplast Symposium was held in Basel (Potrykus et al., 1983). Both conferences focussed on the isolation, culture, fusion, and transformation of protoplasts, with several of the papers presented at these symposia now seen retrospectively as classic publications. The 1980s witnessed many protoplast-based articles, particularly those reporting novel protoplast-to-plant systems for genetic manipulation. During the 1990s, protoplast-based technologies for gene transfer were overshadowed by Agrobacterium and BiolisticsR-mediated gene delivery to plants. However, public antagonism (especially in Europe) to recombinant DNA technologies renewed interest in exploiting protoplasts in somatic hybridisation, cybridisation, protoclonal variation studies, proteomics, and metabolomics. Cells of primary plant tissues possess cellulosic walls with a pectin-rich matrix, the middle lamella, joining adjacent cells. The living cytoplasm of each cell, bounded by the plasma membrane, constitutes the protoplast. Normally, intimate contact is maintained between the plasma membrane and the wall, since this membrane is involved in wall synthesis. However, in hypertonic solutions, the plasma membranes of cells contract from their walls. Subsequent removal of the latter structures releases large populations of spherical, osmotically fragile protoplasts (dnakedT cells), where the plasma membrane is the only barrier between the cytoplasm and its immediate external environment. Protoplast isolation is now routine from a wide range of species; viable protoplasts are potentially totipotent. Therefore, when given the correct chemical and physical stimuli, each protoplast is capable, theoretically, of regenerating a new wall and undergoing repeated mitotic division to produce daughter cells from which fertile plants may be regenerated via the tissue culture process. Protoplast-to-plant systems are available for many species, with an extensive literature relating to their exploitation. It is noteworthy that the basic procedures for protoplast isolation have undergone little change since first reported. However, remarkable progress has been made in the number of species for which protoplast-to-plant systems exist. Furthermore, the later decades of the 20th century witnessed dramatic developments in the genetic manipulation of plants through protoplast fusion and transformation. This review focuses primarily upon more recent and innovative activities involving isolated plant protoplasts. 2. Source material for protoplast isolation The physiological status of the source tissue influences the release of viable protoplasts. Furthermore, seasonal variation, which affects the reproducibility of protoplast isolation from glasshouse-grown plants, can be effectively eliminated using in vitro grown (axenic) shoots, seedlings, and embryogenic cell suspensions. Nevertheless, Keskitalo (2001) reported that protoplast isolation from cultured shoots of M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171 133
134 M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 Tanacetum vulgare and Tanacetum cinerariifolium was most successful during winter and spring (December to April),suggesting the persistence of a seasonal 'clock'even in vitro.In contrast.other workers have not observed seasonal variation in vitro.Mliki et al.(2003)isolated protoplasts from Tunisian varieties of grape (Vitis vinifera)and concluded that the highest yields were from leaves of cultured shoots 45 weeks after transfer of the shoots to new medium.An advantage of seedlings is that protoplasts can be isolated from radicles,hypocotyls,cotyledon tissues,roots,and root hairs within a few days of seed germination.For example,Dovzhenko et al.(2003)reported a reproducible and rapid cotyledon-based protoplast system for Arabidopsis thaliana, which will facilitate molecular studies with this model species.Similarly,Sinha et al. (2003a)found that cotyledons from in vitro-grown seedlings of white lupin gave higher yields compared to leaves,hypocotyls,and roots.Although protoplast yield from cotyledons increased with seedling age,viability declined.Concurrent inves- tigations by Sinha et al.(2003b)optimised protoplast isolation from cotyledons of this legume. 3.Procedures for protoplast isolation Mechanical procedures,involving slicing of plasmolysed tissues,are now rarely employed for protoplast isolation,but are useful with large cells and when limited(small) numbers of protoplasts are required.Recently,this approach has been used successfully to isolate protoplasts of the giant marine alga,Valonia utricularis,for patch clamp analyses of their electrical properties,including physiological changes of the plasma membrane induced by exposure of isolated protoplasts to enzymes normally used to digest cell walls (Binder et al.,2003).When large populations of protoplasts are required,which is the norm,enzymatic digestion of source tissues is essential (Davey and Kumar,1983; Eriksson,1985;Davey et al.,2000a,2003).Interestingly,it was the release of protoplasts by natural enzymatic degradation of cell walls during fruit ripening that stimulated investigations,more than four decades ago,of protoplast isolation from roots of tomato seedlings (Cocking,1960).Subsequently,cellulase and pectinase enzymes became available commercially for routine use.An additional major advance in protoplast isolation involved treatment of tobacco leaves with pectinase to separate the cells, followed by cellulase to remove their walls (Takebe et al.,1968).The procedure was further simplified by a single treatment with a mixture of enzymes (Power and Cocking, 1970).More recently,Gummadi and Panda(2003)discussed the use of pectinases not only in the fruit,paper,and textile industries,but also in plant biotechnology,particularly protoplast isolation.Doi and Tamaru (2001)also presented a comprehensive review of cellulases,focusing upon the cellulase complex,the cellulosome,of Clostridium cellulovorans.Later,the same group demonstrated that culture supernatants from C. cellulovorans readily released protoplasts from cultured cells of A.thaliana and Nicotiana tabacum,with crude extracts from pectin substrate-grown fungi being most active (Tamaru et al.,2002).Sato et al.(2001)exploited light and scanning electron microscopy to observe changes in surface topography during enzymatic isolation of protoplasts from rice callus
Tanacetum vulgare and Tanacetum cinerariifolium was most successful during winter and spring (December to April), suggesting the persistence of a seasonal dclockT even in vitro. In contrast, other workers have not observed seasonal variation in vitro. Mliki et al. (2003) isolated protoplasts from Tunisian varieties of grape (Vitis vinifera) and concluded that the highest yields were from leaves of cultured shoots 4–5 weeks after transfer of the shoots to new medium. An advantage of seedlings is that protoplasts can be isolated from radicles, hypocotyls, cotyledon tissues, roots, and root hairs within a few days of seed germination. For example, Dovzhenko et al. (2003) reported a reproducible and rapid cotyledon-based protoplast system for Arabidopsis thaliana, which will facilitate molecular studies with this model species. Similarly, Sinha et al. (2003a) found that cotyledons from in vitro-grown seedlings of white lupin gave higher yields compared to leaves, hypocotyls, and roots. Although protoplast yield from cotyledons increased with seedling age, viability declined. Concurrent investigations by Sinha et al. (2003b) optimised protoplast isolation from cotyledons of this legume. 3. Procedures for protoplast isolation Mechanical procedures, involving slicing of plasmolysed tissues, are now rarely employed for protoplast isolation, but are useful with large cells and when limited (small) numbers of protoplasts are required. Recently, this approach has been used successfully to isolate protoplasts of the giant marine alga, Valonia utricularis, for patch clamp analyses of their electrical properties, including physiological changes of the plasma membrane induced by exposure of isolated protoplasts to enzymes normally used to digest cell walls (Binder et al., 2003). When large populations of protoplasts are required, which is the norm, enzymatic digestion of source tissues is essential (Davey and Kumar, 1983; Eriksson, 1985; Davey et al., 2000a, 2003). Interestingly, it was the release of protoplasts by natural enzymatic degradation of cell walls during fruit ripening that stimulated investigations, more than four decades ago, of protoplast isolation from roots of tomato seedlings (Cocking, 1960). Subsequently, cellulase and pectinase enzymes became available commercially for routine use. An additional major advance in protoplast isolation involved treatment of tobacco leaves with pectinase to separate the cells, followed by cellulase to remove their walls (Takebe et al., 1968). The procedure was further simplified by a single treatment with a mixture of enzymes (Power and Cocking, 1970). More recently, Gummadi and Panda (2003) discussed the use of pectinases not only in the fruit, paper, and textile industries, but also in plant biotechnology, particularly protoplast isolation. Doi and Tamaru (2001) also presented a comprehensive review of cellulases, focusing upon the cellulase complex, the cellulosome, of Clostridium cellulovorans. Later, the same group demonstrated that culture supernatants from C. cellulovorans readily released protoplasts from cultured cells of A. thaliana and Nicotiana tabacum, with crude extracts from pectin substrate-grown fungi being most active (Tamaru et al., 2002). Sato et al. (2001) exploited light and scanning electron microscopy to observe changes in surface topography during enzymatic isolation of protoplasts from rice callus. 134 M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171
M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 135 Several factors influence protoplast release,including the extent of thickening of cell walls,temperature,duration of enzyme incubation,pH optima of the enzyme solution (Sinha et al.,2003b),gentle agitation,and nature of the osmoticum.Plasmolysis prior to enzymatic digestion of source tissues in salts (Frearson et al.,1973)and/or a sugar alcohol solutions,such as 13%(wt/vol)sorbitol as used for leaves of apricot (Ortin- Parraga and Burgos,2003),reduces cytoplasmic damage and spontaneous fusion of protoplasts from adjacent cells.Addition of glycine to the enzyme mixture was essential in maximising protoplast release from cotyledons and hypocotyls of Cucumis melo and C. metuliferus,although the optimum concentration of glycine depended on the species and cultivar(Sutiojono et al.,2002).Yields from cotyledons were optimised by a 4-day dark treatment before enzyme digestion.Protoplast yield and viability can be further enhanced by slicing of source (preplasmolysed)tissues,manual or enzymatic removal of the epidermis,and conditioning of donor material or its culture on media containing suitable osmotica (Davey et al.,2000a,2004;Power et al.,2004).Fluorescein diacetate remains the standard and most reliable fluorochrome for assessing protoplast/cell viability (Widholm,1972). Recently,Aditya and Baker(2003)described a procedure for isolating protoplasts from salt-stressed calli of the Bangladeshi Indica rice cv.Binnatoa,in which the concentration of mannitol in the wash solution was increased to the same osmotic pressure exerted by sodium chloride in the culture medium as used to maintain source tissues.For many years, embryogenic cell suspensions have been the preferred source of viable protoplasts in cereals,especially rice (Tang et al.,2001).Likewise,in rye,Ma et al.(2003)used fast- growing,friable callus initiated from immature inflorescences to establish embryogenic cell suspensions as a source of totipotent protoplasts.Similarly,in other monocotyledons such as banana,cell suspensions were the preferred source material because of their totipotency,since those from leaf mesophyll and callus were recalcitrant in culture(Assani etal,2002). Following the report that guard cells are a unique source of totipotent protoplasts in sugarbeet(Hall et al.,1996),other workers have performed experiments using guard cell protoplasts.For example,Pandey et al.(2002)reported both large-scale and small-scale procedures to isolate guard cell protoplasts of 4.thaliana for use in physiological studies Other specialised cells from which protoplasts have been prepared include those of the central tissue of root nodules of Vicia faba,where cells infected with Rhizobium bacteroids occur alongside uninfected cells (Peiter et al.,2003).Isolation involved dissection of nodules prior to wall digestion in hypertonic solution,the release of protoplasts into slightly hypotonic solution,and the separation of protoplast fractions by isopycnic density gradient centrifugation.Such protoplasts are useful in physiological investigations of plasma membrane transport. Whilst most studies have focused on protoplasts of higher plants,Hohe and Reski (2002)optimised a semicontinuous bioreactor for the moss,Physcomitrella patens,giving yields of 2.8x10*protoplasts mg dry weight.Yield was increased sixfold by supplementing the medium with 460 mg I ammonium tartrate.Algal protoplasts have also received attention.Thus,Sawabe et al.(1997)isolated protoplasts from the seaweed, Laminaria japonica,followed by their regeneration to plants in a continuous flow culture system
Several factors influence protoplast release, including the extent of thickening of cell walls, temperature, duration of enzyme incubation, pH optima of the enzyme solution (Sinha et al., 2003b), gentle agitation, and nature of the osmoticum. Plasmolysis prior to enzymatic digestion of source tissues in salts (Frearson et al., 1973) and/or a sugar alcohol solutions, such as 13% (wt/vol) sorbitol as used for leaves of apricot (OrtinParraga and Burgos, 2003), reduces cytoplasmic damage and spontaneous fusion of protoplasts from adjacent cells. Addition of glycine to the enzyme mixture was essential in maximising protoplast release from cotyledons and hypocotyls of Cucumis melo and C. metuliferus, although the optimum concentration of glycine depended on the species and cultivar (Sutiojono et al., 2002). Yields from cotyledons were optimised by a 4-day dark treatment before enzyme digestion. Protoplast yield and viability can be further enhanced by slicing of source (preplasmolysed) tissues, manual or enzymatic removal of the epidermis, and conditioning of donor material or its culture on media containing suitable osmotica (Davey et al., 2000a, 2004; Power et al., 2004). Fluorescein diacetate remains the standard and most reliable fluorochrome for assessing protoplast/cell viability (Widholm, 1972). Recently, Aditya and Baker (2003) described a procedure for isolating protoplasts from salt-stressed calli of the Bangladeshi Indica rice cv. Binnatoa, in which the concentration of mannitol in the wash solution was increased to the same osmotic pressure exerted by sodium chloride in the culture medium as used to maintain source tissues. For many years, embryogenic cell suspensions have been the preferred source of viable protoplasts in cereals, especially rice (Tang et al., 2001). Likewise, in rye, Ma et al. (2003) used fastgrowing, friable callus initiated from immature inflorescences to establish embryogenic cell suspensions as a source of totipotent protoplasts. Similarly, in other monocotyledons such as banana, cell suspensions were the preferred source material because of their totipotency, since those from leaf mesophyll and callus were recalcitrant in culture (Assani et al., 2002). Following the report that guard cells are a unique source of totipotent protoplasts in sugarbeet (Hall et al., 1996), other workers have performed experiments using guard cell protoplasts. For example, Pandey et al. (2002) reported both large-scale and small-scale procedures to isolate guard cell protoplasts of A. thaliana for use in physiological studies. Other specialised cells from which protoplasts have been prepared include those of the central tissue of root nodules of Vicia faba, where cells infected with Rhizobium bacteroids occur alongside uninfected cells (Peiter et al., 2003). Isolation involved dissection of nodules prior to wall digestion in hypertonic solution, the release of protoplasts into slightly hypotonic solution, and the separation of protoplast fractions by isopycnic density gradient centrifugation. Such protoplasts are useful in physiological investigations of plasma membrane transport. Whilst most studies have focused on protoplasts of higher plants, Hohe and Reski (2002) optimised a semicontinuous bioreactor for the moss, Physcomitrella patens, giving yields of 2.8104 protoplasts mg1 dry weight. Yield was increased sixfold by supplementing the medium with 460 mg l1 ammonium tartrate. Algal protoplasts have also received attention. Thus, Sawabe et al. (1997) isolated protoplasts from the seaweed, Laminaria japonica, followed by their regeneration to plants in a continuous flow culture system. M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171 135
136 M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 3.1.Stress during protoplast isolation Protoplast isolation per se is a stress-inducing procedure (Papadakis et al.,2001; Papadakis and Roubelakis-Angelaskis,2002),particularly during enzymatic isolation, with accumulation of peroxides and degradation products that induce cell lysis,especially in cereals(Cutler et al.,1989).Attention has refocused on this phenomenon.Commun et al.(2003)reported the production of stilbene phytoalexins in protoplasts of Vitis spp.and detected trans-resveratrol as early as 4 h after the beginning of enzyme digestion,through activation of the vst/gene encoding stilbene synthase.The presence of resveratrol and its derived phytoalexins,episilonviniferin and pterostilbene,may account for loss of protoplast viability.Other studies confirmed that leaf protoplasts of Brassica napus and Petunia hybrida experience stress during isolation (Watanabe et al.,2002),with increase in polyamines initiating senescence,especially in B.napus.The effects of stress during isolation may be long-term,and may account for the recalcitrance of some protoplast systems in culture.Such effects of stress are considered later in this discussion. 4.Culture techniques for isolated plant protoplasts 4.1.Culture media The necessity to develop protoplast-to-plant systems,particularly for economically important species,has demanded a major investment of resources.Typically,isolated protoplasts commenced cell wall regeneration within a short time (often minutes) following introduction into culture.However,they require osmotic protection until their new primary walls can counteract the turgor pressure exerted by the cytoplasm.In some cases,gradual reduction of the osmotic pressure by diluting the culture medium with a solution of similar composition,but of reduced osmotic pressure,is essential for sustaining mitotic division,leading to the formation of daughter cells and tissues. Protoplasts from different species and from different tissues of the same species may vary in their nutritional requirements.Consequently,the optimum medium for long-term culture must be determined empirically.Many media have been based on the MS (Murashige and Skoog,1962)and B5(Gamborg et al.,1968)formulations,with addition of an osmoticum,usually a nonmetabolisable sugar alcohol,such as mannitol,or the somewhat more soluble,sorbitol.Ideally,media should be simple and fully defined to ensure reproducibility between laboratories.An exception is the complex,undefined medium containing coconut milk (Kao and Michayluk,1975)used for the culture of protoplasts at very low densities. The major growth regulators,auxins and cytokinins,are normally essential for sustained protoplast growth,although exceptions exist where only auxin is required,as in carrot and A.thaliana (Dovzhenko et al.,2003).In contrast,auxins and cytokinins are detrimental to growth in citrus(Vardi et al.,1982).The growth requirements of protoplasts often change during culture,necessitating modification of medium composition,typically involving a reduction of the auxin concentration.Phenylurea derivatives,such as N-(2- chloro-4-pyridyl)-N-phenylurea(Sasamoto et al.,2002),and brassinosteroids,which are
3.1. Stress during protoplast isolation Protoplast isolation per se is a stress-inducing procedure (Papadakis et al., 2001; Papadakis and Roubelakis-Angelaskis, 2002), particularly during enzymatic isolation, with accumulation of peroxides and degradation products that induce cell lysis, especially in cereals (Cutler et al., 1989). Attention has refocused on this phenomenon. Commun et al. (2003) reported the production of stilbene phytoalexins in protoplasts of Vitis spp. and detected trans-resveratrol as early as 4 h after the beginning of enzyme digestion, through activation of the vst1 gene encoding stilbene synthase. The presence of resveratrol and its derived phytoalexins, episilonviniferin and pterostilbene, may account for loss of protoplast viability. Other studies confirmed that leaf protoplasts of Brassica napus and Petunia hybrida experience stress during isolation (Watanabe et al., 2002), with increase in polyamines initiating senescence, especially in B. napus. The effects of stress during isolation may be long-term, and may account for the recalcitrance of some protoplast systems in culture. Such effects of stress are considered later in this discussion. 4. Culture techniques for isolated plant protoplasts 4.1. Culture media The necessity to develop protoplast-to-plant systems, particularly for economically important species, has demanded a major investment of resources. Typically, isolated protoplasts commenced cell wall regeneration within a short time (often minutes) following introduction into culture. However, they require osmotic protection until their new primary walls can counteract the turgor pressure exerted by the cytoplasm. In some cases, gradual reduction of the osmotic pressure by diluting the culture medium with a solution of similar composition, but of reduced osmotic pressure, is essential for sustaining mitotic division, leading to the formation of daughter cells and tissues. Protoplasts from different species and from different tissues of the same species may vary in their nutritional requirements. Consequently, the optimum medium for long-term culture must be determined empirically. Many media have been based on the MS (Murashige and Skoog, 1962) and B5 (Gamborg et al., 1968) formulations, with addition of an osmoticum, usually a nonmetabolisable sugar alcohol, such as mannitol, or the somewhat more soluble, sorbitol. Ideally, media should be simple and fully defined to ensure reproducibility between laboratories. An exception is the complex, undefined medium containing coconut milk (Kao and Michayluk, 1975) used for the culture of protoplasts at very low densities. The major growth regulators, auxins and cytokinins, are normally essential for sustained protoplast growth, although exceptions exist where only auxin is required, as in carrot and A. thaliana (Dovzhenko et al., 2003). In contrast, auxins and cytokinins are detrimental to growth in citrus (Vardi et al., 1982). The growth requirements of protoplasts often change during culture, necessitating modification of medium composition, typically involving a reduction of the auxin concentration. Phenylurea derivatives, such as N-(2- chloro-4-pyridyl)-NV-phenylurea (Sasamoto et al., 2002), and brassinosteroids, which are 136 M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171
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 of
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 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
138 M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 the cell-alginate mixture into strontium chloride solution containing the cell wall-digesting enzymes (Aoyagi and Tanaka,1999).In this way,protoplast isolation and gel solidification proceeded simultaneously.These authors claimed that the viability of the immobilised protoplasts was higher using this procedure than with more conventional methods.It will be important to determine the general applicability of this procedure to other higher plant systems.Interestingly,the authors also claimed comparable success with yeast protoplasts by employing this procedure. 4.3.Plating density and protoplast growth in culture The final (overall)density of protoplasts in the culture medium (plating density)is crucial for maximising wall regeneration and concomitant daughter cell formation. Generally,the optimum plating density is in the range 5x10-1x10 protoplasts ml- An excessively high plating density rapidly depletes nutrients,and protoplast-derived cells can fail to undergo sustained division.Cells stimulate mitotic division of adjacent cells by releasing growth factors,including amino acids,into the surrounding medium,a process commonly known as 'medium conditioning'or 'nurse'culture.Consequently, protoplasts fail to undergo sustained division when cultured below a minimum inoculum density threshold.Medium preconditioned by supporting the growth of actively dividing cells for a limited period is valuable in stimulating growth of isolated protoplasts. Similarly,actively dividing cells can promote or 'nurse'the growth of recently isolated protoplasts.Nurse cells can be from the same or different species.For example,division of protoplasts from embryogenic rice cell suspensions was most effectively stimulated by nurse cells of Italian ryegrass (Lolium multiflorum;Jain et al.,1995).Protoplasts/ spheroplasts or cells that have been X-irradiated to inhibit division can also exert a similar nurse effect. Isolated protoplasts may be cultured,using the procedures already described,in liquid medium over semi-solid medium containing the nurse cells.Dispensing protoplasts in a limited volume of medium on a membrane overlaying the semi-solid nurse cell layer promotes division.Chen et al.(2004)described a procedure for regenerating shoots from hypocotyl-derived protoplasts of red cabbage (Brassica oleracea)in which the target protoplasts were mixed in a 1:1 ratio with viable protoplasts of tuber mustard (Brassica juncea),the latter being essential for sustained division and colony formation from red cabbage protoplasts.Light microscopy revealed that regenerated plants had the expected somatic chromosome complement of red cabbage (2n=2x=18),confirming that sponta- neous fusion did not occur between protoplasts of the two species during culture.The applicability of this approach of mixing viable test and nurse protoplasts warrants more extensive evaluation in other systems,especially where protoplast-derived cells fail to undergo sustained mitosis as a monoculture. 5.Totipotent protoplast systems Many of the morphological changes that occur during the development of protoplasts to cells were described in detail in early publications.In contrast,anatomical investigations
the cell–alginate mixture into strontium chloride solution containing the cell wall-digesting enzymes (Aoyagi and Tanaka, 1999). In this way, protoplast isolation and gel solidification proceeded simultaneously. These authors claimed that the viability of the immobilised protoplasts was higher using this procedure than with more conventional methods. It will be important to determine the general applicability of this procedure to other higher plant systems. Interestingly, the authors also claimed comparable success with yeast protoplasts by employing this procedure. 4.3. Plating density and protoplast growth in culture The final (overall) density of protoplasts in the culture medium (plating density) is crucial for maximising wall regeneration and concomitant daughter cell formation. Generally, the optimum plating density is in the range 5104 –1106 protoplasts ml1 . An excessively high plating density rapidly depletes nutrients, and protoplast-derived cells can fail to undergo sustained division. Cells stimulate mitotic division of adjacent cells by releasing growth factors, including amino acids, into the surrounding medium, a process commonly known as dmedium conditioningT or dnurseT culture. Consequently, protoplasts fail to undergo sustained division when cultured below a minimum inoculum density threshold. Medium preconditioned by supporting the growth of actively dividing cells for a limited period is valuable in stimulating growth of isolated protoplasts. Similarly, actively dividing cells can promote or dnurseT the growth of recently isolated protoplasts. Nurse cells can be from the same or different species. For example, division of protoplasts from embryogenic rice cell suspensions was most effectively stimulated by nurse cells of Italian ryegrass (Lolium multiflorum; Jain et al., 1995). Protoplasts/ spheroplasts or cells that have been X-irradiated to inhibit division can also exert a similar nurse effect. Isolated protoplasts may be cultured, using the procedures already described, in liquid medium over semi-solid medium containing the nurse cells. Dispensing protoplasts in a limited volume of medium on a membrane overlaying the semi-solid nurse cell layer promotes division. Chen et al. (2004) described a procedure for regenerating shoots from hypocotyl-derived protoplasts of red cabbage (Brassica oleracea) in which the target protoplasts were mixed in a 1:1 ratio with viable protoplasts of tuber mustard (Brassica juncea), the latter being essential for sustained division and colony formation from red cabbage protoplasts. Light microscopy revealed that regenerated plants had the expected somatic chromosome complement of red cabbage (2n=2x=18), confirming that spontaneous fusion did not occur between protoplasts of the two species during culture. The applicability of this approach of mixing viable test and nurse protoplasts warrants more extensive evaluation in other systems, especially where protoplast-derived cells fail to undergo sustained mitosis as a monoculture. 5. Totipotent protoplast systems Many of the morphological changes that occur during the development of protoplasts to cells were described in detail in early publications. In contrast, anatomical investigations 138 M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171
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 microtubules, 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
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 Pelargoniumhortorum 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