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