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·················· 160pharmaceutical 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