M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 141 6.Innovative approaches for protoplast culture The prime objective in developing effective protoplast-to-plant systems is to maximise cell growth and differentiation,particularly for those systems exploited to generate novel plants,often allied to some form of genetic manipulation.Whilst guidance can be obtained from the literature,a universal protocol does not exist in terms of medium composition and physical parameters to maximise protoplast growth.Consequently,such parameters must be determined empirically.Several novel approaches have been described,some of which have been successful earlier for animal systems.These include electrical treatment of cells (Lynch and Davey,1996)and manipulation of the gaseous environment (Lowe et al., 2003). 6.1.Electrical stimulation of protoplasts Some cells of plants and animals detect voltage gradients and current densities as low as 0.5 uV m-and 5 nA cm-2,respectively,with weak electrical currents of prolonged duration stimulating growth,healing of wounds and bones,and organ regeneration in animals (Goldsworthy,1996).This author provided a comprehensive discussion of the electrostimulation of plants by weak electric currents,including definitive results and more controversial information.The effects of electrical currents are not restricted to intact plants,but also influence cultured cells,including protoplasts.In the forage legume Medicago sativa,protoplasts cultured in weak electric fields developed directly into somatic embryos,prior to plant regeneration.Electrical currents not only alter cell polarity, with calcium ions being important in this respect,but also affect auxin transport.Other reviewers (Davey et al.,1996a and references therein)focused attention on the effects of short-term,high-voltage electrical pulses(250-2000 V,10-50 us),typical of those used to electroporate DNA into isolated protoplasts.Early investigations focused on protoplasts from cotyledons of Glycine canescens and Solanum viarum,cell suspensions of Prunus avium xpseudocerasus,and callus of Pyrus communis.Electrical effects were influenced by several variables,including the voltage and its duration,and protoplast size.The most significant results were that electrotreated protoplasts of Prunus,Pyrus,and Solanum entered mitosis within 5 days of culture compared to untreated protoplasts,which had lag periods of 15,9,and 1 days,respectively.This ability to stimulate growth of protoplasts, particularly those of woody genera,provided impetus for this simple technology to be applied to protoplasts from embryogenic cell suspensions of pearl millet (Pennisetum squamulatum)and tobacco. Experiments with protoplasts and cultured mammalian cells confirmed that electrical pulses stimulate DNA synthesis,which,in tum,is reflected in the earlier onset of mitosis in protoplast-derived cells.A key feature of electrostimulated protoplasts was that enhancement of division was sustained to the callus stage.Moreover,shoot regeneration was stimulated from callus following electrostimulation.Regenerated plants were also more vigorous than controls.Studies with Prunus indicated that the effects of electrostimulation on division and shoot regeneration were long-term over several subcultures,particularly when protoplasts were isolated from cell suspensions,with the latter themselves being initiated from cells derived from electrostimulated protoplasts
6. Innovative approaches for protoplast culture The prime objective in developing effective protoplast-to-plant systems is to maximise cell growth and differentiation, particularly for those systems exploited to generate novel plants, often allied to some form of genetic manipulation. Whilst guidance can be obtained from the literature, a universal protocol does not exist in terms of medium composition and physical parameters to maximise protoplast growth. Consequently, such parameters must be determined empirically. Several novel approaches have been described, some of which have been successful earlier for animal systems. These include electrical treatment of cells (Lynch and Davey, 1996) and manipulation of the gaseous environment (Lowe et al., 2003). 6.1. Electrical stimulation of protoplasts Some cells of plants and animals detect voltage gradients and current densities as low as 0.5 AV m1 and 5 nA cm2 , respectively, with weak electrical currents of prolonged duration stimulating growth, healing of wounds and bones, and organ regeneration in animals (Goldsworthy, 1996). This author provided a comprehensive discussion of the electrostimulation of plants by weak electric currents, including definitive results and more controversial information. The effects of electrical currents are not restricted to intact plants, but also influence cultured cells, including protoplasts. In the forage legume Medicago sativa, protoplasts cultured in weak electric fields developed directly into somatic embryos, prior to plant regeneration. Electrical currents not only alter cell polarity, with calcium ions being important in this respect, but also affect auxin transport. Other reviewers (Davey et al., 1996a and references therein) focused attention on the effects of short-term, high-voltage electrical pulses (250–2000 V, 10–50 As), typical of those used to electroporate DNA into isolated protoplasts. Early investigations focused on protoplasts from cotyledons of Glycine canescens and Solanum viarum, cell suspensions of Prunus aviumpseudocerasus, and callus of Pyrus communis. Electrical effects were influenced by several variables, including the voltage and its duration, and protoplast size. The most significant results were that electrotreated protoplasts of Prunus, Pyrus, and Solanum entered mitosis within 5 days of culture compared to untreated protoplasts, which had lag periods of 15, 9, and 1 days, respectively. This ability to stimulate growth of protoplasts, particularly those of woody genera, provided impetus for this simple technology to be applied to protoplasts from embryogenic cell suspensions of pearl millet (Pennisetum squamulatum) and tobacco. Experiments with protoplasts and cultured mammalian cells confirmed that electrical pulses stimulate DNA synthesis, which, in turn, is reflected in the earlier onset of mitosis in protoplast-derived cells. A key feature of electrostimulated protoplasts was that enhancement of division was sustained to the callus stage. Moreover, shoot regeneration was stimulated from callus following electrostimulation. Regenerated plants were also more vigorous than controls. Studies with Prunus indicated that the effects of electrostimulation on division and shoot regeneration were long-term over several subcultures, particularly when protoplasts were isolated from cell suspensions, with the latter themselves being initiated from cells derived from electrostimulated protoplasts. M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171 141
142 M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 Electrostimulation experiments attracted considerable interest during the 1990s,but this simple approach has received less attention in recent years.Clearly,this is an area that deserves research effort in the future,particularly for protoplast systems that currently remain recalcitrant in culture 6.2.Supplementation of culture media with surfactants,antibiotics,and polyamines A novel approach for enhancing the mitotic division of plant protoplast-derived cells involves supplementation of the culture medium with nonionic surfactants,especially the commercially available copolymer compounds known as Pluronics.One such surfactant, Pluronic F-68,a polyoxyethylene-polyoxypropylene copolymer,is used extensively as a nontoxic,low-cost cell-protecting agent during the culture of both animal and plant cells (Lowe et al.,2001).For example,in experiments with plant protoplasts,supplementation of culture medium with 0.1%(wt/vol)Pluronic F-68 increased the plating efficiency of protoplasts of Solanum dulcamara by 26%over control (Kumar et al.,1992).The beneficial effects of medium supplementation with Pluronic F-68 have also been assessed in cells of several other plant species,including those following recovery from cryopreservation.Lowe et al.(2001)suggested that Pluronic F-68 may exert a stimulatory effect on cell growth and differentiation by promoting the uptake of nutrients, growth regulators,and oxygen. Some antibiotics stimulate the division of protoplast-derived cells.For example,the cephalosporin antibiotic,cefotaxime,promoted mitotic division and cell colony formation of protoplasts isolated from seedling leaves of the woody plant passionfruit (Passiflora edulis)when added to the culture medium at 250 ug ml(d'Utra Vaz et al., 1993).Ciprofloxacin has also been shown to stimulate division in protoplasts isolated from callus of Allium longicuspis (Fellner,1995),with first cell divisions being observed after 2-6 days of culture.In contrast,protoplasts cultured without this antibiotic required at least 10 days in culture before entering division.In both cases,the mode of action of antibiotics is unclear,although cefotaxime may be metabolised to growth regulator-like compounds. Polyamines influence plant cell morphogenesis by regulating DNA replication, transcription,translation,cell division,and differentiation,and are regarded as a new class of plant growth regulators and a reserve of carbon and nitrogen in cultured tissues (Kakkar et al.,2000).Thus,polyamines stimulated DNA synthesis and mitotic activity in oat protoplasts,with arginine also stimulating division in protoplasts of almond.In other reports,leaf protoplasts of cytoplasmic male sterile and male fertile diploid sugarbeet synthesised new cell walls and underwent sustained division to form callus in the presence of the diamine putrescine (Jazdzewska et al.,2000),with this research group having demonstrated previously that spermine promoted division of the same protoplast system (Majewska-Sawka et al.,1997).As expected,inhibition of the conversion of putrescine to spermidine by difluoromethyl arginine,difluoromethyl ornithine,and cyclohexylamine reduced cell division by 30%in protoplasts of the legume Vigna aconitifolia.Since the existing literature is limited,it will be timely to clarify the validity of polyamines as media supplements in stimulating division and morphogenesis of protoplast-derived cells and tissues in a range of species
Electrostimulation experiments attracted considerable interest during the 1990s, but this simple approach has received less attention in recent years. Clearly, this is an area that deserves research effort in the future, particularly for protoplast systems that currently remain recalcitrant in culture. 6.2. Supplementation of culture media with surfactants, antibiotics, and polyamines A novel approach for enhancing the mitotic division of plant protoplast-derived cells involves supplementation of the culture medium with nonionic surfactants, especially the commercially available copolymer compounds known as Pluronics. One such surfactant, PluronicR F-68, a polyoxyethylene–polyoxypropylene copolymer, is used extensively as a nontoxic, low-cost cell-protecting agent during the culture of both animal and plant cells (Lowe et al., 2001). For example, in experiments with plant protoplasts, supplementation of culture medium with 0.1% (wt/vol) PluronicR F-68 increased the plating efficiency of protoplasts of Solanum dulcamara by 26% over control (Kumar et al., 1992). The beneficial effects of medium supplementation with PluronicR F-68 have also been assessed in cells of several other plant species, including those following recovery from cryopreservation. Lowe et al. (2001) suggested that PluronicR F-68 may exert a stimulatory effect on cell growth and differentiation by promoting the uptake of nutrients, growth regulators, and oxygen. Some antibiotics stimulate the division of protoplast-derived cells. For example, the cephalosporin antibiotic, cefotaxime, promoted mitotic division and cell colony formation of protoplasts isolated from seedling leaves of the woody plant passionfruit (Passiflora edulis) when added to the culture medium at 250 Ag ml1 (d’Utra Vaz et al., 1993). Ciprofloxacin has also been shown to stimulate division in protoplasts isolated from callus of Allium longicuspis (Fellner, 1995), with first cell divisions being observed after 2–6 days of culture. In contrast, protoplasts cultured without this antibiotic required at least 10 days in culture before entering division. In both cases, the mode of action of antibiotics is unclear, although cefotaxime may be metabolised to growth regulator-like compounds. Polyamines influence plant cell morphogenesis by regulating DNA replication, transcription, translation, cell division, and differentiation, and are regarded as a new class of plant growth regulators and a reserve of carbon and nitrogen in cultured tissues (Kakkar et al., 2000). Thus, polyamines stimulated DNA synthesis and mitotic activity in oat protoplasts, with arginine also stimulating division in protoplasts of almond. In other reports, leaf protoplasts of cytoplasmic male sterile and male fertile diploid sugarbeet synthesised new cell walls and underwent sustained division to form callus in the presence of the diamine putrescine (Jazdzewska et al., 2000), with this research group having demonstrated previously that spermine promoted division of the same protoplast system (Majewska-Sawka et al., 1997). As expected, inhibition of the conversion of putrescine to spermidine by difluoromethyl arginine, difluoromethyl ornithine, and cyclohexylamine reduced cell division by 30% in protoplasts of the legume Vigna aconitifolia. Since the existing literature is limited, it will be timely to clarify the validity of polyamines as media supplements in stimulating division and morphogenesis of protoplast-derived cells and tissues in a range of species. 142 M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171
M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 143 6.3.Manipulation of respiratory gases during protoplast culture Whilst the composition of the culture medium is undoubtedly the major factor influencing protoplast development,the gaseous environment also plays a fundamental complementary role in growth and differentiation.Importantly,medium,headspace,and plant tissues interact in complex ways that have often been overlooked.The photo- synthetic capability of some tissues and their biomass relative to the capacity of the vessels and the culture room environment contribute to the composition of the gases within culture vessels.There is a need to manipulate headspace gases to ensure that oxygen and carbon dioxide do not deviate dramatically from their normal atmospheric concentrations of 21% and 0.03%(vol/vol),respectively (Buddendorf-Joosten and Woltering,1994),and that ethylene does not accumulate (Zobayed et al.,2001). Oxygen availability limits growth.In recognising this fact,Brandt(1991)calculated the oxygen diffusing to cells in stationary liquid medium,using protoplasts from hypocotyls of B. napus cv.Omega as the experimental material.Adjusting the protoplast density,depth of the medium,and oxygen concentration in the headspace permitted a correlation of the number of protoplast-derived tissues with oxygen availability.Protoplasts died at oxygen concentrations less than 60 uM.At 20%(vol/vol)oxygen and a plating density of 2.0x10protoplasts ml-, only five protoplast-derived tissues developed per milliliter when the medium was 2 mm in depth,compared to 121 per milliliter after reduction of the depth to 1 mm. 6.4.Physical procedures to stimulate gaseous exchange Mention has been made of the culture of protoplasts in small volumes of liquid medium on the surface of filters to increase gaseous exchange,often with nurse cells in an underlying semi-solid layer.Another simple system involves the insertion of glass rods, each approximately 6 mm in diameter and 8 mm in length,into a layer of semi-solid culture medium.Isolated leaf protoplasts of cassava (Manihot esculenta)plated in liquid medium over the semi-solid phase aggregated in the menisci around the glass rods and at the sides of the dishes.Protoplasts in these regions were stimulated to divide,probably because of improved aeration at the menisci(Anthony et al.,1995a). 6.5.Gassing of protoplast cultures Recently,Lowe et al.(2003)reviewed the beneficial effects of oxygen enrichment and manipulating carbon dioxide to inhibit ethylene accumulation on protoplast growth in culture.Division of protoplasts of rice (Oryza sativa cv.Taipei 309),tomato (Lycopersicon esculentum cv.Santa Clara),and jute (Corchorus olitorius)was enhanced by oxygen enrichment of the headspace,achieved by placing culture vessels in screw-capped glass jars with inlet and outlet valves,prior to gassing of the jars with oxygen and sealing of the valves(d'Utra Vaz et al.,1992).Following initial enrichment,the oxygen concentration in the headspace was believed to decline gradually,as slow gaseous exchange was possible through the seals of the vessels.The shoot regeneration frequency from protoplast-derived tissues was also elevated by exposure of protoplasts to an oxygen-enriched atmosphere, indicating a long-term effect.Other workers have reported a similar stimulatory effect of
6.3. Manipulation of respiratory gases during protoplast culture Whilst the composition of the culture medium is undoubtedly the major factor influencing protoplast development, the gaseous environment also plays a fundamental complementary role in growth and differentiation. Importantly, medium, headspace, and plant tissues interact in complex ways that have often been overlooked. The photosynthetic capability of some tissues and their biomass relative to the capacity of the vessels and the culture room environment contribute to the composition of the gases within culture vessels. There is a need to manipulate headspace gases to ensure that oxygen and carbon dioxide do not deviate dramatically from their normal atmospheric concentrations of 21% and 0.03% (vol/vol), respectively (Buddendorf-Joosten and Woltering, 1994), and that ethylene does not accumulate (Zobayed et al., 2001). Oxygen availability limits growth. In recognising this fact, Brandt (1991) calculated the oxygen diffusing to cells in stationary liquid medium, using protoplasts from hypocotyls of B. napus cv. Omega as the experimental material. Adjusting the protoplast density, depth of the medium, and oxygen concentration in the headspace permitted a correlation of the number of protoplast-derived tissues with oxygen availability. Protoplasts died at oxygen concentrations less than 60 AM. At 20% (vol/vol) oxygen and a plating density of 2.0104 protoplasts ml1 , only five protoplast-derived tissues developed per milliliter when the medium was 2 mm in depth, compared to 121 per milliliter after reduction of the depth to 1 mm. 6.4. Physical procedures to stimulate gaseous exchange Mention has been made of the culture of protoplasts in small volumes of liquid medium on the surface of filters to increase gaseous exchange, often with nurse cells in an underlying semi-solid layer. Another simple system involves the insertion of glass rods, each approximately 6 mm in diameter and 8 mm in length, into a layer of semi-solid culture medium. Isolated leaf protoplasts of cassava (Manihot esculenta) plated in liquid medium over the semi-solid phase aggregated in the menisci around the glass rods and at the sides of the dishes. Protoplasts in these regions were stimulated to divide, probably because of improved aeration at the menisci (Anthony et al., 1995a). 6.5. Gassing of protoplast cultures Recently, Lowe et al. (2003) reviewed the beneficial effects of oxygen enrichment and manipulating carbon dioxide to inhibit ethylene accumulation on protoplast growth in culture. Division of protoplasts of rice (Oryza sativa cv. Taipei 309), tomato (Lycopersicon esculentum cv. Santa Clara), and jute (Corchorus olitorius) was enhanced by oxygen enrichment of the headspace, achieved by placing culture vessels in screw-capped glass jars with inlet and outlet valves, prior to gassing of the jars with oxygen and sealing of the valves (d’Utra Vaz et al., 1992). Following initial enrichment, the oxygen concentration in the headspace was believed to decline gradually, as slow gaseous exchange was possible through the seals of the vessels. The shoot regeneration frequency from protoplast-derived tissues was also elevated by exposure of protoplasts to an oxygen-enriched atmosphere, indicating a long-term effect. Other workers have reported a similar stimulatory effect of M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171 143
144 M.R.Davey et al.Biotechnology Advances 23 (2005)131-171 oxygen,with 40%(vol/vol)of the latter stimulating shoot regeneration threefold from rice cells in a bioreactor(Okamoto et al.,1996). There are reports confirming the beneficial effects of manipulation of carbon dioxide in relation to photosynthesis,ex vitro acclimation of regenerated plants (Solarova and Pospisilova,1997;Pospisilova et al.,1999),and prevention of ethylene accumulation, since the latter,even at 0.01 ul I-,acts as a plant hormone that inhibits growth and differentiation (Kumar et al.,1998).The extent to which alteration of the atmosphere with respect to carbon dioxide concentration affects protoplast development has not been investigated in detail,and is another area in need of further study. 6.6.Artificial oxygen carriers:perfluorocarbon liquids (PFCs)and hemoglobin (Hb) solutions An adequate and sustained oxygen supply is crucial to maintain protoplast viability and mitotic division of protoplast-derived cells.Therefore,considerable attention has focused on the beneficial effects of medium supplements that act as'artificial oxygen carriers.'The latter include PFCs and Hb solutions,used either alone or in combination. 6.6.1.Perfluorocarbon liquids PFC liquids are chemically inert and have high gas solubility,enabling them to be exploited in medicine and biotechnology.Such compounds are fluorine-substituted linear, cyclic,or polycyclic hydrocarbons with very high chemical stability (Riess,2001;Lowe, 2003;Alayash,2004).PFC liquids are,however,immiscible and insoluble in aqueous solution,forming a convenient two-phase system with a stable interface.They are unique in dissolving large volumes of respiratory and nonpolar gases,with gas solubilities in the order CO2O2>CO>N2>H2>He.PFCs have been used to regulate gas supply to improve growth of microbial and mammalian cells,with the latter growing at the interface between PFCs and aqueous media (Lowe et al,1998).Fluorocarbon polymers have also been demonstrated to be beneficial in both animal and plant cell systems,including the prevention of ethylene accumulation (Lakshmanan et al.,1997). The effects have been described of PFCs as media supplements,on in vitro growth of plant protoplasts.For example,the mean initial plating efficiency (a measure of early stages of mitotic division)of cell suspension protoplasts of P hybrida was increased by 37%when the protoplasts were cultured at the interface between medium and oxygenated perfluorodecalin (CioFIs;Flutec PP6;F2 Fluorochemicals,Preston,UK).Protoplasts were suspended at 2x10>ml-in 2 ml aliquots of medium in 30 ml screw-capped bottles, either alone(controls)or over 6 ml aliquots of PFC.The latter was gassed with oxygen for 15 min (10 mbar)or,in controls,left ungassed before use.A PFC layer at least 5 mm in depth was required to obtain a stable interface for the protoplasts.Simultaneous supplementation of the medium with the nonionic surfactant,Pluronic F-68,at 0.01% (wt/vol)increased the plating efficiency by more than 50%over control,revealing a synergistic effect of oxygen-gassed PFC and surfactant.Changes in oxygen tension confirmed that the PFC liquid acted as a reservoir for this gas,with the latter diffusing into the medium/cell phase during culture (Anthony et al.,1994a)to significantly enhance protoplast growth in the two-phase system(Anthony et al.,1994b)
oxygen, with 40% (vol/vol) of the latter stimulating shoot regeneration threefold from rice cells in a bioreactor (Okamoto et al., 1996). There are reports confirming the beneficial effects of manipulation of carbon dioxide in relation to photosynthesis, ex vitro acclimation of regenerated plants (Sola´rova´ and Pospisˇilova´, 1997; Pospisˇilova´ et al., 1999), and prevention of ethylene accumulation, since the latter, even at 0.01 Al l1 , acts as a plant hormone that inhibits growth and differentiation (Kumar et al., 1998). The extent to which alteration of the atmosphere with respect to carbon dioxide concentration affects protoplast development has not been investigated in detail, and is another area in need of further study. 6.6. Artificial oxygen carriers: perfluorocarbon liquids (PFCs) and hemoglobin (Hb) solutions An adequate and sustained oxygen supply is crucial to maintain protoplast viability and mitotic division of protoplast-derived cells. Therefore, considerable attention has focused on the beneficial effects of medium supplements that act as dartificial oxygen carriers.T The latter include PFCs and Hb solutions, used either alone or in combination. 6.6.1. Perfluorocarbon liquids PFC liquids are chemically inert and have high gas solubility, enabling them to be exploited in medicine and biotechnology. Such compounds are fluorine-substituted linear, cyclic, or polycyclic hydrocarbons with very high chemical stability (Riess, 2001; Lowe, 2003; Alayash, 2004). PFC liquids are, however, immiscible and insoluble in aqueous solution, forming a convenient two-phase system with a stable interface. They are unique in dissolving large volumes of respiratory and nonpolar gases, with gas solubilities in the order CO2JO2NCONN2NH2NHe. PFCs have been used to regulate gas supply to improve growth of microbial and mammalian cells, with the latter growing at the interface between PFCs and aqueous media (Lowe et al., 1998). Fluorocarbon polymers have also been demonstrated to be beneficial in both animal and plant cell systems, including the prevention of ethylene accumulation (Lakshmanan et al., 1997). The effects have been described of PFCs as media supplements, on in vitro growth of plant protoplasts. For example, the mean initial plating efficiency (a measure of early stages of mitotic division) of cell suspension protoplasts of P. hybrida was increased by 37% when the protoplasts were cultured at the interface between medium and oxygenated perfluorodecalin (C10F18; FlutecR PP6; F2 Fluorochemicals, Preston, UK). Protoplasts were suspended at 2105 ml1 in 2 ml aliquots of medium in 30 ml screw-capped bottles, either alone (controls) or over 6 ml aliquots of PFC. The latter was gassed with oxygen for 15 min (10 mbar) or, in controls, left ungassed before use. A PFC layer at least 5 mm in depth was required to obtain a stable interface for the protoplasts. Simultaneous supplementation of the medium with the nonionic surfactant, PluronicR F-68, at 0.01% (wt/vol) increased the plating efficiency by more than 50% over control, revealing a synergistic effect of oxygen-gassed PFC and surfactant. Changes in oxygen tension confirmed that the PFC liquid acted as a reservoir for this gas, with the latter diffusing into the medium/cell phase during culture (Anthony et al., 1994a) to significantly enhance protoplast growth in the two-phase system (Anthony et al., 1994b). 144 M.R. Davey et al. / Biotechnology Advances 23 (2005) 131–171