Plant Protoplasts: Status and Biotechnological Perspectives

Biotechnology Advances 23 (2005) 131 171
www.elsevier.com/locate/biotechadv
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
* 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/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2004.09.008
132
M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171
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.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Source material for protoplast isolation. . . . . . . . . . . . . . . . . . . . . . . . .
Procedures for protoplast isolation . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Stress during protoplast isolation . . . . . . . . . . . . . . . . . . . . . . . .
4. Culture techniques for isolated plant protoplasts . . . . . . . . . . . . . . . . . . . .
4.1. Culture media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Experimental systems for the culture of isolated protoplasts . . . . . . . . . .
4.3. Plating density and protoplast growth in culture . . . . . . . . . . . . . . . .
5. Totipotent protoplast systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Innovative approaches for protoplast culture . . . . . . . . . . . . . . . . . . . . . .
6.1. Electrical stimulation of protoplasts . . . . . . . . . . . . . . . . . . . . . . .
6.2. Supplementation of culture media with surfactants, antibiotics, and polyamines
6.3. Manipulation of respiratory gases during protoplast culture . . . . . . . . . . .
6.4. Physical procedures to stimulate gaseous exchange . . . . . . . . . . . . . . .
6.5. Gassing of protoplast cultures . . . . . . . . . . . . . . . . . . . . . . . . . .
6.6. Artificial oxygen carriers: perfluorocarbon liquids (PFCs) and
hemoglobin (Hb) solutions . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.6.1. Perfluorocarbon liquids . . . . . . . . . . . . . . . . . . . . . . . . .
6.6.2. Hemoglobin solution . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Exploitation of protoplast-to-plant technologies . . . . . . . . . . . . . . . . . . . .
7.1. Somatic hybridisation to generate novel plants . . . . . . . . . . . . . . . . .
7.1.1. Citrus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.2. Brassica. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.3. Potato and other members of the Solanaceae. . . . . . . . . . . . . .
7.1.4. Cereals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.5. Ornamental plants . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.6. Miscellaneous crop plants . . . . . . . . . . . . . . . . . . . . . . .
7.1.7. Other applications of protoplast fusion . . . . . . . . . . . . . . . . .
7.2. Transformation of protoplasts . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.1. Transformation by DNA uptake into the nucleus of isolated protoplasts
7.2.2. Organelle transformation . . . . . . . . . . . . . . . . . . . . . . . .
7.2.3. Transformation for expression of recombinant proteins
(dmolecular farmingT) . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3. Somaclonal variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Miscellaneous studies with isolated protoplasts. . . . . . . . . . . . . . . . . . . . .
9. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
Three decades have passed since the Centre National de la Recherche Scientifique,
Versailles, hosted the symposium dProtoplastes et Fusion de Cellules Somatiques
Vegetals,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
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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 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 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.
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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.
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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-(2chloro-4-pyridyl)-NV-phenylurea (Sasamoto et al., 2002), and brassinosteroids, which are
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 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. 100150 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
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the cellalginate 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 51041106 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
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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-1,3glucan). 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,
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exploitation in genetic manipulation, physiological investigations, and plantpathogen

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 Pelargonium hortorum 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).
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 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 (2502000 V, 1050 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
avium pseudocerasus, 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.
142
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 polyoxyethylenepolyoxypropylene 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 (dUtra 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 26 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.
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 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 (dUtra 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
144
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 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).
145
Reference has been made to the presence of glass rods in the medium, stimulating
division of cassava leaf protoplasts. Growth of these protoplasts was increased further
by oxygenated perfluorodecalin between the liquid and underlying semi-solid phases
of the medium, generating a triple-layered system (Anthony et al., 1995b).
Oxygenated perfluorodecalin also promoted a fivefold increase in the plating
efficiency of protoplasts from cell suspensions of the Japonica rice cv. Taipei 309.
Importantly, this stimulation was sustained throughout culture, giving a 12% increase
in shoot regeneration from protoplast-derived tissues. Crucially, there were no
detrimental long-term effects of PFCs on regenerated plants, since the latter were
fertile and morphologically similar to seed-derived individuals (Wardrop et al., 1996).
Oxygenated PFC also promoted the plating efficiency of protoplasts of passionfruit
(Passiflora edulis and P. giberti) by up to 62%. Electrofused protoplasts of the two
species had a plating efficiency more than 60% of that of electrofused protoplasts
cultured without PFC (Wardrop et al., 1997a). As in rice, electrofused protoplasts
exhibited a twofold increase in shoot regeneration following culture with oxygengassed PFC.
6.6.2. Hemoglobin solution
Hb in legumes (leghemoglobin) is associated with nitrogen-fixing rhizobia within
nodules, where it facilitates respiratory oxygen flow by the bacteria at concentrations low
enough not to inactivate nitrogenase (Hunt et al., 2001). Hb is also present in dicotyledons
and monocotyledons that do not fix nitrogen (Arredondo-Peter et al., 1997; Sowa et al.,
1999), although its function in these plants is unclear. Each molecule of Hb may bind four
molecules of oxygen, but chemical modifications are required prior to Hb being used as a
gas carrier in experimental systems (Lowe et al., 2003). Such modifications include crosslinking of the alphaalpha, betabeta, and alphabeta chains to decrease the oxygen
affinity of Hb (Jones et al., 1996), polymerisation of Hb molecules with glutaraldehyde
(Nishide et al., 1997), or conjugation to macromolecules such as polyethylene glycol
(Alayash, 2004). Bovine Hb is preferable to that extracted from human blood (Lowe et al.,
2003) since oxygen release depends on chloride ions, rather than the anionic organic
phosphate, 2,3-diphosphoglycerate, with the former being abundant in plant cells. In
general, Hb has received little attention as an artificial oxygen carrier in animal, microbial,
and plant culture systems.
Medium supplementation with the commercial Hb solution, Erythrogenk (Biorelease,
Salem, USA), at 1:50 (vol/vol) increased the plating efficiency of P. hybrida protoplasts by
more than 60% (Anthony et al., 1997b). Interestingly, inclusion of the surfactant
PluronicR F-68 at 0.01% (wt/vol) in the Erythrogenk-containing medium further
stimulated the protoplast plating efficiency to 92% over the control. In extending these
studies to cereals, Azhakanandam et al. (1997) suspended protoplasts of the rice cv. Taipei
309 in liquid medium at 0.5106 ml1 and spread 200 Al aliquots of the suspension on the
surface of cellulose nitrate filters. The latter were placed on 5 ml aliquots of agarosesolidified medium containing nurse cells of L. multiflorum in Petri dishes. Addition of
Erythrogenk at 1:50 (vol/vol) to the aqueous medium containing the rice protoplasts
stimulated their division. As in earlier studies with oxygenated PFC (Wardrop et al., 1996),
shoot regeneration from rice protoplasts cultured in Hb-supplemented medium was
146
increased twofold. Interestingly, tillering was also more prominent in such regenerated
plants.
In other experiments with rice protoplasts, Al-Forkan et al. (2002) employed the same
system, but supplemented the semi-solid phase containing the Lolium dnurseT cells with
Hb at 1:200 (vol/vol). Again, Hb stimulated mitosis in cell suspension protoplasts of the
Indica cvs. Binni and BR26, with increased tillering of regenerated plants. More recently,
Power et al. (2003) also recorded enhanced division of potato (cv. Desiree) cell suspension
protoplasts when Erythrogenk was added to droplets of semi-solid agarose medium
containing the embedded protoplasts. Collectively, these studies indicate that low
concentrations of Hb enhance the growth of protoplasts of monocotyledons and
dicotyledons. Protoplasts of P. hybrida have been used to evaluate any synergistic effects
of PFCs and Hb as media supplements, with division of protoplasts at the PFCmedium
interface being further stimulated when Erythrogenk at 1:50 (vol/vol) was added to the
aqueous medium (Anthony et al., 1997a).
Whilst most investigations have focused on regulating the supply of oxygen, there is
the opportunity to exploit PFCs to regulate carbon dioxide and to scavenge metabolic
by-products, such as ethylene and reactive oxygen species (Rossman et al., 1996),
since the latter may inhibit cell totipotency (Benson, 2000). In detailed studies of
tobacco mesophyll protoplasts in culture, Papadakis et al. (2001) confirmed that
totipotent protoplasts had lower intracellular O2S, hydrogen peroxide, and lipid
peroxidation, reduced forms of ascorbate and glutathione, and increased activity of
antioxidant enzymes, such as superoxide dismutase (SOD), compared to protoplasts
and their derived cells that failed to regenerate shoots. Artificial oxygen carriers can
influence the antioxidant status of protoplasts and protoplast-derived cells. In this
respect, Wardrop et al. (2002) investigated changes in oxygen consumption using a
Clark-type oxygen microelectrode, mitochondrial membrane potential by Rhodamine
123 fluorescence, and intracellular activity of SOD and catalase, in protoplasts of P.
hybrida cultured for 14 days in medium overlaying oxygenated PFC. After 24 h,
cellular oxygen consumption increased by 48% compared to that of untreated cells.
There was more than a 50% increase in mitochondrial membrane potential; SOD and
catalase activities also increased after 37 days in protoplasts with oxygenated PFC.
SOD activity was comparable to that obtained earlier for protoplasts of Salpiglossis
sinuata (Wardrop et al., 1997b).
PFCs are expensive to purchase, but are recoverable and recyclable, giving a prolonged
experimental life. In contrast, Hb solutions, whilst less costly, have a limited shelf life of
ca. 12 years. Nevertheless, both medium supplements warrant more extensive evaluation,
especially in recalcitrant protoplast systems.
7. Exploitation of protoplast-to-plant technologies

7.1. Somatic hybridisation to generate novel plants
A number of recent papers have described the generation of unique plants through
somatic hybridisation by protoplast fusion, together with aspects of the procedures
147
involved in this technology, including the selection of hybrid cells and plants (Davey et al.,
2000b, 2004). Some recent improvements have been described in fusion technology. For
example, De Filippis et al. (2000) compared electrofusion with chemical (polyethylene
glycol; PEG) fusion of pea protoplasts. Electrofusion was preferred in terms of the
maintenance of protoplast viability and reduction in membrane damage, general protoplast
distortion/disruption, and organelle fusion. Similarly, Rakosy-Tican et al. (2001) improved
the microelectrofusion procedure to generate intraspecific hybrids between N. tabacum cv.
Petit Havana and a streptomycin-resistant line, SR1. Tobacco protoplasts were also
featured in experiments to develop laser dtweezersT to fuse protoplasts, with the
distribution of chloroplasts in the parental protoplasts influencing fusion (Li et al.,
1999). Specific genera have been targeted in somatic hybridisation, as indicated in later
discussion. Other recent examples of the application of protoplast fusion to transfer
agronomically useful traits are given in Table 1.
7.1.1. Citrus
Considerable basic and applied research has focused on Citrus because of its global
commercial importance. As Guo and Deng (2001) noted, introgression of genes into
Citrus by sexual hybridisation is often hindered by polyembryony, pollenovule sterility,
sexual and graft incompatibilities, and extended juvenility. A key and generally unusual
feature of Citrus compared to other genera is the ease with which interspecific and
intergeneric somatic hybrids can be produced, with wild relatives of cultivated Citrus
representing an untapped gene pool reservoir to generate novel somatic hybrid and cybrid
plants. In this respect, Liu and Deng (2000a) reported cybrid production involving
irradiated protoplasts of Valencia sweet orange (Citrus sinensis) and iodoacetate-treated
protoplasts of Murcott tangor (C. reticulata C. sinensis), with in vitro grafting being
necessary to maintain the regenerated shoots. Guo et al. (2000) electrofused protoplasts of
cell suspensions of Bonnaza navel orange (C. sinensis) with mesophyll protoplasts from
seedless Red Blush grapefruit (C. paradisi) to generate tetraploid and diploid plants. Some
of the tetraploids exhibited precocious flowering. These authors proposed that somatic
hybrids may be excellent pollen parents for improving seedy pummelo cultivars by
generating triploid seedless hybrids. Other interspecific combinations have included
Hamlin sweet orange (C. sinensis) with rough lemon (C. jambhiri), with mitochondrial
and chloroplast DNAs in the hybrids being from the C. sinensis parent (Liu and Deng,
2000b). Protoplasts of Caipira sweet orange (C. sinensis) have been fused with those of
Rangpur lime (C. limonia) using PEG. The tetraploid plants generated (2n=4x=36) are
potential rootstocks for the citrus industry in Southeast Brazil because they may combine
drought tolerance and growth vigour of lime with blight tolerance of Caipira sweet orange
(Mendes et al., 2001). The same group (Mendes-da-Gloria et al., 2000) generated somatic
hybrids between C. sinensis and C. volkameriana, C. reticulata, and C. jambhiri, and C.
aurantium with C. limonia, again with potential tolerance to blight and tristeza virus.
Cabasson et al. (2001) induced PEG fusion of embryogenic protoplasts of Willow leaf
mandarin (C. deliciosa) with leaf protoplasts of Valencia sweet orange or grapefruit.
Following characterisation by flow cytometry and mitochondrial DNA restriction
fragment length polymorphism (RFLP) analysis, the diploid hybrid plants were propagated
for field evaluation. Similarly, Tusa et al. (2000) from the same research group
148
Table 1
Recent examples of the transfer of useful agronomic traits by protoplast fusion
Species
Brassica
B. napus
B. napus
B. napus
B. napus
Useful traits transferred

(+)
(+)
(+)
(+)
B. rapa
Crambe abyssinica
Orychophragmus violaceus
Sinapsis arvensis
Increased biomass and yield*

Increased erucic acid content in seeds*
Improved fatty acid composition in seeds*
Enhanced resistance to Blackleg
(Leptosphaeria maculans)*
B. oleracea (+) Moricandia arvensis
Introduction of the C3C4
intermediate traity
Raphanus sativus (+) Diplotaxis tenuifolia Introduction of the C3C4
intermediate traity
Citrus
C. amblycarpa (+) Citroncirus
webberri C35
C. limonia (+) C. sunki
cv. Tanaka
C. reticulata cv. Blanco (+)
C. paradisi
C. volkameriana
Poncirus trifoliata
C. sinensis cv. Rohde Red (+)
C. volkameriana
C. sinensis cv. Ruby Blood (+)
C. volkameriana
C. sinensis (+) Fortunella
crassifolia
C. sinensis (+) F. obovata
C. sinensis (+) Clausena lansium
C. unshiu cv. Guoqing No. 1 (+)
C. grandis cv. Buntan Pink
C. reticulata cv. Blanco
C. reticulata C. sinensis
Solanum
S. melongena (+) S. aethiopicum
S. melongena (+) S. sisymbrifolium
S. tuberosum (+) S. etuberosum
S. tuberosum (+) S. nigrum
S. tuberosum (+) S. stenotomum
* Trait transfer confirmed.
y
Trait transfer unconfirmed.
Reference
Qian et al. (2003)
Wang et al. (2003)
Hu et al. (2002b)
Hu et al. (2002a)
Ishikawa et al. (2003)
Bang et al. (2003)
Improved rootstock
for Mexican limey
Tolerance to citrus blight,
tristeza virus, and Phytophthora y
Production of mixoploid plants
tolerant to citrus exocortis virus (CEV)y
Tolerance to citrus blight, tristeza virus,
and Phytophthora y
Resistance to CEVy
Medina-Urrutia et al.
(2004)
Costa et al. (2003)

Tolerance to citrus blight, tristeza virus,
and Phytophthora y
Increased plant vigour*
Costa et al. (2003)
Liu and Deng (2002)

Costa et al. (2003)
Guo et al. (2002)
Costa et al. (2003)

Cheng et al. (2003)

Production of triploid plants*
Generation of seedless cybridsy
Fu et al. (2003)
Guo et al. (2004)
Guo et al. (2004)
Guo et al. (2004)
Resistance to bacterial wilt

(Ralstonia solanacearum)*
Resistance to bacterial and fungal wilts*
Resistance to potato virus Y*
Resistance to potato blight
(Phytophthora infestans)y
Resistance to bacterial wilt
(R. solanacearum)*
Collonnier et al. (2001)
Costa et al. (2003)
Collonnier et al. (2003)

Gavrilenko et al. (2003)
Szczerbakowa et al.
(2003)
Fock et al. (2001)
149
investigated the response of somatic hybrids and cybrids of Valencia sweet orange and
Femminello lemon (C. limonia) to dmal seccoT disease incited by Phoma tracheiphila.
Somatic hybrids and cybrids exhibited intermediate resistance compared to resistant
Monachello lemon and susceptible Femminello lemon.
Intergeneric somatic hybrids have also been reported, for example, by Guo and Deng
(1999), who electrofused protoplasts of Bonnaza navel orange with those of the sexually
incompatible relative, Clausena lansium (Chicken Heart Chinese wampee). Regenerated
plants had a hexaploid chromosome complement of 2n=6x=54 and not the expected
2n=4x=36, indicating that chromosome doubling rather than chromosome elimination
occurred following protoplast fusion. Similarly, Fu et al. (2003) also fused embryogenic
protoplasts of Newhall navel orange with leaf protoplasts of C. lansium. Minigrafting
permitted fusion products to develop to whole plants. Most of the latter were tetraploids, as
expected, although some plants were triploid. Simple sequence repeat (SSR) analysis
revealed the regenerated plants had chromosome fragments of both parents, confirming
their somatic hybridity. Molecular analysis also indicated the chloroplasts to be from the
Clausena parent, while the mitochondria were from the navel orange partner. Triploids
most likely resulted from chromosome elimination from the Clausena parent.
The genus Fortunella has also been exploited in intergeneric somatic hybridisation with
Citrus. Thus, Costa et al. (2003) generated hybrids between Valencia sweet orange and
Fortunella obovata (in addition to Citrus interspecific hybrids) for subsequent evaluation
in rootstock improvement programmes with respect to tolerance to citrus blight, tristeza
virus, and Phytophthora, and for tree stature. Detailed molecular analysis was also
performed on 10-year-old somatic hybrid trees between Valencia orange and Meiwa
kumquat (F. crassifolia) using cleaved amplified polymorphic sequence (CAPS) and
RFLP analyses. Both mitochondrial and chloroplast DNAs were derived from Valencia
orange (the embryogenic cell suspension parent), with the more complex mitochondrial
DNA banding pattern coinciding with increased plant vigour (Cheng et al., 2003). These
authors suggested that mitochondrial DNA pattern may be correlated with phenotypic
abnormality in somatic hybrid plants, and that nuclearcytoplasmic incompatibility may
cause dieback of shoots. C. sinensis has also been used as a partner in asymmetric
hybridisation, with Liu and Deng (1999) fusing iodoacetate-treated protoplasts of Newhall
navel orange with X-irradiated protoplasts of Microcitrus papuana. GFP is a convenient
visual marker to study fusion, regeneration, and selection of somatic hybrid plants in
Citrus (Olivares-Fuster et al., 2002). However, parental plants must be transformed with
the gfp gene prior to fusion, extending the experimental period for completion of these
experiments.
7.1.2. Brassica
Much attention has focused, over several years, on somatic hybridisation in the
Brassicaceae. Qian et al. (2003) generated somatic hybrids between Brassica napus and B.
rapa, and reported that the source of parental material influenced the characteristics of the
resultant somatic hybrid plants. In this respect, B. rapa from China was preferred
compared to germplasms from other global locations for transferring genes for yield into
B. napus. In related studies, Ishikawa et al. (2003) fused leaf protoplasts of Moricandia
arvensis (2n=28) with hypocotyl protoplasts of B. oleracea (2n=18) to generate novel
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hybrids (2n=46) that were confirmed by random amplified polymorphic DNA (RAPD)
analyses. Chloroplast and mitochondrial DNAs were from M. arvensis, with leaf anatomy
having a characteristic C3C4 intermediate trait typical of M. arvensis. Such somatic
hybrids will be useful bridging material to introduce the C3C4 trait into crop brassicas.
Other intergeneric combinations have been reported. For example, Hu et al. (2002a)
produced fertile somatic hybrids between B. napus and Sinapsis arvensis (wild mustard)
in order to enrich the rapeseed gene pool. Symmetric and asymmetric hybrids were
identified by RAPD analysis and nuclear DNA content, with enhanced disease resistance
to Leptosphaeria maculans being present in the hybrids from S. arvensis. Again, this
novel material has potential not only as a bridge to introduce agronomically useful traits
into Brassica crops, but also in breeding new varieties resistant to diseases, such as
blackleg.
Somatic hybrids have potential in bioremediation and environmental clean-up. Brewer
et al. (1999) electrofused protoplasts of the zinc accumulator Thlaspi caerulescens with
those of B. napus and the resulting hybrid cells were selected by their zinc tolerance and,
interestingly, their failure to adhere to the walls of culture vessels. Regenerated hybrid
plants accumulated zinc and cadmium to concentrations normally toxic to B. napus,
showing that the transfer of the trait for metal accumulation in plants is achievable by
protoplast fusion.
7.1.3. Potato and other members of the Solanaceae
Solanum species, particularly potato (Solanum tuberosum), have been featured
extensively in somatic hybridisation programmes to widen the gene pool and to
increase the genetic diversity available to breeders of this major tuberous crop. Indeed,
potato represents one of the first agricultural crops to be cultured in vitro and exploited
to generate somatic hybrids, with a significant contribution in this area of research over
an extensive period resulting from the excellent work of Helgeson et al. (1998).
Hassanein et al. (1998) used cybrids with the S. nigrum genome and a S. tuberosum
plastome to study nuclear/chloroplast interactions, whilst Jansky et al. (1999) evaluated
the fusion products of diploid Solanum species for their resistance to Colorado beetle.
The application of somatic hybridisation to potato breeding programmes has been
discussed with reference to hybrids of S. tuberosum with S. sanctae-rosae (Harding
and Millam, 2000) and S. circaeifolium (Oberwalder et al., 2000). In the latter case,
fertility declined with increase in asymmetry. Johnson et al. (2001) identified in the
field the most promising monoploids of S. phureja and S. chacoense S. phureja for
use in somatic hybridisation experiments, since monoploids may reduce the dgenetic
loadT associated with diploid parental protoplasts. Similarly, agronomic traits have also
been identified under field conditions in tetraploid progeny after selfing somatic
hybrids of S. tuberosum (+) S. commersonii (Cardi et al., 2002). The current status of
potato somatic hybridisation has been presented in a comprehensive review (Orczyk et
al., 2003).
Other somatic hybrids reported in the Solanaceae include those involving the fusion of
protoplasts from kanamycin-resistant nitrate reductase mutants of Nicotiana tabacum with
wild-type protoplasts of N. sanderae (Dragoeva et al., 1997), and Lycopersicon
esculentum with nontuberous Solanum species, such as S. tuberosum. The latter hybrids
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were generated to characterise genetic relationships in the parents and the introgression of
desirable traits into the gene pool of cultivated tomato (Gavrilenko et al., 2001).
7.1.4. Cereals
Intergeneric hybridisation has been attempted in cereals, with somatic hybrids being
generated between rice (O. sativa) and barley (Hordeum vulgare; Kisaka et al., 1998), and
rice with Zizania latifolia (Liu et al., 1999). It is noteworthy that only one asymmetric
hybrid plant was generated in these experiments, between O. sativa and Z. latifolia. At
least three further studies have exploited wheat (Triticum aestivum) as a partner,
generating somatic hybrid material with Haynaldia villosa (Zhou et al., 2001), the
distantly related cold-, drought-, and disease-tolerant Bromus inermis (Xiang et al., 1999),
and Agropyron elongatum (Xia et al., 1999). Asymmetric fusion was useful in
translocating small fragments of DNA into wheat from H. villosa, while small
chromosomes and chromosome fragments of Bromus were detected in the albino somatic
hybrid plants between wheat and B. inermis. Although somatic hybrid plants involving
wheat and Agropyron failed to produce seed, ovaries of the plants could be cultured to give
callus. Importantly, the latter regenerated plants capable of seed production. This
emphasises the necessity of reliable culture procedures for full exploitation of somatic
hybridisation technology.
7.1.5. Ornamental plants
Noteworthy are reports of the generation of somatic hybrids in ornamental species.
Thus, attempts have been made to transfer cold tolerance from Lavatera thuringiaca into
Hibiscus rosa-sinensis (VazquezTello et al., 1996), but plants were not regenerated in this
instance. Subsequently, Horita et al. (2003) generated fertile somatic hybrid plants
between the oriental hybrid lily cvs. Acapulco and Shirotae and L. formolongi cv.
Hikucho. Electrofusion-treated protoplasts divided only in the presence of nurse cells.
Molecular analyses using CAPS markers and flow cytometry confirmed the somatic
hybrid nature of the regenerated plants.
7.1.6. Miscellaneous crop plants
Other studies have characterised, at the molecular level, symmetric and asymmetric
somatic hybrids between sunflower (Helianthus annuus) and H. maximiliani (Binsfeld and
Schnabl, 2002). Both types of hybrids exhibited a recombination of the two parental
genomes to different extents, making some of these plants excellent candidates for
incorporation into conventional breeding programmes. Such studies extended earlier
investigations involving the fusion of protoplasts of H. annuus with those of H. maximiliani,
H. giganteus, and H. nuttallii, in which fertile hybrids were recovered (Henn et al., 1998).
Somatic hybridisation has considerable potential in broadening the gene pool of tropical
crops, such as banana. In this respect, Matsumoto et al. (2002) electrofused protoplasts of
triploid and diploid Musa species, followed by culture with rice nurse cells. Somatic hybrids
were identified using RAPD markers; flow cytometry revealed variation in ploidy in the
somatic hybrid plants. A possible extension of somatic hybridisation technology to
temperate soft fruit species has been proposed, with Mezzetti et al. (2001) reporting PEGinduced fusion of protoplasts of Rubus ideaus (raspberry, 2n=2x=14) with those of R.
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fruticosus (blackberry; 2n=4x=28). Fluorescent in situ hybridisation (FISH) suggested that

large karyotic rearrangements occurred in hybrid cells, which may account for the inability
to regenerate shoots from such tissues. There is one report of the application of protoplast
technology to fruit tree species in the family Ebenaceae, with hybrids between Diospyrus
glandulosa and D. kaki (persimmon) having only chloroplasts from the former parent
(Tamura et al., 1998).
Somatic hybridisation studies involving medicinal Mentha species have been
performed, with the objective of modifying oil composition. This is important because
of the significant commercial value of mint in the food and, especially, flavour industries.
Thus, Sato et al. (1996) combined peppermint (Mentha piperita cv. Blackmint) with
gingermint (M. gentilis cv. variegata). The somatic hybrids synthesised the major volatile
oil components menthone, menthol, and linalol, the last two being from peppermint and
gingermint, respectively. In a later study, Krasnyanski et al. (1998) fused protoplasts of
peppermint (M. piperita cv. Black Mitcham) with those of spearmint (M. spicata cv.
Nature Spearmint), thereby combining the high quality oil property of spearmint with the
disease resistance of peppermint.
Protoplasts of sweet potato (Ipomoea batatas) have been fused electrically with those
of the wild relatives I. trifida and I. lacunose. Interestingly, Belarmino et al. (1996)
reported that protoplasts treated electrically during the fusion process entered mitotic
division and formed cells earlier than PEG-fused protoplasts, in agreement with earlier
observations of the stimulatory effects of electrical treatments on protoplast development
in culture. Somatic hybridisation technology has also been applied to forage legumes (Tian
and Rose, 1999), combining the agronomic characteristics of the annuals, Medicago
scutellata and M. truncatula, by this approach. Other workers initiated somatic
hybridisation studies in jute and analysed the cell lines generated using chloroplast RFLP
markers (Saha et al., 2001).
7.1.7. Other applications of protoplast fusion
Attempts to generate somatic hybrid plants have not been limited to Earth-bound
experiments, since Zheng et al. (2003) electrofused protoplasts of N. tabacum and N.
rustica on the Chinese spacecraft. The authors claimed that the frequency of binucleated
and multinucleated protoplasts increased significantly under microgravity, while the
viability of protoplasts was greater in space than in Earth-bound investigations. Clearly,
similar experiments need repeating in an Earth-bound magnetic levitation facility in which
it is possible to expose protoplasts simultaneously to zero, normal, and increased gravity
(Anthony et al., 2003).
A novel application of protoplast fusion is in attempts to create hybrid chromosomes. In
pioneering experiments, Koshinsky et al. (2000) fused protoplasts of N. tabacum with
those of A. thaliana using the Cre-lox system to mediate recombination between
chromosomes of the two parents. Site-specific recombination allowed a tobacco promoter
to join with a hygromycin resistance-coding region from A. thaliana. The expected
recombination junction was detected in cells of hygromycin-resistant callus, demonstrating
the interspecific transfer of a chromosome arm between plant cells. The feasibility of sitespecific recombination between genomes of different species offers possibilities for
engineering hybrid chromosomes.
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Whilst attention has focused on somatic hybridisation and cybridisation, the fusion of
diploid and haploid protoplasts to generate gametosomatic triploid hybrid plants also has
considerable potential in terms of gene introgression. However, as discussed earlier
(Davey et al., 1996b), this technology has received only limited attention, primarily in the
genus Nicotiana. A possible reason for the lack of interest in this approach may be related
to the general difficulty of obtaining haploid plants from which to induce callus and of
isolating haploid protoplasts from microspores or pollen. Clearly, this is an area worthy of
attention in the future.
7.2. Transformation of protoplasts
7.2.1. Transformation by DNA uptake into the nucleus of isolated protoplasts
Because the plasma membrane has fluid mosaic characteristics, DNA uptake can be
induced by chemical and/or physical procedures. Early reports during the 1970s that
cultured animal cells could take up and express foreign DNA prompted assessments of the
feasibility of this approach for plant protoplasts. Experiments to demonstrate dproof of
conceptT involved the transformation of isolated protoplasts with Ti plasmid from
Agrobacterium tumefaciens, followed by genes carried on simple Escherichia coli-based
cloning vectors. The latter represented the most significant advance in this technology
(Davey et al., 1989), since it confirmed that T-DNA borders were not essential for DNA
integration into the plant genome. Supercoiled (intact) plasmids and those linearised by
restriction enzyme digestion have been used to transform protoplasts; single- and doublestranded DNA can effect transformation.
Treatment of protoplast-plasmid mixtures with PEG and/or electroporation is the
approach normally exploited to induce DNA uptake into protoplasts. However,
transformation frequencies typically remain low (ca. one in 104 protoplasts giving stably
transformed tissues). This reiterates the need for reproducible protoplast-to-plant systems,
with efficient selection to recover transformed cells and tissues. Protoplasts can be
cotransformed with more than one gene carried on the same or separate plasmids. The
stage of the cell cycle of protoplasts influences transformation. Heat shock treatment and
irradiation of recipient protoplasts enhance transformation frequency, probably by
increasing the recombination of genomic DNA with incoming foreign DNA, or the
initiation of repair mechanisms that favour DNA integration. Carrier DNA and the nature
of the plant genome also affect transformation (Davey et al., 2000a).
DNA uptake into protoplasts has been especially important in transforming plants that
are not amenable to other methods of gene delivery, particularly Agrobacterium-mediated
transformation (Rakoczy-Trojanowska, 2002). Many of such studies focused on cereals,
particularly rice, once protoplast-to-plant systems became available for these crops. For
example, Nobre et al. (2000) used PEG to induce DNA uptake into protoplasts isolated
from scutella of the Australian malting barley cv. Clipper. Transformed cells were detected
by the expression of the uidA (gus; beta-glucuronidase) gene, with transformed tissues and
fertile plants being selected on medium containing the antibiotic, geneticin (G418). It is
anticipated that cereal protoplast transformation through DNA uptake will be less relevant
as a result of the successful Agrobacterium-mediated transformation of the major cereals
(Cheng et al., 2004). Nevertheless, DNA uptake into protoplasts may still be appropriate
154
for transgenic plant production in some cases, such as sugarbeet (Hall et al., 1996;
Dovzhenko and Koop, 2003).
Other recent transformation studies utilising protoplasts include a procedure for
efficient delivery of plasmid into suspension culture-derived protoplasts of apple, which
involved raising the temperature during the incubation of protoplasts with DNA to
increase the transfection frequency. Flow cytometry provided a facile procedure to detect
transformed protoplasts (Maddumage et al., 2002). Other workers targeted sweet potato.
For example, Garcia et al. (1998) isolated protoplasts from stems and leaves, used PEG
to induce DNA uptake, and detected transient gene expression by a spectrophotometric
GUS assay. Dhir et al. (1998) electroporated DNA into petiole protoplasts and also
demonstrated expression of the gus gene. In the same paper, they reported a protoplastto-plant system via somatic embryogenesis. Presumably, their intention was to combine
the transformation and regeneration protocols to generate transgenic plants, which, to
date, have not been reported in this crop. In subsequent experiments, expression of the
gfp gene was used to detect transient and stable transformation of protoplasts from leaf
and petiole explants of sweet potato following electroporation-mediated DNA uptake
(Winfield et al., 2001). Similarly, other workers also used GFP to detect transformed
cells. For example, Fleming et al. (2000) induced plasmid uptake into protoplasts
isolated from embryogenic nucellar-derived suspension cells of Itaborai sweet orange
(C. sinensis). Transformed calli were identified by expression of an enhanced gfp
marker gene (egfp) and selected manually from nontransformed tissues. The transgenic
nature of regenerated plants was confirmed by molecular analyses. Regenerated shoots
were micrografted onto sour orange seedling rootstocks in attempts to facilitate
establishment of regenerated shoots ex vitro.
Peppermint (M. piperita cv. Black Mitcham) has been the focus of some attention as a
target not only for somatic hybridisation, but also for transformation. Protoplasts from
stems were transformed initially with a binary vector carrying the neomycin phosphotransferase (nptII) and gus genes to establish the delivery system and, subsequently, the
gus gene was replaced with a limonene synthase gene. Transformed plants were analysed
for their oil profiles (Krasnyanski et al., 1999). In investigations using tobacco,
Kochevenko and Willmitzer (2003) electroporated leaf protoplasts with DNA to target
acetolactate synthase (ALS) genes, using chimeric RNA/DNA and DNA oligonucleotides.
Amino acid substitutions at either of two key locations in the ALS gene resulted in a
chlorsulfuron-insensitive form of the enzyme and, consequently, herbicide-resistant plants
were obtained. In other reports, Sheen (2001) emphasised the importance of mesophyll
protoplast transformation systems for high-throughput screening and systematic characterisation of gene function in order to elucidate plant signal transduction pathways.
As nucleases may impede DNA uptake into isolated protoplasts, experiments have been
undertaken to reduce DNA damage during transformation. In this context, Folling et al.
(1998) studied PEG-mediated DNA transfer into protoplasts of Lolium perenne and
reported that plasmid was protected by a combination of high pH (9.0) and reduced
temperature (0 8C), since such conditions suppressed DNA nicking and improved
transformation efficiency. These authors showed two nucleases usually associated with
isolated protoplasts to be involved with DNA degradation, with one being released into the
medium, while the other was localised to the plasma membrane.
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Most transformation studies have focused on protoplasts of higher plants, but Hohe et
al. (2004) used protoplasts of P. patens as target material. The advantage of this moss is
that, uniquely, it has highly efficient homologous recombination in its nuclear DNA,
making it ideal for single and multiple targeted gene knockouts using the simple procedure
described by these authors. Although the generation of stably transformed plants will
undoubtedly remain the primary objective of most research programmes, isolated
protoplasts will still assume a fundamental role in evaluating gene constructs. This will
be particularly relevant in high-throughput transient expression screening soon after DNA
uptake, as for Arabidopsis and maize (Sheen, 2001), and prior to the use of protoplasts in
longer-term studies. The retention of tissue specificity at the protoplast level is important
because it permits gene constructs to be evaluated in both homologous and heterologous
systems. In this regard, Martens et al. (2002) used protoplasts of tobacco and yeast to
investigate heterologous expression of dihydroflavanol 4-reductases from various plants,
such as Gerbera. This was further emphasised in the related work of Cheng et al. (2001),
who expressed an Arabidopsis calcium-dependent protein kinase in maize protoplasts and
highlighted the value of protoplast transient expression systems as powerful tools for
determining and screening for plant gene functions using genomic and proteomic
approaches.
7.2.2. Organelle transformation
In general, transformation studies have targeted the nuclear genome of recipient
protoplasts. However, it is likely that plastome engineering will assume greater importance
as the procedure progresses from model systems such as tobacco, which have been used to
evaluate the expression of recombinant proteins on a commercial scale in plastids, to major
crops (Maliga, 2003). The advantages of plastid transformation, as compared to nuclear
transformation, include high foreign protein synthesis in these organelles and the absence
of epigenetic effects in regenerated plants (Bock, 2001; Daniell et al., 2002). Chloroplast
engineering was first demonstrated following PEG-mediated DNA uptake into isolated
protoplasts of tobacco (Golds et al., 1993; ONeill et al., 1993), with subsequent work
improving this procedure (Koop et al., 1996). In this respect, these authors used a vector
carrying the aminoglycoside 3V-adenyltransferase (aadA) gene targeting foreign sequences
to the small single-copy region of the plastome, followed by selection of transplastomic
cells on medium containing spectinomycin and streptomycin. Other dominant selectable
markers that have been used for plastid transformation in higher plants include the nptII
and aphA-6 genes (both detoxifying kanamycin), and the badH gene in combination with
betaine aldehyde (Klaus et al., 2003). The latter investigators described a novel system that
combined the use of antibiotic resistance with restoration of pigmentation and photosynthesis in plastid mutants. In these experiments, protoplast-derived microcolonies, rather
than recently isolated protoplasts, were used as target cells for biolistics-mediated gene
delivery, as reported earlier (Huang et al., 2002). Crucially, the transplastomic plants
generated by these workers were fertile and exhibited maternal inheritance of transgenes in
their progeny.
One key attraction of chloroplast engineering is the apparent lack of transgene
transmission through pollen, as emphasised by Ruf et al. (2001). However, some caution
must be exercised in this respect, since Stegemann et al. (2003) demonstrated that this
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might not always be true. Indeed, the latter authors demonstrated that the escape of genetic
material from the chloroplast to the nucleus occurs more frequently than was once
believed, and such DNA transfer may contribute to intraspecific and intraorganismic
genetic variation. Klaus et al. (2004) summarized the procedures to generate marker-free
transplastomic plants following DNA delivery to target cells. It is important to note that
most of the plastid transformants, as cited by Klaus et al. (2003), were generated by
particle bombardment of leaf tissues, which is the procedure preferred by most workers,
since it circumvents the requirement for labour-intensive protoplast-to-plant systems. The
recent success in engineering plastomes suggests that engineering of mitochondrial DNA
is also likely to be realised in the future, with isolated protoplasts or protoplast-derived
cells possibly being two of the recipient systems.
7.2.3. Transformation for expression of recombinant proteins (dmolecular farmingT)
In recent years, there has been growing interest in the use of plants as expression
systems for the production of recombinant proteins (Walmsley and Arntzen, 2003). At
present, several plant-derived pharmaceutical proteins are at an advanced stage of
commercial development, with examples including antibodies, vaccines, human blood
products, hormones, and growth regulators (Schillberg et al., 2003; Twyman et al., 2003).
In the case of antibody production, for example, the general eukaryotic protein synthesis
pathway appears to be well conserved between plants and animals. Consequently, plants
can fold and synthesise full-size antibodies and secrete immunoglobulin A molecules
(Larrick et al., 2001). Such use of plants as so-called dcell factoriesT has distinct
advantages over conventional animal or microbial systems, including lower production
costs compared to fermentation systems, significant commercial scale-up potential, and,
importantly, the absence of associated pathogens, thus eliminating the need for costly
downstream processing. The additional advantage of using plants and their cells is that
they provide ethically acceptable protein expression systems that are not attracting the
public hostility that has often been associated with genetically modified crops. Approaches
for transforming plant cells have included Agrobacterium-mediated gene delivery
(Valentine, 2003) and particle bombardment (Somers and Makarevitch, 2004), but the
efficiency of these techniques is variable, with the selection and regeneration of whole
plants being time-consuming. In contrast, electroporation and/or PEG treatment of
protoplasts is an alternative approach that permits rapid screening of constructs in transient
expression systems prior to stable transformation (Sheen, 2001).
7.3. Somaclonal variation
Somatic hybridisation and transformation technologies provide reliable approaches for
combining interspecific and intergeneric traits and targeted modification of genomes.
Superimposed on these procedures may occur somaclonal variation or, in the case of
protoplasts, so-called protoclonal variation. Somaclonal/protoclonal variation may involve
expression and release of naturally occurring genetic variation as a result of culture and
may be regarded as a simple form of genetic manipulation (Kantharajah and Golegaonkar,
2004). Interestingly, this should not be seen in any adverse way by those with concerns
relating to genetic manipulation technologies. Thus, Mizuhiro et al. (2001) reported that
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protoplast-derived plants of Primula malacoides showed alterations in morphological

characteristics compared to plants from in vitro-germinated seed. Specifically, such
changes involved significantly reduced petioles and variation in floral morphology,
particularly pedicle and peduncle lengths, inflorescence number, and corolla size. In
general, protoplast-derived plants formed compact inflorescences with multiple small
flowers. Red pigmentation at the centre of flowers was paler than in controls, while the
periphery of some petals had a green pigmentation. Similarly, Jelenic et al. (2001) reported
that potato plants regenerated from protoplast-derived callus were all morphological and
cytological chimeras, with alterations in general appearance, leaf morphology, and tuber
characteristics.
Where maximum genetic variation is required, somaclonal variation provides a useful
adjunct to the more technologically demanding approaches of somatic hybridisation and
transformation. The attraction of protoclonal variation is that it requires no knowledge of
the genetic basis of specific traits, it negates recombinant DNA techniques, it does not
require mutagenic agents, specialised apparatus, or containment measures, and it can be
exploited through routine culture procedures.
8. Miscellaneous studies with isolated protoplasts

Protoplasts provide a basic experimental system for ultrastructural, genetical, and
physiological studies, several recent examples of which are summarised in Table 2, in
addition to those discussed here in the text. They take up macromolecules other than DNA,
enabling them to be exploited in investigations of endocytosis/pinocytosis at the plasma
membrane, and virus uptake and replication. Controlled lysis of protoplasts permits the
isolation of subcellular fractions, including membranes, organelles, and vacuoles, with the
latter being used to study accumulation of compounds, such as sugars. Barley aleurone
protoplasts contain two distinct types of vacuoles, namely protein storage vacuoles and a
lysosome-like organelle, the secondary vacuole (Swanson et al., 1998). Both of these are
acidic, lytic organelles. Uptake of fluorescent probes into these vacuoles requires ATPdependent tonoplast transporters. Other workers have employed patch clamp techniques,
similar to those developed for animal cells, to investigate ion transport through the plasma
membrane and regulation of the osmotic balance of plant cells. For example, Fan et al.
(2003) used pollen protoplasts of Brassica chinensis to study the regulation of potassium
ion channels by external and internal protons. Additionally, Binder et al. (2003)
investigated the control of membrane potential in relation to turgor in protoplasts of the
marine alga, Valonia utricularis. Other studies, not employing patch clamping, have
investigated sodium fluxes through cation channels in the plasma membrane of
Arabidopsis root protoplasts (Demidchik and Tester, 2002). Biologically active synthetic
peptides, carrying a hydrophobic membrane-permeable sequence as a carrier for import
through the plasma membrane, have been incorporated into protoplasts (Cormeau et al.,
2002). This approach complements microinjection, or the use of membrane permeabilizing
reagents, to study in vivo proteinprotein or DNAprotein interactions. Related experiments, such as those reported by Takeda and Kasamo (2001), targeted the transmembrane
distribution of phospholipids in mung bean hypocotyl protoplasts. These workers observed
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Table 2
Recent examples of the applications of plant protoplasts
Species
Application
Reference
Arabidopsis thaliana
Gene recognition mechanisms

involved in plant pathogenicity
Elucidation of plant signal
transduction mechanisms
Electrophysiological studies
of outward K+ channels
Electrochemical assays of metabolic
flux; enzyme (peroxidase) activity
Viral pathogenicity
Synthetic peptide import through
the plasma membrane
Viral replication processes
Comparison of stress mechanisms
in plants vs. human cancer cells
Viral recombination and replication
Genetic basis of developmental
regulation and specificity
Regulation of osmotic water transport
across cell membranes
Membrane permeability and
tolerance to Al3+
Leister and Katagiri (2000)
A. thaliana/Zea mays
Brassica chinensis
Bryopsis plumosa
(marine green alga)
Cucurbita pepo
Helianthus annuus
Hibiscus cannabinus
Hordeum vulgare
Nicotiana benthamiana
Nicotiana plumbaginifolia
Nicotiana tabacum
Oryza sativa/Pisum sativum/
Sorghum bicolor/
Triticum vulgare/Z. mays
Phaseolus vulgaris
Raphanus sativus
Vicia faba
Vigna radiata
V. unguiculata
Z. mays
Electrophysiological studies of
inward-rectifying K+ channels
Immunocytochemical evaluation
of aquaporin accumulation
Fluorometric analysis of photosynthetic
electron transport
Intracellular responses to drought
and salinity stress
Studies on plasma membrane organisation
Transient gene expression and proteomics
Sheen (2001)
Fan et al. (2003)
Yasukawa et al. (2002),
Zhou et al. (2003)
Choi et al. (2003)
Cormeau et al. (2002)
Liang et al. (2002)
Zhou et al. (2000)
Shapka and Nagy (2004)
Chesnokov et al. (2002)
Ding et al. (2004)
Ishikawa et al. (2001)
Etherton et al. (2004)

Suga et al. (2003)
Goh et al. (2002)
Kim et al. (2004)
Vermeer et al. (2004)
Cheng et al. (2001)
striking variation in the distribution of different classes of phospholipids throughout the

plasma membrane. Petal protoplasts have also been exploited as experimental material,
with Kim et al. (2002) using those of P. hybrida to isolate a cDNA clone encoding
osmotin. These authors demonstrated that the latter is developmentally regulated and
involved in wound stress signal transduction.
Protoplasts provide convenient material to investigate plant cell responses to growth
regulators, with, for example, volume changes in those from maize coleoptiles and
Arabidopsis hypocotyls being recorded in response to auxin treatment (Steffens et al.,
2001). Subsequently, Komis et al. (2003) studied protoplasts in leaf cells of Chlorophytum
comosum, which were able to regulate their protoplast volume and shape during
plasmolysis. A gliding movement of protoplasts within cells was observed during
plasmolysis, which ceased following treatment with an antiactin filament drug or myosin
inhibitor, confirming that the actomyosin system is involved in regulating protoplast
159
volume. Other workers (Suga et al., 2003) studied the distribution of aquaporins in
different cells of radish and discussed their physiological role in the osmotic permeability
of protoplasts from different tissues. Leaf protoplasts of tobacco were used by
Augustynowicz et al. (2001) to study the light-induced movement of chloroplasts. It
was concluded that the contribution of the actin cytoskeleton in such migratory behaviour
is independent of the presence of the plant cell wall. Other aspects of cell wall
development were reported by Zhao et al. (2004), who utilised pollen protoplasts in
studies of plant reproduction. Specifically, they observed that the newly formed walls of
cultured pollen protoplasts mirrored those of pollen tube tips as the latter grow down the
style in Lilium longiflorum. The newly formed wall is rich in arabinogalactan proteins and
higher esterified pectins. Such protoplasts may be useful in isolating the partner for stigma/
style cysteine-rich adhesion.
Several workers have exploited protoplasts to improve our understanding of plant
virus interactions. One example is the assembly of an insect virus in protoplasts of N.
plumbaginifolia (Gordon et al., 2001). Related studies include experiments on the
infectivity of the flexible filamentous shallot virus X (SLVX) in protoplasts of Beta
vulgaris (Vishnichenko and Zavriev, 2001), producing infectious viral particles within
the host protoplasts. Liang et al. (2002) developed a leaf protoplast system for kenaf
(Hibiscus cannabinus) in order to study the replication of Hibiscus chlorotic ringspot
virus (HCRSV), with viral coat protein synthesis occurring 72 h following transfection.
Rubio et al. (2002) investigated the generation of defective RNAs (D RNAs) from
genomic RNA2 of lettuce infectious yellow virus (LIYV). GFP, in addition to being
exploited in cell transformation, is also a useful marker with which to visualise the
assembly of movement protein tubules of Cowpea mosaic virus (CPMV) within infected
protoplasts. The movement protein was shown to become localised in peripheral
punctate and long tubular structures extending from the surfaces of isolated protoplasts,
suggesting the role for a plasma membrane host factor in tubule formation (Pouwels et
al., 2002). The applicability of isolated protoplasts as a system for investigating viral
infectivity was highlighted by Choi et al. (2003), who infected protoplasts of Zucchini
squash (Cucubita pepo) with infectious and noninfectious strains of Cucumber mosaic
virus (CMV). It was concluded that the inability of some strains to infect plants was
related to poor migration of the virus within plant cells, rather than reduced viral
replication.
Protoplasts have also played a role in plantfungal interactions, albeit at the cell
stage and not immediately after isolation. Thus, Leprince et al. (2001) used protoplastderived cells of N. plumbaginifolia to investigate aspects of retrotransposon movement
in tobacco species, while Melayah et al. (2001) focused attention on plants regenerated
from tobacco mesophyll protoplasts to study the mobility of the tobacco Tnt1
retrotransposon in response to stress-induced transcriptional activation by fungal
factors.
Isolated protoplasts provide simple systems that may be developed as viable
alternatives to the traditional use of animals and animal cells in toxicological assessments
(Lowe et al., 1995). Totipotent protoplasts are potentially valuable for assaying the longerterm effects of chemicals (e.g., agrochemicals, cosmetics, food additives, and pharmaceuticals) or physical factors (e.g., radiation). In this respect, protoplasts from rapid
160
cycling plants (e.g., A. thaliana) will permit the evaluation of any adverse effects of such
agents over several generations through seed progeny. Clearly, this approach is not
possible with animal cells because they lack totipotency.
9. Conclusions
It is now over 100 years since Klercker (1892) first made crude preparations of plant
protoplasts. Since that time, enormous progress has been made in refining the
methodologies for protoplast isolation, culture, and genetic manipulation through somatic
hybridisation and transformation. Currently, protoplasts provide systems for investigating
most aspects of plant cell physiology and genetics, including proteomic and genomic
studies. Despite the enormous progress achieved in this area, several important challenges
remain. These include the recalcitrance of some protoplast systems to express their
totipotency, with leaf protoplasts of cereals being a classic example. The advent of the new
millennium has seen a marked resurgence of interest in protoplast technology with a
particular focus on the generation of novel somatic hybrid and cybrid plants that cannot be
produced through conventional breeding.
Protoplasts are naked cells that are the equivalent, in general terms, to cultured
animal cells. However, unlike the latter, protoplasts exhibit the unique property of
totipotency. Consequently, protoplasts provide a cell system that can be manipulated
readily, using physiological and pharmacological perturbations, and such experimentation can be followed through differentiation pathways to the whole (plant) organism and
subsequent generations. It is clear that future research using protoplasts will be
informed, to some extent, by developments in animal cell culture and that closer liaison
between animal and plant biologists will increasingly become a feature of 21st century
biology. One relevant example is the exciting developments in the field of dmolecular
farmingT in which human therapeutic proteins are synthesised in plant cells. Protoplasts
will increasingly provide an underpinning technology for these developments to be more
fully realised.
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Biotechnology Advances 23 (2005) 131 171

Research review paper

Plant protoplasts: status and biotechnological

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

2. Source material for protoplast isolation

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

3. Procedures for protoplast isolation

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

3.1. Stress during protoplast isolation

4. Culture techniques for isolated plant protoplasts

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

5. Totipotent protoplast systems

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

exploitation in genetic manipulation, physiological investigations, and plantpathogen

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

6. Innovative approaches for protoplast culture

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

6.3. Manipulation of respiratory gases during protoplast culture

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

7. Exploitation of protoplast-to-plant technologies

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

Useful traits transferred

Increased biomass and yield*

Tolerance to citrus blight,

Costa et al. (2003)

Liu and Deng (2002)

Costa et al. (2003)

Tolerance to citrus blight,

Generation of seedless cybridsy

Guo et al. (2004)

Generation of seedless cybridsy

Guo et al. (2004)

Resistance to bacterial wilt

Collonnier et al. (2001)

Costa et al. (2003)

Collonnier et al. (2003)

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

fruticosus (blackberry; 2n=4x=28). Fluorescent in situ hybridisation (FISH) suggested that

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

protoplast-derived plants of Primula malacoides showed alterations in morphological

8. Miscellaneous studies with isolated protoplasts

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

Gene recognition mechanisms

Leister and Katagiri (2000)

Etherton et al. (2004)

striking variation in the distribution of different classes of phospholipids throughout the

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171

M.R. Davey et al. / Biotechnology Advances 23 (2005) 131171