Journal of Experimental Botany, Vol. 48, No. 307, pp. 255-263, February 1997
Journal of
Experimental
Botany
Sugar beet guard cell protoplasts demonstrate a
remarkable capacity for cell division enabling
applications in stomatal physiology and molecular
breeding
Robert D. Hall1'5, Tjitske Riksen-Bruinsma1, Guy Weyens2, Marc Lefebvre2, Jim M. Dunwell3'4,
Arjen Van Tunen1 and Frans A. Krens1
1
Department of Cell Biology, DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO),
Postbox 16, 6700 AA Wageningen, The Netherlands
2
SES Europe NV/SA, Industriepark 15, B-3300 Tienen, Belgium
Zeneca Seeds, Jealott's Hill Research Station, Bracknell, Berkshire RG126EY, UK
3
Received 28 May 1996; Accepted 30 August 1996
Abstract
A highly-efficient protocol for the large-scale isolation
of guard cell protoplasts from sugar beet (Beta vulgaris
L.) has been developed. Optimization of conditions
for culturing these protoplasts resulted in extensive
cell division and colony formation, at frequencies
exceeding 50%. Plants can subsequently be regenerated from these guard cell-derived colonies. This provides definitive confirmation that, in sugar beet leaf
protoplast populations, only guard cells are the source
of totipotent protoplasts. These findings are the outcome of a directed, non-empirical approach to overcoming plant cell recalcitrance which was initiated by
exploiting computer-assisted microscopy to couple in
vitro response to cell origin. The results reaffirm the
conclusion that, in plants, extreme degrees of cytodifferentiation need not entail terminal specialization.
The responsive nature of this system can be ascribed
to the unique use of cultures essentially comprising a
single in vivo cell type. A uniform model system has
thus been created with potential for widespread
application. Their distinct morphological (and mechanical) features make guard cells a valuable choice for
studying various fundamental aspects, not only of
stomatal physiology, but also of plant cell (de)differentiation, differential gene expression etc. Furthermore,
an applied value for such a system can also be envis4
5
aged. Results indicate that these cells are highly
amenable to genetic manipulation techniques. The
importance of these observations to our understanding
of plant cell function and behaviour is discussed.
Key words: Beta, guard cells, stomatal physiology, totipotency, transformation.
Introduction
Only a detailed knowledge and understanding of in vitro
cell culture systems permits a denned and directed
approach towards the improvement of cellular responses
in vitro to produce reliable, uniform and reproducible
experimental systems. However, the in vitro response of
plant cells and tissues is, even in non-recalcitrant systems,
distinctly heterogeneous. In protoplast populations it is
usual that only a minority of cells proceed to divide and
form viable colonies (for references, see Roest and
Gilissen, 1993). Countless empirical attempts have been
made to improve this situation but with limited success.
In a previous publication, the novel application of computer-assisted microscopy to track down and subsequently
identify the origin of rare totipotent cells in heterogeneous
protoplast populations isolated from sugar beet leaves
was reported (Hall el cil., 1995). In this highly recalcitrant
system only a tiny proportion (<0.5%) of the protoplasts
Present address: Department of Agricultural Botany, University of Reading, PO Box 211, Reading RG6 AS, UK
To whom correspondence should be addressed. Fax: +31 317 418094. E-mail: [email protected]
© Oxford University Press 1997
256
Hall et al.
were capable of cell division and, in turn, only a fraction
of the microcalli obtained subsequently proved to be
totipotent. Results revealed that just two, morphologically
distinct cell types divided in these cultures, only one of
which produced regenerable callus. This single totipotent
cell type was identified, surprisingly, as having been
derived from stomatal guard cells.
Considering the unique morphological and physiological specializations of guard cells, the confirmation that
such cells can be induced to re-express their full
embryogenic and thus genetic potential has considerable
scientific implications (Sahgal et al., 1994; Hall et al.,
1995). Consequently, the development of a system for
the isolation and culture of this single cell type could
prove of enormous value in providing a new inroad into
studies on (de) differentiation processes and guard cell
physiology. Furthermore, in addition to this fundamental
value, such a system, in overcoming in vitro recalcitrance,
could then be applied to other aims including, for
example, the development of a suitable genetic transformation protocol for sugar beet. This crop is of major
agricultural importance in the northern hemisphere and
presently suffers from a range of limitations in terms of
disease susceptibility, climatic tolerance and product quality (Doney, 1993). Sugar beet is, therefore, an ideal target
for genetic modification via biotechnological techniques.
This paper describes how the previously gained knowledge on totipotency has been directly exploited to
improve substantially the in vitro response of sugar beet
protoplast cultures. A protocol for guard cell isolation
and culture has been developed and, in so doing, the
productivity of the system has been enhanced to an extent
where, for the first time in sugar beet, its efficient application becomes feasible. One such use, to form the basis of
a transformation protocol for sugar beet, has already
proven very successful and is the subject of another paper
(Hall et al., 1996). However, this novel system has more
widespread value and the results obtained are discussed
in relation to their scientific implications for research into
guard cell physiology.
Materials and methods
beaker containing 50 ml cold (4°C) Ficoll medium (lOOg 1 '
Ficoll, 735 mg I" 1 CaCl 2 .2H 2 0, l g P 1 PVP40, autoclaved).
The epidermal fragments were then recovered on a 297 /xm
nylon mesh (Nybolt, Zurich, Switzerland), washed with 500 ml
sterile tap water and then rinsed from the mesh into a 9 cm
Petri dish using 10 ml CPW9M containing 3.8% (w/v)
CaG 2 .2H 2 O (Krens et al., 1990). Any remaining leaf fragments
were removed and dishes were preincubated for 1 h at room
temperature.
Guard cell protoplast isolation from enriched epidermis fractions
To recover the epidermis fraction, the suspension was centrifuged for 1 min at 55 xg and the supernatant discarded. The
pellet was resuspended in 50 ml digestion medium and 5 ml
aliquots were transferred to each of 10, 6 cm Petri dishes
(Greiner, TC quality), sealed with Parafilm and incubated
overnight at 25 °C in darkness with gentle agitation. The
digestion medium consisted of CPW9M supplemented with
0.5% (w/v) Cellulase RS and 3% (w/v) Macerozyme R10 (both
Yakult Honsha, Tokyo, Japan), pH 5.8. After 16 h, many
protoplasts were generally to be seen floating near the surface
of the medium. After gentle agitation of the suspensions using
a sterile pipette to release protoplasts adhering to cuticle
fragments, the digests were pooled and passed through 297 and
55 /urn nylon filters (Nybolt, Zurich, Switzerland). The filtrate
was mixed with an equal volume of iso-osmotic Percoll
containing 15% (w/v) sucrose (Percoll 15S) and divided over
12x12 ml centrifuge tubes (Greiner, TC quality). In each tube,
first 1 ml CPW15S (Krens et al., 1990) and then 0.5ml 9%
(w/v) mannitol containing 1 mM CaCl 2 (9 M) were carefully
layered on top of the protoplast suspension. After centrifugation
at 55 x g for 10 min (MSE Centaur 2, Fisons UK, swing-out
rotor), the viable guard cells had collected in the 9 M layer. To
concentrate the protoplasts, these layers were pooled and mixed
with Percoll 15S to give a final volume of 16 ml. This was then
divided between two centrifuge tubes, layered as above and
recentrifuged. Careful removal of the 9 M layers yielded the
enriched guard cell fraction for subsequent counting using a
haemocytometer.
Guard cell protoplast culture
Enriched guard cell protoplast populations were embedded in
calcium alginate and cultured in modified K8P medium as
previously described (Hall et al., 1993). After 18 d, the alginate
discs were cut into 3 mm wide strips for transfer to solid PGo
medium (de Greef and Jacobs, 1979) containing 1 fiM BAP
(PG1B). Two weeks later, individual calli were transferred to
the same medium and after a further 2 weeks these calli were
again subcultured and transferred to the light (3000 lx, Philips
TLD fluorescent, 16/8 h light/dark photoperiod) at 25 °C.
Regeneration occurred within the next 3 x 2 week subculture
periods.
Plant material
Three sugar beet breeding lines (populations), Bv-NF (Krens
et al., 1990), SES 1 and SES 3. were used. Shoot cultures were
initiated to provide a reusable and uniform source of starting
material and were maintained with a 4-weekly subculture period
as described previously (Hall et al.. 1993).
Large-scale isolation of sugar beet epidermis
A modified version of the blender method described by Kruse
et al. (1989) was used. For each isolation, 2 g leaves (with the
midribs removed) from 4-week-old shoots was blended in a
Waring blender (model 32BL79. Wareing, Connecticut, USA)
at maximum speed (23 000 rpm) for 60 s in a 250 ml metal
Plant establishment
Embryos emerging from totipotent calli were first transferred
on to PG1B medium until they were of sufficient size ( ± 5 mm)
to be grown individually in pots. Rooting was induced in halfstrength Murashige and Skoog medium (Murashige and Skoog,
1962) supplemented with 3% (w/v) sucrose. 0.8% (w/v) agar
(Daichin. Brunschwig. Amsterdam, NL) and 25 ^M IBA, for
later transfer to soil.
Transformation experiments
Particle bombardment: Plant tissue was bombarded using a
PDS-1000 helium gun (Bio-Rad Corporation, Richmond. CA,
Sugar beet guard cell protoplasts
USA), employing 1550 psi rupture discs and with a distance of
9 cm between the plant material and the stopper plate. Gold
particles (1 /urn) were coated with pPG5 plasmid DNA (5/^1
washed particles: 5 fx.g DNA) according to the manufacturer's
protocol. pPG5 is a pUC18-based construct containing the pat
gene fused to the CAMV35S promotor and terminator and the
gus gene fused to an enhanced CAMV35S promotor and the
CAMV35S terminator. Sugar beet epidermis fragments were
produced using the blender method and were placed in a single
layer on PG1B medium solidified with 0.8% (w/v) agar. As a
control, whole leaf segments ( 5 x 5 mm) were taken from in
v/7ro-grown tobacco plants and placed with their abaxial side
uppermost in groups of 9 in the centre of a 9 cm Petri dish.
After bombardment, the plant tissue was transferred to fresh
dishes and cultured for 2 d prior to histochemical staining for
GUS activity. Observations on the epidermis fragments could
be made without the need for a decoloration step. For the
tobacco tissue, chlorophyll was removed using several washings
in 96% ethanol before examination.
PEG-mediated transformation of protoplasts: The protocol
detailed by Krens et al. (1982) was applied using 5 x 105 cells
suspended in 1 ml 9 M medium. DNA (50 ^g) was added and
immediately followed by 0.5 ml 40% (w/v) PEG medium. After
30 min incubation at room temperature, the suspension was
sequentially diluted at 5 min intervals, 4 times, with 2 ml F
medium. The cells were then recovered by centrifugation for
subsequent culture as described above.
Histochemical staining: Cells and tissues were stained for GUS
activity by incubation in substrate buffer, overnight, at 37 °C.
This buffer contained 50 mM TRIS-HC1, 125 mM CaCl 2 .2H 2 O,
400 mM
mannitol,
0.5 mM
K 3 (Fe(CN) 6 ),
0.5 mM
K 4 (Fe(CN) 6 ), 0.3% (v/v) Triton-X-100, and 0.75 m g m P 1
X-Gluc substrate at pH 7.0. Protoplasts could be stained while
still embedded in the calcium alginate without any loss in
detection of activity.
Results
Large-scale isolation of sugar beet epidermis
The Waring blender method proved to be very appropriate for obtaining substantial amounts of epidermal
tissue from sugar beet leaves. The filtered fraction consisted of epidermal fragments, essentially detached from
all mesophyll tissue (Fig. 1A), in association with fragments of vascular bundles. Typically, after optimization
of the protocol, the epidermis fraction from 2 g leaf
material, after centrifugation, constituted a pellet of
2.5-3.5 ml Packed Cell Volume.
Isolation of guard cell protoplasts and optimization of
recovery
Several cellulolytic and pectolytic enzymes (e.g. Cellulase
RS, Cellulase R10, Cellulase TC, Macerozyme R10,
Pectinase, Pectolyase Y23, Driselase), were tested in a
wide range of combinations and concentrations to determine the optimum mix for maximum yield of guard cell
protoplasts. For practical reasons, a mixture was sought
which was appropriate for overnight digestion.
257
A high pectinase concentration, in combination with a
lower cellulase concentration, was necessary for optimum
guard cell protoplast release. While 3% Macerozyme was
generally sufficient, double this concentration was, however, found to be necessary with some enzyme batches.
Other pectinases proved to be toxic, even at very low
concentrations. It was critical to design an enzyme cocktail which did not simultaneously enable the release of
viable cambial protoplasts from the contaminating vascular fragments. The mixture chosen fulfils this criterion in
producing large populations of guard cells essentially
devoid of cambial protoplasts (Fig. IB). The amount of
epidermis per isolation proved to be of critical importance
in determining successful protoplast release with the
optimum being equivalent to 200 mg leaf and 5 ml digestion medium per 6 cm Petri dish.
Guard cell protoplasts, in contrast to mesophyll protoplasts, were found to have a lower buoyant density than
most protoplast media and were usually observed to float
even in the digestion mixture. Accordingly, difficulties
were initially encountered concerning protoplast recovery.
Adding sucrose to the media before centrifugation
increased yields, but the use of Percoll in addition to
sucrose enabled maximum recovery for all three genotypes
and doubled colony production (results not presented).
Other manipulations, such as preincubating the source
plants in darkness for 24 h prior to harvesting, substituting sorbitol for mannitol in the protoplast media and
increasing the osmolality of the digestion/washing solutions from +700 to +900 mOsm kg" 1 were all found to
have no influence on protoplast yield.
After combining all the improvements concerning protoplast release and recovery into the single, optimized
protocol detailed in the Materials and methods, high
protoplast yields were routinely obtained, irrespective of
genotype and with a high frequency (70-90%) of guard
cell protoplasts (Table 1). The remaining protoplasts are
derived either from epidermis or mesophyll tissue.
Protoplast culture
Calcium alginate: As was found for leaf protoplasts,
enriched populations of guard cell protoplasts budded
extensively and divided only sporadically in liquid culture
medium (Fig. 1C). However, immobilization in calcium
alginate stabilized the protoplasts, prevented budding
almost entirely (Fig. ID) and supported extensive cell
division to produce large numbers of friable, guard cellderived colonies (Fig. IE). Filter-sterilized sodium alginate supported a 3-fold higher plating efficiency than
autoclaved alginate (results not presented).
n-Propyl gallate (nPG): This antioxidant was routinely
added to all protoplast isolation and culture media. A
pilot experiment revealed that cell division was greatly
• h"
•
*
-*§:•
s
^k,
Sugar beet guard cell protoplasts
Table 1. Average vields (+SD) of sugar beet guard cell (GC)
protoplasts after optimization of the isolation and purification
protocols
Results are presented in relation to the fresh weight of leaf material
used, after removal of the midribs. « = number of individual experiments.
Genotype
n
Total protoplast
(yield g" 1 )
GC protoplasts Total yield (GC
(%)
protoplasts g" 1 )
NF
SES 1
SES3
15
15
4
1.1 ±0.23 x 106
1.1 ±0.21 x 106
1.2±0.24xl0 6
80
77
73
8.7±1.9xl05
8 5 ± 1 . 4 x 105
8.8 + 3.0x 105
reduced in culture medium lacking nPG and concentrations ^0.5 mM proved toxic (not presented). Further
tests indicated that, for both SES 3 and N F genotypes,
the highest frequencies of guard cell division were
obtained when using 0.25 mM nPG. Under optimum
conditions a division frequency of 51% was obtained for
NF guard cell protoplasts and 29% for those of SES 3.
For SES 1, 0.1 mM nPG was optimal giving maximum
plating efficiencies of 23%. In these cultures, despite the
high in vitro cytokinetic response, no cells other than
guard cell protoplasts were observed to divide. However,
under suboptimal conditions, a few compact calli may
arise from contaminating cambial protoplasts.
The overall improvement in in vitro response, resultant
from the switch to isolating protoplasts from epidermis
fractions instead of whole leaves is clearly illustrated in
Fig. IF and Table 2. The introduction of the additional
purification steps resulted in an approximately 200-fold
enhancement in cytokinetic response for all three
genotypes.
259
The influence of bovine serum albumin: The inclusion of
filter-sterilized BSA in the enzyme mix was found to
enhance overall protoplast yields. Preliminary experiments revealed that a supplement of 1% BSA was optimal
and this concentration was used in subsequent experiments. However, although BSA could enhance protoplast
yields up to 2-fold, for each genotype division of guard
cell protoplasts after plating was halved (results not
presented). Direct inclusion of BSA in the culture medium
prevented all growth of sugar beet guard cells. Therefore,
BSA was subsequently omitted from all media.
Regeneration response: Regeneration in these cultures
occurred relatively rapidly after individual calli were
transferred to the light (Fig. 1G). Regenerants were
entirely derived from somatic embryos and, after removing the radicle, shoots could routinely be grown on for
in vitro multiplication or for rooting and transfer to soil.
Regeneration responses are still suboptimal (Table 3) and
work is in progress both to improve these frequencies
Table 3. Average regeneration frequencies of calli derived from
guard cell protoplasts isolated from sugar beet epidermis
For each experiment, 100 or 200 calli were tested. Regeneration
frequency is estimated as the percentage of plated calli which produced
at least one embryo.
Regeneration frequency
Genotype
NF
SES 1
SES 3
10
3
1
Table 2. The influence of preisolation of epidermis prior to isolating protoplasts from sugar beet leaf tissue on subsequent plating
efficiencies (PE)
Results are the means of several (n) experiments.
Genotype
Whole leaf protoplast isolates
(% PE)
Epidermis protoplast isolates
(% PE)
Improvement factor
NF
SES 1
SES 3
0.10 (n = 5)
0 07 (« = 4)
0.09 (n = 3)
21.65 («=13)
13.80 (n = 3)
15.54 (« = 7)
217x
197 x
172 x
Fig. 1. (A) Detail of a fragment of sugar beet (Beta vulgaris) leaf epidermis obtained via the Waring blender method (Bar = 50 ^m). (B) An enriched
population of guard cell protoplasts obtained from a blended epidermis isolate, (Bar = 20 ^m). (C) Abnormal development of a guard cell protoplast
after 7 d culture in liquid medium. Extensive budding is evident and division is sporadic (Bar = 20fitn). (D) Early stages of cell division of a guard
cell protoplast after stabilization in calcium alginate and culture for 7 d in the same liquid medium as used in (C). No budding occurs, cell wall
formation is more pronounced and cross walls are clearly evident (arrowed) (Bar = 20^m). (E) Detail of the extensive and uniform colony
formation in a fragment of alginate-embedded guard cell protoplast culture 16 d after plating (Bar = 25Ofim). (F) Cultures of sugar beet leaf
protoplasts (right) and purified guard cell protoplasts (left) after 32 d in culture. A small number of friable calli (approximately 15 per dish) are
present in the leaf culture in comparison to approximately 12 000 in the guard cell culture. (Bar = 500/*m). (G) Regeneration of sugar beet plantlets
from guard cell protoplast-derived callus via somatic embryogenesis. (Bar = 100 ^m). (H) High-level expression of the gus gene in a stomatal guard
cell in an epidermis fragment 2 d after bombardment using pPG5-coated gold particles (P = visible particles) (Bar = 25^m). (I) Histochemical
staining for gus activity in the abaxial surface of tobacco leaf tisue following bombardment as in (H). The majority of the intensely-stained spots
consisted primarily of a central highly-expressing guard cell (arrowed). (J) Guard cell protoplasts exhibiting transient expression of the gus gene 2
d after PEG-mediated transformation using the construct pPG5 (Bar = 20fim).
260
Hall et al.
and to enhance the number of plantlets derived from each
totipotent callus.
Transformation experiments
Particle bombardment: Before embarking on a detailed
investigation into the possible use of guard cell protoplasts
for sugar beet transformation, an experiment was
designed to determine whether the construct chosen for
use was appropriate for sugar beet and, specifically, to
confirm that sugar beet guard cells are in a physiological
state conducive to transgene expression. Epidermis fragments were bombarded with plasmid-coated gold particles
and stained for GUS expression 2 d later. In parallel, a
control bombardment of tobacco leaf segments was performed. Histochemical staining revealed intense coloration of individual beet guard cells (1-3 per epidermis
fragment) in the apparent absence of obvious staining of
any of the surrounding epidermal cells (Fig. 1H). This
high propensity of guard cells to express the GUS gene
was further emphasized when, upon close examination of
the blue spots in bombarded tobacco control tissue, it
was revealed that these also consisted primarily of highlyexpressing guard cells (Fig. II). Staining in non guard
cell-containing spots was notably less intense.
PEG - protoplast transformation: To determine whether
sugar beet guard cell protopasts might be an appropriate
starting point for guard cell-associated transient expression studies and also for the development of a transformation system for sugar beet, a standard, published
technique for PEG-mediated DNA uptake was applied
to determine if these rather fragile cells can withstand the
rigours of such a protocol. Results clearly indicate that,
following this procedure and before any optimization,
very satisfactory levels of transient transgene expression
can be obtained (Fig. U ) . Plasmid concentration is
strongly influential to the success of the treatment and
cannot be compensated for by the inclusion of carrier
DNA (Table 4). In this and other experiments, maximum
transient expression of the GUS gene occurred 2 d after
transformation. At this time > 2 % of cells were GUS +
with 83% of these being guard cell protoplasts.
Discussion
By designing a protocol specifically around stomatal
guard cells, recalcitrance in sugar beet to protoplast-based
technology has been overcome. In addition, the results
detailed here represent the successful development of a
single system which can also function both as an invaluable tool for carrying out fundamental research into the
processes of stomatal physiology and plant cell
(de)differentiation and, additionally, as an ideal starting
point in the development of a genetic manipulation protocol for what is presently one of the most recalcitrant
crop species.
It has proven possible to isolate sugar beet guard cell
protoplasts with high yields and at high degrees of purity
with remarkable ease. Under optimum conditions these
protoplasts can be induced to undergo division at frequencies of > 50% and ultimately can also proceed to regenerate plants. These results compare favourably with those
reported by Tallman for Nicotiana glauca which, presently, is the only other species for which guard cell
totipotency has been successfully demonstrated (Sahgal
et al., 1994). The previous report that protoplast division
in this system was specifically linked to a single tissue in
the source material (Hall et al., 1995) paved the way for
this directed approach to overcoming the limited response
of these cultures. Exploiting this knowledge has enabled
improvements to be introduced into the existing protocol
in a defined manner to instigate an overall enhancement
in culture efficiency. In so doing it has been possible to
avoid the usual empiricism frequently associated with this
type of research.
Division frequency in these cultures increased in parallel
with the proportion of guard cells present. This, combined
with a total failure to demonstrate division in virtually
every other cell type (Hall et al., 1995), provides the
definitive confirmation that these cells are the sole progenitors of totipotent callus in this system. This is doubly
remarkable considering that decades of research into
guard cell division, which began in the 1920s (Thielman,
1925), proved fruitless (Dehnel, 1960) while division of
epidermis or mesophyll cells is commonplace (Pillai et al.,
1991, Park and Wernicke, 1993; D'Ultra-Vaz et al., 1993;
Creemers-Molenaar et al., 1994). The obvious question
arising from these observations is 'why guard cells?'.
Table 4. Transient expression of the GUS gene in sugar beet cells after transforming protoplasts with the plasmid pPG5
Histochemical staining was performed 1. 2 or 3 d after transformation. Protoplasts were plated in the standard way with 62 500 cells/dish, of which
65% were guard cell protoplasts. Calf thymus DNA was used as carrier Results are the means of three repicates. n.a. = result not available.
Genotype
NF
^g DNA/5 x 105 protoplasts
Blue-stained GUS + cells (guard cells iin parentheses)/dish
pPG5
Carrier DNA
di
d2
d3
10
50
40
0
11 (5)
n.a.
108(86)
1267 (1045)
4(2)
298(276)
Sugar beet guard cell protoplasts
The culture conditions used are, in general terms, quite
standard and ought to be suitable for most species, tissues
and cell types (Wei and Xu, 1990; Bhadra et al., 1994).
However, in developing a culture protocol for a recalcitrant system, it may be that only a highly-specialized cell
type can ultimately respond. In this sugar beet system,
the cause underlying the general non-responsive nature
of leaf protoplasts may be so dominant that cells with
unique properties are required before the barriers determining in vitro behaviour are overcome. There are a
number of specific features of stomatal guard cells which
may enable their survival under conditions which remain
inappropriate for that of their neighbours. Guard cells
lack plasmodesmata and thus have, in vivo, a more
isolated existence than most plant cells. They are also the
only cells in the leaf which are accustomed to regular
fluctuations in osmotic potential—this in accordance with
the key role which osmotic pressure plays in stomatal
function (Willmer, 1993, Hedrich et al., 1994). As such,
guard cells may, inherently, be better equipped to deal
with the inevitable osmotic shocks associated with protoplast isolation and purification. However, this cannot be
the entire explanation as in mixed protoplast populations,
cells derived from, for example, mesophyll or the epidermis, while not dividing, also do not die, but remain
viable for considerable periods (Hall et al., 1995).
Nevertheless, it may be that during preparation, their
physiology is disturbed to such an extent that they are
subsequently unable to respond effectively to the culture
environment. Furthermore, cell division in cambial protoplasts has been observed in this system but, despite
intensive efforts over a number of years, it has never
proven possible to achieve even an indication of a regenerative response from the calli obtained (Pedersen et al.,
1993; Schlangstedt et al., 1994; Hall et al., 1995).
It is perhaps more likely that it is the typical physiological state of guard cells which makes them most amenable
to expressing their full potential for cell division and
totipotency. Despite their specialized function (Sack,
1987; Mansfield et al., 1990), guard cells are particularly
versatile in their responsiveness to both environmental
factors and signals from within the plant (Assmann, 1993;
Mansfield, personal communication). This must entail a
dependance on many more genes remaining active than
in most (or all) other cells in the leaf. As such, guard
cells may paradoxically, be well suited to re-expressing
full genetic potential. It would, therefore, be fruitful to
investigate this point using other recalcitrant and even
non-recalcitrant systems to determine whether guard cells
remain responsive in these cultures also. In this regard,
computer-assisted microscopy, as described previously
(Hall et al., 1995), could provide the ideal approach for
such work as it would entail no extra requirement for
guard cell isolation protocols prior to experimentation.
The one distinguishing feature of the culture protocol
261
for sugar beet protoplasts is the inclusion of the antioxidant nPG in all media. This was previously found to be
essential for supporting cell division in these cultures
(Krens et al., 1990; Hall et al., 1994). nPG inhibits
lipoxygenase (Peterman and Siedow, 1983) which,
through fatty acid oxidation, initiates free radical chain
reactions in biological systems (Vliegenthart and Veldink,
1982). Resultant (differential) membrane damage could
subsequently determine cellular response through altered
uptake properties or the loss of critical (hormone) receptors. However, and perhaps more importantly, free radicals in plants lead to the formation of ethylene
(Kacperska and Kubacka-Zebalska, 1989; Ievinsh, 1992)
which has known wide-ranging, physiological effects on
plant cells (Abeles et al., 1992). It has recently been
demonstrated that nPG significantly inhibits the release
of ethylene in sugar beet leaf protoplast systems (Krens
et al., 1994). Guard cells are the first line of defence in
plants regarding gaseous exchange. It is therefore feasible
to suggest that they may have a greater ethylene tolerance.
As such, this cell type may again be better equipped to
deal with what might be an unusually hostile environment.
The idea of an influential role for ethylene in guard cell
development is not unprecedented. Recently, Roberts
et al. (1995) proposed a key role for ethylene in determining the eventual cellular response of their tobacco guard
cell protoplast system.
It would be valuable to determine if the level of ethylene
produced or the degree of sensitivity of sugar beet cells
to it, is significantly different in this system in comparison
to other recalcitrant and non-recalcitrant ones.
Furthermore, now that a sugar beet guard cell protoplast
isolation protocol is available, direct comparisons between
mixed leaf protoplast populations (recalcitrant) and purified guard cell protoplasts (non-recalcitrant) can easily be
made. Perhaps therefore, the more important question
which should be addressed is not 'why guard cells?', but
rather 'why not the expected cell types?'. It is quite
surprising that while guard cells can be induced to divide
at frequencies of up to 60% (Hall et al., 1995), virtually
every other cell fails to divide. The central cause must be
the inability of such cells to re-express or switch off
critical genes. The factors influential in this process are
likely to differ in different systems. Investigating a potential role for ethylene in determining the ultimate behaviour
of cells in in vitro systems might shed some additional
light on this problem.
Guard cells in plants, despite their functional specialization, are clearly not irreversibly differentiated. Their
uniqueness of form concerns not only morphological
features such as cell wall structure and cytoplasmic organization, but also a wide range of physiological and
metabolic processes (Zeiger, 1983; Sack, 1987). As such,
a guard cell culture system lends itself to potential applications concerning gene expression, guard cell function and
262
Hall et al.
the general phenomenon of (de) differentiation in plant
cells. The frequent observation of stomatal failure in in
vitro-grown plants, which can cause extensive complications in the micropropagation industry (Kozai, 1991),
could also be effectively investigated using this system.
However, potentially the most fruitful application of a
guard cell culture protocol is its use for studies on basic
guard cell physiology. In this regard, a cautionary note
is warranted concerning the observed effects of BSA as
reported above. While BSA has generally become a
standard ingredient in media used for isolating guard cell
protoplasts destined for physiological studies (Weyers
and Meidner, 1990), results presented here clearly indicate
that despite the significant advantageous effect on protoplast recovery, BSA has a distinct detrimental effect on
subsequent cell development. Consequently, the advisability of employing this compound in such studies is brought
into serious question.
The ability of guard cells actively to express alien genes
has previously been shown in transgenic Arctbidopsis
plants using the GUS reporter gene driven by putative
guard cell specific promoters (Nakamura et al., 1995;
Taylor et al., 1995). The results detailed here support
these findings and indicate that a guard cell protoplast
system could be exploited in transient expression studies
concerning gene expression associated with guard cell
physiology and metabolism. The high metabolic activity
and great uniformity of such a system augur well for the
development of such a procedure. Sugar beet breeders
are still in search of an efficient genetic manipulation
protocol for this difficult crop (Steen and Pedersen, 1993).
On the basis of the results of the preliminary investigation
described here, a commercial application for this stomatal
guard cell system has also become feasible (Hall et al.,
1996).
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