Plant Cell, Tissue and Organ Culture 26: 127-133, 1991.

© 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Measurement and effects of gel matric potential and expr sibility on

pr uction of morphogenic callus by cultured sugarbeet leaf discs

Lowell D. Owens & Chris A. Wozniak ~

Plant Molecular Biology Laboratory, Agricultural Research Service, United States Department of
Agriculture, Beltsviile, MD 20705, USA (1present address: Northern Crop Science Laboratory,
United States Department of Agriculture, Agricultural Research Service, State University Station,
Fargo, ND 58105, USA)
Received 6 December 1990; accepted in revised form 8 April 1991

Key words: Beta vulgaris, gel expressibility, matric potential, morphogenic potential, regeneration
potential

Abstract

During studies to optimize production of morphogenic callus from cultured leaf discs of sugarbeet (Beta
vulgaris L.) large differences were observed associated with the gelling agent employed. Water
availability, as determined mainly by gel matric potential, was found to be the dominant factor. A
simple method was devised to measure the relative matric potential of different gels. A precisely
moistened filter-paper disc was placed on the gel surface, allowed to equilibrate, removed and weighed.
The relative gain or loss of water from the paper disc was a measure of the matric potential of the gel
and varied with both gel type and concentration. Leaf disc expansion and production of callus-derived
embryos and shoots were shown to be directly proportional to gel matric potential. Water availability
may also be affected by the ease with which liquid is expressed from gels in response to localized
pressure caused by explant expansion and contortion. This property, called gel expressibility, was easily
measured with a weight and capillary pipette and shown also to vary with gel type and concentration.
Validity of the technique for measuring relative matric potential was verified physiologically by culturing
leaf discs on filter-paper overlays to eliminate expressibility differences among gels. Additionally,
comparison of leaf disc growth on uncovered gel surfaces versus filter-paper overlays demonstrated the
contribution of liquid expression to overall water availability. Expression of liquid by explants on
uncovered gel surfaces greatly enhanced the production of morphogenic callus.

Introduction cal responses are sometimes postulated to reflect

In plant tissue culture research both the gelling
agent and gel concentration are often varied to
achieve a certain tissue culture objective. In this
manner, explant or callus growth and shoot mul-
tiplication may be increased (Romberger &
Tabor 1971; Stoltz 1971; Brown et al. 1979;
Singha 1982; Debergh 1983; Pasqualetto et al.
1986; Mackay & Kitto 1988) or vitrification re-
duced (Debergh et al. 1981; Mackay & Kitto
1988; Pasqualetto et al. 1988). These physiologi-
the availability of water in gelled medium, as
determined by the matric potential of the gel.
However, reliable measurements of the matric
potential of gels at concentrations normally used
in tissue culture have not been reported.
In analyzing the relationship between gel
properties and the growth and regeneration of
sugarbeet leaf discs, we identified two physical
properties, matric potential and expressibility,
that together appear to largely determine the
water availability of gels. Matric potential refers
128
to the tenacity with which water is held by the
solid phase of the gel and expressibility to the
ease with which water is expressed in response to
mechanical deformation of the gel by the ex-
plant. Here we describe simple techniques for
measuring the relative matric potential and ex-
pressibility of gels and demonstrate their in-
dividual contributions to explant growth and pro-
duction of morphogenic callus.
Materials and methods
Relative matric potential measurements
Gels were prepared using Bacto agar (Difco),
Gelrite (Kelco division of Merck), Phytagar
(GIBCO) and HGT and SeaPlaque agarose
(FMC) in RV medium (Freytag et al. 1988)
modified to contain 1 mg 1-1 BA (N 6-
benzyladenine as the sole growth regulator and
3% sucrose. Media were adjusted to pH 5.8,
sterilized by autoclaving at 121°C for 15 min,
distributed into Petri dishes (100 x 25 mm;
35 ml/dish) and allowed to solidify for about 21 h
at room temperature. Air-dry filter paper discs
(Whatmon no. 3; 83mm dia., 4.4% moisture)
were cut, individually weighed and precisely
moistened by adding to the gel surface (86 mm
dia.) an amount of liquid medium equal to 2.60
times the air-dry weight of the disc and placing
the paper disc in the liquid. The resulting mois-
ture level of the paper was about 90% of satura-
tion. The dish was covered, wrapped with poly-
vinyl chloride plastic film and incubated at 25°C
for about 21 h, following which the paper was
carefully removed with forceps and weighed.
Slits (1 mm) cut in opposite edges of the disc
aided in this removal. Any liquid remaining on
the gel surface was assumed to have been associ-
ated with the paper and, consequently, was col-
lected by tipping the dish 13° from horizontal
and blotting the accumulated liquid with a tared,
dry piece of paper (avoiding gel contact) and
weighed. The combined weights of liquid were
normalized to the amount of liquid initially
added to the dish and plotted as a function of gel
concentration.
Gel expressibility measurements
A 5 g weight (11 mm dia.) was gently placed on
the surface of gel media prepared as described
above and covered. The weight slowly settles
into the gel without breaking the gel surface, and
liquid is expressed from the compressed gel
under and around the weight as a function of
time. Expression occurs at a gradually decreasing
rate for more than an hour. After an arbitrarily
chosen time of 30 min, liquid in the depression
adjacent to the weight was removed with a 10/xl
tared capillary pipette or, for greater volumes,
with a micropipettor and weighed. The osmolali-
ty of liquid expressed in this manner was mea-
sured with an osmometer by freezing point de-
pression.
Plant material
A shoot culture of sugarbeet (Beta vulgaris L.)
clone REL-1 was obtained from J. Saunders,
East Lansing, Michigan. Shoots were maintained
in polystyrene Petri dishes ( 1 0 0 x 2 5 m m ; 2
shoots/dish) on MS (Murashige & Skoog 1962)
medium containing 3% sucrose and 0.25 mg 1-1
BA (Saunders & Shin 1986) and solidified with
Bacto-agar (Difco; 0.7%). Shoots were sub-
cultured at 3-week intervals. High regenerability
was maintained by explanting some adventitious-
ly shooting petioles at each subculture such that
shoots used were not more than four transfers
from initiation.
Leaf disc culture
Leaf discs (4 or 7 mm dia.) were prepared with a
cork borer from sugarbeet shoot cultures 10 to
14 d after transfer. Discs were plated with lower
(abaxial) side in contact with the RV medium
described above at a density of five 7-mm or ten
4-mm discs per dish. The dishes were wrapped
and incubated at 25°C and 45% relative humidity
under continuous (unless otherwise stated)
flourescent light (cool white, 30/xmol m -2 s-l).
Discs were transferred to fresh medium at 14 and
28 d. Leaf disc diameters were measured at 18 d,
at which time expansion was determined to be
substantially complete. At about 7 to 8 weeks

the disc-edge callus was teased apart for enume-
ration of somatic embryos and organogenic shoot
buds. Data were analyzed by a standard statisti-
cal procedure for comparing multiple means
(Dunnett 1964).
When filter paper overlays were employed, the
paper discs (Whatman No. 3; 83 mm dia.) were
weighed, washed in three changes of the RV
liquid medium described above and autoclaved
while submersed in the same liquid. Each paper
disc was lifted with a forceps, allowed to drain
vertically for 15 s, touched twice to the lip of the
dish to dislodge drops of liquid and transferred
to a tared Petri dish containing gel-solidified
medium prepared as above. The dish was re-
weighed, and liquid medium was added to adjust
the total liquid weight to 2.60 times the air-dry
weight of the paper disc.
Results
Physiological effects of gel type and
concentration
During studies to optimize the production of
regenerable callus from cultured leaf discs of
sugarbeet we observed large differences associ-
ated with the gelling agent employed (Table 1).
The concentrations of gels selected for these
experiments were those commonly found in the
literature. We noted, however, that leaf discs on
0.3% Phytagar tended to cause puddles of liquid
Table 1. Effect of gel type and concentration on production
of callus-derived embryos and shoots from cultured sugarbeet
leaf discs."
Gelling agent Total embryos
plus shoots
No. / plate b
Bacto agar (0.9%) 12 -+ 2.6
HGT agarose (0.7%) 56 -+ 4.7**
Phytagar (0.3%) 17 -+ 3.2
Gelrite (0.3%) 61 -+ 11.9"*
"Five leaf discs (7 mm dia.) were plated per dish and cul-
tured with a 16 h daily photoperiod. Data were recorded at
42 d.
b Means - S E of 21 replicates from 3 experiments.
**Significantly different from standard treatment (Bacto
agar), p = 0,01.
129
to form around the margin, in response to disc
expansion and contortion during growth, appar-
ently causing oxygen-deficiency in this instance.
Expression of liquid occured to lesser but differ-
ing extents on the other gels, as observed with a
dissecting microscope, and led us to devise a way
of measuring the physical property responsible
for this effect.
Gel expressibility measurement
Expression of liquid from gels in response to
mechanical pressure was found to be inversely
related to gel concentration (Fig. 1). The rela-
tionships were curvilinear for all gels tested,
however, in marked contrast to the agar family
of curves, the relationship for Gelrite ap-
proached linearity at concentrations normally
used for tissue culture. The expressibility curves
for Gelrite and Bacto agar closely resemble the
inverse of gel-strength curves as measured with a
penetrometer and plotted as a function of gel
concentration (from data reported by Mackay &
Kitto 1988). Obviously, gel expressibility is in-
versely related to gel strength.
Gel expressibility- physiological effects
Leaf discs were cultured on 0.3% Gelrite with
and without filter-paper overlays. Discs on filter-
5OO
Gelrife
4OO, SeaPlaque agarose
._=
E
300.
"B
GT agarose
200-
e \ . ~ Phytagar
• 1
"o
"5
g
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Gel concentration (%)
Fig 1. Relation of gel expressibility to gel type and concen-
tration. Plotted are means of two replicate measurements
with SE bars shown when larger than symbol.
130

paper overlays expanded less than on uncovered 1.4

gel surfaces and were less productive in terms of
I•I
~
. Phytagar
morphogenic callus and pooled numbers of re- 1.3-
generative structures, i.e., somatic embryos and -g Bacto agar

shoots arising from organogenesis (Table 2). The 1.2-

..... ',..', _ .05~. SeaPlaque
moistened filter-paper overlay acted as a passive
o
transmitter of liquid from the gel to the explants 1.1-
but, owing to the high rigidity of paper as com- i:'
t I i
:
f
pared to the gel, virtually eliminated expressed 1.0-
' '' ~HGT
liquid as a source of water. This allowed the gel ,x~,~ i i "~agarose
matric potential to mainly control water availa- 0.9-
Gelrite
bility and impose a water stress. In contrast, i i I

explants in direct contact with the gel were ob- 0.8 i ! i

served to compress the gel, by a combination of
expansion and contortion while adhering to the
gel, express liquid and thereby reduce water
stress. This was reflected in greater disc expan-
sion, callus growth and production of embryos
and shoots (Table 2).
Matric potential measurement
The relative matric potential of gels was mea-
sured using precisely moistened filter paper as a
standard matrix. The degree of moistening, 2.60
times the air dry weight of the paper, was select-
ed based on the amount of liquid remaining after
saturated paper discs are drained in a vertical
position for about 15 s. In Fig. 2 the relative
matric potential is expressed as the fraction of
liquid gained or lost from the filter paper relative
to the amount initially added (=1.0). Equilib-
rium between the paper and gel was reached
within a few hours, but measurements at 21 h
was chosen for convenience. At equal gel con-
centrations, Gelrite exhibited a much lower rela-
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
Gel concentration (%)
Fig. 2. Relation of relative matric potential to gel type and
concentration. Plotted are means of at least 3 replicate
measurements with SE bars shown when larger than symbols.
High relative potential values represent potential values ap-
proaching that of pure free water (conventionally assigned a
value of 0), and low values represent increasingly negative
(stressed) potentials. The dashed lines indicate the concen-
trations of different gels that display a relative matric poten-
tial equal to that of 0.70% Bacto agar; namely, 0.12%
Gelrite, 0.46% HGT agarose, 0.62% Phytagar and 1.06%
SeaPlaque agarose.
tive matric potential (less available water) than
did the agar or agarose gels, again pointing to a
fundamental difference of this gel. SeaPlaque
agarose also differed from the rest in displaying
little change in relative matric potential with gel
concentration and low heat stability (failed to gel
after autoclaving for 25 min). Attempts to use
this technique with lower gel concentrations than
those shown in Fig. 2 led to artifacts, apparently

Table 2. Effect of gel expressibility on sugarbeet leaf disc expansion and production of morphogenic callus and callus-derived
embryos and shoots?
Explant in Disc diameter Morphogenic Embryos
contact with at 18 d callus plus
(ram) dry weight shoots
(rag) (no. / plate)
Gel surface 11.0 + 0.4 124 - 10 118 +- 9

Filter-paper
overlay 9.5 -- 0.3** 28 --- 4** 40 +- 4**
a Medium was solidified with 0.3% Gelrite. Ten leaf discs (4 mm dia.) was cultured per plate. Callus and embryos plus shoot data
were taken at 48 d. Values are means of 4 replicates +-SE.
** Significantly different from control (bare gel surface), p = 0.01.
 131

caused by partial collapse of these weak gels,

even though reliable expressibility data could be
obtained. E
lo
o~

Matric potential - physiological effects

Leaf discs were cultured on filter-paper overlays

to eliminate differences in expressibility of gels
of different strengths. Leaf disc expansion and
production of callus-derived embryos and shoots pier

were inversely proportional to gel concentration []

0
(Fig. 3) and thus directly proportional to matric eger agarose sgamse
potential. Significant disc expansion was not ob- 0.70 % 0.46 % 0.62 % 0.12 % 1.06 %
mOsm 184 212 195 194 199
served on any gel concentration beyond the 18 d
scoring period. Fig. 4. Leaf disc expansion and production of callus-derived
embryos and shoots on various gels at concentrations giving
The thickness of the film of liquid at the point equal matric potentials. Ten discs (4 mm dia.) were plated
of contact between the leaf disc margin and per dish on filter-paper overlays. Graphed are means of 3
paper support was dramatically affected by gel replicates with half SE bars shown.
concentration. It was easily perceived with the a Significantly different from the standard treatment (0.7%
naked eye on 0.1% Gelrite but inperceptible on agar) at p = 0.10 only.
0.7% Gelrite.
The physiological applicability of matric general, leaf discs expanded equally and pro-
potential measurements was tested using sugar- duced similar number of embryos and shoots on
beet leaf discs. Five different gels were prepared different gels adjusted to the same relative mat-
at concentrations predicted, from the data in Fig. ric potential (Fig. 4). The one exception, appar-
2, to give equal matric potentials using 0.7% ently lower ( p = 0.10, only) embryo and shoot
Bacto agar as the standard. Filter-paper ovelays production on SeaPlaque agarose, may indicate
were used to eliminate differences in gel expres- the presence of inhibitors in this gel.
sibility among the different strength gels. In

Discussion
13. 200

~ 12. The dramatic response on cultured tissue to gel

Disc diameter
y = 13.307- I0.241x R A 2 = 0 . 9 8 8 150 6
type and concentration, as shown in this study,
"0 II. has been reported previously for a variety of
lo
~0 explants (see Introduction). These physiological
15 io. '¢1"
]00 1~ responses to gel type and concentration are due
• 9. mainly to water potential. The total water poten-
E
.go tial may be expressed as the sum of several
._~ 50 ÷ component potentials; osmotic, matric, gravita-
"O 8 tional, pressure and overburden (Papendick &
o. ,o Campbell 1980). In tissue culture studies the
E
6. 0 m matric potential is often considered to be the
0 0.I 0.2 0,3 0.4 0.5 0.6 0.7 0.8
only water potential component altered by gel
Gelrite c o n c e n l t a f l o n (%)
type and concentration.
Fig. 3. Effect of matric potential, as represented by gel We propose that in tissue culture systems there
concentration, on leaf disc expansion and production of
is another factor involved-an interaction be-
somatic embryos and shoots from disc-edge callus. Ten discs
(4 mm dia.) were plated per dish on filter-paper overlays. tween the explant and the gel-which affects
Plotted are means of 4 replicates with SE bars shown when water availability. The explant may cause water
larger than symbols. to be expressed from the gel by exerting local-
132
ized pressure through expansion or contortion
during growth. The amount of water expressed is
a function of gel expressibility, explant weight
and, most importantly, the amount of mechani-
cal pressure exerted by explant expansion and
contortion. The degree of mechanical pressure
realized from explant growth appears also to be
affected by the degree to which the explant
adheres to the gel surface. The interactive phe-
nomenon between explant and gel is related to
both pressure and overburden potentials as de-
fined by Papendick & Campbell (1980), but a
precise definition of its nature awaits further
analysis. We show in Table 2 that the contribu-
tion of liquid expression to water availability is
substantial in the case of leaf disc explants.
Air-dry filter paper has long been used as a
standard matrix to measure matric potentials in
soil (Campbell & Gee 1986). Calibration curves
have been developed to relate the water content
of paper, at equilibrium with soil moisture, to
water potential (McQueen & Miller 1968). A
modification was used in this study. Paper mois-
tened to a level somewhat below saturation was
used as the standard matrix rather than air-dry
paper. Use of air-dry paper with the range of gel
concentrations used here gave relative matric
potential values that were less accurate in pre-
dicting physiological responses. Probably, the
large volume of water taken up by air-dry paper
caused the gels to partially collapse, giving rise
to artifactual data.
It should be noted that when moisture ex-
change between the gel and paper is through
liquid phase flow, then the matric potential is
measured. However, if the exchange is entirely
in the vapor phase, as is the case with ther-
mocouple psychrometer measurements, then the
potential measured is the sum of matric and
osmotic potentials (Campbell & Gee 1986). Our
attempts to measure differences between the
combined matric and osmotic potentials of Gel-
rite and agar gels with a thermocouple psych-
rometer were unsuccessful-possibly due to the
extremely narrow range of humidities created in
the vapor chamber (Papendick & Campbell
1980) by the range of gel concentrations used in
this study, together with their intrinsic high mat-
ric potentials. From the calibration curve of
McQueen & Miller (1968) we estimate the gel
matric potentials to be in the range of a negative
few thousandths of a bar, i.e., near the potential
of free water.
Cell enlargement, the primary means by which
leaf discs and other explants increase in size, is
quite sensitive to water potential (Boyer 1985).
Nevertheless, it is surprising that such a narrow
range of matric potentials has been such a mark-
ed effect on the growth of cultured tissues. Per-
haps the rate of water transport through the gel
and, hence, across films of water at points of
explant-gel contact is controlled predominantly
by matric potential.
As documented by Debergh (1983), gelling
agents contain salts and other impurities that
decrease water availability by lowering the os-
motic potential. This is confirmed for the gelling
agents examined in this study. At gel concen-
trations giving equal matric potentials the os-
molalities of expressed liquids varied by as much
as 15% (Fig. 4). Nevertheless, this variation did
not appear to affect the physiological responses
observed (Fig. 4). These findings are further
supported by the data of MacKay & Kitto (1988)
who used washed agar in their experiments. The
physiological responses obtained with a concen-
tration series of presumably salt-free agar were
similar to those reported in Fig. 3. In both
instances the reduced water availability observed
at high gel concentrations appears to be associ-
ated primarily with gel matric potential and not
osmotic potential.
The simple techniques described here should
be of use to the tissue culturist faced with the
bewildering array of gelling agents now commer-
cially available but poorly described. For ex-
plants that cannot significantly deform gels, such
as shoot tips, meristems or embryos, matric
potential measurements alone should be suffici-
ent to predict the water availability of one gelling
agent relative to another. For explants capable
of deforming gels, such as leaf discs, gel expres-
sibility may be the more useful measurement for
predicting water availability. In gene-transfer ex-
periments, explants are often cocultured with
agrobacteria on filter-paper overlays, either with
(Horsch et al. 1988) or without (Jia et al. 1989) a
nurse culture. In these instances attention should
be addressed mainly to the matric potential of
the gel since the filter-paper overlay virtually

eliminates liquid expression and permits the gel
matric potential to determine water availability.
Acknowledgement
The authors thank Gaylon Campbell for en-
lightening discussions and gratefully acknowl-
edge the expert technical assistance of Debra
Eberts and Susan Park.
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