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Starch Gel Electrophoresis of Ferns: A Compilation of Grinding Buffers, Gel and Electrode
Buffers, and Staining Schedules
Author(s): Douglas E. Soltis, Christopher H. Haufler, David C. Darrow and Gerald J. Gastony
Source:
American Fern Journal,
Vol. 73, No. 1 (Jan. - Mar., 1983), pp. 9-27
Published by: American Fern Society
Stable URL: http://www.jstor.org/stable/1546611
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AMERICAN FERN JOURNAL:
VOLUME
73 NUMBER
1 (1983)
Starch Gel Electrophoresis of Ferns:
A Compilation of Grinding Buffers,
Gel and Electrode Buffers, and Staining Schedules
DOUGLAS E. SOLTIS*, CHRISTOPHER H. HAUFLER**,
DAVID C. DARROW***, and GERALD J. GASTONY***
The homosporous pteridophytes have been largely uninvestigated by electrophore-
sis, despite the fact that they offer many exciting research possibilities (Soltis et al.,
1980). The paucity of electrophoretic studies of ferns and fern allies may be due in
large part to the high concentrations of condensed tannins that many species contain
(Cooper-Driver, 1976 and pers. comm.). These compounds render enzymes inactive
by binding with them following cellular disruption, thereby frustrating researchers
who have attempted electrophoretic analysis utilizing standard methods of sample
preparation.
The method of sample preparation developed by Kelley and Adams (1977a, b) in
their analysis of enzyme variation in Juniperus was an important procedural
breakthrough in overcoming the difficulties that result from the liberation of large
amounts of phenolic compounds during tissue preparation. Recently, a simplified
version of that method was applied by Soltis et al. (1980) to fern leaf tissue,
facilitating rapid preparation
of active enzyme samples and thereby making electro-
phoretic analyses of large numbers of individuals more feasible.
In an attempt to improve methods of analysis of fern enzymes in starch gel
electrophoresis, we have experimented with modifications of the method of sample
preparation
outlined by Soltis et al. (1980). We also have examined several different
methods of sample preparation such as those of Gottlieb (1981a), Mitton et al.
(1979), and Werth et al. (1982), and have evaluated the relative merits of each with
fern tissue. Finally, during the course of our electrophoretic investigations of ferns
we found that standard gel and electrode buffers and staining schedules, such as
those of Brewer (1970) and Shaw and Prasad (1970), often provided unsatisfactory
results when applied to ferns. We have determined gel and electrode buffers, as well
as staining schedules, that provide clear starch gel enzyme banding for 22 enzyme
systems in ferns. Requests for advice resulting from the recent surge of interest in
fern enzyme electrophoresis have prompted
us to compile our procedural
data so that
other researchers can take advantage
of our experimentation. We hope that these data
will stimulate more extensive electrophoretic investigation
of pteridophytes
and other
electrophoretically difficult taxa.
Gottlieb (1981b) recently reviewed aspects of enzyme electrophoresis primarily in
gymnosperms and angiosperms. His discussion is equally relevant to understanding
the potential applications and limitations of electrophoretic evidence in pterido-
phytes. Since homosporous pteridophytes have high chromosome numbers, it is
tempting to invoke polyploidy in interpreting
their enzyme band patterns. It is well
*Department of Biology, University of North Carolina, Greensboro, NC 27412.
**Department of Botany, University of Kansas, Lawrence, KS 66045.
***Department of Biology, Indiana University, Bloomington, IN 47405.
9
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TABLE 1. ELECTRODE AND GEL BUFFER RECIPES USED SUCCESSFULLY IN ELECTROPHORETIC ANALYSIS OF FERNS (Gram
amounts are given for one liter final volume of buffer except where noted).
Electrode Buffer
1. 0.400 M Citric Acid, trisodium salt;
1-17.64 g Citric Acid, trisodium salt
dihydrate, 1.0 M HCI to pH 7.0
2. 0.135 M Tris, 0.032 M Citric Acid;
16.35 g Trisa, 6.10 g Citric Acidb
3. 0.135 M Tris, 0.017 M Citric Acid;
16.35 g Trisa, 3.35 g Citric Acidb
4. 0.223 M Tris, 0.086 M Citric Acid;
27.00 g Trisa, 16.52 g Citric Acidb,
NaOH to pH 7.5
5. 0.223 M Tris, 0.069 M Citric Acid;
27.00 g Trisa, 13.33 g Citric Acidb
6. 0.100 M NaOH, 0.300 M Boric Acid;
4.00 g NaOH, 18.55 g Boric Acidb
7. 0.038 M LiOH, 0.188 M Boric Acid;
1.60 g LiOH-H20, 11.60 g Boric Acidb
Adjust to pH 8.3 with dry components
pH
(a 22?C
7.0
8.0
8.5
7.5
7.2
8.6
8.3
Gel Buffer
0.020 M Histidine-HCl; 4.19 g L-Histidine-HCl
monohydrate, 1.0 M NaOH to pH 7.0
0.009 M Tris, 0.002 M Citric Acid;
dilute 67 ml of electrode buffer to I liter
0.009 M Tris, 0.001 M Citric Acid;
dilute 67 ml of electrode buffer to 1 liter
0.008 M Tris, 0.003 M Citric Acid;
dilute 35 ml of electrode buffer to I liter
0.008 M Tris, 0.002 M Citric Acid;
dilute 35 ml of electrode buffer to I liter
0.015 M Tris, 0.004 M Citric Acid;
1.84 g Trisa, 0.69 g Citric Acidb
0.045 M Tris, 0.007 M Citric Acid,
0.004 M LiOH, 0.019 M Boric Acid;
Tris-citrate buffer (5.45 g Trisa,
1.28 g Citric Acidb, bring volume to
900 ml), add 100 ml electrode buffer
to give 9:1 ratio, 1.0 M NaOH to pH 8.3
pH
(a 22?C
7.0
8.0C
8.5c
7.5'
m
7.2' g
m
7.8 o
2C
.
8.3 ;
r-
ci
m
Oo
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8. 0.039 M LiOH, 0.263 M Boric Acid;
1.64 g LiOH-H20, 16.23 g Boric Acidb
9. 0.065 M L-Histidine free base, ca.
0.015 to 0.016 M Citric Acid;
10.09 g L-Histidine free base, Citric Acidb to
pH 5.7 (= ca. 2.9 to 3.1 g)
10. 0.180 M Tris, 0.004 M EDTA, 0.100 M
Boric Acid; 21.80 g Trisa, 1.52 g
EDTA tetrasodium salt dihydrate,
6.18 g Boric Acidb, Boric Acid to
pH 8.6
11. 0.400 M Citric Acid, trisodium salt;
117.64 g Citric Acid, trisodium salt
dihydrate, 1.0 M HC1 to pH 7.0
8.0
5.7
8.6
7.0
0.042 M Tris, 0.007 M Citric Acid;
0.004 M LiOH, 0.025 M Boric acid;
5.04 g Trisa, 1.25 g Citric Acidb,
0.16 g LiOHH2O, 1.56 g Boric Acidb,
1.0 M HC1 to pH 7.6
0.009 M L-Histidine, 0.002 M Citric Acid;
dilute 140 ml of electrode buffer to 1 liter
0.045 M Tris, 0.001 M EDTA, 0.025 M Boric Acid;
dilute 250 ml of electrode buffer to 1 liter
0.005 M Histidine-HCl; 1.05 g L-Histidine-HCl
monohydrate, 1.0 M NaOH to pH 7.0
aTrizma-base (Sigma T1503; Sigma T1378 works as well and is much less expensive).
bAmounts of Citric Acid and Boric Acid are given in grams of anhydrous free acid per liter.
CCheck
pH of solution after mixing; pH of Tris buffers will change with dilution.
Buffer systems 1 and 11 are from Gottlieb (198 la).
Buffer systems 2 and 3 are modifications of buffer system I of Shaw and Prasad (1970).
Buffer systems 4 and 5 are modifications of buffer system XII of Shaw and Prasad (1970).
Buffer system 6 is from Mitton et al. (1977).
Buffer system 7 is a modification of the buffer system of Gottlieb (1981a).
Buffer system 8 is a modification of the buffer system of Adams and Joly (1980).
Buffer systems 9 and 10 are from Gottlieb (pers. comm.).
7.6 a
C,,
m
7.6 m
-
r-
I
m
C)
m
C)
7.0
C/,
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AMERICAN FERN
JOURNAL:
VOLUME
73 (1983)
known, however, that multiple forms of a given enzyme may be coded by different
alleles at a single locus (allozymes) or by genes at more than one locus (isozymes).
Furthermore,
many standardly assayed plant enzymes have isozymes located in two
or more subcellular compartments (e.g., the cytosolic and chloroplastic isozymes of
PGI and PGM discussed by Gottlieb, 1981b, 1982-for interpretation
of enzyme
symbols see Table 2). When found in pteridophytes, multiple, subcellularly com-
partmentalized
isozymes should not be misinterpreted
as products of duplicated loci
resulting from polyploidy.
According to Chapman et al. (1979), attempts to determine the amount of
heterozygosity per locus in homosporous pteridophytes
are complicated by recombi-
nation between homoeologous duplicated loci. An obvious prerequisite
to recombina-
tion of this kind is the actual presence of such loci. Gastony and Gottlieb (1982)
developed a means of demonstrating whether duplicated loci are, in fact, present in
pteridophytes. By electrophoretically analyzing sporophytes taken from nature and
individual gametophytes grown from spores of these sporophytes, segregational
analysis of sporophytic enzyme banding patterns can be conducted. This permits
genetic interpretation
of parental sporophytic enzyme phenotypes without the time-
consuming crossing programs required by total reliance on sporophytic tissue. Use
of this methodology enables the investigator to determine whether apparent
heterozygosity of sporophytes is coded by alleles at a single locus or by genes at
duplicated (homoeologous) loci. Our application of this methodology to several fern
genera (e.g., Athyrium, Bommeria, and Pellaea) has demonstrated that the genetic
variability observed in these taxa results from allelic diversity and segregation at
single, not duplicated, loci.
MATERIALS
Living sporophytes of Athyrium filix-femina, Bommeria ehrenbergiana,
B. hispida,
B. pedata, B. subpaleacea, Botrychium virginianum, Ceratopteris thalictroides,
Cystopteris bulbifera, C. dickieana, C. fragilis, C. laurentiana, C. protrusa, C.
reevesiana, C. tennesseensis, C. tenuis, Isoetes butleri, I. engelmannii, Lvgodium
japonicum, Nephrolepis exaltata, Ophioglossum engelmannii, Pellaea andromedi-
folia, P. atropurpurea, P. glabella, Polypodium polypodioides, P. virginianum,
Polystichum acrostichoides, Pteridium aquilinum, Woodsia obtusa, and W. oregana
were maintained in greenhouse culture and utilized in this investigation. Living
gametophytes representing the species of Athyrium,
Bommeria, Cystopteris, Pellaea
and Pteridium listed above were cultured on nutrient
agar (Gastony & Haufler, 1976)
and also provided material for this study.
GRINDING BUFFER SOLUTIONS
We routinely utilize modifications of either the phosphate grinding buffer-poly-
vinylpyrrolidone (PVP) solution employed by Mitton et al. (1979), the Tris-maleate
grinding buffer-PVP solution of Soltis et al. (1980), or the Tris-HCl grinding
buffer-PVP solution of Gottlieb (1981a) with PVP substituted for polyvinylpoly-
pyrrolidone (PVPP), as described below. Recipes for preparing
these grinding buffer
solutions are provided below; molarity or percent volume values are provided and
12
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D.
E.
SOLTIS
ET
AL.: STARCH
GEL ELECTROPHORESIS
OF FERNS
gram
or milliliter amounts
required
to prepare
25 ml of buffer
solution are given in
parentheses.
Phosphate grinding buffer-PVP solution.-0.029 M (0.28 g) sodium tetraborate,
0.017 M (0.08 g)
sodium metabisulfite, 0.20 M (1.0 g) L-ascorbic acid sodium salt, 0.016 M (0.07 g) diethyldithiocar-
bamic acid sodium salt, 4% w/v (1.0 g) PVP average molecular weight 40,000 (Sigma PVP 40T), or
36-40% w/v (9.0-10.0 g) PVP average molecular weight 10,000. Dissolve gram amounts in 25 ml of
0.10 M phosphate buffer pH 7.5 (to make 100 ml of phosphate buffer dissolve 1.36 g KH,PO4 in H,O.
add 9.0 ml 1M NaOH, and bring volume to 100 ml with H20) and then add 0.25 ml (1%)
2-mercaptoethanol.
Tris-maleate grinding buffer-PVP solution.-0.20 M (1.91 g) sodium tetraborate, 0.02 M (0.095
g) sodium metabisulfite, 0.25 M (1.24 g) L-ascorbic acid sodium salt. 0.026 M (0.113 g)
diethyldithiocarbamic acid sodium salt, 0.10 M (0.29 g) maleic acid, 0.10 M (0.30 g) Tris, 4% w/v (1.0
g) PVP average molecular weight 40,000 or 32-40% w/v (8.0-10.0 g) PVP average molecular weight
10,000. For 25 ml of buffer, dissolve amounts indicated in 19 ml distilled water; mix thoroughly and
crush out lumps; adjust to pH 7.5 with 1.0 M HCI; add 0.025 ml (0.1%) 2-mercaptoethanol;
add H2O to
25 ml. Originally (when we utilized PVP 10,000) it was necessary to allow the PVP to hydrate
overnight
before using the grinding buffer solution. When employing PVP 40,000, it is possible to prepare the
grinding buffer solution immediately prior to sample preparation
by stirring the PVP into solution.
Tris-HCI grinding buffer-PVP solution.-0.1% v/v (0.025 ml) 2-mercaptoethanol, 0.001 M
(0.010 g) EDTA (tetrasodium salt), 0.010 M (0.019 g) potassium chloride. 0.010 M (0.050 g)
magnesium chloride hexahydrate, 4 or 20% w/v (1 or 5 g) PVP 40,000, 25 ml 0.10 M Tris-HCl buffer,
pH 7.5. Stir the PVP into solution or allow it to hydrate in the buffer overnight.
TABLE 2. GEL AND ELECTRODE BUFFER SYSTEMS THAT WE HAVE FOUND TO YIELD THE
BEST BANDING IN FERNS WE HAVE ASSAYED.
Enzyme Symbol Gel and Electrode Buffer System
(from Table 1)
Acid phosphatase APH 6, 7
Aconitase ACN 1, 5, 9
Aldolase ALD 1-5, 7, 10, 11
Aspartate aminotransferase AAT (or GOT) 6, 7, 8
Catalase CAT 4, 7, 8, 10
Esterase (Colorimetric) EST 6, 7
Esterase (Fluorescent) FE 8
Fructose-
1,6-diphosphatase F1,6DP 1, 11
Glucose-6-phosphate dehydrogenase G6PDH 4, 5, 6, 7
Glutamate dehydrogenase GDH 3, 5, 7
Glyceraldehyde-3-phosphate
dehydrogenase G3PDH 1, 11
Hexokinase HK 2, 3, 5, 11
Isocitrate dehydrogenase IDH 1, 2, 3, 4, 5
Leucine aminopeptidase LAP 3, 7, 8
Malate dehydrogenase MDH 1, 4, 5, 9
Malic enzyme ME 7, 10
Peroxidase PER 2, 7
Phosphoglucoisomerase PGI 5, 6, 7
Phosphoglucomutase PGM 3, 5, 6, 7, 8
6-Phosphogluconate dehydrogenase 6-PGD 1, 2, 4, 5
Shikimate dehydrogenase SkDH 1, 2, 4, 5, 11
Triosephosphate isomerase TPI 2, 5, 6, 7, 8
13
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AMERICAN FERN
JOURNAL:
VOLUME 73 (1983)
Comparison of these three grinding buffer solutions indicates that for most
enzymes the staining results are highly comparable. For some enzymes, however,
the Tris-HCl-PVP solution seems to improve enzyme band clarity (e.g., PGI,
PGM, LAP), while for other enzymes the reverse is true. In Pellaea andromedifblia,
for example, PGI banding was sharp with the Tris-HCI-PVP solution but was
inhibited by the presence of ascorbic acid and sodium tetraborate in the
Tris-maleate-PVP buffer. In Athyrium filix-femina, however, the Tris-maleate-PVP
solution gives superior banding for PGI and one more observable EST locus when
compared to the Tris-HCl-PVP solution. The phosphate-PVP solution often result-
ed in reduced APH activity when compared to the other two grinding buffer
solutions.
In working with species of Asplenium, Werth et al. (1982) used a method of
sample preparation
that uses caffeine, but not PVP. Following this technique, equal
weights of tissue and caffeine were ground in a 0.1 M HEPES pH 7.0 buffer with
0.2% 2-mercaptoethanol
and 0.5% sodium metabisulfite to produce
a slurry.
We found
that for virtually all enzyme systems investigated, the method of Werth et al.
provided results roughly comparable to those obtained with grinding buffers contain-
ing PVP. The presence of 0.5% sodium metabisulfite in the grinding buffer appears
to be of great importance. When this ingredient is omitted from the buffer, activity
is noticeably reduced or almost totally lost for many enzymes. Significantly, the full
complement of PGI enzyme bands obtained with PVP was not expressed in most of
the fern taxa investigated when sodium metabifsulfite was omitted. These results are
in agreement with similar observations of Werth
et al. (1982).
It should be emphasized that to obtain the best results, the appropriate
amount of
PVP depends upon the taxon under investigation and in part upon the molecular
weight of the PVP employed. Soltis et al. (1980) utilized a grade of PVP with an
average molecular weight of 10,000 (although this is not stated in their report) and
incorporated
40% w/v PVP (4 g PVP in 10 ml of Tris-maleate grinding buffer). At
present, we routinely employ a grade of PVP with an average molecular weight of
40,000. Both molecular weights of PVP are suitable, but less PVP 40,000 is
required than PVP 10,000. The amount of PVP used is an important
consideration
because, as reported by Soltis et al. (1980), use of excessive amounts of PVP in the
preparation of grinding buffer-PVP solutions frequently results in a decrease or
complete loss of enzyme activity. Although PVPP is effective with a wide range of
angiosperms, its substitution for PVP failed to produce banding for PGI in Pellaea
andromedifblia whether the PVPP was hydrated in the grinding buffer or added
directly to the leaf tissue during grinding.
This discussion stresses the importance of selecting optimal grinding buffer
components and procedures for the taxon under investigation. As noted above,
certain components of these buffers may inhibit band expression, whereas in other
cases the full complement of components may be required to obtain clear banding.
We have found that LAP activity is greater if sodium ascorbate, sodium tetraborate,
and sodium metabisulfite are eliminated from the grinding buffer. On the other
hand, activity for some enzymes, (e.g., SkDH, G6PDH, MDH, CAT, 6PGD) may
be very noticeably reduced when a buffer solution incorporating only PVP is
14
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D.
E.
SOLTIS ET
AL.:
STARCH
GEL ELECTROPHORESIS
OF FERNS
utilized. A further complication in some taxa is that when a grinding buffer lacking
2-mercaptoethanol is employed, artifactual ("ghost") bands proliferate for some
enzymes and can hinder accurate interpretation
of the band patterns. Given these
examples, we strongly encourage comparative experimentation with grinding buffer
solutions when initiating electrophoretic studies.
SAMPLE PREPARATION AND ELECTROPHORESIS
Leaf samples to be analyzed electrophoretically were taken from mature sporo-
phytes. The methods discussed herein, however, are applicable to gametophytic as
well as sporophytic tissue. In our original electrophoretic analyses of ferns, leaf
samples were prepared according to the method of Soltis et al. (1980) in which
small amounts of leaf tissue are ground under liquid nitrogen with a porcelain
mortar and pestle until a fine dry powder is obtained. The powder is quickly mixed
with grinding buffer to form a thick slurry. This slurry is absorbed directly into thick
paper wicks (made from Whatmann 3 MM chromatography paper or some other
suitable wick paper).
It should be emphasized that the use of the grinding buffer-PVP solutions alone
(outlined above), without the use of liquid nitrogen in the preparation
of samples,
provide clear enzyme banding in all of the fern taxa that we have examined so far.
Therefore, at present we utilize a simplified method of sample preparation
in which
liquid nitrogen has been eliminated. In standardizing our grinding procedure by
consistently homogenizing 100 mg of leaf material in 0.5 ml of grinding buffer, we
obtained roughly equivalent staining intensity for all samples, which facilitates
comparing individuals and scoring gels. If small amounts of tissue are being ground,
it may be possible to substitute 43 mm2 plastic weigh boats for mortars and glass or
plexiglass rods for pestles. As discussed by Gastony and Gottlieb (1982), it is
possible to obtain enzyme banding from single gametophytes. Their technique
involves smearing individual gametophytes onto wicks pre-moistened with extraction
buffer-PVP solution. With all of the described grinding/extraction procedures, the
amount of grinding buffer and the type and size of wicks should be tailored to the
taxon and tissues under investigation. Once the plant material has been absorbed
into wicks, the wicks are inserted into a vertical slit in the starch gel and subjected
to horizontal electrophoresis at 4?C. Band definition may be improved
by removing
wicks from the gel after the first 10-20 minutes of electrophoresis. It is important
to
maintain electrical contact across the slit in the gel. We routinely place some sort of
spacer (e.g., strips of Whatmann paper, a plastic straw, a thin piece of plexiglass) at
the cathodal end of the gel to compress the gel slightly and to insure that the slit
does not open.
To permit comparison of banding patterns between gels, it is important to
maintain a constant starch concentration throughout a study. We have experimented
with a variety of starch concentrations throughout
the practical range of 11.5 to 15%
and have found concentrations of 12 to 13.2% most suitable, although in some
instances alternative concentrations may improve band resolution. Connaught, Fish-
er, and Sigma starch have been used and all provide clear enzyme banding, provided
appropriate gel and electrode buffer systems and staining schedules are utilized.
15
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AMERICAN FERN JOURNAL:
VOLUME
73 (1983)
In order to facilitate determining the location of the anodal front, it is useful
to insert a wick bearing the marker dye Bromphenol Blue (Sulfone Form, Sigma
B0126; 0.04% w/v in 95% ethanol). Depending on the gel and electrode buffer
system employed, as well as the taxon under investigation, the enzyme bands will
migrate varying distances behind the Bromphenol Blue marker. Optimal running
times for each enzyme system will therefore have to be determined empirically.
GEL AND ELECTRODE BUFFER RECIPES
We have found that standard gel and electrode buffer systems, such as those of
Shaw and Prasad (1970), and Brewer (1970), often yield unsatisfactory
results with
fern tissue. We have determined gel and electrode buffer systems that provide clear
banding for 22 enzymes in ferns. Recipes for the gel and electrode buffers we most
commonly employ are provided in Table 1.
Several of the gel and electrode buffer systems that we employ are modifications
of those of Shaw and Prasad (1970). It should be noted, therefore, that errors are
present in several of the recipes they provided. For example, in Shaw and Prasad
buffer system I (see page 299, Table 1 of their report), utilization of 16.35 g of Tris
yields an electrode buffer with a molarity of 0.135 (see present report, Table 1,
electrode buffers 2 and 3), rather than 0.155 as reported by Shaw and Prasad.
Similarly, use of 27.0 g of Tris in the preparation
of the electrode buffer of Shaw
and Prasad system XII yields a molarity of not 0.233 but 0.223 (see present report,
Table 1, electrode buffers 4 and 5). Furthermore,
use of the electrode buffer recipe
of Shaw and Prasad system XII yields a buffer of approximately pH 5.5, which
differs significantly from the estimate of pH 7.0 listed by Shaw and Prasad. Use of
the amounts of Tris and citric acid given for system 4 in Table I of the present report
will also yield an electrode buffer of pH 5.5; sodium hydroxide is then added to
adjust the buffer to pH 7.5.
Of the 11 gel and electrode buffer systems for which recipes are provided in Table
1, some clearly are preferable to others for certain of the 22 enzymes for which we
have obtained sharp banding. For example, best enzyme banding for MDH is
obtained when system 1, 4, 5, or 9 is used. Which system works best is dependent,
in part, on the taxon under investigation. We have found, for example, that the best
systems for Athyrium and Cystopteris differ somewhat from those that supply clear
enzyme banding for Bommeria. The systems provided here, however, should provide
an excellent framework for conducting electrophoretic analyses of most ferns. The
gel and electrode buffer systems that we have found to yield the clearest banding for
each of the 22 enzymes with which we have experimented are listed in Table 2.
STANDARD STAINING SCHEDULES
During the course of our electrophoretic investigations of ferns, we found that
many published staining schedules, such as those of Shaw and Prasad (1970),
yielded unsatisfactory results. Enzyme banding often can be improved dramatically
when these standard
recipes are modified. One very important
modification that we
employ involves increasing the pH of the buffer in staining schedules for ALD,
GDH, G6PDH, HK, IDH, MDH, ME, PGI, PGM, and 6PGD. Tris-HCl staining
buffers of pH 7.0 or 7.1 typically are used in staining for these enzymes (Shaw &
16
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D.
E.
SOLTIS ET AL.:
STARCH GEL
ELECTROPHORESIS OF FERNS
Prasad, 1970). We have found, however, that in ferns the pH optima for these
enzymes are in the 8.0-8.5 range, and we therefore use a Tris-HCl staining buffer
of pH 8.0-8.5 for them. For some enzymes, this simple modification improves
staining dramatically. For example, standard
MDH staining recipes use a buffer of
pH 7.0. Application of such a protocol to ferns, however, often results in very little
or inconsistent staining. Well stained bands are obtained consistently for MDH when
a Tris-HCl staining buffer of pH 8.0-8.5 is used (provided an appropriate
gel and
electrode buffer system is employed; see Table 2).
It also should be noted that we have consistently been unable to obtain observable
activity with fern leaf tissue for some enzymes, despite considerable experimenta-
tion. These include Alcohol dehydrogenase, Alkaline phosphatase, o-Glycerophos-
phate dehydrogenase and Lactate dehydrogenase. There are several enzymes for
which we have obtained observable activity with ferns, but have not yet obtained
clear banding, such as Diaphorase and Peptidase, and these are therefore not
included in this report. In addition, there are a number of enzyme staining schedules
that we have not yet attempted. We hope that the staining schedules provided below
will stimulate additional experimentation in this regard.
The staining schedules that we utilize for the 22 enzymes for which we obtain
clear banding are provided below in alphabetical order. The final volume for all stain
recipes is 100 ml. Depending on gel size and staining container capacity, it may be
possible to assay two gel slices simultaneously with each of the following 22
solutions. Alternatively, when assaying single gel slices, the recipes may be halved
to 50 ml final volume. Readers are encouraged to consult Enzyme Nomenclature
1978 (International Union of Biochemistry, Nomenclature Committee, 1979 Ihere-
after referred to as I.U.B., 1979]) for details of enzyme specificity and Gottlieb
(1981b) for a recent review of the electrophoretic technique and its application to
plant populations. It should be noted that enzymes such as peroxidases, esterases,
and acid phosphatases operate on a large number of substrates in vitro; therefore,
standard staining protocols for these enzymes result in the staining of various
numbers of isozymes whose homologies are not apparent. Although the utility of
these isozymes in between-species comparisons is therefore limited, their banding
patterns can be useful in assessing variability within populations.
Several different organisms serve as the source for commercial preparations of
Glucose-6-phosphate dehydrogenase. This enzyme typically is specific for NADP,
but some forms are capable of utilizing either NADP or NAD. It should be
emphasized that NADP is much more expensive than NAD, hence selecting a
Glucose-6-phosphate dehydrogenase type that can use NAD will save considerable
money. Therefore, in those recipes that call for added Glucose-6-phosphate dehydro-
genase, the notation NAD(P)* reflects this option.
Acid phosphatase (EC 3.1.3.2)
0.05 M sodium acetate buffer, pH 5.0 100 ml
1.0 M MgC12 0.5 ml
o-naphthyl acid phosphate, sodium salt 100 mg
Fast garnet GBC salt 80 mg
We have obtained results with buffer molarities ranging from 0.05 M to 0.2 M
and within a pH range of 5.0 to 6.0. Stain at room temperature;
a modification of
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JOURNAL:
VOLUME 73 (1983)
Scandalios (1969). Gottlieb (1981b) noted that plants may have a dozen or more
acid phosphatases. We consistently have observed one very intense zone of acid
phosphatase activity, with several fainter bands occasionally evident.
Aconitase (EC 4.2.1.3.; = Aconitate hydratase
in I.U.B., 1979)
1.0 M Tris-HC1
buffer, pH 8.5 10 ml
H0O 90 ml
cis-aconitic acid 70 mg
Isocitrate ( = Isocitric) dehydrogenase 7 units
1.0 M MgCI2 1 ml
NADP 10 mg
MTT 5 mg
PMS 1 mg
Stain in the dark at 30?C; a modification of Shaw and Prasad (1970).
Aldolase (EC 4.1.2.13; = Fructose-biphosphate
aldolase in I.U.B., 1979)
1.0 M Tris-HCl buffer, pH 8.5 10 ml
H,O 90 ml
Fructose-
1,6-diphosphate,
tetra(cyclohexylammonium)
salt 500 mg
or
trisodium salt 200 mg
1.0 M arsenic acid, sodium salt 1 ml
Glyceraldehyde-3-phosphate dehydrogenase 200 units
NAD 20 mg
MTT 20 mg
PMS 5 mg
Stain at room temperature;
a modification of Shaw and Prasad (1970).
Aspartate aminotransferase (EC 2.6.1.1.; = Glutamate oxaloacetate trans-
aminase)
1.0 M Tris-HCl buffer, pH 8.0 10 ml
H20 90 ml
L-aspartic acid 100 mg
cx-ketoglutaric
acid 100 mg
Adjust pH to 8.0 with 1.0 M NaOH as necessary, then add:
Pyridoxal-5'-phosphate 5 mg
Fast
blue BB salt 100 mg
Stain in the dark at room temperature; a modification of Gottlieb (1973a) and
Selander et al. (1971, appendix). Our results show that this enzyme is very sensitive
to the pH of the staining buffer; very little staining is observed at pH 7.0 or at pH
9.0. The number of loci coding for proteins that are capable of this aminotransferase
reaction is known to vary in plants. Although we typically encounter a single locus,
Gottlieb (1981b) has reported the likelihood of specific subcellular localizations for
the several dimeric isozymes.
Catalase (EC 1.11.1.6)
3% H202 5 ml
0.1 M phosphate buffer, pH 7.0 10 ml
0.06 M Na2S203-5H2O 7 ml
H20 78 ml
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19
D.
E.
SOLTIS
ET AL.:
STARCH GEL ELECTROPHORESIS OF FERNS
Incubate in this solution at room temperature for 1-2(30) minutes; pour off the
solution, rinse several times with distilled water, then add:
0.09 M KI 50 ml
H2O 50 ml
Catalase activity will appear as white bands on a dark blue background; a
modification of Shaw and Prasad (1970). For some of the buffer systems in Table 1,
it may be necessary to add several drops (approximately 2 ml) of glacial acetic acid
in order to induce staining. An alternative method is to substitute 50 ml 0.05 M
sodium acetate buffer (pH 5.0) for the 50 ml H20. The gel may turn completely blue
in a very short time. Be prepared either to photograph the gel or score it while
staining. One locus has been observed in ferns. The protein is reported to be
tetrameric (Scandalios, 1969).
Esterase (Colorimetric; EC 3.1.1.-)
a-naphthyl acetate 40 mg
B-naphthyl
acetate 40 mg
dissolved in acetone 2 ml
1.0 M phosphate buffer, pH 6.0 10 ml
H20 90 ml
Fast blue RR salt 100 mg
Stain at room temperature;
a modification of Gottlieb (1974).
Esterase (Fluorescent; EC 3.1.1.-)
4-methylumbelliferyl acetate 42 mg
dissolved
in acetone 25 ml
1.0 M sodium acetate
buffer,
pH 5.0 18 ml
H20 57 ml
Stain in the dark at room temperature and observe under long-wave ultraviolet
light; staining schedule of Mitton et al. (1979). Observe immediately after staining
(bands fade quickly). Caution should be exercised in scoring gels because flavonoid
"bands" may also be visualized under UV light. To determine whether flavonoid
"bands" are present, the gel slice should be observed under UV light prior to
staining.
Fructose-1,6-diphosphatase (EC 3.1.3.11; = Fructose-biphosphatase in I.U.B.,
1979)
1.0 M Tris-HCl buffer, pH 8.0 10 ml
H20 90 ml
Fructose-
1,6-diphosphate,
tetra(cyclohexylammonium) salt 250 mg
or
trisodium salt 100 mg
1.0 M MgC12 1 ml
Phosphoglucoisomerase 50 units
Glucose-6-phosphate dehydrogenase 50 units
NAD(P)* 10 mg
MTT 5 mg
PMS 1 mg
Stain in the dark at 30? C; a modification of Shaw and Prasad (1970).
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(1983)
Glucose-6-phosphate
dehydrogenase
(EC 1.1.1.49)
1.0 M Tris-HCl buffer, pH 8.0 or 8.5 10 ml
H,O 90 ml
Glucose-6-phosphate, disodium salt 100 mg
NADP 20 mg
MTT (or NBT) 10 mg
PMS 2 mg
Stain in the dark at 37? C; a modification of Shaw and Prasad (1970).
Glutamate
dehydrogenase
(EC 1.4.1.2)
1.0 M Tris-HCl buffer, pH 8.0 10 ml
H,O 70 ml
1.0 M L-glutamic acid, pH 8.0 (use free acid
or monosodium salt and add NaOH to pH 8.0) 20 ml
NAD 20 mg
MTT (or NBT) 10 mg
PMS 2 mg
Stain in the dark at room temperature;
a modification of Gottlieb (1973b) and of
Shaw and Prasad (1970).
Glyceraldehyde-3-phosphate
dehydrogenase
(EC 1.2.1.12; = Glyceraldehyde-
phosphate dehydrogenase
in I.U.B., 1979)
1.0 M Tris-HCl buffer, pH 8.0 10 ml
H,O 90 ml
Fructose-1,6-diphosphate, trisodium salt 100 mg
Aldolase 10 units
Incubate above mixture approx. 30 min. at 30-37? C, then add:
1.0 M arsenic acid, sodium salt 1 ml
NAD 10 mg
MTT 5 mg
PMS 1 mg
Stain in the dark at 30? C; a modification of Shaw and Prasad (1970).
Hexokinase
(EC 2.7.1.1)
1.0 M Tris-HCl buffer, pH 8.5 10 ml
H,O 90 ml
Glucose 90 mg
1.0 M MgC1, 5 ml
EDTA, tetrasodium salt, dihydrate 40 mg
NAD(P)* 10 mg
MTT (or NBT) 15 mg
Glucose-6-phosphate dehydrogenase 40 units
PMS 2 mg
ATP 25 mg
Stain in the dark at room temperature;
a modification of Shaw and Prasad (1970).
Isocitrate dehydrogenase
[NADP+] (EC 1.1.1.42)
1.0 M Tris-HCl buffer, pH 8.0 10 ml
H,O 85 ml
Isocitric acid, trisodium salt 100 mg
1.0 M MgCl, 5 ml
NADP 10 mg
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ET
AL.: STARCH GEL ELECTROPHORESIS
OF FERNS
MTT (or NBT) 15 mg
PMS 2 mg
Depending on the taxon being analyzed, results have been obtained by using
buffers ranging from pH 7.2 to pH 8.5. Stain in the dark at room temperature;
a
modification of Shaw and Prasad (1970).
Leucine aminopeptidase
(EC 3.4.11.-)
L-leucine-p-naphthylamide (free base or
acid salt) 20 mg
dissolved in dimethyl
formamide 5 ml
1.0 M phosphate
buffer,
pH 6.0 10 ml
H,O 90 ml
Black
K salt or fast black K salt 50 mg
Stain in the dark at room temperature;
a modification of Gottlieb (1973c). This
enzyme has broad activity; it can cleave a number of N-terminal amino acids.
Although commonly referred to as Leucine aminopeptidase ("LAP"), it is more
precisely referred to as Aminopeptidase. There are a number of aminopeptidases,
some of which are specific for certain N-terminal amino acids. Aminopeptidase
(cytosol; EC 3.4.11.1) is activated by heavy metals, whereas Aminopeptidase
(microsomal; EC 3.4.11.2) is not activated by heavy metals (I.U.B., 1979). It may
be necessary to modify the pH of the stain in order to increase staining intensity.
One isozyme is usually observed and a second is occasionally apparent; both are
monomeric.
Malate dehydrogenase
(EC 1.1.1.37)
1.0 M Tris-HCl
buffer,
pH 8.0 or 8.5 10 ml
2.0 M DL-malic
acid, pH 8.0 (add
NaOH to pH 8.0) 10 ml
H20 80 ml
NAD 10 mg
MTT
(or NBT) 10 mg
PMS 2 mg
Stain in the dark at room temperature;
a modification of Shaw and Prasad (1970).
Some investigators add 38 mg EDTA (tetrasodium salt, dihydrate)
to this recipe. We
use 2.0 M DL-malic acid rather than 1.0 M L-malic acid as most standard
staining
schedules require because DL-malic acid is much less expensive than purified
L-malic acid, which is the actual substrate. There are at least four malate NAD+
dehydrogenases reported (I.U.B., 1979). The one most frequently reported in
routine electrophoresis is L-Malate: NAD + oxidoreductase (EC 1.1.1.37), of which
at least two putative isozymes have been observed. The protein is dimeric; subcellu-
lar compartmentalization
of the isozymes is discussed by Gottlieb (1981b).
Malic enzyme (EC 1.1.1.40; = Malate dehydrogenase [Oxaloacetate-decar-
boxylating, NADP+] in I.U.B., 1979)
1.0 M Tris-HC1 buffer, pH 8.0 or 8.5 10 ml
H2O 80 ml
2.0 M DL-malic acid, pH 8.0 (prepare as in Malate dehydrogenase) 10 ml
1.0 M MgC12 2 ml
NADP 20 mg
MTT (or NBT) 20 mg
PMS 2 mg
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Stain in the dark at room temperature; a modification of Richmond (1972 and
pers. comm.).
Peroxidase
(EC 1.11.1.7)
3-amino-9-ethyl carbazole 65 mg
dissolved in dimethyl formamide 5 ml
0.05 M sodium acetate buffer, pH 5.0 95 ml
0.1 MCaC12 2 ml
3% H,O, 2 ml
Incubate the gel in a refrigerator
until the bands appear
(30-60 min); a modification
of Shaw and Prasad (1970) and of Gottlieb (1973b).
Phosphoglucoisomerase
(EC 5.3.1.9; = Glucosephosphate
isomerase in I.U.B.,
1979)
1.0 M Tris-HC1 buffer, pH 8.0 10 ml
H20 90 ml
1.0 M MgCl2 1 ml
Fructose-6-phosphate, disodium salt 30 mg
Glucose-6-phosphate dehydrogenase 40 units
NAD(P)* 10 mg
MTT (or NBT) 20 mg
PMS 2 mg
Stain in the dark at 37?C or at room temperature;
a modification of Shaw and
Prasad (1970). This dimeric enzyme has numerous synonyms (I.U.B., 1979)
including: Phosphohexose isomerase, Phosphohexomutase, Oxoisomerase, Hexose
phosphate isomerase, Glucose-6-phosphate isomerase, Phosphosaccharomutase,
and
Phosphohexoisomerase. Cytosolic and chloroplastic isozymes should be expected
(Gottlieb, 1981b, 1982).
Phosphoglucomutase
(EC 2.7.5.1)
1.0 M Tris-HC1 buffer, pH 8.0 or 8.5 10 ml
H,O 90 ml
1.0 M MgCl2 2 ml
Glucose-l-phosphate, disodium (Sigma G7000)
or dipotassium (Sigma G6875) salt 100 mg
1.7 x 10-4 M ca-D-glucose-l,6-diphosphate,
tetra(cyclohexylammonium) salt 5 ml
Glucose-6-phosphate dehydrogenase 40 units
NAD(P)* 10 mg
MTT (or NBT) 20 mg
PMS 2 mg
A modification of Shaw and Prasad (1970). An equally suitable alternate recipe
which eliminates the need to purchase a-D-glucose-l,6-diphosphate is:
1.0 M Tris-HC1 buffer, pH 8.0 10 ml
H20 90 ml
1.0 M MgCl2 2 ml
Glucose-l-phosphate, disodium salt
(Sigma G1259) 50 mg
Glucose-6-phosphate dehydrogenase 40 units
NAD(P)* 10 mg
MTT 10 mg
PMS 2 mg
Stain in the dark at 37?C or at room temperature.
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SOLTIS
ET AL.: STARCH GEL ELECTROPHORESIS
OF
FERNS
6-Phosphogluconate dehydrogenase (EC 1.1.1.44)
1.0 M Tris-HC1
buffer,
pH 8.0 10 ml
H20 90 ml
6-phosphogluconic
acid, barium
salt 40 mg
1.0 M MgC12 2 ml
NADP 10 mg
MTT 10 mg
PMS 2 mg
Stain in the dark at room temperature or at 37?C; a modification of Shaw and
Prasad (1970).
Shikimate dehydrogenase (EC 1.1.1.25)
1.0 M Tris-HC1
buffer,
pH 8.5 10 ml
H20 90 ml
Shikimic acid 100 mg
NADP 10 mg
MTT
(or NBT) 20 mg
PMS 2 mg
Stain in the dark at room temperature
or at 37?C.
Triosephosphate isomerase (EC 5.3.1.1)
1.0 M Tris-HCl
buffer,
pH 8.0 10 ml
H20 90 ml
Dihydroxyacetone phosphate,
lithium salt 10 mg
EDTA,
tetrasodium
salt, dihydrate 38 mg
NAD 30 mg
MTT 10 mg
PMS 2 mg
Arsenic
acid, sodium salt 460 mg
Glyceraldehyde-3-phosphate
dehydrogenase 300 units
Stain in the dark at 37?C; Gottlieb (pers. comm.). An alternative and much less
expensive method of staining for TPI follows (also see agarose recipe):
1.0 Tris-HCl buffer,
pH 8.0 10 ml
H20 90 ml
DL-a-glycerophosphate 200 mg
Pyruvic
acid, sodium salt 100 mg
a-Glycerophosphate dehydrogenase 100 units
Lactate
dehydrogenase 100 units
NAD 20 mg
Incubate the above solution for 2 hours at 30-37?C. At the end of the incubation
period, inactivate the enzymes by adjusting the solution to pH 2.0 with 1.0 M HC1
(taking care not to go below pH 2.0) and then re-adjust to pH 8.0 with 1.0 M
NaOH. Add:
1.0 M arsenic
acid, sodium salt 1 ml
Glyceraldehyde-3-phosphate dehydrogenase 100 units
NAD 20 mg
MTT 5 mg
PMS 1 mg
Stain in the dark at 30?C. The bands will be dark blue on a light blue background.
Readers who follow this protocol must first show the lack of cx-Glycerophosphate
dehydrogenase activity in their taxa. This can be done simply by staining a gel slice
with:
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1.0 M Tris-HCl buffer, pH 8.0 10 ml
H,0 90 ml
DL-a-glycerophosphate 200 mg
NAD 20 mg
MTT 5 mg
PMS I mg
AGAROSE STAINING SCHEDULES
Several of the reagents commonly required in enzyme electrophoresis, such as
NADP, are expensive. Costs may be reduced by using staining schedules employing
agarose (modified from Mitton et al., 1979 and Gaines, pers. comm.), which
require much smaller quantities of most ingredients than do standard staining
schedules but yield comparable results.
Agarose staining schedules are provided below for ALD, G6PDH, GDH, IDH,
MDH, PGI, PGM, 6-PGD, SkDH, and TPI. The following general procedure is
employed to stain one starch gel slice using the agarose technique: dissolve all
ingredients required for enzyme staining (see below) in 6 ml of Tris-HC1 buffer; in
a second flask mix 0.06 g agarose (Agarose type II, Sigma A6877) with 6 ml
distilled water (a 1% agarose solution) and bring this solution to a boil while stirring
constantly; combine the two solutions and very quickly apply to surface of one gel
slice (it may be necessary to increase ml amounts of buffer and 1% agarose
depending on the size of the gel slice); stain all slices in the dark at room
temperature.
Aldolase (EC 4.1.2.13)
PMS 1 mg
MTT 5 mg
NAD 10 mg
Fructose-1,6-diphosphate, trisodium salt
or tetra(cyclohexylammonium) salt 75 mg
Arsenic acid 25 mg
Glyceraldehyde-3-phosphate dehydrogenase 40 units
0.1 M Tris-HC1 buffer, pH 8.5 6 ml
1% agarose 6 ml
Glucose-6-phosphate dehydrogenase
(EC 1.1.1.49)
PMS 1 mg
MTT 4 mg
NADP 4 mg
EDTA, tetrasodium salt, dihydrate 6 mg
Glucose-6-phosphate, disodium salt 5 mg
0.1 M Tris-HCl buffer, pH 8.0 6 ml
1% agarose 6 ml
Glutamate
dehydrogenase
(EC 1.4.1.2)
PMS 1 mg
MTT 4 mg
NAD 3 mg
L-glutamic acid, monosodium salt 100 mg
0.1 M Tris-HCl buffer, pH 8.0 6 ml
1% agarose 6 ml
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D.
E.
SOLTIS
ET AL.:
STARCH GEL ELECTROPHORESIS
OF
FERNS
Isocitrate dehydrogenase
[NADP+] (EC 1.1.1.42)
PMS 1 mg
MTT 4 mg
NADP 4 mg
MgC 12 40 mg
Isocitric acid, trisodium salt 10 mg
0.1 M Tris-HC1 buffer, pH 8.0 6 ml
1% agarose 6 ml
Malate dehydrogenase
(EC 1.1.1.37)
PMS 2 mg
MTT 7 mg
NAD 7 mg
L-malic acid 16 mg
0.25 M Tris-HC1 buffer, pH 8.6 6 ml
1% agarose 6 ml
Keep PMS and L-malic acid powders separate until adding the buffer.
Phosphoglucoisomerase
(EC 5.3.1.9)
PMS I mg
MTT 4 mg
NAD(P)* 1 mg
0.1 M Tris-HCl buffer, pH 8.0 4 ml
10% MgC12 1 ml
0.018 M fructose-6-phosphate, disodium salt I ml
Glucose-6-phosphate dehydrogenase 10 units
1% agarose 6 ml
Phosphoglucomutase
(EC 2.7.5.1)
PMS 1 mg
MTT 5 mg
NAD(P)* 4 mg
0.1 M Tris-HCl buffer, pH 8.0 2 ml
0.05 M glucose-l-phosphate, disodium salt (Sigma G7000) 2.5 ml
Glucose-6-phosphate dehydrogenase 10 units
0.001 M fructose- ,6-diphosphate, trisodium salt 1 ml
10% MgC12 1 ml
1% agarose 6 ml
6-Phosphogluconate
dehydrogenase
(EC 1.1.1.4.4)
PMS 1 mg
MTT 4 mg
NADP 4 mg
6-phosphogluconic acid, barium salt 10 mg
10% MgC12 0.5 ml
0.1 M Tris-HCl buffer, pH 8.5 5 ml
1% agarose 6 ml
Dissolve the 6-phosphogluconic acid barium salt in 1.0 ml of 0.1 M Tris-HC1
buffer (pH 8.5) approximately 15 minutes before mixing in other ingredients.
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Shikimate dehydrogenase (EC 1.1.1.25)
PMS 1 mg
MTT 4 mg
NADP 4 mg
Shikimic acid 10 mg
0.1 M Tris-HCl buffer, pH 8.0 6 ml
1% agarose 6 ml
Triosephosphate isomerase (EC 5.3.1.1)
PMS 1 mg
MTT 4 mg
NAD 10 mg
EDTA, tetrasodium salt, dihydrate 10 mg
Arsenic acid 150 mg
Dihydroxyacetone phosphate 1.5 mg
Glyceraldehyde-3-phosphate dehydrogenase 75 units
0.1 M Tris-HCl buffer, pH 8.0 6 ml
1% agarose 6 ml
GEL FIXATION
AND DOCUMENTATION
AAT and PER gel slices are fixed in 50% glycerol; all other gel slices are fixed in
50% ethanol.
In order to keep a permanent record of results, we routinely photograph
gel slices
using Kodak Technical Pan Film 2415 following the exposure index and developing
procedure recommended for high contrast.
ACKNOWLEDGEMENTS
D.E.S. and C.H.H. thank Jeff Atwood, Harriet Blanton, Leslie Gottlieb, James
Hamrick, Terry Lastovicka, and Andrew Torres for laboratory
assistance and,helpful
comments and Charles Werth for providing details of his sample preparation
methodology. G.J.G. is grateful to Leslie Gottlieb for the use of laboratory
facilities
and advice during work with Pellaea andromedifolia. Grants-in-aid of research to
D.E.S. from the Research Council of the University of North Carolina at Greens-
boro, to C.H.H. from General Research Fund Grants 3005 and 3588 of the
University of Kansas, and to D.C.D. from the Department of Biology of Indiana
University, NSF Doctoral Dissertation Improvement
Grant DEB-8117239, and the
Society of the Sigma Xi are gratefully acknowledged.
LITERATURE
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CHAPMAN, R. H., E. J. KLEKOWSKI, Jr., and R. K. SELANDER. 1979. Homoeologous
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GASTONY, G. J. and L. D. GOTTLIEB. 1982. Evidence for genetic heterozygosity in a homosporous
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, and C. H. HAUFLER. 1976. Chromosomes and apomixis in the fern genus Bommeria
(Gymnogrammaceae). Biotropica 8:1-11.
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D.
E.
SOLTIS ET AL.:
STARCH
GEL ELECTROPHORESIS OF FERNS
GOTTLIEB, L. D. 1973a. Genetic control of Glutamate oxaloacetate transaminase isozymes in the
diploid plant Stephanomeria
exigua and its allotetraploid
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1973b. Genetic differentiation, sympatric speciation, and the origin of a diploid species of
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MITTON, J. B., Y. B. LINHART, J. L. HAMRICK, and J. S. BECKMAN. 1977. Observations on the
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, Y. B. LINHART, K. B. STURGEON, and J. L. HAMRICK. 1979. Allozyme polymorphism
detected in mature needle tissue of ponderosa pine. J. Hered. 70:86-89.
RICHMOND, R. C. 1972. Enzyme variability in the Drosophila willistoni group. II. Amounts of
variability in the superspecies, D. paulistorum. Genetics 70:87-112.
SCANDALIOS, J. G. 1969. Genetic control of multiple molecular forms of enzymes in plants: a
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SELANDER, R. K., M. H. SMITH, S. Y. YANG, W. E. JOHNSON, and J. B. GENTRY. 1971. IV.
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