64 B. M.

van den Berg Electrophoresis 1991, 12, 64-69

Bartel M. van den Berg A rapid and economical method for hybrid purity testing
Royal Sluis, Enkhuizen of tomato (Lycopersiconesculentum L.) F 1 hybrids
using ultrathin-layer isoelectric focusing of alcohol
dehydrogenase variants from seeds
In the cultivated tomato (Lycopersicon esculenturn L.), two electrophoretic variants
of the enzyme alcohol dehydrogenase (ADH) encoded by the alleles Adh-I and
Adh-1' are found. A rapid and economic method for testing the hybrid purity of
tomato F 1 seeds, based on the expected presence of Adh- 1 alleles, was developed. The
method is based on the analysis of the A D H variants by ultrathin-layer isoelectric
focusing, p H range 3-10, of crude extracts from imbibed seeds, followed by enzyme
activity staining. The isoelectric points (prs) of the ADH variants were estimated to
be 5.5 and 5.7 for Adh-I+and Adh-l', respectively. Using the procedure described
and a newly developed sample applicator strip, it is possible for one person to routine-
ly analyze 1 152 seeds per day using only a single electrophoresis unit. An investiga-
tion of a large number of inbred lines, both experimental and commercial hybrids,
together with open-pollinated varieties, showed the potential of the method. Among
F 1 hybrids, a higher frequency of the Adh-1 I allele was found than among open
pollinated varieties, suggesting that F 1 hybrid breeding has resulted in a higher fre-
quency of Adh-l I alleles by selection of linked genes.

1 Introduction
Hybrid breeding has been increasingly applied for horti-
cultural crops. Hybrid varieties offer several potential ad-
vantages over open-pollinated varieties, e.g., crop uniformity,
heterotic vigor, and protection of the breeders' material by
virtue of the inbred lines. In the case of the tomato, commercial
F 1 hybrids are derived from single crosses of two parental
lines. In the production of F 1 hybrid seeds of the tomato, most
procedures can be controlled well enough to ensure the genetic
purity of the seeds produced. However, self-pollination may
occur due to errors in the process of emasculation and pollina-
tion of the female lines, which would result in the presence of
undesired inbred seeds among the F1 seeds L11.
In most cases the hybrid purity of tomato F 1 hybrids can be
tested by a detailed morphological analysis of mature plants
raised from a seed sample. However, if the parents differ in
genotype for an electrophoretic character [2,31, seed lots can
be tested for inbreds by electrophoretic analysis of single seeds
or seedling extracts. The latter method is more rapid, cheaper,
and, in cases where the female parent and theF 1 have a similar
plant morphology, more reliable 1, 21. An electrophoretic
test for hybrid purity can be carried out when the F 1 hybrid is
heterozygous for a gene encoding an electrophoretically de-
tectable protein and when both F 1 parents are homozygous
for their respective allelic variants.
Until now, only three genetic protein polymorphisms that
could be used for hybrid purity testing have been reported for
Correspondence: Dr. B. M. van den Berg, Royal Sluis, Research and De-
velopment Department, Section Plant Biochemistry, P. 0. Box 22, 1600
AA Enkhuizen, The Netherlands
Abbreviations: ADH, alcohol dehydrogenase; Bis, N,N'- methylenebis-
acrylamide; %C, crosslinking agent (g) x 100/%T; NADP, p-nicotin-
amide adenine dinucleotide; NBT, Nitro Blue Tetrazolium; PAGE, poly-
acrylamide gel electrophoresisis; pZ, isoeletric point; PMS, N-methylphen-
azonium methosulfate; SGE, starch gel electrophoresis; %T, [acrylamide
(g) + bis (g)1/100 mL; TCA, trichloroacetic acid; UTLIEF, ultrathin-layer
isoelectric focusing; Vh, Volt x hour.
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 199 1
the cultivated tomato. These are polymorphisms for the genes
encoding the enzymes acid phosphatase (4, 51 and alcohol
dehydrogenase (ADH) 16-81, and for the gene encoding the
seed protein PRS-1 Ill.
Two allelic variants of the gene Adh-1 have been demonstrat-
ed in the cultivated tomato 16, 81. Tanksley and Jones [ 8 l
reported the use of ADH analysis in inbred testing. They
showed that 9 of the 25 commercial F 1 hybrids investigated
could be tested for inbreds because both Adh-1 alleles were
present. With starch gel electrophoresis (SGE), an inbred in-
dividual can easily be detected by the absence of one of the
A D H variants due to self-pollination of the female parent. A
drawback of SGE is the low number of individuals that can
be analyzed per day by one person, whereas the analysis
of several hundred individuals per day needs several elec-
trophoresis units. The investigation presented here aimed
at the development of an ultrathin-layer isoelectric focusing
(UTLIEF) procedure to test the hybrid purity of seed lots by
A D H analysis, with emphasis on reducing the cost of equip-
ment, materials, and reagents, and increasing the number of
seeds that can be tested per day per person. In addition, a large
number of F 1 hybrids and open-pollinated varieties were
analyzed to find furher possibilities for the application of the
UTLIEF procedure.
2 Materials and methods
2.1 Plant material
The Royal Sluis F1 hybrid seeds were from experimental as
well as commercial seed lots. Seeds of parental lines were
derived from at least the 7th generation of inbreeding. Seeds of
tomato F 1 hybrid varieties from several large breeding com-
panies were taken from commercial seed samples.
2.2 Chemicals
Water was purified by the Milli-Q system from Millipore
(Bedford, USA). Acrylamide, ammonium persulfate, carrier
0 173-0835/9 1/010 1-0064 $3.50+.25/0
Electrophoresis 1991,12,64-69
ampholytes (Servalyt pH 3- 10, aspartie acid, N,N'- methyl-
enebisacrylamide (Bis), cathode fluid 10 (based on arginine,
lysine and ethylenediamine), Coomassie Brilliant Blue R-250
(Serva Blue R), glutamic acid, and kerosene were from Serva
(Heidelberg, Germany). Glycine, trichloroacetic acid (TC A),
and p l protein kit (range 4.75-10.4) were from BDH (Poole,
UK). Acetic acid, NADP, Nitro Blue Tetrazoliume (NBT),
N- methylphenazonium methosulfate (PMS) and chemicals
not mentioned here were from Sigma (St. Louis, USA).
2.3 Materials
Gel-Fix for polyacrylamide gel electrophoresis (PAGE) and
covers(265 x 193 mm),andelectrodewicks(300 x 6 x 1 mm)
were purchased from Serva. Flat-bottom microtiter plates (96
wells) were from Costar (Cambridge, USA). Adhesive tape
(Tesa, 100 pm) was from Beun de Ronde (Abcoude, NL).
Silicone rubber applicator strips were made from 1 mm
silicone rubber sheets (from Eriks, Alkmaar, NL) by punching
with a stainless steel device.
2.4 UTLIEF
2.4.1 Gel preparation
UTLIEF gels were essentially prepared according to the flap
technique as described by Radola 191. The polymerization
mixture that was poured on the Gel-Fix film contained 1.68
mL acrylamide solution (40 %T + 5 %C), 0.6 mL Servalyt
pH3-10,5.72mLof 16 %glycerol,andO.4mLofl.S %am-
monium persulfate. The final acrylamide concentration in the
UTLIEF gel mixture was 8 %. The gel mixture was degassed
thoroughly on a vacuum line for 1-2 min. After flapping and
polymerization, the gels were either used immediately or
stored in humid boxes at 4 O C for a maximum of one week.
2.4.2 Applicator strip
Silicone rubber applicator strips were made from silicone rub-
ber sheets (1 mm thick). A stainless steel devicewasdeveloped
in order to punch ellipsoids with axes of 3.5 and 1.5 mm. Ellip-
soid holes were punched out of the sheet 1.0 mm apart from
one another.
2.4.3 Sample preparation and application
Tomato seeds were put in microtiter plates and the wells were
filled with water. After incubationovernight( 16-20 h),inorder
to induce de novo synthesis of ADH-1 under anaerobic condi-
tions 171, the water was removed and 80 pL fresh water was
added to each well. Then the seeds were homogenized using a
laboratory-made device. After prefocusing, an aliquot of 7 pL
was taken from the homogenate and pipetted onto the gel
surface in an applicator strip hole that was layered about 5 mm
from the anode.
2.4.4 UTLIEF running conditions
IJTLIEF was performed 'in a Desaga electrophoresis unit
(Desaphor HE 200), equipped with a 200 x 265 x 1mm cool-
ing glass plate, connected to a Pharmaciaconstant power sup-
ply (ECPS 3000/150) and aLaudaRC3 cooling apparatus (at
10 "C). The gels were placed on the cooling plate with a few
Tomato hybrid purity testing with UTLIEF 65
drops of kerosene to ensure good contact. The electrode wicks
were soaked in 0.5 M aspartic acid and 0.5 M glutamic acid
(anodic wicks) and cathode fluid 10 (cathodic wicks). The
electrodes were placed on the wicks and covered with a glass
plate as weight to ensure close electrical contact. Three elec-
trodes were placed on the gel when the gel was divided into
two parts for the analysis of 2 x 96 lanes. Prefocusing was car-
ried out at settings of 10 W, 1500 V and 17 mA; it started at
about 280 V until the voltage reached about 720 V (150 Vh).
After sample application, focusing was continued at the same
settings for 50-60 min (2000 Vh). Four electrodes were placed
on one gel when the gel was divided into three parts for analysis
of 3 x 96 lanes. Then prefocusing was carried out at settings of
7 W, 1250 V and 30 mA; it started at about 180 V until the
voltage reached about 400 V (150 Vh). After sample applica-
tion, focusing was continued for 5 Vh at 50 V, 7 W, 1250 V,
then at prefocusing settings for 1 h and 15 min (1500 Vh).
2.4.5 Staining for ADH activity
Staining for ADH activity was performed immediately after
isoelectric focusing by incubating the gel in a solution ofO. 1M
Tris-HC1, pH 7.5, containing 4 % ethanol, 4 mg PMS, 30 mg
NBT and 30 rng NADP (P-nicotinamide adenine dinucleo-
tide), at room temperature in the dark. After 5 min, when the
stained bands were visible, the gel was washed for 1 min with a
solution consisting of ethanol (40 %),water (50 %) and acetic
acid (10 %)to prevent background staining. For preservation,
the gels were air-dried.
2.4.6 Total protein staining
Immediately after focusing, the gels were incubated in 20 %
TCA for 5 min. The proteins were stained in a solution of
0.25 % Serva Blue R in solution A (10 % acetic acid, 40 %
methanol, 50 % water, v/v) for 10 min. The gels were de-
stained several times, for 5 min, in solution A until the back-
ground was clear and then air-dried.
2.5 Nomenclature
Symbols for the ADH-1 allozymes and Adh-l alleles were
used in conformity with their use in other research reports
16-81 and in conformity with the rules of the Tomato Genetic
Cooperative t101.
3 Results
3.1 Analysis of ADH- 1 variants by UTLIEF
Two alleles of the gene Adh-1 were demonstrated in the
cultivated tomato by Tanksley using SGE [61. They were
designated A d h - l + and Adh-1 I. The allele from the open-
pollinated variety Marglobe was named Adh-l+; other var-
iants are numbered consecutively, in accordance with to-
mato nomenclature [lo]. In order to identify both variants
and to use the same nomenclature, the variety Marglobe and
the F 1 hybrid Arletta (which contains both ADH- 1 variants)
were analyzed by UTLIEF. A pH gradient of 3-10 gave the
best results. Figure 1 shows the results of the analysis of both
Marglobe and Arletta and the parents ofArletta. It can be con-
66 B. M. van den Berg Electrophoresis 1991,12, 64-69

cluded that Adh-l ’ codes for the ADH-1 variant with the In addition to the ADH-I and ADH-l’bands, a third promi-
+

lowest pl. Using reference proteins run in parallel with the
tomato samples, followed by cutting the gel and staining the
reference proteins with Coomassic Brilliant Blue, the p l o f the
variants could be estimated to be 5.5 and 5.7 for ADH-1 +and
ADH-I I , respectively. For proper estimationoftheprs theex-
periment was performed three times.
nent band can be seen (Figs. 1-3), which results from
dimerization of the two ADH-1 variants. In addition to these
three prominent bands, several faint bands are visible, which
most probably correspond to those which Tanksley called
shadow bands. These bands were explained as being secon-
dary modification products [71.

Figure 1 . Analysis of crude extracts from imbibed tomato seeds ofthevarieties Marglobe and Arletta and the parents ofArletta as well as plmarker proteins
by UTLIEF, pH 3- 10. After electrophoresis the gel was cut into 3 parts and stained for A D H and protein. ( I ) Female parent of Arletta;(2) male parent of
Arletta; (3) F1 Arletta; (4) pImarker proteins; (5) Marglobe. In the case of No. I , 2,3 and 4, ten elliptoid holes were filled with extract from different seeds.
Note the absence of ADH staining activity for some samples; a, denotes the sample application point.

Figure 2 . UTLIEF gel showing the analysis of 2 x 96 seeds of the F 1 hybrid Arletta by division of the gel into two parts; a, denotes the sample application
point. The vertical arrows indicate places on the gel that lack staining. The horizontal arrow indicates an inbred individual.
Electrophoresis 1991, 12,64-69
3.2 Technical improvements to increase the efficiency and
economy of the inbred testing method
3.2.1 Development of a sample applicator strip
The number of seeds to be tested on one gel could be increased
by developing a new applicator strip made from a 1 m m
silicone rubber sheet. In this applicator strip of 19 cm length,
96 elliptic holes were cut with a laboratory-made tool. Elliptic
holes proved to have several advantages over other types of
holes. First, the width ofthe lanes could be made as small as 1.5
The phenotypes of the different individuals are clearly visible
on the gel. Most lanes show the expected triple-banded
phenotype of F 1 plants. An inbred phenotype can also be dis-
cerned. A few lanes lack staining. These are seeds that were no
longer able to synthesise the ADH-1 enzyme. Most probably
these seeds were dead (see Figs. 1-3).
3.2.2 Division of the UTLIEF gel into three parts
By dividing the UTLIEF gel of size 265 x 196 mm into two
parts of size 265 x 95 mm, and by using the 96-hole sample ap-
plicator strip and three electrodes, 192 lines could be analyzed
simultaneously. To increase the number of lines further, gels
Figure3. UTLIEFgelshowing theresultsoftheanalysisof3 x 96seedson asinglegel bydivisionofthegelintothreeparts;a,denotesthesample
point. No inbreds were detected here.
Tomato hybrid purity testing with UTLIEF 67
were divided into 3 parts by using four electrodes. This
resulted in three gel parts of size 265 x 80 mm. By adjusting
the power supply it was possible to gain results similar to those
from experiments with two gel parts (Fig. 3). Using the Desaga
electrophoresis unit, type H E 200, that can have two gels run-
ning simultaneously, 1152 seeds can be tested on a routine
scale by running two gels twice a day.
3.3 DistributionofAdh-lallelesamong F 1 hybrids, parental
lines and open-pollinatedvarieties from several sources
20 seeds individually, taken from the various inbred lines,
showed that they are homozygous for the gene Adh-I. Also,
experimental F 1 hybrids and their parental lines were ana-
lyzed. Table 2 gives the results of the analysis of both the com-
mercial and experimental F 1 hybrids, as well as their parental
lines. Only a minute number of F 1 hybrids with genotype
Adh-I ‘1Adh-l was found. On the basis of the distribution of
the Adh-l allele over the inbred lines, a higher number would
have been expected. Table 3 shows the genotypes of F 1
hybrids from 9 breeding companies. The genotype was deter-
mined after analyzing 5 seeds. No inbreds were detected. Only
8 out of 33 F 1’s were heterozygous. Table 4 shows the
genotypes of 28 open-pollinated varieties. For each variety, at
least 12 seeds were analyzed; no heterozygosity was found.
-
application
68 B. M. van den Berg Electrophoresis 199 I, 12, 64-69

Table 1. Adh-I genotypes of commercial F 1 hybrid varieties from Royal Table 4. Adh-1 genotypes of open-pollinated tomato varieties
Sluis
Ace 55 V F Adh-1 +/Adh-l+
FI hybrid Adh-1 genotype F1 variety Adh-1 genotype Cal Ace VF Adh-I +JAdh-I+
Cal JVF Adh-I'JAdh-I'
Antilope Adh-Il/Adh-I1 Leopard0 Adh-I IjAdh-' C hico I11 Adh-1 ' JAdh-1'
Arletta Adh-I'lAdh-I + Mandel Adh-1 /Adh-1 Cceur du Bcef Adh-1 JAdh-l+
Arno Adh-l+ JAdh-l+ Marina Adh-I' JAdh-I+ Columbia Adh-1 JAdh-I+
Bonset Adh-1 iJAdh-I+ Multiset Adh-l+/Adh-li Early Pak Adh-I+JAdh-l+
Bornia Adh-IIJAdh-I+ Narita Adh-I'JAdh-l+ Feston Adh-I'/Adh-I'
Corindo Adh-I' JAdh-I Parana Adh-I+ JAdh-I+ Flor adel Adh-I'lAdh-1'
Crisolita Adh-I+JAdii-I+ Proset Adh-I'lAdh-1' Heinz 1370 F Adh-I JAdh-I +
+

Diamon Adh-llJAdh-I+ Robin Adh-l'/Adh-l+ Lima Adh-1 '/Adh-1'

Esla Adh-I+JAdh-I+ Romulus Adh-l+JAdh-1' Marmande Adh-11 JAdh-I+
Euroset Adh-I-JAdh-I+ Royesta Adh-l'JAdh-li Moneymaker Adh-l+JAdh-I+
Everset Adh-I+JAdh-I+ Sidonia Adh-I'JAdh-I+ Monprecos Adh-I JAdh-I
+ +

Florenta Adh-l+JAdh-I+ Tegula Adh-I'JAdh-I+ Napoli Adh-I+ JAdh-I+

Floriset Adh-If JAdh-I + Tobol Adh-liJAdh-I+ Pearson improved Adh-I JAdh-1
+

Forset Adh-I'lAdh-l+ Turmalina Adh-I'lAdh-l+ Pritchard Adh-1 +JAdh-l+

Fortaran Adh-I1lAdh-l+ Turquesa Adh-l'lAdh-l+ Red Cloud Adh-l+JAdh-I+
Goldset Adh-1 JAdh-I t Verge1 Adh-I'/Adh-l+ Rio Grande Adh-I'JAdh-1'
Jacinto Adh-I'JAdh-I+ Wilset Adh-l+IAdh-l' Roma Adh-1 +/Adh-I'
Roforto Adh-I +JAdh-I+
Roza Adh-I+ JAdh-I+
Table 2. Genotype of commercial and experimental F 1 hybrids and San Marzano Adh-li jAdh-I+
parental lines from Royal Sluis Santa Kruz Kada Adh-I+/Adh-I+
Genotypes St. Pierre Adh-I JAdh-I
U C 134 Adh-1'JAdh-1'
Adh-I'JAdh-1' Adh-I'JAdh-I+ Adh-lf /Adh-l+ Urbana VF Adh-I JAdh-l+
+

Ventura F R Adh-I'JAdh-I+
Inbred lines 33 - 87 Walter F2S Adh-1 JAdh-I '
F 1 hybrids 2 34 36

Table 3. Adh-1 genotypes of commercial tomato F 1 hybrids of 9 seed Only 5 out of 28 varieties had genotype Adh-1' IAdh-1 Since '.
companies (only + and - are given to indicate the genotype) all F 1 hybrids are derived by crossing open-pollinated
varieties, these results indicate that F1 breeding results in a
Company F 1 hybrid Genotype selection in favor of the Adh-1' allele. In general, the results
Asgrow Solar Set +1 show a wide abundance of both Adh-l alleles, which indicates
Bruinsma Dombito ++ a great potential application of the UTLIEF method for deter-
Enza Amfora ++ mining inbred frequencies in commercial seed lots.
Buffalo ++
Ophir ++
Hazera FA 121 I1
Moran Blazer +I 4 Discussion
Northrup Quick Pick ++
Valerie +I Our aim was to develop an electrophoretic method to test for
Pet0 Seeds Contessa +1 inbreds among tomato F 1 hybrid varieties using an ADH
Count 11 variation that is cheaper and faster than electrophoresis based
Duke +1 on starch or polyacrylamide gels. The method described here
Early Cascade +1 possesses several advantages over horizontal SGE and
Floritalia ++ vertical PAGE, which are commonly used for inbred testing.
Ferry Morse Jackpot +1
Rijk Zwaan Marcella ++ First, only one electrophoresis unit is necessary for UTLIEF,
de Ruiter Aratino ++ whereas with PAGE and SGE several units are needed in
Bolero ++ order to analyze more than a thousand seeds per day. Second,
Caruso ++ the application of UTLIEF is cheaper: less chemicals, es-
Counter ++ pecially NADP, are needed because less staining medium per
Estafette ++ square cm gel is required and more samples are present per
Evita 11 square centimeter of gel. Third, using UTLIEF the number of
Garanto ++ samples that can be analyzed per day and per person can be in-
Larma ++ creased dramatically: 1152 seeds with one electrophoresis
Laura ++
Liberto ++ unit. Therefore, the application of UTLIEF results in an ad-
Rondelo +1 ditional reduction in cost by reducing the investment in equip-
Sonatine ++ ment, chemicals and staff. The method has been applied for
Vilmorin Dona ++ two years and the cost for the analysis of one seed (on the basis
Sluis and Groot CarPY ++ of routine application and including overhead costs) has been
Elcy ++ estimated to be $0.20 per seed.
Dario ++
Darus ++ Further research will be directed at the following three points.
G c 779 ++ First, development of equipment for faster homogenization of
Electrophoresis 1991,12,69-74 Detection of P-glucanases in polyacrylamide gels 69

iarge numbers of single seeds; second, development of a com- 5 References

bination of sample applicator strip and multipipet so that 12
samples can be pipetted in one handling; and third, investiga-
tion into the possibility of dividing the gel into four or five
parts.
I thank my colleagues Mr.J. TamboerandMr.B. Grapendaal
for their help in a few o f t h e experiments, and Dr. J. van
Brederode (University of Utrechtlfar valuable comments dur-
ing the preparation of the manuscript.
Received August 23, 1990
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Rick,C.M.andFobes,J.,Rept. TomatoGenet. Coop. 1914,24,25.
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Franqois C8th Detection of P-glucanase activity on various

Souad El Ouakfaoui
Alain Asselin p- 1,3 and p- 1,4-glucans after native and denaturing
DCpartement de Phytologie,
polyacrylamide gel electrophoresis
UniversitC Laval, Quebec
P-Glucanases were detected after polyacrylamide gel electrophoresis under native
and denaturing conditions using various p-1,3- and p- 1,4-glucans, including mixed
glucans (laminarin, pachyman, carboxymethyl cellulose, lichenan and barley p-
glucan). After electrophoresis and incubation of gels, substrates incorporated into
polyacrylamide gels were stained with specific fluorochromes, Sirofluar for p- 1,3
linkages and Calcofluor White M2R for 0- 1,4 linkages. Under UV illumination, lysis
zones appeared as dark bands against a fluorescent background. Enzymes of
bacterial, fungal and plant sources could be revealed sequentially in gles containing
mixed p-( 1,3)( 1,4)-glucans by staining first with sirofluor followed by staining with
Calcofluor White M2R. Active profiles were more diverse when substrates were
stained with sirofluor. The use of purified sirofluor at pH 11.5 compared with Aniline
Blue at pH 8.6 allowed better detection of P- 1,3-glucanase activities. In gels contain-
ing laminarin stained with sirofluor, bands exhibiting a more intense fluorescence
than the background fluorescence were observed in addition to dark nonfluorescent
bands. It is postulated that these two types of p-1,3-glucanase activities differ bytheir
enzymatic action (partial versus extensive hydrolysis). Analysis offungal extracts us-
ing denaturing gels embedded with various P-glucans displayed lysis bands migrating
between 32 and 35 kDa.

1 Introduction have been reported under normal developmental conditions

and under stress. Glucanases acting on p-( 1,3)( 1,4)-glucans
P-Glucanases represent a wide group of enzymes acting on have also been detected in germinating grains of barley [21. In
diverse P-linked glucans such as p-1,3 and Pl,4-glucans. many filamentous fungi, enzymes can either hydrolyze p- 1,3-
These glucans are widely distributed and often occur as struc- glucansin anendoorinanexomanner [31.Detectionofp-1,3-
tural components of cell walls or storage polysaccharides. The glucanase activity is primarily determined by hydrolysis of
structure of the glucan backbone and the nature of substi- laminarin (isolated from an alga, Laminariu digitata)using a
tution residues are highly variable 111. In plants, P-1,3- reducing sugar colorimetric assay and, as of recently, after
glucanases (laminarinases) and 0- 1,4-glucanases (cellulases) native polyacrylamide gel electrophoresis (PAGE) and iso-
electric focusing (IEF) 141. Detection of p- 1,4-glucanase has
Correspondence: Dr. Alain Asselin, Departement de Phytologie, Faculte been reported after IEF using carboxymethyl (CM) cellu-
des Sciences de l’agriculture et de l’alimentation, Universite Laval, Quebec lose as substrate, stained with Congo Red or iodine 151, or
(Quebec), Canada, GlK7P4
p-anisidine 161. Detection of cellulase has also been reported
Abbreviations: CM, carboxymethyl; IEF, isoelectric focusing; IF, inter- after sodium dodecyl sulfate (SDS)-PAGE with CM-cellulose
cellular fluid; PAGE, polyacrylamide gel electrophoresis; SDS, sodium as substrate in addition to mixed p-(1,3)(1,4) barley glucan
dodecyl sulfate; TMV, tobacco mosaic virus; Tris, tris(hydroxy- and lichenan stained with Congo Red [71. We have recently
methy1)aminomethane developed a P-1,3-glucanase assay in native PAGE in gels
0VCH Verlagsgesellschaft mhH, D-6940 Weinheim, 199 I 0173-0835/9 10101-0069 %3.50+.25/0