824 B. M.

van den Berg Eleclrophoresis 1990,11,824-829

5 References 1101 Kass,L. andMunster,D.,Am.J. Clin.Pathol. 1979,72,611-613.

11 Bennett, J. M., Catovsky, D., Daniel, M. T., Flandrin, G., Galton, D.
A. G., Gralnick, H. R. and Sultan, C., Brit. J. Haematol. 1976.33,
45 1-458.
121 Hayhoe, F. G. J. and Quaglino, D., in: Hayhoe, F. G. J. and Quaglino,
D. (Eds.), Haematological Cytochemistry, Churchill Livingstone,
Edinburgh, 1980, pp. 259-308.
131 Flandrin, G. and Daniel, M. T., in: Catovsky, D. (Ed.), TheLeukemic
Cell, Churchill Livingstone, Edinburgh 1981, pp. 29-48.
141 Bozdech,M. J. and Bainton,D. F., J.Exp.Med. 198 1,153,182- 195.
151 Li, C. Y.,Lam,K. W. andYam,L.T.,J. Histochem. Cytochem. 1973,
21,l-12.
161 Radzun, H. J., Parwaresch, M. R., Kulenkampff, C., Staudinger. M.
and Stein, H., Blood 1980,55, 891-897.
171 Yourno, J . and Mastropaolo, W., Blood 1981,58,939-946.
181 Scott, C. S., Linch, D. C., Bynoe, A. G., Allen, C., Hogg, N., Ainley,
M. J., Hough, D. and Roberts, B. E., Blood 1984,63, 579-587.
I91 Kass, L. and Peters, C . L., Am. J. Clin. Pathol. 1978,69,57-61.
[ 111 Drexler, H. G. and Gaedicke, G., Leuk. Res. 1983, 7,599-609.
!121 Drexler, H. G., Gaedicke, G. and Minowada, J., Leuk. Res. 1985,9,
209-229.
I131 Scott, C. S., Hough, D., Bynoe, A. G., Jones,D. B. and Roberts, B. E.,
J. Histochem. Cytochem. 1984,32,579-584.
[141 Yourno, J., Blood 1986,68,479-487.
[I51 Cohn, P. D., Emanuel, P. D. and Bozdech, M. J., Blood 1987,69,
1574- 1579.
1161 Yourno, J., Walsh, J., Kornatowski, G., O’Connor, D. and Kumar,
S. A., Blood 1984,63.238-241.
t171 Yourno, J., Burkart, P., Mastropaolo, W., Lizzi, F. andTartaglia, A.,
J. Histochem. Cytochem. 1986,34, 727-733.
1181 Drexler, H. G., Gignac, S. M. and Minowada, J., Blut 1988, 57,
327-339.
[191 Blumberg, P. M., Cancer Res. 1988,48, 1-8.
1201 Koemer, H. P., Blood 1983,62,709-721.
I21 I Yourno, J., Burkart, P., Lizzi, F. and Tartaglia, A., Blood 1982,60,
304-308.
1221 Greaves, M. F., Science 1986,234, 697-704.

Bartel M. van den Berg Inbred testing of tomato (Lycopersicon esculentum L.)
Royal Sluis, Enkhuizen F 1 varieties by ultrathin-layer isoelectric focusing of
seed protein
T o test seed lots of tomato F 1 hybrid varieties for the presence of undesirable inbred
seeds by electrophoresis, a method has been developed on the basis of ultrathin-layer
isoelectric focusing. The method is based on the genetic variation of the seed protein
PRS-1 which could be visualized by isoelectric focusing of a 5 mMNaC1-soluble seed
protein extract in a pH 6-9 gel followed by protein staining. Two genetic variants of
the PRS-1 protein, PRS-1 +and PRS-1 l , were found among open-pollinated varieties,
as well as among F 1 hybrid varieties. The isoelectric points (pl) ofthe PRS- 1 proteins
are 7.1 and 6.1 for PRS- 1 and PRS- 1l , respectively. The PRS- 1 protein is unique to
+

seed tissue and is located primarily in the embryo. A genetic 1:2: I segregation of the
gene Prs-1 among several F2 populations shows monogenic inheritance. Analysis of
commercial F 1hybridvarieties from several seed companies indicated that the Prs-1
allele, in contrast to the Prs-l+allele, is primarily present in gene pools of “Money-
maker type” tomatoes. The described method is generally applicable to all tomato F 1
varieties that are heterozygousfor the gene Prs-I. With the described method one per-
son can routinely analyze more than 768 seeds per day.

1 Introduction desired fertilization or mixing of seeds from different genetic

The delivery of genetically pure seed lots is of upmost impor-
tance for seed companies. They must be able to characterize
varieties because genetic impurities can have severe effects on
the quality of the crop. These impurities may arise from un-
Correspondence: Dr. B. M. van den Berg, Royal Sluis, Research and
Development Department, Section Plant Biochemistry, P.O. Box 22,
NL-1600 AA Enkhuizen, The Netherlands
Abbreviations:Bis, A’,”-methylenebisacrylamide; %C,crosslinking agent
(9) x 100/%T; PAGE, polyacrylamide gel electrophoresis; p2, isoelectric
point; PRS, protein seed (number); %T, acrylamide (9) + bis (g)/lOO mL;
TCA, trichloroacetic acid; UTLIEF, ultrathin-layer isoelectric focusing;
Vh, volthour
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
origin. The genetic impurities present in basic seeds that are
used to produce commercial seeds may cause problems as well
[ l , 21. Proteins, as the primary products of structural genes,
can serve as genetic markers in order to characterize varieties
since their phenotype (thee1ectrophoretogram)is not influenc-
ed by anything other than genetic factors [3,41. Seed lots are
routinely analyzed biochemically to test the genetic quality of
several agricultural crops such as maize, wheat, barley and
other cereals. Testing the genetic quality of these crops re-
quires many markers in order to differentiate between the
many varieties [5,61. For many horticultural crops the devel-
opment of F 1 hybrids called for the development of methods
for inbred testing. In this special case of genetic purity
testing (inbred testing), only one variable genetic marker is
0173-083519010909-0824 %3.50+.25/0
Electrophoresis 1990, I I , 824-829 Inbred testing of tomato F I by UTLIEF 825

needed [I]. Tests for the determination of inbred frequencies Table 1. Prs-1 genotypes of tomato F1 hybrid and open-pollinated
based on electrophoretic analysis have been developed for varietiesa)
several horticultural crops, e. g., Brassica crops [ 1, 2, 7-1 11 F 1 hybrid v,arieties from Royal Sluis
and tomatoes [ 12- 151. The electrophoretic method has sev-
eral advantages over the morphological tests for genetic purity Arletta +/+ Forset +/+ Royesta +/+
+I+ Sidonia +I+
r~21.
Bonset
Bornia
+/1
+/1
Grosset
Jacinto +/I Tegula +/+
Corinto +/+ Mandel +I+ Tobol +/+
The parents of tomato F 1 hybrids often differ in number and/ Crisolita +/+ Narita +/ + Turquesa +/+
or kind of resistance genes. Therefore, the presence of inbred Euroset +/1 Parana +I+ Verge1 +/ 1
seeds, e. g., caused by mistakes in the process of hand emas- Everset +/1 Proset +/ + Wilset +I+
culation and pollination, can affect the genetic quality of seed Floriset +/1 Robin +I+ Zircon +/ 1
lots. As a consequence, inbred frequencies need to be deter- Open-pollinated varieties
mined. Polymorphism of enzyme loci in tomato and its wild
relatives has been studied in great detail by Rick and co- Ace 55 V F +/+ Moneymaker 1/1 Strain B +/+
workers [ 161. Their studies showed little enzyme polymor- Columbia +/+ Napoli l/l UC 82 +I+
ChicoIII +/+ RedCloud +/+ UC 134 +I+
phism among the cultivated tomato. Only two cases of en- Floradel CFS +/+ Rio Grande +/+ Urbana VF +/+
zyme polymorphism have been reported, namely for the en- Heinz 1 3 7 0 F +/+ RomaVF +/+ Ventura FR +/+
zymes acid phosphatase from leaf tissue [ 12,131 and alcohol Marmande +/+ SanMarzano +/+ VFN8 +I+
dehydrogenase from imbibed seed [14, 151. These enzyme Monprecos +/+ St. Pierre +/+ Walter F2S +/+
polymorphisms can be used to test for inbreds. However, for a
given protein marker only a limited number of F 1 hybrid a) Only + and 1 signs are given to indicate genotypes.
varieties can be tested since the F 1 parents must differ in
genotype for the marker protein. The availability of only two 250 (Serva Blue R), kerosene, and p1 protein kits (snake
variable loci to test inbreds from tomato F 1 hybrid seed lots venom and mix 9) from Serva (Heidelberg, Germany); acetic
necessitated the search for additional protein markers. Since acid, glycine, glycerol, a plprotein kit range 4.75-10.4, and
seed proteins have not yet been studied in great detail for this trichloroacetic acid (TCA) from BDH (Poole, UK).
purpose, this study focused on the analysis of variability of
proteins from the tomato seed. The principal aim of this in-
vestigation was to screen for electrophoretic variation in order 2.3 Materials
to develop additional methods for inbred testing of seed lots of
tomato F 1 varieties. Gel-Fix for covers and Gel-Fix for polyacrylamide gel elec-
trophoresis (PAGE; 265 x 193 mm) and electrode wicks (300
The mainly used biochemical technique for the characteriza- x 6 x 1 mm) were from Serva. Flatbottom 96-well microtiter
tion of varieties has been protein electrophoresis 171. A recent- plates were from Costar (Cambridge, USA). Adhesive tape
ly developed protein separation technique is ultrathin-layer (Tesa, 100 pm) was from Beun de Ronde (Abcoude, NL).
isoelectric focusing (UTLIEF) [ 17-191. Compared to the
techniques that are more frequently used on a routine basis,
e. g., starch and acrylamide gel electrophoresis, UTLIEF 2.4 UTLIEF
possesses a greater discriminative and resolving power, has
low costs in chemicals and has a shorter running time due to 2.4.1 Preparation of gels
the possibility of applying a higher voltage 117-191. There-
fore, this study was focused on the analysis of speed protein by UTLIEF gels were prepared according to the flap technique as
UTLIEF in order to reveal as much genetic protein variability described by Radola 1201 and Frey et al. (211. Adhesive tape
as possible. was used as spacer along each sideofthe Gel-Fix film(265 mm
x 193 mm). Both the Gel-Fix and cover films were rolled onto
glass plates with a few drops of water between the glass plate
and the film. The polymerization mixture contained 1.32 mL
2 Materials and methods acrylamide (30 % mono w/v, 3 % Bis w/v); 5.72 mL of 16 %
glycerol; 0.6 mL carrier ampholytes, pH 6-9; and 0.4 mL of
2.1 Plant material 1.5 % ammonium persulfate. The final acrylamide concen-
tration was 5 % T and 3 % C. The mixture was degassed
The seeds used from open-pollinated and F1 hybrid varieties thoroughly on a vacuum line for 3 min. After flapping and
listed in Table 1 were produced by Royal Sluis. The parental polymerization, the gels were used (after at least 1 h) or stored
inbred lines used to produce the F 1 hybrids were from at least at 4 "C for no longer than one week. Using one anodal and two
the 7'h generation of inbreeding. Seeds of commercial F 1 cathodal electrodes in the middle and on the sides, respective-
hybrid varieties from seed companies other than Royal Sluis ly, two gel parts of size 250 x 90 mm could be run simul-
were derived from commercially available seed samples. taneously on one Gel-Fix film.

2.2 Chemicals 2.4.2 Sample preparation and application

The water used was purified by the Milli-Q system (Millipore,
Bedford, USA). The chemicals used were: acrylamide, N,N'-
methylenebisacrylamide (Bis), Cathode Fluid 10 (0.44 %
arginine; 0.36 % lysine and 12 % ethylene diamine), carrier
ampholytes (Servalyt p H 6-9), Coomassie Brilliant Blue R-
Tomato seeds were put in microtiter plates (96 wells with a
volume of 380 pL) and the wells were filled with 80 pL of a
5 mM NaCl solution (4 "C). The seeds were homogenized us-
ing a home-made device attached to a drilling machine (in
preparation). After prefocusing, an aliquot of either 7 pL or
826 B. M.van den Berg
20 pL (for 2 x 96 or 2 x 36 samples per gel, respectively) was
taken from the homogenate and pipetted onto the gel surface.
Pipetting was carried out in a hole of a slot former made from
silicone rubber layered approximately 5 mm from the anode.
2.4.3 Running conditions
UTLIEF was performed in a Desaga electrophoresis unit
(Desapher H E 200, Heidelberg, Germany) equipped with a
200 x 265 x 1 mm cooling glass plate, connected to a Phar-
macia constant power supply, ECPS 3000/150, and a Lauda
RC3 cooling apparatus, operated at a temperature of 10 "C.
The gels were placed on the cooling plate with a few drops of
kerosene to ensure good contact. The electrode wicks were
soaked in 0.5 M glycine (anodal wicks) and Cathode Fluid 10
(cathodal wicks). The electrodes were placed on the wicks and
then covered with a glass plate to ensure close electrical con-
tact. Prefocusing was carried out at limit settings of 10 W,
2000 V and 12 mA. Prefocusing started at about 240 V and
3.5 W until the voltage reached about 800 V (15-20 min).
After application of the samples the focusing was continued at
the same settings for 3000 Vh (80-90 rnin).
2.4.4 Protein staining
After focusing, the gels were soaked in 20 % TCA for 5 min
followed by incubation for 10 min in a solution containing
10 % acetic acid, 40 % methanol, and 50 96 water, v/v/v, and
0.05 % Serva Blue R (Serva Blue R was first dissolved in 10
mL of methanol). The gels were destained in three washesdur-
ing 5 min in the same solvent (without dye) until the back-
ground staining was completely removed. After rinsing with
water the gels were air-dried and stored.
Figure 1. UTLIEF (pH 6-9) o f 5 mM NaCI-soluble tomato seed proteins from open-pollinated varieties and F1 hybrid varieties and their parents. Open-
pollinated varieties (1-8): (1) VFN8: (2) Walter FS; (3) Moneymaker; (4)Marglobe; ( 5 ) Marrnande; (6) Roma VF; (7) St. Pierre; Royal Sluis F1 hybrid
varieties and their parents (left, female: middle, F1 hybrid, which are numbered; right, male): (8) Bonset: (9) Bornia: (10) Jacinto: (1 1) Zircon.
Electrophoresis 1990, I I , 824-829
2.5 Analysis of organ specificity of the PRS-1protein
Seeds of the F 1 hybrid variety J acinto were soaked in water for
60 min. The seeds were dried with tissue paper and cut lateral-
ly. The embryo parts were easily isolated with tweezers. The
remaining tissue contained both the endosperm and the seed
coat [231.
2.6 Nomenclature
A symbol for the protein that was subject of this study has
been chosen in conformity with the rules of the Tomato
Genetic Cooperative [231. The protein has been designated
with three capitals and a number: PRS-1 (PRS denotes Pro-
tein from Seed). This number was given since genetic variants
of other seed proteins have been found as well (unpublished
results). The variety Marglobe, chosen as reference [23l, con-
tains the allele Prs-I that codes for the protein PRS-I+ (the
+
protein with a p l o f 7.1). Other alleles and their encoding pro-
teins will be numbered consecutively: the open pollinated
variety Moneymaker has the allele Prs-l that codes for the
protein PRS-1'.
3. Results
3.1 Detection and electrophoretic variation of the PRS-1
proteins
An electrophoretic study ofthe water-soluble (or 5 mMNaC1)
proteins from seeds of open-pollinated tomato varieties by
UTLIEF in the p H range of 6-9 revealed, among many other
Electrophoresis 1990, I I . 824-829
bands, two variable protein bands. The variable protein in the
reference variety Marglobe has been named PRS- 1 (see Sec- +
tion 2.6) and the other variant, found in the open-pollinated
varieties Moneymaker and Napoli, has been designated PRS-
I'(Fig. 1;Table l).ThepZsofbothproteins wereestimatedus-
ing reference proteins with known plunder standardized elec-
trophoretic conditions. The pls for PRS-1+ and PRS- 1 were
found to be 7.1 and 6.1, respectively (Fig. 2).
The intensity of the PRS-1 band of the crude extract from a
single tomato seed was too low for proper routine detection of
the PRS-1' variant when water was used as extraction
medium. This was even more the case for plants that contain
both PRS-I variants. Further, the PRS- 1 protein tends to
precipitate near its isoelectric point, as was indicated by thick,
faint bands. Therefore, several possibilities were investigated
to guarantee a reliable detection of the PRS- 1 protein. The
+
use of urea and a high salt concentration in the homogeniza-
tion medium resulted in the liberation of much more protein
and, consequently, the appearance of numerous protein bands
in the gel, which made unambigious identification ofthe PRS-
1 proteins impossible. The use of a homogenization medium
with low ionic strength and the addition of glycerol resulted in
better solubility of the PRS-1 proteins and in improved stabili-
ty ofthe PRS-1 +variantaround its pzduring isoelectric focus-
ing.
3.2 Variability of the PRS-1 protein among Royal Sluis
varieties
The occurrence of both PRS-I variants among open-pol-
h a t e d and F1 hybrid varieties and their parents was in-
vestigated. Plants of the open-pollinated varieties and parents
Figure 2. Isoelectric points ofthe PRS-1 proteins after UTLIEF (pH 6-9).
(1) plmarker proteins, test kit snake venom from Serva; (2)pimarker pro-
teins, test kit protein Mix 9 from Serva; (3) F1 variety Jacinto.
Inhrcd testing of tomato t 1 by UTLIEF 827
of the F 1hybrid varieties contained only one ofthe PRS- 1 pro-
tein variants,eitherPRS-I'or PRS-l', butthelatterwith alow
frequency. Among the F 1 hybrid varieties two phenotypes
were found, namely a phenotype with only one PRS- 1 protein
and the other phenotype with both PRS-1 proteins (Fig. 1;
Table 1). Of the three PRS-1 phenotypes that were to be ex-
pected among the F 1 varieties, the absence of one can be ex-
plained by the low frequency of the PRS- 1 ' protein among the
parental lines. T o test for homogeneity of the open-pollinated
varieties as well as of the parental lines of the Fl hybrid
varieties for the phenotypes found, at least 20 seeds were in-
dividually analyzed for their PRS-1 phenotype. All of the
tested varieties and inbred lines were homogeneous for the
PRS-1 phenotype.
3.3 Organ-specificity of the PRS-1 protein
The presence of the PRS-1 protein in seeds demanded further
investigation ofthe organ specificity. The PRS- 1protein could
not be detected in seedlings or parts from mature flowering
plants other than seeds. A separate analysis of embryo and
remaining tissue (endosperm and seed coat) for the presence of
the PRS-1 protein by UTLIEF is shown in Fig. 3. The PRS- 1
protein was found in both the embryo and the remaining
tissue, but most of the PRS-1 protein present in seeds comes
from the embryo. Specific proteins for both the embryo and
the remaining tissue (see Fig. 3)indicate clear separation ofthe
embryo and remaining tissue and therefore exclude con-
tamination of both seed parts.
Figure 3. UTLIEF of the embryo and remaining tissue proteins (after
removal oftheembryo from the seed).(l)EmbryofromJacint0;(2)remain-
ing tissue from Jacinto: (3) F 1 variety Jacinto. M, p i marker proteins from
BDH. Note the tissue-specific bands near the cathode indicated with
arrows.
828 B. M . van den Berg Electrophoresis 1990, 11, 824-829

-PRS- I+
-PRS-I I

Figure 4 . UTLIEF (pH 6-9) of the F2

0 progeny of the F 1 hybrid variety Jacintofor
the genetic segregation of the gene Prs-I.

3.4 Mendelian segregation of gene Prs-1 3.5 Prs-I genotypes of F1 hybrid varieties from Western
European and American seed companies
From the specific occurrence of the PRS-I proteins as de-
scribed, it can be suggested that a single locus is involved in the A number of commercial F1 hybrid varieties have been
three PRS-1 phenotypes observed and that we are dealing with analyzed for distribution of PRS- 1 alleles. The results inTable
two alleloproteins. To give more conclusive proof for the in- 3 indicate that the presence of the Prs-l allele is restricted to
volvement of a single locus, an F 2 segregation (1 :2: 1) for the the F1 hybrids developed by Dutch seed companies. In the
gene had to be demonstrated 1241. Therefore, several F2 pop- Royal Sluis material it was found that the Prs-1‘ allele has a
ulations were analyzed for genetic segregation ofthe gene Prs- much higher frequency among “Moneymaker type” tomatoes
I (Fig. 3). The results presented in Table 2 show monogenic for the fresh market than among “industrial types”. This is
inheritance. also the case among the commercial F 1 hybrids from other
seed companies that are listed in Table 3. These findings sup-
Table 2. Segregation of the gene Prs-1 in three F2 generations showing port the conclusion that the electrophoretic method presented
monogenic inheritances) can generally be applied to all tomato varieties to be tested for
Cross Number of phenotypes found inbreds.
selfing o f F l +/+ I/+ 111 x2 (l:2:l) P
F2 Jacinto 50 116 12 4.21 0.13 4 Discussion
F2 Turquesa 49 114 71 4.67 0.11
F2 Bornia 52 120 71 3.00 0.22
A morphological test for genetic purity in most cases needs
a) Only + and 1 are given to indicate genotypes. several months before final results are gained because mature

Table 3. Prs-I genotypes (indicated here with + and 1 signs) of F 1 hybrid varieties from other seed companies
than Royal Sluis

Company F 1 hybrid Genotype Company F 1 hybrid Genotype

Asgrow Solar Set +I+ de Ruiter Aratino +I1
Sunny +I+ Bolero +/1
Bruinsma Candela +/+ Caruso +/1
Dombito +/+ Counter +/1
Enza Amfora +/ 1 Estafette 1/1
Buffalo +/i Evita 111
Ophir +/I Garanto 111
Hazera F A 121 +/+ Larma +/+
Moran Blazer +I+ Laura +/+
Northrup Quick Pick +I+ Liberto 111
Valerie +I+ Rondelo +/1
Peto Seeds Contessa +I+ Sonatine 111
Count +I+ Vilmorin Dona +I+
Duke +I+ Sluis en Groot Carpy +I+
Early Cascade +/+ Elcy +/1
Floritalia +I+ Dario +I+
Ferry Morse Jackpot +/+ Darus +/l
Rijk Zwaan Marcella lil Gc 719 +I+
Electrophoresis 1990, I I , 524-829
plants, bearing mature fruits, have to be analyzed. It needs a
large area of land or glass-house spacing, is labor-intensive
and, in cases where female lines are morphologically similar to
the F1 hybrid, the morphological test for inbreds may be
unreliable. The application of the electrophoretic method
based on UTLIEF has several advantages over using a detail-
ed morphological analysis of plants grown under standardiz-
ed conditions. First, an electrophoretic purity test is less time-
consuming when carried out on seeds or seedlings. As soon
as the seeds are available, one person can analyze at least
8 x 96 seeds on a routine basis in one day. This can be accom-
plished by running two gels simultaneously twice a day.
Second, the electrophoretic method is cheaper than the anal-
ysis of mature plants. Although electrophoresis is a relatively
expensive technology, routine application showed areduction
in the costs of inbred testing by about 80 %. Third, since elec-
trophoretic results on the genetic quality of seed lots are gained
much earlier, the seeds can be sold earlier, which consequent-
ly leads to a reduction in the cost of seed storage. The electro-
phoretic method of inbred testing has been applied until now
for more than 2 years by the Royal Sluis Production Depart-
ment. An evaluation of the application of the method describ-
ed here showed that the advantages ofthe methods as describ-
ed above have been proven in practice.
Organ-specificity was investigated since the PRS- 1 protein
might have been unique to endosperm tissue (which is triploid,
having two identical alleles from the mother parent). Then, a
genetic dosage effect could be expected, leading to a different
ratio in the concentration of the two PRS-I variants 125,261.
This ratio might interfere with determination of inbred fre-
quencies on aroutine scale, all the more so because the mature
tomato seed contains only a small amount of endosperm. We
therefore investigated the organ-specificity of the PRS- 1 pro-
tein, which showed that the PRS- 1 protein is located primarily
in the embryo. The PRS-1 protein could not even be detected
in seedlings. Therefore, it is only possible to determine the
PRS-1 phenotype of an individual, without destroying it, by
analysis of the seeds of mature fruits.
Of the several functional types of plant proteins, the enzymes
1271 and storage proteins [281 possess considerable polymor-
phism in several species. However, just as was found for en-
zymes, seed proteins from tomato showed little polymor-
phism. This confirms the narrow genetic base ofthe cultivated
tomato. Though open-pollinated varieties showed several
genetic variants (unpublished results), the parents of the F 1
hybrids showed even less protein polymorphism. This search
for genetic variability of seed proteins will be continued with
the use of isoelectric focusing in immobilized p H gradients
[291.
I thank the tomato breeders Ir. P.C.C.J. Hermans and Mr.
A .C.M. Struys f o r helpful discussions during the research,
Inbred testing of tomato FI by UTLIEF 829
my colleague Dr. R. Weges, and Dr. J. van Brederode (Uni-
versity of Utrecht)for suggestions that improved the manu-
script.
Received May 11, 1990
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