50 PNL Volume 19 1987 RESEARCH REPORTS
J. Przybylska1, S. Blixt2, Z. Zimniak-Przybylska1,
and J. George1
1- Institute of Plant Genetics, Polish Academy of Sciences,
Poznan, Poland
2- Weibullsholm Plant Breeding Institute, Landskrona, Sweden
Two procedures, called here Method A and Method B, were used
to detect variation in electrophoretic patterns of pea seed amy-
lases (2,3). In Method A proteins were separated in 9% polya-
crylamide gels, in a discontinuous buffer system according to
Davis (1); following electrophoresis gels were incubated for 5 hrs
in 1% solution of soluble starch in 0.2 M acetate buffer, pH 5.3,
and then stained with I2-KI solution(2). In Method B the electrode
buffer was 0.125 M Tris-H3B03, pH 8.9, instead of Trisó
glycine, pH 8.5, and hydrolyzed starch was incorporated into gels
instead of being put into the incubation mixture (3). In both
procedures, electrophoresis was continued until after the dye mar-
ker (bromophenol blue) had reached the anodic edge of a gel.
Zymograms obtained with Method A showed two anodic variant
zones of enzyme activity; the faster moving bands, forming variant
zone Amy-1, were well defined while the slower bands of Amy-2
variant zone were very faint, scarcely discernible (2). In Method
B separation and detection of amylase-variants were significantly
improved but Amy-1 variants were not revealed; marked destaining
near the start of migration was at first not attributed to the
Amy-1 variant zone (3).
In further investigations with the use of Method B different
commercial products of hydrolyzed and soluble starch were tried.
Results obtained with the hydrolyzed starch and with the soluble
starch "Analar" from BDH were as reported earlier. However, zymo-
grams obtained with the use of a soluble starch purchased from
POCh, Gliwice, Poland, showed slow migrating bands forming an ad-
ditional variant zone. The additional zone seemed to correspond
to the Amy-1 zone on zymograms obtained with the use of Method A
(Fig. 1). It was therefore assumed that under conditions of
Method A and Method B variants forming Amy-1 and Amy-2 zones mi-
grate in a reverse order. This was confirmed by eluting the Amy-1
and Amy-2 variant bands from a gel obtained with Method A, fol-
lowed by re-electrophoresis under conditions of Method B (Fig. 2).
It should be mentioned, however, that in the case of the Amy-2
variant, besides the characteristic fast moving band two addi-
tional slow moving bands could be seen. These additional bands
are probably artifacts, possibly aggregations.
The order of electrophoretic mobilities of the respective
Amy-1 variants in gels obtained with Method A and Method B seems
to be similar (see Fig. 1). However, on screening a wider plant
material differences may be found.
1. Davis, B. J. 1964. Ann. New York Acad. Sci. 121:404-427.
2. Przybylska, J., S. Blixt, H. Parzysz, and Z. Zimniak-
Przybylska. 1982. Genetica Polonica 23:103-121.
3. Zimniak-Przybylska, Z., S. Blixt, and J. Przybylska. 1985.
Genetica Polonica 26:303-306.
This work was supported by the Polish Academy of Sciences under
the Project CPBP 05.01.

Fig. 1. Zymograms of seed amylases from several Pisum lines, obtained
under conditions described as Method A (A) and Method B (B)
(see text).
1-5 -- extracts from the following lines:
P. sativum W 110, P. elatius W 226, P. sativum W 809,
P. sativum W 1201, P. sativum W 1998.
Fig. 2. Amy-1 and Amy-2 variants from Pisum sativum W 110
separated using Method A (A), individually eluted
from unstained gel fragments, and subjected to
re-electrophoresis using
Method B (B).