ELECTROPHORETIC ANALYSIS OF PISUM SEED AMYLASES

PNL Volume 15

1983

RESEARCH REPORTS

ELECTROPHORETIC ANALYSIS OF PISUM SEED AMYLASES^1/

Zimniak-Przybylska, Z

J. Przybylska

and

Institute of Plant Genetics

Polish Academy of Sciences, Poznan, Poland

Zymograms of pea seed amylases were reported to display two anodic

variant zones of enzyme activity (2); bands forming the faster moving

zone Amy-1 were well-defined while those in the slower moving zone Amy-2

were faint. In 108 accessions, representing different Pisum forms, six

single-banded phenotypes in each zone were distinguished (2).

Separation and detection of Amy -2 variants have since been

improved. In a modified technique, electrophoretic separation i3 per-

formed in a discontinuous buffer system; resolving and stacking

polyacrylamide gels are prepared according to Davis (1) and 0.125 M

tris-borate buffer, pH 8.9 (3) is used as an electrode buffer. Starch

(0.3%) is incorporated into gels. After electrophoresis, gels are in-

cubated in 0.2 M acetate buffer, pH 5.3, for 5 hrs and then stained with

I₂-KI solution. Under the above experimental conditions zone Amy-1 is

not revealed.

The 108 Pisum accessions previously examined have now been

reanalyzed with this modified technique. The comparative

electrophoretic analysis was performed in slab gels and the distinction

of Amy-2 variants was based on the observed differences in migration

distance. In total, eleven Amy-2 variants could be distinguished. It

should be stressed, however, that differences in electrophoretic

mobility among some of the successive variants are so small that the

bands could not be resolved if a mixture of the respective extracts were

subjected to electrophoresis. The distinguished Amy-2 variants

(phenotypes) are shown in Fig. 1. Variants designated now as 2c₁, 2c₂,

and 2c₃were not separated in the previous investigation and were clas-

sified as variant 2c. Similarly, variants 2e₁, 2e₂, 2e₃, and 2e₄ were

previously classified as variant 2e.

The modified technique has revealed an additional polymorphism of

the Amy-2 zone in ecotypes el.atius, humile, and sativum. The most com-

monly occurring Amy-2 phenotypes were variants 2c₂, and 2e₃. Variant 2c₂

was found in 40 Pisum forms classified as P. humile, P. sativum,

P. abyssinicum. and P. fulvum. Variant 2e₃ was observed in 3 2 P.

sativum accessions and in P. elatius. W 805. "Variants 2a, 2b, 2c₃, and

2d were found in single accessions indicated in Fig. 1. The distribu-

tion of Amy-2 phenotypes seems to show no correlation to this taxonomic

scheme.

1. Davis, B. J. 1964. Ann. New York Acad. Sci. 121:404-427.

2. Przybylska, J., S, Blixt, H. Parzysz, Z. Zimniak-Przybylska. 1982.

Genetica Polonica 23:103-121.

3. Stegemann, S. , H. Francksen, and V. Kacko. 1973. Z. Naturforsch.

28:722-732.

^1/ Cooperative investigation with Dr. S. Blixt from the Weibullsholm

Plant Breeding Institute, Landskrona, Sweden, performed under an

Agreement between the Royal Swedish Academy of Sciences and the

Polish Academy of Sciences. The project involves cooperation with

Dr. Ch. Lehmann from "Zentralinstitut fur Gene tik und

Kulturpflanzenforschung" of German Academy of Sciences, Gatersleben,

GDR.

PNL Volume 15 1983

RESEARCH REPORTS <> I

mi an

Fig. 1. Amy-2 phenotypes in pea seeds. The phenotypes shown are

produced by the following accessions: a - The accession of

P. fulvum obtained from the Hebrew University of Jerusalem; b -

Gat. 2 55, P. elatius; c₁ - W 809, P. sativum; c₂ - W 1 998,

P. sativum; c₃ - W 1951, P. sativum; d - JI 224a, P. fulvum ;

e₁ - W226, P. elatius: e₂ - W 1447, P. elatius: e₃ - W 1201,

P. sativum: e₄ - W 1968, P. sativum: f - W 1647, P. sativum.

Editor's Note: In order to maintain a consistent system of nom-

enclature for the amylase variants observed, the authors have

used subscripts to designate further variation within mobility

classes (a-e) originally reported in Przybylska, et al. Genetica

Polonica 23:103, 1982. This somewhat unconventional format may

lead to very cumbersome allelic designations should additional

cryptic variation be revealed in further studies. We respect-

fully suggest that after the investigators have completed their

analysis of amylase phenotypes and the genetic basis for the

observed variation has been determined, the allelic designations

be modified to a more conventional and convenient form. The

rules for genetic symbols (PNL 9:67-70) do not explicitly deal

with this problem, but since more and more biochemical markers

are being reported, it is important that we develop a consistent

system which is acceptable to most, if not all, Pisum genetic-

ists.