**3. Separation of isozymes by gel electrophoresis**

Isoenzymes can be separated by electrophoresis or isoelectric focusing. The isozymes under given proper circumstances — show peculiar patterns in the gel, which are called zymogramm (Hunter and Markert 1957).

Electrophoresis is a type of chromathography. The power for the separation of proteins is the difference in voltage between the two ends of the gel. The movement of proteins in the electric field is effected by their weight, shape and charge (Smith, 1960; Bálint and Bíró in Bíró, 1989).

The gel for the separation can be made from starch, agarose or acrilamide (Fig 2.). A standardized method of starch gel electrophoresis is used by UPOV (1996) for the analysis of identity of plant cultivars by isozyme analysis (Baum, 1986).

Advantages of the starch gel are that it is non-toxic, and more isozymes can be analysed by the slicing of a thick gel. More recently polyacrylamide gels are used because of their larger resolution. The porous structure of poliacrilamide gel is formed through a process of polymerization of acrylamide (CH2=CH-CO-NH2) and bis-acrylamide (CH2=CH-CO-NH-CH2-NH-CO-CH=CH2). As a result of polimerisation a colourless, diaphanous, flexible, and consistent gel arises, which is resistant to scalding or chilling. The density, viscosity and size of poles are determined by the concentration of acrylamide and bis-acrylamide. (Hajósné Novák és Stefanovitsné Bányai in Hajósné Novák, 1999).

Gel Electrophoresis of Grapevine (*Vitis vinifera* L.) Isozymes - A Review 71

Fig. 3. Isoelectric focusing patterns of horseradish (HP), and grapevine (GP) peroxidases. IEP isoelectric points, P1-P7 names of isoenzyme bands, AP application zone, PK cathodic peroxidase. -a) anodic and cathodic pattern of peroxidases obtained from different organs of *Vitis riparia*. L Leaf, R root, C callus. The bands of horseradish peroxidases are distorted due

"The utility of isozymes as genetic markers is generally attributed to their frequent polymorphism, codominance, single gene-Mendelian inheritance, rapid, simple and relatively inexpensive assay and their ubiquity in plant tissues and organs (even in embrios and pollen). Although the selective neutrality of isozymes has been debated, it seems highly probable that they are adaptive under certain circumstances." (Bretting and Widrlechner

Other advantages of isozyme analysis are the rapid analyses of samples, a small amount of plant material is sufficient. Young plants can be tested and selected based on their genotypes for features, which morphologically appear later. These can mean significant temporal and financial savings in the case of the breeding of annual plants. Now the best

Disadvantages of isozyme analysis as against the DNA markers, that they are organ-, tissueand developmental stage-specific (Fig 4.). They often go through post-transcriptional

to a margin effect on the slab. -b) Shematic diagram of the anodic part of peroxidase

**5. Advantages and disadvantages of isozyme analyses** 

cost-efficient markers are the isozymes (Bretting and Widrlechner, 1995b).

modifications, which limit their usage (Staub et al., 1996).

patterns. (Bachmann and Blaich, 1988)

1995a).

Fig. 2. Segregation of isozyme bands in PGM zone 4. Each column shows representative individuals of a selfed population of Cherry Bell-4. (Nomura et al., 1999.)
