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M O L EC U LA R B IO LOGY OF PLANT C E L L S IN V ITRO

THE COMPARATIVE STUDYOFMITOCHONDRIALGENOMEAND

ESTERASE ISOENZYMECOMPOSITION INMORPHOGENICAND

NONMORPHOGENIC CALLUS TISSUES

OFPISUM SATIVUM

A.G.Mardamshin, M .I.Gubaidullin, F.A.Ishtiryakova, K.R.Ziyakaeva, R.D .Valieva

Department ofBiochemistry and Cytochemistry, UfaScientific Centre, Russian Academy

o f Sciences, 450054, Ufa, Prospekto fOktyabrya, 69, Russia, E-Mail:

The comparative analysis of endonuclease restriction profiles of mtDNA

and esterase isoenzyme composition was carried out in 14-month pea callus

tissues which differed in their capacity to morphogenesis. Both types of

callus tissues were grown in darkness on Murashige and Skoog nutrient

medium.

Extracts for determination of esterase activity were prepared by crushing

callus tissue samples in cooled mortar in ratio 1:1 with extraction buffer

containing 0,01 M Tris, 0,25 M sucrose, 0,005 M EDTA. pH of medium was

adjusted to 6,8 with help o f ascorbic acid. The homogenate was centrifuged

at 5000 rpm. Electrophoretic separation of proteins was conducted in 7,5%

polyacrylamide gel. Relative electrophoretic mobility (REM) calculated with

reference to bromphenol blue. Electrophoretic analysis of esterase isoenzyme

composition showed that 7 components were present in two types of callus

tissues. Among them the isoforms with relative electrophoretic mobility 0,5;

0,67; 0,7 have approximately the same activity. The differences in activity

were observed among isoforms with REM 0,6; 0,8; 0,87.

Mitochondrial DNAs from callus tissues were isolated using the method

o f phenol deproteinization. mtDNA digestion by restriction endonucleases

EcoRI, Hind III, Pstl with the following electrophoretic separation in 0,8%

agarose gel was carried out conventional method. Restriction endonuclease

analysis of mtDNA preparations of morphogenic and nonmorphogenic callus

tissues using the mentioned above enzymes revealed no differences.

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