engleski

The identification and charachterization of esterases related to morphogenesis in Mammillaria gracillis tissue culture

Cactus Mammillaria gracillis Pfeiff. (Cactaceae), cultivated in vitro, spontaneously switch from an organised to unorganised way of growth, producing a habituated organogenic callus which regenerates normal and hyperhydric shoots without the addition of growth regulators. Tumour tissues TW and TR, induced by A. tumefaciens strain B6S3 and GV3101, do not express any organogenic potential. The esterase (arylesterase EC 3.1.1.2 and carboxylesterase EC 3.1.1.1) activity and isoenzyme pattern of cactus tissues (shoot, callus, hyperhydric regenerant, tumours TW and TR) were tested every 7 days during the period of one subculture (28 days). Two esterase substrates, 1- and 2-naphthylacetate, were used. In shoots and tumours, the highest esterase activity for both substrates was measured on the 21st day, while in the callus and hyperhydric regenerants it had the highest value on the 7th day. Esterases were separated electrophoretically in polyacrylamide gradient gels under native conditions. In total, 12 isoesterases, reacting with both substrates, were resolved. No differences in isoenzyme profile were noticed in correlation to the age of tissues. A selective inhibitor for carboxylesterase, bis-p-nitrophenyl phosphate (BNPP, 100 mM) was used to better distinguish the esterases. The higher degree of inhibition (50%) was obtanied in all samples when 1-naphthylacetate was used as a substrate than when 2-naphthylacetate was used (30%). No color reaction was found on the gel for the isoesterase E10 with BNPP and 1-naphthylacetate or for isoesterase E5 with BNPP and 2-naphthylacetate.

Mammillaria gracillis; esterases

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