CHANGES IN THE LEVELS OF ENDOGENOUS PHENOLICS, AROMATIC MONOAMINES, PHENYLALANINE AMMONIA-LYASE, PEROXIDASE AND AUXIN OXIDASE ACTIVITIES DURING INITIATION OF ALFALFA EMBRYOGENIC AND NONEMBRYOGENIC CALLI

Culture of alfalfa (Medicago sativa) leaf explants on the medium suppl emented with 100 mu M 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 mu M kinetin induced extensive cell division and callus proliferation. Al though the exposure of explants to the high 2,4-D level evoked a large increase in phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity, t he total content of phenolic acids in cultured explants dramatically d ecreased during the first 6 days. Culture on high 2,4-D medium induced in leaf segments significant alterations in the proportional contribu tion of free, soluble ester-, glycoside- and cell wall-bound phenolic acids to their total content. Significant increase in the content of f ree phenolic acids and marked decrease in the content of phenolics inc orporated into the cell walls during 6 days of explant culture (period of dedifferentiation and intensive cell division) was determined. Mar kedly increased levels of free, soluble bound and insoluble bound tyra mine and phenylethylamine determined in explants coincided with the pe riod of strongly promoted cell division. Primary calli were transferre d from the induction medium to the different auxin supplemented media leading to the formation of either embryogenic (EC) or nonembryogenic (NEC) callus cultures. The PAL activity 20 days after subculture was i n EC twice as high as the activity determined in NEC but there was no difference between their total contents of phenolic acids. Higher prop ortion of cell wall-bound phenolic acids was observed in embryogenic c ells (about 10%) compared with both nonembryogenic and primary callus cells (5-6%). Significantly higher total extracted peroxidase (PO, EC 1.11.1.7) activity was found in NEC than in EC. Although EC showed a l ow total PO activity, ionically bound PO represented 20% of the total activity (in NEC only 7%). The level of aromatic monoamines was higher in EC, especially the content of soluble tyramine was 3 times higher. A correlation between aromatic monoamine levels and differentiation p rocesses occurring in EC was observed.