Hairy root cultures of red beet (Beta vulgaris) were permeabilized applying different physical factors-pH, sonication, temperature, oxygen starvation and osmotic stress for recovery of betalaines. Exposure of hairy roots to acidic nutrient media of pH 2.0. 3.0 and 4.0 released about 70, 15, and 10% of the betalaines respectively to the medium within 30 min with 90% loss of viability at pH 2.0 and no loss of viability at higher pH. Reduction of treatment time at pH 2.0-5, 10, 15 and 20 min released 4.5, 9.2, 14.9 and 20.7% of the pigments with slight improvement in viability. Further, water at pH 2.0 effluxed betalaine more rapidly than culture medium of the same pH. pH also imparted a substantial shift in absorbance and hue value of the effluxed pigment. Sonication, tried for the first time for beet hairy roots, with continuous ultrasound of 0.02 MHz for periods 15, 30 and 60 s released only 8% of the pigments and 120 s treatment effluxed 12% without the loss of viability. However, post-sonicated roots could be used for the second time sonication and recovery of about 10% pigments after 10 days after which the viability was totally lost in all the treatments. Temperatures of 40, 45 and 50 degreesC released about 5.4, 31 and 47% colour in 30 min and about 13.4, 40.2, and 47.5% in 60 min where the viability was drastically affected at 40 degreesC with no viability at higher temperatures. Osmotic stress did not release any betalaines even at a high level of 5 M sorbitol treatment for 24 h. Oxygen stress, induced by cessation of gyration, released about 25% of pigment only in the presence of light over a period of 50 h. In all the treatments the effluxed betalaine was more stable, without any drastic reduction of the hue, when the culture medium was used for permeabilization instead of water. Treatment of permeabilized hairy roots with additional calcium (20 mM) in the medium for improvement of cell membrane integrity did hardly impart any positive effect in terms of viability. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.