Senescence development of Brassica napus leaf protoplast during isolation and subsequent culture

Putrescine, ammonia and ethylene increased throughout isolation of leaf protoplasts from an inbred line of Brassica napus, suggesting that they are parts of a senescence response. There was a 2.7-fold increase in the putrescine: spermidine + spermine ratio of leaf protoplasts; the ratio remained higher during the subsequent culture. Swelling of Brassica napus leaf protoplasts occurred in both MS (Murashige and Skoog, 1962) and an ammonium nitrate-free MS (MS(-NH4+)) medium, but did not undergo sustained divisions. Concentrations of soluble protein and chlorophyll gradually decreased in the cultured protoplasts, bur there was an increase in ethylene release, which are apparently signs of senescence development. Culture conditions had no significant effect on the progress of senescence-associated symptoms. There was a rapid decline in glutamine synthetase (GS, EC 6.3.1.2). In contrast, glutamate dehydrogenase (GDH, EC 1.4.1.2.) activities increased progressively over the first two days. Increases in glutamate content and GDH activity of freshly prepared leaf protoplasts occurred in parallel.