The Large-scale Isolation of Protoplasts from Immature Tomato Fruit

It has long been realised that the study of isolated protoplasts offers a potentially new approach to problems of plant cell biology. For this potential to be fully realised, intact protoplasts must be produced, free from contamination by cell debris, on a scale sufficiently large to enable biochemical and physiological experiments to be performed. This aim, though not impossible as has been suggested (1), is not easily achieved because the extreme fragility and physical characteristics of intact protoplasts prevent them from being handled by normal techniques such as centrifugation, filtration, or even pouring from one vessel to another. Recently it was shown that plant protoplasts could be isolated in large numbers by treating the parencbymatous placental tissue of immature tomato fruit with commercially available pectinase enzymes in a plasmolysing medium (2). This communication will now describe the apparatus and techniques developed for the large-scale production of these protoplasts. Because of the complex composition of the plant cell wall, the effect of limited enzymic treatment was to weaken, rather than completely dissolve, the treated walls. However the weakened walls of pectinase-treated, plasmolysed parenchymatous placental tissue of tomato could be teased apart so gently as to release protoplasts in enormous numbers compared with the few obtained by the more vigorous teasing necessary for untreated plasmolysed tissues (3-6). To produce an uncontaminated preparation of protoplasts on any scale required the execution of three operations: (a) enzyme treatment; (b) the release of protoplasts fi'om within the weakened cell walls; and (c) separation of the intact protoplasts from cell debris.