Isolation of leaf protoplasts from Pancratium maritimum L. and two other dune plants: Possible applications

Protoplast techniques can be applied for studying physiological and biochem ical processes in plant cells. In order to utilize these techniques, a meth od for isolation of viable protoplasts from dune plants was established. Us ing a combination of cellulase R-10 Onozuka, hemicellulase and pectinase pr otoplasts were isolated from younger leaves of Pancratium maritimum. Field- grown plants harvested in autumn or winter yielded approximately six times as many protoplasts per g of fresh tissue as did plants collected in spring (3.6-5.3 x 10(6) versus 9 x 10(5) protoplasts per g of fresh tissue). No p rotoplasts were released from plants harvested in summer. The production of protoplasts from cultivated plants reached high yields of protoplasts (3.6 x 10(6)) independent to the season. The viability of these protoplasts was 89.2% (+/- 1.1). Within 2-3 days of liquid culture 88.6% (+/- 3.6) of the protoplasts were able to regenerate cell-walls. First divisions were detect ed after 5-7 days of culture. By using the same procedure, yields of 2.31 x 10(6) and 1.28 x 10(4) protoplasts were obtained from field-collected leav es of Cakile maritima and Ammophila arenaria, respectively. Viability of pr otoplasts were 87.5% (+/-4.3) and 75.1% (+/-2.4), respectively. The potenti al applications of this method are discussed.