A rapid method for isolation and purification of protoplasts from epidermal tissue of Vicia faba L

BARRANTES FJ; ZANELLO LP; CURVETTO NR

Mircen Journal Of Applied Microbiol Biotechnol

Springer Netherlands

Lugar: Amsterdam; Año: 1988 vol. 4 p. 275 - 283

A method has been developed for isolating and purifying epidermal and guard cell protoplasts (ECPs and GCPs) from leaves ofVicia faba L. This method has three essential characteristics: 1) requires only small quantities of initial plant tissue; 2) is rapid, being based on a two-step enzymatic digestion and purification by discontinuous density gradient centrifugation using Percoll, and 3) gives a high viability of purified protoplasts. The procedure yielded about 6% ECPs and 10% GCPs on the basis of their occurrence on epidermal foliar tissue, the final suspension of protoplasts being 100% pure. Cell viability was assessed by the ability of each protoplast type to accumulate neutral red in their vacuoles. Values of 90% and 95% were obtained for ECPs and GCPs respectively. The complete lack of cell wall after enzymatic treatment was checked at the light microscope level by the absence of Calcofluor fluorescent staining of cellulosic material. Representative counts for purified ECPs and GCPs obtained at the interfaces of 20/30% and 40/80% Percoll gradients were 1.32×105 and 3.7×105 elements/ml, which represents a yield of 930 and 2,200 protoplasts/cm2 of epidermal tissue respectively. The integrity of the plasma membrane and organelles after the isolation procedures was confirmed by transmission electron microscopy and by the ability of protoplasts to exclude Evans blue.