ISOLATION AND CULTURE OF PROTOPLAST FROM LEAVES OF POTATO (Solanum tuberosum L.)

Abstract

Leaves taken from 3-4 week-old cuttings of Solanum tuberosum L. AVRDC1297.19 were used as a source for mesophyll protoplast. Protoplasts were prepared by digestion in 0,05% (w/v) cellulase (Sigma) and 0,025% (w/v) pectolyase (Sigma) in 6% sorbitol. Protoplasts were first placed in liquid medium suppplemented with 250mg/l polyethylen glycol (PEG, PM 6000), 0,05mg/l benzyl adenine (BA), Img/l a-naphthalene acetic acid (NAA), and 0,02mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), in the dark, at 27°C for 7 days. The protoplasts reformed the cell wall within 2-4 days and underwent sustained divisions leading to the formation of small clusters of cells within 7 days. Afterwords, in order to stimulate the growth of calli, the cultures were trasferred onto different media, at 16-h light regime, 2500-3000 lux, and 27°C. Small granular callus were obtained on hormone-free MS medium.