IMPROVED ISOLATION AND CULTURE OF PROTOPLASTS FROM S. chacoense AND POTATO: MORPHOLOGICAL AND CYTOLOGICAL EVALUATION OF PROTOPLAST-DERIVED REGENERANTS OF POTATO CV. DESIREE

The study reports the establishment of an improved and repeatable protoplast-to-plant protocol using axenic cultures of wild diploid species S. chacoense Bitt. and potato cv. Desiree and cell suspensions alone of cv. Desiree, as a pre-requisite to somatic hybridization. The protoplast isolation was influenced by type and age of explants, composition of enzyme and wash solutions, and duration of explant incubation in enzymes. Five different enzyme mixtures having varied combinations of Cellulase R10, Macerozyme, Hemicellulase, Cellulase RS, Pectolyase Y23 and Xylanase were used. The Xylanase solution produced the highest yield of viable protoplasts in both the species. Leaf explants were taken from 10, 20 and 30 days old axenic cultures, with 20 days old cultures being the highest protoplast yielders (7.61 +/- 0.31 and 9.74 +/- 0.50 10(6) protoplasts/ g fresh weight for S. chacoense and cv. Desiree, respectively). A decline in the yield and viability of protoplasts was also observed with the cell suspension age. The suspensions three days after subculture produced the highest yield (10.20 +/- 1.13 10(6) protoplasts/g f.wt.) of viable protoplasts (96.66 +/- 0.72%). Out of four plating densities tested (0.5, 1.0, 1.5 and 2.0 x 10(5) protoplasts ml(-1)), culture of protoplasts at 1 x 10(5) protoplasts ml(-1) proved efficient for enhanced mitotic division of protoplasts. Mesophyll protoplasts of S. chacoense being non-totipotent, failed to regenerate. Whereas, the protoplasts of cv. Desiree regenerated plants with cytological and morphological variations. Cell suspension protoplast-derived regenerants had higher somatic chromosome numbers and a wide range of aneuploidy (2n=4x=48+2-to-8). However, a much higher percentage of leaf protoplast-derived regenerants had 2n=4x=48 chromosomes and variants had close to normal chromosomal complement (2n=4x=48+2). The protoplast-to-plant regeneration of potato under the same culture conditions where leaf protoplasts of S. chacoense remained non-totipotent, may be exploited as a unilateral selection strategy for possible somatic hybrids of S. chacoense and potato.