Abstract
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±0.50 106 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
106 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 105 protoplasts ml-1), culture of protoplasts at 1 x 105 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.
