To isolate protoplast, a pre-treatment was completed with the order to reduce and
identify the phenolic contents round the year to encourage the isolation of protoplasts. Protoplasts
from in vivo mesophyll leaves of apple cultivar “Anna” was isolated from 15 days old leaves by
plasmolying in medium containing 90 g L-1 mannitol for half hour, then 130 g L-1 mannitol for
half hour. Then using enzymatic mixture involving (1.5% cellulase + 0.5% pectianase + 1.5%
Macrozyme) Prior to isolation. Anyhow, diverse factors, for example, Osmotic pressure, incubation
period, sieve pore size, centrifugation period and hormonal balance was estimated using the
techniques for isolation. The quantity of cells was computed as the quantity of cells per square
on haemocytometer. A considerable higher yield of protoplast formation was noted in the CPW
medium using a pore size of 25 μm with using incubation period for 20 hours. Moreover, the best
protoplast regeneration with using of protoplast density of 2.0 x 105 in MS medium supplemented
by 1.0 mg L-1NAA and 0.3 mg L-1BAP. We believed that our protocol might encourage the plant
recovery using in apple somatic hybridization programs.