When the surviving protoplasts were cultivated, they did not divide
and approximately 80% died after 5–14 days. Older cell suspension
cultures also contained more compact cell clumps than friable cell
clusters, the former being unable to undergo somatic embryogenesis.
Our data agrees with earlier studies of woody plant species,
in which friable cell clusters were the most useful for protoplast
isolation and plant regeneration.
The efficient regeneration of protoplasts from different plant
species depends strongly on the medium composition. KM8P
medium is frequently used to culture protoplasts from woody plant
species, and this was based on the protoplast culture medium
developed for Vicia hajastana [17]. Although Y3-based media have
not been used to cultivate oil palm protoplasts before, we found
that Y3A medium was the most suitable for this purpose. In contrast,
MS-based media and A medium [16] achieved only a low
frequency of protoplast division. The macroelements and microelements
in our Y3-based media were similar to the original Y3
medium, although we added KH2PO4 to Y3A and Y3D–F, which
increased the frequency of protoplast division compared to Y3-
based media without KH2PO4 (Y3, Y3B and Y3C). Protoplasts