Dalbergia sissoo
is an important timber tree facing mysterious die back and wilting problem. In
case of die back,
Dalbergia is facing the threat of destruction in its natural habitats due to lack of potential
pathogenicity test which is the major bottleneck in pathogen assessment and tree improvement programmes.
Isolation of protoplasts was attempted to produce an effective source for the pathogenicity test. This study
described a procedure for the rapid isolation, in high yield, of photosynthetically active mesophyll protoplasts from
young leaves of
D. sissoo. The present study reports the isolation of protoplasts from leaf mesophyll of
D. sissoo.
Leaf strips were suspended in the enzyme solution for the isolation of protoplast. Different concentrations of
enzymes were used to optimize the suitable combination for the protoplast isolation. Enzyme solution turned green
after a gentle swirling motion, which indicates the release of protoplasts. Release of protoplast was checked in the
solution under the microscope. A combination of filtration, centrifugation and washing was used to purify the
protoplasts. The optimum combination of enzymes for protoplast isolation was 1.5% cellulase R-10+ 0.5 %
pectinase R-1 after incubation for 6 hr at 28° C. The isolated protoplasts were round and filled with chloroplasts.
The size of protoplasts was 20~35 µm. The protoplast yield was 2 × 10
5 per g of leaf tissue. The protoplast
viability as assessed by 0.01% Phenosaphranine staining was 77%.
