Isolation of protoplasts from seedlings

For efficient genetic manipulation, the isolation and culture of protoplasts and plant regeneration are important. Our objective in the protoplast study was to use complementation experiments with mutants blocked at specific steps in anthocyanin biosynthesis. Initial experiments were conducted to see the efficiency of protoplast isolation from different explants (leaves, stem sections, root sections, suspensions and callus cultures). Leaves and stems collected from 7-8 day germinated seedlings of A188 under normal conditions of light at 25 ± 1 C were sliced into thin sections prior to enzyme treatment (1 gm tissue/10 ml mixture) with cellulase (0.2%), macerozyme (0.1%), pectinase (0.1%) and mannitol (0.3M). The incubation time varied for leaf and stem sections. Within 2-3 hrs of incubation, stem protoplasts of various sizes were observed, whereas leaf mesophyll protoplasts were observed after 5-6 hrs. Best yields of protoplasts (1X106/gm tissue) were obtained from stem sections compared to leaf sections. After the incubation, the suspension was filtered through cheese cloth to separate larger pieces of leaf and debris, followed by centrifugation at 200 g for 5-10 min to sediment protoplasts. The sediment was later rinsed in washing solution (medium containing 0.3M mannitol). The protoplast sediments were suspended in 25% sucrose solution and centrifuged at 100g for 5-10 min. Intact protoplasts, which float on the upper layers, were collected and plated at required density onto P2 culture medium (Potrykus et al., TAG, 347-350, 1977). The optimization of culture conditions for callus initiation is in progress.

P. Suprasanna, K.V. Rao and G.M. Reddy

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