Protoplast isolation, culture and regeneration in Mango (Mangifera indica L.)

Abstract

Attempts were made to standardise techniques for isolation, culture and regeneration of protoplasts in mango, during 1998-1999 at the Plant Molecular Biology and Biotechnology Centre, College of Agriculture, Vellayani. Efforts were made to standardise the source of explant, optimum concentration, temperature and duration of incubation and osmolarity of the enzyme mixture. Callus induced from nucellus of immature fruits was found to be the best plant material for protoplast isolation. The cell wall digesting enzymes Cellulase 'Onozuka' R-I0 and Macerozyme R-lO at l.0 and 0.5 per cent respectively at a pH of 5.8 yielded the highest number of protoplasts (101 protoplasts per " field). The optimum time of incubation was found to be eight hours. Pre- plasmolysis of the callus was found to be not beneficial. Treatment without pre-plasmolysis and with 9.0 per cent osmolarity recorded the highest yield. Incubation temperature of 28.0 "c was found to be optimum for the best yield of protoplasts. Cell wall formation and microcalli development from the protoplast was observed when Murashige and Skoog medium with half strength of major salts, supplemented with BAP 3.0 mg r', NAA 0.1 mg r '

along with sucrose or glucose 90.0 g r ' was used.

Combinations of

osmoticums like sucrose 70.0 g r', mannitol 10.0 g r' and glucose 10.0 g r' as !. well as sucrose 70.0 g r', mannitol 10.0 g r' and inositol 10.0 g r' were found to be ideal for cell wall formation and micro calli development.