Influence of Hydrolytic Enzymes On Protoplast Isolation in Carica Papaya L. Var. Maradol

Papaya (Carica papaya L.) is one of the most important fruit crops grown in Mexico, however, this crop is highly susceptible to Papaya ringspot potyvirus (PRSV-P). Somatic hybridization by means of the fusion of protoplasts from papaya and its well known PRSV-P-resistant relatives represents one of the most important efforts to reduce the PRSV-P incidence in this plant species, therefore, efficient protocols for isolation and purification of viable protoplasts from these species are necessary. The aim of this work was to evaluate several concentrations of mixtures of the hydrolytic enzymes cellulase, pectinase and driselase in order to assess their response in yield and viability of protoplasts isolated from somatic embryos of papaya variety Maradol. Papaya somatic embryos were placed in Petri dishes containing 10 mL of each of the solutions composed of the enzyme mixtures, and then incubated on a rotary shaker at 33 rpm, under darkness at 25ºC for 16 h. After that, protoplasts contained in the obtained suspension were isolated and purified through filtration and centrifugation, followed by resuspension in sucrose. Viability of the protoplasts was assessed by staining them with methylene blue dye, and counting up was made using a hematocytometer under light microscopy. The mixture of hydrolytic enzymes composed of 1% cellulose + 0.2% pectinase + 0.2% driselase produced the highest protoplast yield (1.22 x 10⁶ protoplasts per gram of fresh weight of somatic embryos). On the other hand, 34.24-60.76% were the two highest viability values obtained with all combinations with the three enzymes, except those with 3% cellulase, where the lowest viability values (0-9.95%) were obtained. Hydrolytic enzymes which have more influence in these two evaluated variables were cellulose from Aspergillus niger and driselase from Basidiomycetes spp., whereas the effect of pectinase from Rhizopus sp. was not significant.