Factors that influence the yield and viability of protoplasts from Carica papaya L.

Abstract

The effects of four main factors (the concentration of cellulase R-10, macerozyme R-10 and D-mannitol, and duration of enzyme incubation) determining yield and viability of protoplasts isolation were investigated using Taguchi experimental design to optimize protoplasts isolation conditions from the leaves of Carica papaya L. seedlings. The results showed that the concentration of cellulase R-10 and incubation time were key factors affecting the protoplast yield; D-mannitol concentration played a significant role in protoplast viability. Moreover, the result of single factor tests revealed that pH of the enzyme solution significantly influenced both yield and viability of protoplasts. The highest yield of more than 1.5 × 107protoplasts g-1 fresh weight (FW) with more than 90% viability were consistently obtained by the optimum isolation conditions: enzyme combination of 1.2% cellulase R-10, 0.3% macerozyme R-10 and 0.44 M D-mannitol, pH 5.8 and incubation for 13 h at 26°C with a shaker speed of 60 rpm in darkness. This study will facilitate to obtain sufficient yields and viability of protoplasts for subsequent novel applications in papaya biotechnology such as the gene functional analysis and mechanism of PRSV infection in papaya protoplasts. Key words: Carica papaya L., isolation conditions, protoplasts, Taguchi experimental design, viability, yields.