Protoplast isolation from different explant sources in pitahaya (Selenicereus costaricensis, Cactaceae): insights from roots, callus, and shoot tissues

Efficient protocols for protoplast isolation in succulent plants, including cacti, are limited. In this study, we developed a protocol for isolating protoplasts from different tissues of pitahaya (Selenicereus costaricensis), including in vitro shoots and roots, greenhouse shoots, and callus cultures. We analyzed the effects of tissue source on protoplast yield and optimized the protocol by evaluating factors such as the composition of the enzyme solution and its pH, cell wall digestion time, shaking speed, and sucrose concentration during purification. Callus cultures yielded the highest protoplast numbers (2.5 x 10(6) protoplasts/g fresh weight), followed by greenhouse plant shoots (1.6 x 10(6) protoplasts/g fresh weight), while in vitro roots yielded the lowest (< 4 x 10(4) protoplasts/g fresh weight). Calcium oxalate crystals, mainly raphides, were observed in purification solutions containing damaged protoplasts from in vitro and greenhouse shoots, indicating a presumed detrimental effect on protoplast integrity. Conversely, no crystals or mucilaginous cells were observed in protoplast preparations from in vitro roots and callus cultures, facilitating the recovery of intact protoplasts. Protoplasts isolated from in vitro roots and shoots could not be effectively purified. Notably, greenhouse shoots digested exclusively with food-grade pectinase produced relatively high protoplast yields.