Isolation, purification and fusion of protoplasts of babaco (V. heilbornii) and jigacho (V. stipulata)

Abstract

In a first stage, disinfection and induction protocols about callous and in vitro leave were standardized; it’s a necessary material for the isolation, purification and fusion of protoplast. Protoplast isolation stage was evaluated for three enzymatic solutions with cellulose: Onozuka R-10 (1,5 2,25 & 3% p/v), macerozyme: R-10 (1,5 2,25 & 3% p/v) and pectolyase: Y-23 (0,2 0,3 & 0,4% p/v) above callus and leaves in both species; higher yields of protoplast number and times was achieve with enzymatic solution: cellulase-macerozyme (3%) and pectolyase (0,4%), it was incubated at 26-28oC during 33 hours for leave and 7 hours for callous. Protoplasts isolation were successfully purified using Jadán (2000) techniques that consist in three purification types (filtration, direct centrifugation and aqueous two-phases systems), in this stage, BH3 media (specific by protoplast) was used, sucrose and mannitol were used as osmotic stabilizers. Babaco callous protoplast purification with jigacho leaves protoplast where fused with a PEG solution (polyethylene glycol) and a A+B solution was used for the cellular membrane osmotic stabilizer. In addition, BH3 media was used in some washes to eliminate PEG, after finished the fusion process, cells were observed in an optic microscopy. A successful application for technique of fusion protoplasts demands a protocol for plant regeneration from these “new protoplasts”.