Embryo and Protoplast Isolation from Barlia robertiana Seeds (Orchidaceae)

In the present study, it is aimed to investigate embryo and protoplast isolation from orchid seeds, because of know-how deficiency. As material Barlia robertiana seeds were subjected enzymatic process. Globular embryos with suspensor were fully removed by enzymatic maceration for between 100-180 min of incubation. The highest obtained embryo yields were found in Rapidase EX Color and Rapidase Vino Super, with degree of 89.2-90.3 × 103·g-1 MF and 86.4-91.3 × 103·g-1 MF, respectively. According to multicomponent maceration tests, a treatment comprising 150 min of the enzymatic reaction with 0.18% (E/S) at 50℃ was the most optimal for obtaining high embryo yields. Regarding protoplast isolation tests, enzyme combinations C (1% Cellulase “Onozuca” R-10 + 0.2% Macerozyme R-10 and 0.1% Driselase) and F (1% Cellulase “Onozuka” + 1% Macerozyme + 0.5% Driselase) after 15 hours produced the best results for protoplast isolation and were significantly different compared to other enzyme combinations. Greater protoplast yields were obtained using a rotatory system with protoplasts incubated in the dark. Also the present findings were discussed from the point of view of in vitro plant manipulation and orchid cultivation in cultural media and then their importance emphasized in molecular biological/genetical studies.