Protoplast isolation, fusion and plant regeneration in African violet (Saintpaulia ionantha)

In this study, protoplast isolation and fusion were performed in vivo and in vitro grown purple and white flowering African violets. Also, in vitro micropropagation protocol was obtained. At the results of surface sterilization experiments, success rate of sterilization is achieved as 94.7% in leaf explants by STK2 sterilization method. Direct shoot regeneration was obtained as 100% success rate from these sterilized leaf explants cultivated in MS nutrient media (Murashige and Skoog, 1962) that contains 2mg/L BAP, 1mg/L NAA. After 8 weeks culture time, shoots were taken rooted on MS medium with successful rate of 100%. Rooted plantlets were transferred into a mixture of peat/perlite (1:2) for acclimatization. The best result of the protoplast isolation experiments established from the leaves of African violets having purple and white flowers grown in vitro and in vivo conditions, was obtained as 130x103 by incubation within enzyme mixture containing 1% Pectinase, 2% Cellulase and 2% Hemicellulase under dark conditions for 16 hours without pre-application from in vivo grown purple African violet leaves. The highest microcallus regeneration (50 microcallus number /10mL) was established in protoplasts originated from in vivo. Protoplasts isolated from leaves of purple and white flowering African violets grown in vivo and in vitro conditions were colored with fluorescent dye Höechst333422 before the PEG fusion. At the results of this fusions, 397 multiple fusions in PEG 8000 and 69 binary fusions. in PEG 6000 were observed in protoplasts originated from in vivo.