Efficient protoplast isolation from ovule-derived embryogenic callus in Citrus volkameriana

Authors : Berken CİMEN

Pages : 567-576

View : 13 | Download : 5

Publication Date : 2020-12-01

Article Type : Research Paper

Abstract :The present study reports on the isolation of viable protoplast from ovule-derived embryogenic calli of Volkameriana insert ignore into journalissuearticles values(Citrus volkameriana L.);, which is a rootstock in high demand for lemon production.Ovules of C. volkameriana isolated at 3 different immature fruit stages, comprising4, 8, and 12 weeks after anthesis insert ignore into journalissuearticles values(WAA);, were cultured on5 different media in order to produce embryogenic callus lines as a source material for protoplast isolation. EME medium insert ignore into journalissuearticles values(MT basal medium + 0.5 gLinsert ignore into journalissuearticles values(-1); malt extract);, with the addition of phytohormones [kinetin insert ignore into journalissuearticles values(KIN);, 2,4-dichlorophenoxyacetic acid insert ignore into journalissuearticles values(2,4-D); and 6-benzylaminopurine insert ignore into journalissuearticles values(BAP);] at different concentrations, were tested for callogenesis. According to 2-way ANOVA, significant effects were determined as a result of the immature fruit stage and type of culture media insert ignore into journalissuearticles values(P <= 0.01); on the callogenesis and embryogenic callus induction frequency. First, callus formation was recorded after 4 WAA on medium comprising EME + 2,4-D insert ignore into journalissuearticles values(1.0 mg L-1); + BAP insert ignore into journalissuearticles values(0.5 mg L-1);. Callus induction frequency was the highest insert ignore into journalissuearticles values(90.00%); in the same culture medium when the ovules were culturedat8 WAA. In addition, culturing the ovules isolated from 12 WAA immature fruits of C. volkameriana resulted in the highest indirect somatic embryogenesis insert ignore into journalissuearticles values(24%);. Embryogenic callus initiation was the highest insert ignore into journalissuearticles values(25.56%); using EME + KIN insert ignore into journalissuearticles values(1.0 mg L-1); and ovules cultured at 8 WAA insert ignore into journalissuearticles values(14%); resulted in the highest embryogenic callus formation. Effects of different enzyme concentrations on the efficiency of protoplast isolation were calculated using the hemocytometer cell counting method. The combination of 2% cellulase and 0.2% pectinase gave the highest numbers of protoplasts, at 12.33 x 10insert ignore into journalissuearticles values(5); protoplast/mL. Embryogenic callus lines obtained by culturing ovules of C volkameriana yielded high-quality protoplasts after isolation and could be useful as a protoplast source for further somatic hybridization studies.
Keywords : Citrus, rootstock, embryogenic callus, protoplast isolation