Preparation and Fusion of Citrus sp. Microprotoplasts

Large-scale production of microprotoplasts from `Ruby Red' grapefruit ( Citrus paradisi Macf.) and from the Citrus L. sp. relative Swinglea glutinosa (Blanco) Merr., was performed after treatment of suspension cells with APM. An average of 75.2% of the microprotoplasts contained a single chromosome, followed by 17.1% with two, 4.6% with three, and 2.0% with four. Only 1.1% had more than five chromosomes. Maximum chromosome number observed was eight and the average yield was 2 × 10 6 of total microprotoplasts per gram of suspension cells. Flow cytometry analysis confirmed low DNA content. The polyethylene glycol fusion method was used to fuse microprotoplasts from `Ruby Red' grapefruit with protoplasts of `Succari' sweet orange [ Citrus sinensis (L.) Osbeck], and microprotoplasts from S. glutinosa with protoplasts from sour orange ( C. aurantium L.). Embryos or suspension cells from the recipient species with a few additional chromosomes were obtained; however, embryogenesis of the fusion products was reduced or inhibited. Chemical name used: amiprophos-methyl (APM).