Plant regeneration from protoplast of Brazilian citrus cultivars

A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were is olated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolatio n was composed of mannitol (0.7 M), CaCl2, (24.5 mM), NaH2PO4 (0.92 mM), ME S (6.15 mM), cellulase (Onozuka RS - Yakult, 1%), macerase (Onozuka R10 - Y akult, 1%) and pectolyase Y-23 (Seishin, 0.2%). Protoplast culture in liqui d medium after chemical fusion lead to the formation of callus colonies fur ther adapted to solid medium. Somatic embryo formation occurred spontaneous ly after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA(3) (2.0 mu M) and malt extract (500 mg/L). Plant regen eration was also achieved by adventitious shoots obtained through direct or ganogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L), BAP (1.32 mu M), NAA (1.07 mu M) and coconut w ater (10 mL/L). Plantlets were transferred to root medium. Rooted plants we re transferred to a greenhouse for further adaptation and development.