Cryopreserved callus, a source of protoplasts for citrus improvement

The availability and maintenance of embryogenic callus is a major limitatio n for large-scale application of somatic hybridization for citrus breeding. The suitability of cryopreserved callus as source of protoplasts was evalu ated. Sweet orange callus were frozen by slow cooling and stored for two ye ars in liquid nitrogen. Cryopreserved callus were fast thawed and used as s ource of material for protoplast isolation, protoplast fusion and plant reg eneration, in comparison with control non-cryopreserved callus. No differen ces were found in protoplast yield, quality, growth and regeneration capaci ty between both callus types. Protoplasts isolated from cryopreserved callu s were also successfully used in somatic fusion assays. Plants regenerated from protoplasts of the two sources had the same phenotypic characters and no differences were detected by microsatellite analysis. Availability of cr yopreserved callus facilitates the development of breeding programmes based on somatic hybridization, avoiding the risks and high labour needs associa ted with standard maintenance by periodical subcultures.