Cryopreserved callus, a source of protoplasts for citrus improvement
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The availability and maintenance of embryogenic callus is a major limitation for large-scale application of somatic hybridization for citrus breeding. The suitability of cryopreserved callus as source of protoplasts was evaluated. Sweet orange callus were frozen by slow cooling and stored for two years in liquid nitrogen. Cryopreserved callus were fast thawed and used as source of material for protoplast isolation, protoplast fusion and plant regeneration, in comparison with control non-cryopreserved callus. No differences were found in protoplast yield, quality, growth and regeneration capacity between both callus types. Protoplasts isolated from cryopreserved callus 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 cryopreserved callus facilitates the development of breeding programmes based on somatic hybridization, avoiding the risks and high labour needs associated with standard maintenance by periodical subcultures.