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.