A protoplast-to-tree system in Microcitrus based on protoplasts derived from a sustained embryogenic callus

Adventitious embryos derived from a zygotic embryo in an in vitro cultured ovule of Microcitrus were transferred several times on solidified medium containing benzyladenine and 3-indoleacetyl-L-alanine to induce embryogenic callus. This callus was maintained for several years on medium devoid of growth regulators without losing its embryogenic capacity. Exposure of this callus to maceration enzymes led to protoplast suspensions. Purified protoplasts were plated in solid medium devoid of growth regulators. Somatic embryos were derived efficiently from individual protoplasts and most of these could be regenerated into mature trees bearing normal flowers and typical fruits. This system differs from the Citrus protoplast-to-tree system. In the latter embryogenic callus was derived from the nucellus of polyembryonic species while Microcitrus is monoembryonic and required hormone-induced callus formation from proliferating zygotic embryos.