SOMATIC EMBRYOGENESIS IN IMMATURE EMBRYOS AND PROTOPLASTS OF PINUS-CARIBAEA

Somatic enbryogenesis was obtained from cultured immature embryos of Pinus caribaea Morelet var. hondurensis on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), N6-benzyladenine (BA) and/or kinetin. The stage of zygotic embryo development for optimal somatic embryogenesis was found to be the early stage of polyembryony. Embryogenic calli have been subcultured for more than ten months and have retained their embryogenic ability. Different callus lines (different genotypes) expressed distinct nutritional requirements for growth. Embryos bearing well differentiated cotyledons were recovered after transfer of calli on a modified Hakman and von Arnold (J. Plant Physiol., 121 (1985) 149-158) medium supplemented with abscisic acid and devoid of organic nitrogen. Rooted plantlets were obtained after transfer to an hormone-free medium. Protoplasts were isolated from embryogenic cells suspensions. Sustained divisions of protoplast-derived cells and regeneration of somatic proembryos were achieved using first the medium disigned for the culture of cotyledon protoplasts of the same species (E. Lainé et al., Physiol. Plant., 72 (1988) 374-379) and then dilution with the cell suspension medium. The appearance and nutritional requirements of the established protoplast-derived cell suspension were similar to those of the original cell suspension. Cell aggregates were transferred to solid medium for callus development. Fully differentiated embryos with cotyledons and hypocotyl were obtained 5 months after protoplast isolation. © 1990.