Methods for controlled regeneration of cucumis sp plants in vitro from protoplasts.

摘要

Generation of competent Cucumis sp. embryoids, plantlets and plants (including seeds) from protoplasts, e.g., from cotyledonary , root, leaf or stem tissue, by isolation in enzyme solution followed by cell wall formation, formation of multicellular colonies, formation of minicalli with induction of embryoids, plating of minicalli with maturation of embryoids, and plantlet development from the embryoids in a series of media, CPM-1, -2 and -3, and CTM-4, containing two or more of NAA, ZR, 2,4-D, BAP and GA3 under defined temperature, time and light conditions. Serial transfer to novel enzyme and Cucumis protoplast induction and expression media for defined periods results in cell wall formation, cell division, competent minicalli formation, and induction and expression (maturation) of competent embryoids and plantlets. Transfer to hormone-free development media permits plantlet development, which in turn progress to mature normal, flowering, fruiting plants. The process is applicable to producing encapsulated embryoids from single genotypes or from somatic hybrids as "artificial seeds". The process applied to increase of F-1 hybrids permits production of "artificial seeds" (encapsulations) in 1/2 to 1/10 the time it would take to produce natural hybrid seeds. Techniques involving px/cy somatic hybridization, freeze storage, and post-thawing development are disclosed. Sexual sterility barriers between plants in the genus Cucumis preventing genetic transfer of important characteristics are overcome.