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Development of axillary shoots was induced when isolated embryonic axes of chestnut (Castanea sativa Mill.) were cultured on a defined medium containing 6‐benzyl‐aminopurine (BAP). The optimal concentrations of BAP were determined for development of axillary shoots from both embryonic axes and subcultured shoots. After shoot multiplication a great number of shoots have been maintained sequentially without significant change in the proliferative rate for one year. Limited rooting has been obtained with excised shoots. Indole‐3‐butyric acid (IBA) at 2 mg/l was used to induce root primordia. After 8 days of treatment the plantlets were transferred to an auxin‐free medium for root development.

Vitrified shoots were produced in multiplication cultures of Castanea sativa Mill. when Murashige and Skoog mineral medium was used during successive subcultures, whereas normal shoots were obtained when Heller's macronutrient formula was used with or without addition of 1 mM (NH4)2SO4. Vitreous and normal plantlets were compared both anatomically and chemically. Xylem and sclerenchyma tissues are less differentiated and lignified in vitrified than in normal shoots. On the other hand, there is a hypertrophy in the cortical and pith parenchyma. Leaves of vitrified shoots are characterized by the lack of clear differentiation between the palisade and spongy mesophyll, where a system of large intercellular spaces develops. The cytohistological zonation of vitrified apices is characterized by the comparatively small meristem tissue in the apical dome. Phenolic content of explants, assayed by high pressure liquid chromatography, was lower in vitreous than in normal cultures. Gallic acid was identified in both types of cultures.