Multiple shoot-bud formation and plantlet regeneration on Castanea sativa Mill. seeds in culture.

Primordial initiation and development of shoot-buds has been accomplished by using shoots derived from chestnut (Castanea sativa Mill) seedlings cultured with added 6-benzylaminopurine (BAP). Germination of chestnut seeds in the presence of BAP (4 - 40 μM) stimulated varying numbers of shoot-buds in those areas of the main axis that were favorably altered. When excised single shoots from these treated seeds were subcultured on a fresh medium containing BAP (4 - 40 μM) continual shoot production was observed. Bud growth and shoot elongation were stimulated by transferring cultures to a reduced concentration of BAP (2 μM) plus indole-3-butyric acid (IBA 0.4 μM). Plant regeneration occurred in the presence of IBA (0.8 μM) after a preconditioning treatment in which naphthaleneacetic acid (NAA 50 μM) and kinetin (k 2 μM) were applied to the tissue culture shoots for 7 days in light.