The effects of the mineral media and the carbon source on the proliferation capacity of different Castanea sativa x C. crenata cultivars were the focus of the research reported here. Using the commercial cultivar Marigoule, the addition of riboflavin to the rooting medium did not improve the rooting rates recorded. It did, however, positively affect the survival of regenerated plantlets after weaning. By measuring certain physiological parameters, the beneficial effect of high concentrations of CO₂ on the acclimatization of chestnut regenerated plantlets was recorded. However, general protocols for large-scale micropropagation of specific cultivars could not be defined.
Our research has determined for the first time the developmental window in which somatic embryogenesis induction is possible from ovaries, ovules and/or zygotic embryos in C. sativa. Induction is possible between the 2^nd and the 10^th weeks post-anthesis, giving an overall frequency of 4.5%. Somatic embryogenesis induction in chestnut was not possible from mature tissues; however, embryogenesis was achieved using leaf tissue from shoot multiplication cultures. This indicates, for the first time, that material from explants other than zygotic chestnut embryos is competent for somatic embryogenesis.
The effect of thidiazuron on the ability of different seedling explants of chestnut to induce multiple shoots was also evaluated: cotyledonary node explants, which contain preformed meristematic tissue, were the only responsive explants.