A protocol for culturing chestnut axillary shoots by temporary immersion in liquid medium was developed. The influence of type of explant, support material, bioreactor, and immersion was investigated for five artificial hybrids and five natural hybrids of Asian and European chestnut selected for resistance to ink disease. The type of explant influenced shoot quality and proliferation rates, and basal explants with callus produced more and longer shoots than apical and nodal segments. Use of rockwool cubes as support material prevented hyperhydricity and allowed proliferation of explants in Murashige and Skoog medium with half-strength nitrates supplemented with 0.22 lM BA and 3 % sucrose, cultured both in plantform TM and RITA Ò vessels with three or six immersions per day and additional aeration of 1 min per hour in the case of plantform TM bioreactors. Basal explants cultured in plantform TM for 5 weeks produced long shoots suitable for rooting, whereas apical and nodal explants cultured in plantform TM or RITA Ò produced shorter shoots that were suitable for maintenance of stock. For most of the clones, similar or higher proliferation rates were observed when cultured in liquid medium than in semisolid medium, with the additional benefit of cost-reduction of the former system. Shoots developed in liquid medium were submitted to ex vitro root induction by dipping in indole-3-butyric acid, and acclimatized under greenhouse conditions. This is the first demonstration of the production of chestnut plantlets from shoots cultured in liquid medium, and the protocol presented here shows good potential for application in large-scale propagation.

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A protocol for culturing chestnut axillary shoots with the basal sections continuously immersed in liquid medium was developed and the influence of various parameters was assessed: explant type and size, support material, bioreactor size and aeration regime. Shoots excised from eight selected chestnut genotypes were successfully cultured in 10 L bioreactors in liquid Murashige and Skoog medium with half strength nitrates and supplemented with 0.05 mg/L N 6-benzyladenine and 30 g/L sucrose. Forced ventilation and the use of a support material were essential for producing healthy, non-hyperhydric shoots. High proliferation rates were achieved and good quality shoots ready for multiplication, rooting and acclimatization were obtained. Larger leaves showing higher levels of photosynthetic pigments were observed in shoots cultured in bioreactors, suggesting a certain degree of photoautotrophy in shoots cultured under forced ventilation. This is the first demonstration of the production of chestnut shoots in stationary liquid medium, indicating the feasibility of the method for large-scale propagation of the species.

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In this study, results of photomixotropic and photoautotrophic chestnut micropropagation are compared. The aim is to increase the quality of micropropagated plants as well as to reduce costs due to plant loss during acclimatization. A successful protocol has been developed for the proliferation phase without exogenous sucrose added to the medium that has been tested with 4 genotypes. For this, shoots were exposed to decreasing sugar levels for several months to ensure their viability in sucrose-free medium. In vitro rooting in photoautotrophic conditions was applied to 15 chestnut genotypes. Every genotype formed vigorous plants which were easy to acclimatize. More than 6000 shoots were rooted with a rooting and acclimatization average value of 71% for the best treatment. This result was better than the one obtained in conventional micropropagation.