A temporary immersion system for micropropagation of axillary shoots of hybrid chestnut

Abstract

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 µM BA and 3 % sucrose, cultured both in plantform™ and RITA® vessels with three or six immersions per day and additional aeration of 1 min per hour in the case of plantform™ bioreactors. Basal explants cultured in plantform™ for 5 weeks produced long shoots suitable for rooting, whereas apical and nodal explants cultured in plantform™ or RITA® 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.