Rooting and preventing shoot-tip necrosis of in vitro cultured horse chestnut shoots

Abstract

Efficient bud regeneration was achieved from germinating horse chestnut (Aesculus hippocastanum L.) somatic embryos cultivated on 1-10 μM benzyladenine (BA). Adventitious buds were detached from the mother tissue and used to establish permanent shoot cultures on 0-20 μM BA. Secondary buds were regenerated from the shoot base of the explants. Bud multiplication was very poor (1.9) and shoot-tip necrosis was very high (100%) on plant growth regulator (PGR)-free medium. The highest multiplication was achieved on 5 and 10 μM BA (16.8 and 18.7, respectively), with no shoot-tip necrosis, while hyperhydration was rather frequent on shoots cultivated on BA above 5 μM. Individual shoots were elongated on medium with 1 μM BA and 500 mg/l polyvinylpyrrolidone (PVP MW40 000) for 4 weeks. However, it was necessary to reduce BA level below 1 μM for shoot rooting and that caused mass shoot-tip necrosis. As classical rooting methods failed, the basal part of each elongated shoot was first wounded by cutting with a sterile blade and then dipped into a 0, 5 or 10 mM indole-3-butyric acid solution for 1 min and cultivated on solid half-strength MS PGR-free medium with 0.02% activated charcoal for 2-3 weeks. To prevent shoot tip necrosis during this phase, a BA solution was applied directly on apical meristem. Shoot-tip necrosis was completely eliminated by weekly application of 10 μl of 1 μM BA. As soon as the root initials were observed, the shoots were transferred to MS medium supplemented with 500 mg/l PVP and 5 μM BA. The frequency of rooting was 23%, and further optimisation of root-inducing phase is needed.