Rooting and preventing shoot-tip necrosis of in vitro cultured horse chestnut shoots
2012
Zdravković-Korać, Snežana (Institute for Biological Research Siniša Stanković, Belgrade (Serbia)) | Tubić, Ljiljana (Institute for Biological Research Siniša Stanković, Belgrade (Serbia)) | Milojević, Jelena (Institute for Biological Research Siniša Stanković, Belgrade (Serbia)) | Devrnja, Nina (Institute for Biological Research Siniša Stanković, Belgrade (Serbia)) | Kostić, Igor (Institute for Biological Research Siniša Stanković, Belgrade (Serbia)) | Ćalić, Dušica (Institute for Biological Research Siniša Stanković, Belgrade (Serbia))
Efficient bud regeneration was achieved from germinating horse chestnut (Aesculus hippocastanum L.) somatic embryos cultivated on 1-10 M benzyladenine (BA). Adventitious buds were detached from the mother tissue and used to establish permanent shoot cultures on 0-20 M 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 M BA (16.8 and 18.7, respectively), with no shoot-tip necrosis, while hyperhydration was rather frequent on shoots cultivated on BA above 5 M. Individual shoots were elongated on medium with 1 M BA and 500 mg/l polyvinylpyrrolidone (PVP MW 40 000) for 4 weeks. However, it was necessary to reduce BA level below 1 M 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 l of 1 M BA. As soon as the root initials were observed, the shoots were transferred to MS medium supplemented with 500 mg/l PVP and 5 M BA. The frequency of rooting was 23%, and further optimisation of root-inducing phase is needed.
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