Somatic embryogenesis from stamen filaments of Aesculus flava Sol. and peroxidase activity during the transition from friable to embryogenic callus

Abstract

Species of the genus Aesculus are among the most attractive ornamental woody plants. Conventional propagation methods of these species are either inefficient (stem cuttings) or unsuitable for clonal propagation (seeds). The aim of the present study was to develop an efficient protocol for clonal propagation of elite specimens of yellow buckeye (Aesculus flava) by somatic embryogenesis. For this purpose, stamen filaments of yellow buckeye were cultivated on media supplemented with 1, 5 or 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D) combined with 0, 1, 5 or 10 μM 6-furfurylaminopurine (Kin), either under light or dark conditions, for 8 weeks, and then subcultivated on plant growth regulator (PGR)-free medium with 400 mg/l of glutamine. The highest somatic embryo (SE) initiation rates were achieved for the explants cultivated in darkness on medium containing 1 μM 2,4-D + 10 μM Kin during callus induction (CI) phase. Embryogenic calli (EC) were initiated from friable calli, starting from the 7th week of culture initiation, while SEs appeared two weeks later, following a week of subcultivation of the explants on PGR-free medium. EC and SEs were observed only in the explants grown in darkness during CI phase. Minimal duration of CI phase and darkness necessary for SE induction was four weeks, while the highest embryogenic response was achieved when each lasted for 8–10 weeks. Obtained SEs were efficiently multiplied on medium supplemented with 0.05 μM 2,4-D + 5 μM Kin by recurrent somatic embryogenesis. SEs at globular stage of development exhibited the highest capacity for secondary SE regeneration. High germination and conversion rates were attained in cotyledonary-stage SEs cultivated on medium with 0.05 μM 2,4-D + 5 μM Kin, but this phase needs to be further optimised, since the obtained plants failed to acclimatize to greenhouse conditions. During the transition of calli from friable to embryogenic state, total peroxidase (POX) activity significantly decreased, indicating their involvement in the acquisition of embryogenic capacity. The presented protocol is suitable for clonal propagation and genetic transformation of this ornamental species, and POX activity may be used as a marker for SE initiation.