A combined pathway of somatic embryogenesis and organogenesis to regenerate radiata pine plants

This study describes for the first time in Pinus genus a plant regeneration system via a combined pathway of somatic embryogenesis and organogenesis from immature seeds of radiata pine. Somatic embryos were obtained from embryogenic line 2162 of Pinus radiata D. Don on EDM basal medium containing $60{\mu}M$ ABA and 6% sucrose. The explants used for organogenesis experiments were either freshly collected somatic embryos or somatic embryos germinated for 1 week. Germination medium was half-strength LP medium, supplemented with 0.2% activated charcoal. Different induction periods and BA concentrations were assayed for shoot induction. After induction treatments, explants were elongated on the same medium used for germination stage. Rooting medium was quarter-strength LP medium supplemented with three different auxin treatments: $1.5mg\;L^{-1}$ 1-naphthalene acetic acid (NAA), $1.5mg\;L^{-1}$ indole-3-butyric acid (IBA) and $1mg\;L^{-1}$ IBA with $0.5mg\;L^{-1}$ NAA (MIX). The effect of the photon flux ($120mmol\;m^{-2}\;s^{-1}$ and darkness) in the first week of the explants in the rooting media was also tested. This methodology could offer an alternative to overcome some problems associated with somatic embryogenesis such as the seasonality of embryogenic tissue (ET) initiation or a low embryo production from the ET, a particularly important issue in the case of genetically transformed ETs.