Improved micropropagation protocol for maritime pine using zygotic embryos

Pinus pinaster Ait. is a conifer widely distributed through Spain. It is considered one of the major forest species because of wood and resin production. This fact explains the efforts of companies for capturing and enhancing the genetic gain in breeding programs. Micropropagation is a useful strategy to obtain identical plants from selected material, but nowadays, organogenesis protocols are the unique techniques to obtain clonal plants from a high number of elite genotypes. In this work, an improved micropropagation protocol for maritime pine using zygotic embryos as initial explant was developed. To optimize the protocol, the study of culture medium and exogenously applied cytokinins was needed. Embryos cultured on 1/2 DCR as basal medium exhibited high organogenic responses during induction, elongation, and rooting periods. Explants cultured on medium with 2.2 mu M meta-topolin showed the highest values in all parameters analyzed along the different micropropagation phases, from induction to acclimatization. These explants showed a survival percentage of 90%, embryos forming shoots of 96.7%, and bud forming capacity and shoot elongation capacity of 4.38 and 3.39, respectively. Moreover, 70% of rooted shoots were obtained. All rooted plants survived in the acclimatizing stage after two months under ex vitro conditions. Use of 1/2 DCR as basal medium may provide an efficient and simplified in vitro protocol from whole zygotic embryos, ensuring the large-scale production of Pinus pinaster superior genotypes.