Embryogenesis-related genes ( LdBBM, LdLEC1, LdWOX2 and LdSERK ) were confirmed in sequence and expression abundance for Larix decidua —these findings are valid for somatic as well as for zygotic embryo development.S omatic embryogenesis is a reliable source of high-quality genotypes as it presents an advantageous alternative for conifers in forestry, independent from seed production. Although this propagation method is already being applied, molecular factors initiating and controlling the process remain to be understood. The embryogenesis-associated genes BABYBOOM (BBM), LEAFY COTYLEDON1 (LEC1), WUSCHEL-related HOMEOBOX2 (WOX2) and SOMATIC EMBRYOGENESIS RECEPTOR-like KINASE (SERK) were identified and analyzed in somatic embryos of the European larch, L. decidua Mill. Subsequent comparisons with annotated sequences displayed similarities with angiosperm homologs. Transcript accumulation of the identified genes during embryogenesis has been analyzed. LdLEC1 and LdWOX2 are mainly expressed during early embryogenesis, whereas LdBBM and LdSERK reveal increased expression during later development. Temporal and spatial expression studies revealed a specific LdLEC1 signal in the outer cell layer of young embryo heads, whereas mature embryos showed a homogeneous expression. The overexpression of LdLEC1 in Arabidopsis influences germination and cotyledon formation, thus indicating the interspecific importance of LEC1 for proper embryo and specifically cotyledon development. Our data support a conserved role of principal regulators during plant embryogenesis that may be used as molecular markers for embryogenicity and to further determine initiating processes of somatic embryogenesis.

Although full sequence data of several embryogenesis-related genes are available in conifers, their functions are still poorly understood. In this study, we focused on the transcription factor WUSCHEL-related HOMEOBOX 2 (WOX2), which is involved in determination of the apical domain during early embryogenesis, and is required for initiation of the stem cell program in the embryogenic shoot meristem of Arabidopsis. We studied the effects of constitutive overexpression of Pinus pinaster WOX2 (PpWOX2) by Agrobacterium-mediated transformation of P. pinaster somatic embryos and Arabidopsis seedlings. Overexpression of PpWOX2 during proliferation and maturation of somatic embryos of P. pinaster led to alterations in the quantity and quality of cotyledonary embryos. In addition, transgenic somatic seedlings of P. pinaster showed non-embryogenic callus formation in the region of roots and subsequently inhibited root growth. Overexpression of PpWOX2 in Arabidopsis promoted somatic embryogenesis and organogenesis in a part of the transgenic seedlings of the first and second generations. A concomitant increased expression of endogenous embryogenesis-related genes such as AtLEC1 was detected in transgenic plants of the first generation. Various plant phenotypes observed from single overexpressing transgenic lines of the second generation suggest some significant interactions between PpWOX2 and AtWOX2. As an explanation, functional redundancy in the WOX family is suggested for seed plants. Our results demonstrate that the constitutive high expression of PpWOX2 in Arabidopsis and P. pinaster affected embryogenesis-related traits. These findings further support some evolutionary conserved roles of this gene in embryo development of seed plants and have practical implications toward somatic embryogenesis induction in conifers.

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Douglas fir (Pseudotsuga menziesii) is one of Europe’s most important non-native tree species due to its drought tolerance as well as timber quality and yield. To obtain superior seed from selected parental trees, breeding programs had been established in seed orchards. Douglas fir seed is used as source material for somatic embryogenesis with the aim to select elite genotypes invaluable for clonal mass propagation. To improve given protocols for somatic embryo initiation, we used immature Douglas fir zygotic embryos as explants and abscisic acid (ABA) as plant growth regulator in contrast to the application of auxins and cytokinins. With ABA supplementation, induction frequencies were slightly but in mean higher than with auxin/cytokinin, showing also a strong genotype effect. This offered the possibility to capture SE cultures from otherwise recalcitrant crosses. Furthermore, we observed remarkable differences between the two sets of plant growth regulators concerning the morphological development of the explants, including the absence of non-embryogenic callus by using ABA as inducer. This simplifies the detection of events and the handling of the obtained cultures. Nevertheless, a histological approach suggested, that the same competent cells are addressed by the different hormonal stimulation. Besides, we studied the influence of different points in time of cone harvest, two different basal media and different genetic backgrounds of the explants as well as the maturation ability of the induced embryogenic cultures. In sum, we were able to improve the first steps of somatic embryogenesis and to maintain a significantly higher number of high-value genotypes.