Constitutive Overexpression of a Conifer WOX2 Homolog Affects Somatic Embryo Development in Pinus pinaster and Promotes Somatic Embryogenesis and Organogenesis in Arabidopsis Seedlings.

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.

Identification of putative homologs of Larix decidua to BABYBOOM (BBM), LEAFY COTYLEDON1 (LEC1), WUSCHEL-related HOMEOBOX2 (WOX2) and SOMATIC EMBRYOGENESIS RECEPTOR-like KINASE (SERK) during somatic embryogenesis.

MAIN CONCLUSION: 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.

IgG stability in fresh and conditioned medium of tobacco (Nicotiana tabacum) and larch (Larix decidua) embryogenic suspension cultures.

To explore the feasibility of larch (Larix decidua Mill.) embryogenic cell culture as alternative plant expression system, protein stability in fresh and conditioned medium was characterized in this study and compared to tobacco BY2 suspension culture. Fresh and conditioned media were spiked with 1 microg human IgG and IgG content was determined by ELISA after 24 h incubation. In fresh media, IgG recovery rate decreased to 12-23%. Adsorption on vessel walls probably is the best explanation for this IgG loss and EDTA in the medium strongly influenced wall adsorption. A high IgG recovery rate occurred in all conditioned cell culture media (7 or 14 days after inoculation). Changes in the low molecular weight-constitution of conditioned medium, rather than co-secreted polymers, are responsible for IgG stability in the cell suspension cultures.

Zygotic and somatic embryo morphogenesis in Pinus pinaster: comparative histological and histochemical study.

We compared morphogenesis and accumulation of storage proteins and starch in Pinus pinaster Ait. zygotic embryos with those in somatic embryos grown with different carbohydrate sources. The maturation medium for somatic embryos included 80 microM abscisic acid (ABA), 9 g l(-1) gellam gum and either glucose, sucrose or maltose at 44, 88, 175 or 263 mM in the presence or absence of 6% (w/v) polyethylene glycol (PEG) 4000 MW. Maturation medium containing 44 or 88 mM of a carbohydrate source produced only one or no cotyledonary somatic embryos per 0.6 g fresh mass of culture. The addition of PEG to the basal maturation medium resulted in a low yield of cotyledonary somatic embryos that generally showed incomplete development and anatomical abnormalities such as large intercellular spaces and large vacuoles. High concentrations of maltose also induced large intercellular spaces in the somatic embryonic cells, and 263 mM sucrose produced fewer and less developed cotyledonary somatic embryos compared with 175 mM sucrose, indicating that the effect of carbohydrate source is partially osmotic. Zygotic embryos had a lower dry mass than somatic embryos at the same stage of development. Starch granules followed a similar accumulation pattern in zygotic and somatic embryos. A low starch content was found in cotyledonary zygotic embryos and in somatic embryos developed in the presence of 175 mM maltose or 263 mM glucose. In zygotic embryos and in PEG-treated somatic embryos, protein bodies appeared later and were smaller and fewer than in well-developed somatic embryos grown without PEG. We propose that storage protein concentration might be a marker of embryo quality.