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.

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.

Immature and mature zygotic embryos were used as source of explants for induction of somatic embryogenesis in Araucaria angustifolia. Embryogenic cultures (EC) were only obtained from immature zygotic embryos. Basic medium, carbon source, and genotype showed a significant influence on the formation of stage I somatic embryos (SE). When EC were submitted to maturation conditions, SE continued their individual development until stage II, but mature embryos were not obtained. Proteins secreted by embryogenic cultures were, to a certain degree, genotype specific and included an extracellular class IV chitinase and β-1-3-glucanase.

Protoplasts isolated from embryogenic suspension cultures of European larch (Larix decidua Mill.) were cultured in thin alginate layers using a nylon mesh to enable a monitoring of the development of single cells. The patterns of cell division and differentiation are characterized and compared with zygotic embryogenesis to which homologies can only be drawn to some extent when the protoplasts grow in an auxinfree environment. Already at 2.5 μM both 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid cause vacuolation and elongation of individual cells, thus disturbing the process of somatic embryogenesis which generally lacks the precise quantitative patterns occurring in vivo. Prior to the formation of an embryo, a proembryonal mass develops. Oligonucleated products of spontaneous protoplast fusions are able to cellularize even without preceding karyokinesis and perform a normal embryogenic program.

Protocols for genetic transformation of maritime pine (Pinus pinaster Sol. ex Aiton) embryogenic tissues were developed using the Agrobacterium C58pMP90/pPCV6NFGUS. This is the first report of Agrobacterium-mediated T-DNA integration in P. pinaster confirmed by Southern blot analysis. The omission of casein hydrolysate from culture medium during cocultivation and subsequent subculture was crucial to control Agrobacterium growth. Two different transformation protocols were compared: (1) bacterial drops were spread over embryogenic clumps; (2) a mixture of bacterial and embryogenic cell suspensions was plated on filter paper.The highest frequency of transformation (22 independent transformed lines per g fresh weight, for embryogenic clone 31/668/00) was obtained with Protocol 2. The same basic procedure allowed transformation of embryogenic cell suspensions, which was dependent on subculture age. From 52 hygromycin-resistant independent lines obtained, 47 showed stable uidA gene expression and were PCR-positive for uidA gene and 42 for hpt gene. No residual Agrobacterium was detected in the transformed lines. Transgene integration was achieved using both protocols, as confirmed by Southern hybridization. From 38 (90%) transformed lines successfully cryopreserved and recovered, 71% regrown replicates have maintained the frequency of cell aggregates and early-formed embryos with uidA expression. Maturation of 44 transformed lines gave rise to 3 mature somatic embryos, each one coming from a different transformed line. Our results show the high potential of Protocol 2 for application to different culture systems.