Steps Of Somatic Embryogenesis Research Articles

Abscisic acid induces somatic embryogenesis and enables the capture of high-value genotypes in Douglas fir (Pseudotsuga menziesii [MIRB.] Franco)

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.

Genotype-dependent changes of gene expression during somatic embryogenesis in oil palm hybrids (Elaeis oleifera x E. guineensis).

To understand the molecular processes triggered during the different steps of somatic embryogenesis (SE) in oil palm, the expression of 19 genes associated to SE identified in proteomic and transcriptomic studies was investigated by qRT-PCR. To evaluate the differential expression of these genes, two interspecific hybrid genotypes (Elaeis oleifera x Elaeis guineensis) contrasting for the acquisition of embryogenic competence were used. Aclorophyllated leaves of both hybrids, one responsive (B351733) and the other non-responsive (B352933) to SE were submitted to callus induction and collected at different time points: 0 (before induction), 14, 30, 90 and 150 days of callus induction (doi). The results obtained showed that all evaluated genes were downregulated at 14 doi in the responsive genotype when compared to the non-responsive. It was also possible to observe that most of the genes changed their expression behavior at 30 doi and were upregulated thereafter until 150 doi, with the exception of the pathogenesis-related PRB1-3-like (PRB1-3) gene, which did not show differential expression at 30 doi and was downregulated at 90 and 150 doi when compared to the non-responsive hybrid. These results indicate that 30 doi is a turning point in gene expression, probably associated to embryogenic competence acquisition. We also show that the expression behavior of the responsive genotype is more stable than that of the non-responsive when the different induction time points are compared to 0 doi (before induction). Moreover, the results obtained in this study corroborate our hypothesis that the regulation of genes involved in the control of oxidative stress and energy metabolism are crucial for the acquisition of embryogenic competence in oil palm.

Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies

Suitable internal control genes to normalize qPCR data from different stages of embryo development and germination were identified in two representative conifer species. Clonal propagation by somatic embryogenesis has a great application potentiality in conifers. Quantitative PCR (qPCR) is widely used for gene expression analysis during somatic embryogenesis and embryo germination. No single reference gene is universal, so a systematic characterization of endogenous genes for concrete conditions is fundamental for accuracy. We identified suitable internal control genes to normalize qPCR data obtained at different steps of somatic embryogenesis (embryonal mass proliferation, embryo maturation and germination) in two representative conifer species, Pinus pinaster and Picea abies. Candidate genes included endogenous genes commonly used in conifers, genes previously tested in model plants, and genes with a lower variation of the expression along embryo development according to genome-wide transcript profiling studies. Three different algorithms were used to evaluate expression stability. The geometric average of the expression values of elongation factor-1α, α-tubulin and histone 3 in P. pinaster, and elongation factor-1α, α-tubulin, adenosine kinase and CAC in P. abies were adequate for expression studies throughout somatic embryogenesis. However, improved accuracy was achieved when using other gene combinations in experiments with samples at a single developmental stage. The importance of studies selecting reference genes to use in different tissues or developmental stages within one or close species, and the instability of commonly used reference genes, is highlighted.

Sulphur depletion altered somatic embryogenesis in Theobroma cacao L. Biochemical difference related to sulphur metabolism between embryogenic and non embryogenic calli

Somatic embryogenesis is a useful tool for Theobroma cacao improvement and propagation. Depending on culture medium composition, different morphogenetic structures (including somatic embryo) occur in response to alteration of genes expression patterns and biochemical changes. The effect of SO 4 2 - ion deficiency in culture media on somatic embryogenesis was studied through sequential replacement of MgSO 4 and K 2 SO 4 by MgCl 2 and KCl, respectively, at different steps of somatic embryogenesis. It appears that explants gradually lost their embryogenic competence as the period of exposition to sulphur free medium increases. These results suggest that, sulphur availability and the duration to sulphur exposition might modulate the expression of genes involved in somatic embryo differentiation in T. cacao . Cysteine, glutathione, reducing sugars, cysteine synthase and cysteine desulfurase activities were analysed in different morphogenetic structures obtained in vitro . Cysteine and reducing sugars contents appeared to be higher in embryogenic calli than their nonembryogenic homologues, whereas glutathione content appears to be lower in embryogenic calli. Cysteine synthase activities also discriminate the embryogenic calli from non embryogenic calli. In the embryogenic calli, the ratio cysteine synthase/cysteine desulfurase activities were above unit. The assimilation of exogenous sulphur (sulphate) for the synthesis of cysteine might hence be crucial for somatic embryogenesis in T. cacao . This explains the reduction and the absence of somatic embryo response observed during sulphur depletion in culture media. Sulphur nutrition is therefore critical in cacao somatic embryogenesis. Keyswords: Cacao, embryo, sulphate, cysteine synthase, glutathione, deficiency African Journal of Biotechnology Vol. 9(35), pp. 5665-5675, 30 August, 2010

PROTEIN SECRETION BY CELL CULTURES: AN ESSENTIAL BIOLOGICAL ISSUE

All the tissues cultures in vitro secrete a vast array of molecules into the culture media. The secretion of high molecular weight compounds, such as proteins, has showed an active role in different biological phenomena. Indeed, several reports have made allusion to the extracellular proteins as a pivotal point in the initiation of the signal cascade which induces the early steps of somatic embryogenesis. Some protein markers of these phenomena have been identified in carrot. However, little is known about the biological function of the extracellular proteins secreted during this process. The models already suggested always fall in speculation and controversy. In this review, a comprehensive analysis of extracellular proteins secreted into the culture media is made, with an emphasis in those that could be involved in the early induction of somatic embryogenesis.

Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences ( P ≤ 0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.

Enzyme Markers for Somatic Embryogenesis in Vitis

Summary A protocol has been detailed for somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris. Isozymic patterns of acid phosphatase, alcohol dehydrogenase, esterase, gluconate-6-phosphate dehydrogenase and phosphoglucomutase were analyzed during precise steps of somatic embryogenesis from callus to plantlet. Among the five enzymes investigated, alcohol dehydrogenase, acid phosphatase and esterase gave different results, either in their specific activity, as in the case of alcohol dehydrogenase, or in the number and isoelectric point of the isoforms expressed, as in that of acid phosphatase and esterase. Non-morphogenic calli were clearly different from embryogenic calli, and from embryos. Moreover, the calli dedifferentiated from somatic embryos did not retain the level and types of isozymes that characterize the embryo state. Our analysis of the electrophoretic patterns of specific enzymes proves to be an effective approach to the characterization of the main steps of Vitis rupestris somatic embryogenesis.