Cloning and Molecular Characterization of a SERK Gene Transcriptionally Induced During Somatic Embryogenesis in Ananas comosus cv. Shenwan

A somatic embryogenesis receptor-like kinase (SERK) gene, designated as AcSERK1, was isolated from pineapple (Ananas comosus cv. Shenwan). AcSERK1 shared all the characteristic domains of the SERK family, including five leucine-rich repeats, one proline-rich region motif, transmembrane domain, and kinase domains. Somatic embryogenic cultures of pineapple were established following transfer of callus cultures to Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid. The role of AcSERK1 during establishment of somatic embryogenesis in culture was investigated. The AcSERK1 was highly expressed during embryogenic competence acquisition and global embryo formation in culture. These findings were obtained along with morphological changes in callus cultures exhibiting embryogenic potential. Overall, levels of expression of AcSERK1 were lower in nonembryogenic tissues and organs than in embryogenic callus. In situ hybridization analysis revealed that AcSERK1 expression was detected in embryogenic tissues, including single competent cells, meristematic centers wherein embryogenic structures are formed, and global embryos. These results suggested that AcSERK1 expression was associated with induction of somatic embryogenesis and that it could be used as a potential marker gene to monitor the transition of pineapple callus tissues into competent and embryogenic cells and tissues.     

Cell architecture during gametophytic and embryogenic microspore development in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

In this work, the cell architecture of the microspore following both gametophytic and embryogenic developmental pathways in vitro was compared with the gametophytic development in vivo in Brassica napus, at both light and electron microscopy level. The microspore reprogramming to embryogenesis involves defined changes affecting cell activities and structural organization which can be considered as markers of the microspore embryogenic pathway, but less is known about others developmental programmes followed by the microspore in vitro after both, inductive and non-inductive conditions. Low-temperature processing of the samples, cytochemical and immunocytochemical approaches to identify various cell components were performed. Differences in specific cellular features such as cellular size and shape, nuclear architecture, starch accumulation, presence of vacuoles and ribosomal population were studied to characterize sequential stages of microspore embryogenesis and other pathways occurring in vitro. The presence of abundant starch grains in a defined cytoplasmic region appeared as a specific feature of the in vitro gametophytic development, as well as of the non-induced microspores of in vitro cultures under embryogenic-inductive conditions.     

Selection of Brassica napus L. embryogenic microspores by flow sorting

Flow cytometry can be used to select and sort microspore subpopulations of Brassica napus cv. Topas. Data obtained from embryogenic microspore populations were used to identify potentially embryogenic microspores from developmentally heterogeneous microspore populations based on differences in forward light scatter and green autofluorescence. Culture enrichment for embryogenic microspores is possible. Frequencies of 8 and 14% microspore embryogenesis were obtained when selected 16 h and 72 h after culture initiation. This represents 5- and 13-fold increase in microspore embryogenesis compared to non-sorted controls.     

Expression of knotted1 marks shoot meristem formation during maize embryogenesis

The formation of shoot and root meristems that ultimately give rise to all tissues of the plant body occurs for the first time during embryogenesis. Meristem formation has traditionally been defined in terms of the appearance of histological features of meristems; this approach has led to varying interpretations of the timing of meristem formation relative to other events in embryogenesis. Markers that would provide more objective criteria for the analysis of meristem formation have not been widely available. The maize homeobox gene, knotted1 (kn1), is expressed in shoot meristems throughout postembryonic stages of shoot development. In order to determine whether this gene is expressed in the shoot meristem from its earliest inception, we examined the expression of kn1 in embryos at a series of stages by in situ hybridization to kn1 mRNA and immunolocalization of KN1 protein. Our results show that the onset of kn1 expression is temporally and spatially coincident with the earliest histologically recognizable signs of shoot meristem formation in the embryo, and thus provides a valuable marker for this process. © 1995 Wiley-Liss, Inc.     

Expression of the QrCPE gene is associated with the induction and development of oak somatic embryos

Somatic embryogenesis is a powerful tool for plant regeneration and also provides a suitable material for investigating the molecular events that control the induction and development of somatic embryos. This study focuses on expression analysis of the QrCPE gene (which encodes a glycine-rich protein) during the initiation of oak somatic embryos from leaf explants and also during the histodifferentiation of somatic embryos. Northern blot and in situ hybridization were used to determine the specific localisation of QrCPE mRNA. The results showed that the QrCPE gene is developmentally regulated during the histodifferentiation of somatic embryos and that its expression is tissue- and genotype-dependent. QrCPE was strongly expressed in embryogenic cell aggregates and in embryogenic nodular structures originated in leaf explants as well as in the protodermis of somatic embryos from which new embryos are generated by secondary embryogenesis. This suggests a role for the gene during the induction of somatic embryos and in the maintenance of embryogenic competence. The QrCPE gene was highly expressed in actively dividing cells during embryo development, suggesting that it participates in embryo histodifferentiation. The localised expression in the root cap initial cells of cotyledonary somatic embryos and in the root cap of somatic seedlings also suggests that the gene may be involved in the fate of root cap cells.     

A novel stress-related gene in developing pepper anthers

To study gene expression patterns and to find genes related with microspore embryogenesis during pepper (Capsicum annuum L.) anther development, mRNA expression patterns were investigated at four developmental stages distinguished according to the size of flower bud, the color of anthers, and the cytological feature of microspores. Through GeneFishing using 120 random primers, 81 genes were found to be differentially expressed as anthers develop. We directly sequenced seven of them, which were either up- or down-regulated at stage 2, since microspores at stage 2 are known to be responsive to the induction signals for microspore embryogenesis. Nucleotide sequence analysis of the isolated differentially expressed genes (DEGs) and the comparison of these sequences with the GenBank data indicate that DEG13 is a novel gene, which is highly homologous to a stress-related gene of potato, POACT88 (≈91%) and to alcohol dehydrogenase gene of Arabidopsis (≈70%), whose expression is also tightly related to stresses. In vitro data also showed that DEG13 was more abundantly expressed in heat-treated microspores than in untreated microspores. Here, we report developmental stage-specific gene expression patterns during anther development and a novel stress-related gene, DEG13, which may be involved in microspore embryogenesis in response to heat treatment.     

Zinc sulphate improved microspore embryogenesis in barley

The effect of ZnSO₄ concentration on barley (Hordeum vulgare L.) microspore embryogenesis was investigated using cultivars of different androgenetic response. Concentrations from 0 (control) to 600 μM in the stress pre-treatment medium alone or in combination with 30 (control) to 600 μM in the embryo induction medium were assayed in anther culture. Incorporation of Zn²⁺ in the pre-treatment medium itself did not affect microspore embryogenesis. The optimum concentration in the stress pre-treatment and induction media was 180 μM for cultivars (cvs.) Igri and Reinette, and 90 μM for cv. Hop. A significant increase of 30 and 300% in cv. Igri and Reinette, respectively, were produced with 180 μM ZnSO₄ in both the number of embryos and green plants. In order to confirm the effect of Zn²⁺ on microspore embryogenesis this micronutrient was incorporated in the induction medium of isolated microspore cultures of cv. Igri. Concentrations of 90–300 μM ZnSO₄ resulted in an increase of 40–53% in the number of embryos and green plants. All these results indicate that the beneficial effect of Zn²⁺ is exerted mainly during the culture phase, increasing the number of embryos, leading to an increased number of green plants, but it had no effect on percentage of regeneration or green plants.     

斑马鱼胚胎发育过程中TGF-1基因的表达特征分析

为研究转化生长因子 (Transforming growth factor , TGF)1对斑马鱼胚胎发育的调控作用, 通过NCBI获得TGF-1基因序列, TGF-1 cDNA全长1571 bp, 编码377个氨基酸。系统进化树分析发现, TGF-蛋白按照不同的类型严格聚类, 斑马鱼TGF-1与其他鱼类的TGF-1聚集到一个分支, 在进化中非常保守。对斑马鱼胚胎进行RT-PCR和Real-Time PCR检测显示, TGF-1基因为母源表达基因, 在分节期之前的表达水平比较低, 而从咽囊期开始持续高水平的表达。胚胎整体原位杂交发现, TGF-1基因在斑马鱼24 hpf 胚胎中开始有特异信号出现, TGF-1基因的表达主要分布在腮弓、侧线原基、耳囊、嗅觉基板、心脏和前肾等处, 表明TGF-1基因可能参与斑马鱼胚胎免疫调节、循环系统发育和侧线形成。用低氧处理斑马鱼胚胎, 发现低氧处理24h后斑马鱼胚胎发育延迟。利用Real-Time PCR和胚胎整体原位杂交检测发现, 低氧处理后发育延迟的斑马鱼胚胎中TGF-1 mRNA表达量较常氧组显著降低。以上结果表明, TGF-1基因参与斑马鱼胚胎发育调控, 并且可能与低氧处理后斑马鱼胚胎发育延迟有关。研究结果将为深入研究斑马鱼TGF-1基因的功能奠定基础。       

NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores.     

Characterization of expressed sequence tags obtained by SSH during somatic embryogenesis in <Emphasis Type="Italic">Cichorium intybus</Emphasis> L

Background  

Somatic embryogenesis (SE) is an asexual propagation pathway requiring a somatic-to-embryonic transition of differentiated somatic cells toward embryogenic cells capable of producing embryos in a process resembling zygotic embryogenesis. In chicory, genetic variability with respect to the formation of somatic embryos was detected between plants from a population of Cichorium intybus L. landrace Koospol. Though all plants from this population were self incompatible, we managed by repeated selfing to obtain a few seeds from one highly embryogenic (E) plant, K59. Among the plants grown from these seeds, one plant, C15, was found to be non-embryogenic (NE) under our SE-inducing conditions. Being closely related, we decided to exploit the difference in SE capacity between K59 and its descendant C15 to study gene expression during the early stages of SE in chicory.     

Changes in protein pattern during different developmental stages of somatic embryos in chickpea

Mature embryonic axes were used for chickpea (Cicer arietinum L.) regeneration via somatic embryogenesis. Qualitative and quantitative estimation of protein profile during somatic embryogenesis by SDS-PAGE and densitometric analysis showed differential expression of various storage proteins at different stages of somatic embryo development, which was compared with the profile of developing seeds. Total protein content in somatic embryos of chickpea increased from globular stage [2.9 μg mg⁻¹(f.m.)] to cotyledonary stage [4.8 μg mg⁻¹(f.m.)] and then started decreasing during onset of maturation and germination [up to 1.5 μg mg⁻¹(f.m.)]. Differential expression of seed storage proteins, late embryogenesis abundant (LEA) proteins and proteins related with stress response were documented at different stages of somatic embryogenesis. Germinating somatic embryos showed degradation products of several seed storage proteins and the appearance of new polypeptides (76.8, 67.6, 49.9 and 34.2 kDa), which were absent during differentiation of somatic embryos. A low molecular mass (17.7 kDa) polypeptide was uniformly present during all stages of somatic embryogenesis and it may belong to a group of stress-related proteins. This study describes the expression of true seed storage proteins like legumin, vicilin, convicilin and their subunits at different stages of somatic embryogenesis, which may serve as excellent markers for embryogenic pathway of regeneration in chickpea.     

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter--glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.