Novel dye for detection of callus embryo by confocal laser scanning fluorescence microscopy.

In the present study a new luminescent dye 3-N-(2-pyrrolidinylacetamido)benzanthrone (AZR) was synthesized. Spectroscopic measurements of the novel benzanthrone 3-aminoderivative were performed in seven organic solvents showing strong fluorescence. The capability of the prepared dye for visualization has been tested on flax, red clover and alfalfa to determinate the embryo in plant callus tissue cultures. Callus cells were stained with AZR and further analysed utilizing confocal laser scanning fluorescence microscopy. Performed experiments show high visualization effectiveness of newly synthesized fluorescent dye AZR that is efficient in fast and relatively inexpensive diagnostics of callus embryos that are problematic due to in vitro culture specificity.

Altered callose deposition during embryo sac formation of multi-pistil mutant (mp1) in Medicago sativa.

Whether callose deposition is the cause or result of ovule sterility in Medicago sativa remains controversial, because it is unclear when and where changes in callose deposition and dissolution occur during fertile and sterile embryo sac formation. Here, alfalfa spontaneous multi-pistil mutant (mp1) and wild-type plants were used to compare the dynamics of callose deposition during embryo sac formation using microscopy. The results showed that both mutant and wild-type plants experienced megasporogenesis and megagametogenesis, and there was no significant difference during megasporogenesis. In contrast to the wild-type plants, in which the mature embryo sac was observed after three continuous cycles of mitosis, functional megaspores of mutant plants developed abnormally after the second round of mitosis, leading to degeneration of synergid, central, and antipodal cells. Callose deposition in both mutant and wild-type plants was first observed in the walls of megasporocytes, and then in the megaspore tetrad walls. After meiosis, the callose wall began to degrade as the functional megaspore underwent mitosis, and almost no callose was observed in the mature embryo sac in wild-type plants. However, callose deposition was observed in mp1 plants around the synergid, and increased with the development of the embryo sac, and was mainly deposited at the micropylar end. Our results indicate that synergid, central, and antipodal cells, which are surrounded by callose, may degrade owing to lack of nutrition. Callose accumulation around the synergid and at the micropylar end may hinder signals required for the pollen tube to enter the embryo sac, leading to abortion.

[Agrobacterium-mediated genetic transformation of secondary somatic embryos in alfalfa].

We describe a genetic transformation method of secondary somatic embryogenesis in alfalfa through cotyledon-stage somatic embryos of alfalfa infected by Agrobacterium strain GV3101. The Agrobacterium strain GV3101 contained binary vector pCAMBIA2301 that had gus gene as reporter and npt II gene as selectable marker. The infected primary embryos were induced through series of medium under 75 mg/L kanamycin selection. We obtained the transgenic alfalfa plants. Then, GUS expression in different tissue of transgenic alfalfa was tested by GUS histochemical analysis. Further, the stable integration and transformation efficiency were tested by polymerase chain reaction and Southern blotting hybridization. The result showed that GUS expression was different in different organs of transgenic alfalfa; the copy number of integrated npt II gene was from 1 to 4; the transformation efficiency via primary somatic embryogenesis was 65.82%.

Characterization of the mannan synthase promoter from guar (Cyamopsis tetragonoloba).

Guar seed gum, consisting primarily of a high molecular weight galactomannan, is the most cost effective natural thickener, having broad applications in the food, cosmetics, paper, pharmaceutical and petroleum industries. The properties of the polymer can potentially be enhanced by genetic modification. Development of suitable endosperm-specific promoters for use in guar is desirable for metabolic engineering of the seed gum. A ~1.6 kb guar mannan synthase (MS) promoter region has been isolated. The MS promoter sequence was fused with the GUS reporter gene and overexpressed in the heterologous species alfalfa (Medicago sativa). The potential strength and specificity of the MS promoter was compared with those of the constitutive 35S promoter and the seed specific ß-phaseolin promoter. Quantitative GUS assays revealed that the MS promoter directs GUS expression specifically in endosperm in transgenic alfalfa. Thus, the guar MS promoter could prove generally useful for directing endosperm-specific expression of transgenes in legume species.

Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.).

Peroxidase (POD) and superoxide dismutase (SOD) enzyme activities were analyzed in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.) carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I antisense (OC-Ias), or hygromycin phosphotransferase (hpt) genes. All of the transformed lines analyzed had elevated levels of all POD isoforms. Three POD isoforms with pI values of approximately 4.5, 4.8, and 8.4, and one additional pair of isoforms with a pI value of approximately 8.8 were separated from tissue extracts of all transgenic lines. Isoelectrofocusing patterns revealed the induction of one isoform of SOD with a pI of about 5.6 in all transgenic lines compared with non-transformed embryogenic tissue. These results indicate that the process of transformation may disrupt redox homeostasis in alfalfa tissues.

In vitro reconstitution of legumin (11S) mRNA and binding proteins as related to post-transcriptional regulation of protein synthesis in developing alfalfa embryos.

There is undetectable transcription of 11S storage protein (medicagin) mRNA by nuclei isolated from pre-cotyledonary-stage somatic embryos of alfalfa (Medicago sativa L). However, this message exists at steady-state levels in the embryos at this stage of development without concomitant synthesis of the storage protein. At the pre-cotyledonary stage, therefore, the transcriptional rate for 11S mRNA is low; what message is transcribed is sequestered in the form of mRNP complexes and is not recruited into polysomes in vivo (33). Both transcription (in vivo and in isolated nuclei) and translation of the 11S mRNA are evident at the onset of cotyledon development in somatic and zygotic embryos, reaching a maximum during expansion of the cotyledons and then declining as the embryos mature. Pre-cotyledonary-stage somatic embryos which do not utilize the 11S-mRNA in polysomes lack certain mRNA-binding proteins (32, 36 and 38 kD) which are present at later stages of development. These mRNA-binding proteins may be responsible for the initiation of large polysome formation since they were exclusively present in the translational extracts of cotyledonary somatic and zygotic embryos in which there was no repression of storage protein synthesis. In contrast, the pre-cotyledonary somatic embryos contained a different set of 11S-mRNA-binding proteins (28, 50, 55, and 62 kD) whose presence in the cotyledonary stage embryos was very rare or non-existent; these could be responsible for preventing translation.

Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derived alfalfa cells.

Alfalfa leaf protoplast-derived cells can develop into somatic embryos depending on the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the initial culture medium. In order to reveal gene expression changes during the establishment of embryogenic competence, we compared the cell types developed in the presence of 1 and 10 microM 2,4-D, respectively, at the time of their first cell divisions (fourth day of culture) using a PCR-based cDNA subtraction approach. Although the subtraction efficiency was relatively low, applying an additional differential screening step allowed the identification of 38 10 microM 2,4-D up-regulated transcripts. The corresponding genes/proteins were annotated and representatives of various functional groups were selected for more detailed gene expression analysis. Real-time quantitative PCR (RT-QPCR) analysis was used to determine relative expression of the selected genes in 2,4-D-treated leaves as well as during the whole process of somatic embryogenesis. Gene expression patterns confirmed 2,4-D inducibility for all but one of the 11 investigated genes as well as for the positive control leafy cotyledon1 (MsLEC1) gene. The characterized genes exhibited differential expression patterns during the early induction phase and the late embryo differentiation phase of somatic embryogenesis. Genes coding for a GST-transferase, a PR10 pathogenesis-related protein, a cell division-related ribosomal (S3a) protein, an ARF-type small GTPase and the nucleosome assembly factor family SET protein exhibited higher relative expression not only during the induction of somatic embryogenesis but at the time of somatic embryo differentiation as well. This may indicate that the expression of these genes is associated with developmental transitions (differentiation as well as de-differentiation) during the process of somatic embryogenesis.

Nitric oxide is required for, and promotes auxin-mediated activation of, cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures.

It is now well established that nitric oxide (NO) serves as a signaling molecule in plant cells. In this paper experimental data are presented which indicate that NO can stimulate the activation of cell division and embryogenic cell formation in leaf protoplast-derived cells of alfalfa in the presence of auxin. It was found that various NO-releasing compounds promoted auxin-dependent division (as shown by incorporation of bromodeoxyuridine) of leaf protoplast-derived alfalfa cells. In contrast, application of NO scavenger or NO synthesis inhibitor inhibited the same process. Both the promotion and the inhibition of cell cycle activation correlated with the amount and activity of the cognate alfalfa p34cdc2 protein Medsa;CDKA;1,2. The effect of l-NG-monomethyl-L-arginine (L-NMMA) was transient, and protoplast-derived cells spending more than 3 days in culture become insensitive to the inhibitor as far as cell cycle progression was concerned. L-NMMA had no effect on the cell cycle parameters of cycling suspension-cultured cells, but had a moderate transient inhibitory effect on cells re-entering the cell cycle following phosphate starvation. Cycling cultured cells, however, could respond to NO, as indicated by the sodium nitroprusside (SNP)- and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-dependent accumulation of the ferritin protein. Based on these observations, it is hypothesized that L-NMMA-sensitive generation of NO is involved in the activation, but not the progression of the plant cell division cycle. In addition, SNP promoted and L-NMMA delayed the exogenous auxin [2,4-dichlorophenoxyacetic acid (2,4-D)] concentration-dependent formation of embryogenic cell clusters expressing the MsSERK1 gene; this further supports a link between auxin- and NO-dependent signaling pathways in plant cells.

Immunolocalization of carbonic anhydrase and phosphoenolpyruvate carboxylase in developing seeds of Medicago sativa.

To investigate the role of carbonic anhydrase (CA; EC 4.2.1.1) and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) during Medicago sativa seed development, the distribution of both proteins was examined using an immunohistological approach. Both enzymes are co-localized in most ovular and embryonic tissues. In early stages of seed development, both proteins were abundant in embryo and integuments, while at subsequent stages both proteins are accumulated in endosperm, nucellus and integuments. At late stages of seed development when both endosperm and nucellus are degraded, significant accumulation of both proteins was observed in the embryo proper. Chlorophyll was found to accumulate in embryos after the heart stage and reached a maximum at mature stage. It is suggested that CA and PEPC play a role in respiratory carbon dioxide refixation while generating malate to support amino acid and/or fatty acids biosynthesis.

Endogenous protein kinase-C activity and phosphorylated proteins in messenger ribonucleoprotein complexes of developing embryos of alfalfa.

Developing somatic and zygotic embryos of alfalfa (Medicago sativa L.) exhibited endogenous protein kinase activity and protein acceptors of phosphate groups using both cell-free translational extracts and oligo(dT)-cellulose-column-purified mRNPs. The cell-free-translation extracts from pre-cotyledonary-stage somatic embryos had approximately 50- and 100-fold more protein kinase activity than cotyledonary-stage somatic and zygotic embryos. Several polypeptides were phosphorylated; some of them were unique to the early stage and some to the late-stage developing embryos. A 65 kDa protein was phosphorylated heavily in pre-cotyledonary-stage somatic embryos. This phosphorylated protein was comprised of three main components, two of which were phosphorylated heavily. Heat-shock treated-embryos lost their exitant kinase activity and at the same time another form of protein kinase activity was activated which phosphorylated a novel 28 kDa protein. Endogenous protein kinase activity was also observed within the mRNPs of polysomal and non-polysomal fractions of developing embryos, and this phosphorylated only 65, 43 and 30 kDa proteins within these fractions. A 30 kDa protein from the pre-cotyledonary-stage somatic embryos showed a higher affinity for accepting phosphate groups than the proteins from cotyledonary-stage somatic or zygotic embryos. The activity of protein kinase was largely c-AMP-independent, but was dependent on Ca2+, phospholipid and phorbol ester. The enzyme belongs to the protein kinase-C family; the 65 kDa protein cross-reacts with antibodies made against protein kinase-C (alpha- and beta-isoforms) and it may be an autophosphorylated protein.

The Role of auxin, pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa.

Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 microM) or high (10 microM) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mM) in the presence of 1 microM 2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 microM 2,4-D-containing culture medium by 10 mM 2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.

Expression of MsLEC1- and MsLEC2-antisense genes in alfalfa plant lines causes severe embryogenic, developmental and reproductive abnormalities.

Although it has been proposed that plant lectins play a number of roles, the function of these proteins in normal plant growth and development has been unclear. To analyze the functions of putative alfalfa lectin genes, lines of transgenic alfalfa plants expressing approximately half of the open reading frame of MsLEC1 or MsLEC2, in the antisense or sense orientation, were established and analyzed. The antisense plants displayed severe abnormalities in embryogenesis, and both vegetative and reproductive development were perturbed. Some differences were observed between MsLEC1- and MsLEC2-antisense plants, and abnormalities were especially severe during the early stages of development in both the primary and secondary transgenic generations. In contrast, vector-control and sense-transgene plants exhibited normal growth and development. MsLEC1 and MsLEC2 mRNA accumulation levels were reduced in cognate antisense plants, especially during the later stages of embryogenesis, but also tended to be low in MsLEC1 sense-transgene plants. However, correlated with the phenotypic abnormalities observed in the MsLEC1-antisense plants was the specific reduction in the accumulation of a candidate MsLEC1 protein. Our results suggest that the MsLEC1 and MsLEC2 gene products, in addition to being important for embryogenesis, are required throughout alfalfa development.

The MADS-domain protein AGAMOUS-like 15 accumulates in embryonic tissues with diverse origins.

AGL15 (AGAMOUS-like 15), a member of the MADS-domain family of regulatory factors, accumulates preferentially in the organs and tissues derived from double fertilization in flowering plants (i.e. the embryo, suspensor, and endosperm). The developmental role of AGL15 is still undefined. If it is involved in embryogenesis rather than some other aspect of seed biology, then AGL15 protein should accumulate whenever development proceeds in the embryonic mode, regardless of the origin of those embryos or their developmental context. To test this, we used AGL15-specific antibodies to analyze apomictic embryogenesis in dandelion (Taraxacum officinale), microspore embryogenesis in oilseed rape (Brassica napus), and somatic embryogenesis in alfalfa (Medicago sativa). In every case, AGL15 accumulated to relatively high levels in the nuclei of the embryos. AGL15 also accumulated in cotyledon-like organs produced by the xtc2 (extra cotyledon2) mutant of Arabidopsis and during precocious germination in oilseed rape. Furthermore, the subcellular localization of AGL15 appeared to be developmentally regulated in all embryogenic situations. AGL15 was initially present in the cytoplasm of cells and became nuclear localized before or soon after embryogenic cell divisions began. These results support the hypothesis that AGL15 participates in the regulation of programs active during the early stages of embryo development.

Characterization of somatic embryogenesis-related cDNAs from alfalfa (Medicago sativa L.).

Messenger RNAs from cultures of embryogenic and non-embryogenic alfalfa (Medicago sativa L.) genotypes were used to differentially screen a cDNA library prepared from embryogenic cell masses of somatic embryo cultures to identify early-stage embryo transcripts. The three alfalfa somatic embryogenesis-specific transcripts cDNAs (ASET1, ASET2 and ASET3) identified by this screen were enriched in RNA samples from embryogenic tissues of the embryogenic genotype but were absent from petioles or mature embryos of an embryogenic genotype as well from tissue cultures of a nonembryogenic genotype. The ASET clones did not cross-hybridize and showed different patterns of expression in northerns of RNA from various fractions of alfalfa somatic embryo cultures. The ASET clones did not hybridize with the soybean embryogenesis-specific clone (Sbh1) which was shown to be expressed in embryogenic and non-embryogenic alfalfa tissue cultures. Sequencing showed ASET1 to be a partial transcript 595 nucleotides long. ASET2 was a complete transcript of 1193 nucleotides. From a comparison of the predicted open reading frame with the GenBank protein database it was concluded that ASET2 was a novel transcript. The protein predicted by the ASET2 sequence has several potential membrane-spanning domains and a potential phosphorylation site. In addition, the ASET2 cDNA had a long 5' region that contained two upstream reading frames (URFs) which could potentially code for 30 and 6 amino acid polypeptides.

Identification of a RAPD marker associated with somatic embryogenesis in alfalfa.

The current study was conducted to identify random amplified polymorphic DNA (RAPD) markers linked to genes controlling somatic embryogenesis in alfalfa. Segregation analyses of the somatic embryogenesis trait and the RAPD markers in an F1 population of 83 plants, derived from a cross between embryogenic A70-34 and non-embryogenic Arrow36 alfalfa plants, identified a polymorphic band that is associated with somatic embryogenesis. Based on the assumptions that somatic embryogenesis in alfalfa is controlled by two dominant genes with complementary effects and that the genotypes of A70-34 and Arrow36 are AAaaBbbb and aaaabbbb, respectively, the segregation data for the marker and the somatic embryogenesis trait in the F1s indicate that the marker is linked to the A locus. The maximum recombination fraction estimated for the linkage between the marker and the gene is 36.3%.

Constitutive expression of the beta-phaseolin gene in different tissues of transgenic alfalfa does not ensure phaseolin accumulation in non-seed tissue.

Phaseolin is a glycoprotein that constitutes the major storage protein in bean seeds. The phaseolin gene promoters function in a seed-specific manner. In an attempt to understand if events following transcription of the gene also contribute to the seed-specific accumulation of the phaseolin protein, we studied the effect of substituting the constitutive CaMV-35S promoter for the beta-phaseolin gene promoter on expression of the phaseolin gene in different plant organs. A chimeric gene consisting of the 35S promoter, the coding sequence of the beta-phaseolin gene (all five introns and six exons) and the 3'-flanking region of the beta-phaseolin gene, was introduced into alfalfa via Agrobacterium tumefaciens. While all organs examined shared high levels of phaseolin transcripts, the only organ that showed significant accumulation of the phaseolin protein were the mature seeds. Co-migration of the major immunoreactive polypeptides from the non-seed organs with the authentic beta-phaseolin polypeptides on SDS-PAGE indicates that the protein in non-seed organs undergoes correct post-translational processing and modification, but are more unstable in a non-seed environment.

Alfalfa heat shock genes are differentially expressed during somatic embryogenesis.

We have isolated two cDNA clones (Mshsp18-1; Mshsp18-2) from alfalfa (Medicago sativa L.) which encode for small heat shock proteins (HSPs) belonging to the hsp17 subfamily. The predicted amino acid sequences of the two alfalfa proteins are 92% identical and a similar degree of homology (90%) can be detected between Mshsp18-2 and the pea hsp17. In comparison to various members of small HSPs from soybean amino acid sequence similarities of 80-86% were identified. The alfalfa HSPs share a homologous stretch of amino acids in the carboxy terminal region with hsp22, 23, 26 from Drosophila. This region contains the GVLTV motif which is characteristic of several members of small HSPs. At room temperature alfalfa hsp18 mRNAs were not detectable in root and leaf tissues but northern analysis showed a low level of expression in microcallus suspension (MCS). The transcription of Mshsp18 genes is induced by elevated temperature, CdCl2 treatment and osmotic shock in cultured cells. In alfalfa somatic embryos derived from MCS a considerable amount of hsp18 mRNA can be detected during the early embryogenic stages under normal culture conditions. The differential expression of these genes during embryo development suggests a specific functional role for HSPs in plant cells at the time of the developmental switch in vitro.