Histological analysis of direct somatic embryogenesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected.     

Leafy cotyledon genes are essential for induction of somatic embryogenesis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

The capacity for somatic embryogenesis was studied in lec1, lec2 and fus3 mutants of Arabidopsis thaliana (L.) Heynh. It was found that contrary to the response of wild-type cultures, which produced somatic embryos via an efficient, direct process (65–94% of responding explants), lec mutants were strongly impaired in their embryogenic response. Cultures of the mutants formed somatic embryos at a low frequency, ranging from 0.0 to 3.9%. Moreover, somatic embryos were formed from callus tissue through an indirect route in the lec mutants. Total repression of embryogenic potential was observed in double (lec1 lec2, lec1 fus3, lec2 fus3) and triple (fus3 lec1 lec2) mutants. Additionally, mutants were found to exhibit efficient shoot regenerability via organogenesis from root explants. These results provide evidence that, besides their key role in controlling many different aspects of Arabidopsis zygotic embryogenesis, LEC/FUS genes are also essential for in vitro somatic embryogenesis induction. Furthermore, temporal and spatial patterns of auxin distribution during somatic embryogenesis induction were analyzed using transgenic Arabidopsis plants expressing GUS driven by the DR5 promoter. Analysis of data indicated auxin accumulation was rapid in all tissues of the explants of both wild type and the lec2-1 mutant, cultured on somatic embryogenesis induction medium containing 2,4-D. This observation suggests that loss of embryogenic potential in the lec2 mutant in vitro is not related to the distribution of exogenously applied auxin and LEC genes likely function downstream in auxin-induced somatic embryogenesis.     

The role of BAP in somatic embryogenesis induction from seed explants of <Emphasis Type="Italic">Arachis</Emphasis> species from Sections <Emphasis Type="Italic">Erectoides</Emphasis> and <Emphasis Type="Italic">Procumbentes</Emphasis>

Somatic embryogenesis was induced from seed explants of Arachis archeri, A. porphyrocalix (Section Erectoides) and A. appressipila (Section Procumbentes) in response to 6-benzylaminopurine (BAP). Embryo axes first developed into single shoots in response to 4.4 μM BAP. Friable embryogenic calluses were produced from the hypocotyl region of these explants in response to different BAP concentrations. Embryonic leaflets also gave rise to friable calluses, but somatic embryos were only observed in explants of A. archeri and A. appressipila. Histological analyses revealed the presence of heart-shaped, torpedo and cotyledonary stages embryos, both as isolated and fused structures. A low frequency of embryo-to-plant conversion was achieved by inducing shoot development on medium solidified with 0.5% phytagel and supplemented with 1.5% or 3% sucrose. Rooting was induced on MS supplemented with indole-3-acetic acid (IAA).     

BREVIS RADIX is involved in cytokinin-mediated inhibition of lateral root initiation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In contrast to auxin, relatively little is known about the molecular mechanism of cytokinin (CTK) inhibition of lateral root initiation. Previous studies demonstrated that BREVIS RADIX (BRX), a protein of unknown biochemical function, maintains a rate-limiting brassinosteroid biosynthesis enzyme expression to keep brassinosteroid biosynthesis above a critical threshold. Here, we show that the brx-2 mutant is insensitive to exogenous CTK-induced inhibition of lateral root initiation and that this can be restored by embryonic brassinosteroid treatment. However post-embryonic brassinosteroid treatment can not rescue brx-2 mutant phenotype in the presence of CTK. Meanwhile the brassinosteroid receptor defective mutant bri1-6 shows normal CTK-mediated inhibition on LR growth. These results suggest the CTK-mediated inhibition of LR initiation is not directly dependent on brassinosteroid level. Furthermore, compared with wild type, brx-2 exhibits altered auxin response in presumptive founder cells, lateral root primodia and primary root tip in the presence of exogenous CTK. We concluded that CTK inhibition on lateral root initiation depend on specific auxin response loss in presumptive founder cell. The aberrant primary root growth caused by the embryonic brassinosteroid shortage can indirectly result in the lateral root phenotype of brx-2 in presence of CTK. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and Functional Analysis of Phosphoproteins Regulated by Auxin in <Emphasis Type="Italic">Arabidopsis</Emphasis> Roots

The phytohormone auxin modulates diverse aspects of plant growth and development. Protein phosphorylation is believed to play a key role in regulating auxin-mediated responses. To determine the phosphoproteins affected by auxin in Arabidopsis, we used phospho-specific antibodies to analyze their profiles on two-dimensional gels, then identified them by mass spectrometry. We found two phosphoproteins, enolase and the beta subunit of succinyl-CoA synthetase (SCS-beta), and noted that their phosphorylation was increased by auxin. To investigate their importance in auxin-mediated processes, we characterized Arabidopsis knockout mutants of the two genes. A homozygous null mutation in the gene for SCS-beta conferred embryo lethality. The enolase knockout mutants showed defects in root development similar to those of auxin-related mutants such as alf3 and xbat32. Therefore, we suggest that enolase is involved in auxin-regulated processes.     

Proteomic analysis of somatic embryogenesis in <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Two dimensional gel electrophoresis coupled to mass spectrometry has been used to study the somatic embryogenesis in Vitis vinifera, by comparing embryogenic and non embryogenic calluses of the Thompson seedless cv. More than 1,000 spots were reproducibly resolved in colloidal Coomassie brilliant blue stained gels over a pI nonlinear range of 3–10 in the first dimension and using homogeneous 12.5% polyacrylamide gels in the second dimension. The expression pattern of 35 spots differed significantly between the two samples. These spots were processed by mass spectrometry analysis and the protein identity was assigned by using both the non-redundant protein and EST databases. Several responsive proteins, some already known to be involved in the somatic embryogenesis process while others, for the first time put into relation with this process, have been described. Moreover, they have been subdivided in functional categories, and their putative role is discussed in terms of their relevance in the somatic embryogenesis process.     

Polyethylene glycol and abscisic acid improve maturation and regeneration of<Emphasis Type="Italic"> Panax ginseng</Emphasis> somatic embryos

Embryogenic culture was initiated from mature zygotic embryos of Panax ginseng. Multiple somatic embryos formed and proliferated on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2.26 M) and kinetin (0.046 M). Mature as well as immature somatic embryos grew into plantlets lacking roots on the same media. Histomorphological analysis of somatic embryos treated with abscisic acid (ABA) and polyethylene glycol (PEG 4000) showed a slight improvement in the root meristem organization of torpedo-stage embryos (embryos were more compact and their cells exhibited a lower degree of vacuolation). Shoot regeneration of non-treated somatic embryos was 31% while that for somatic embryos treated with PEG 4000 and ABA was 70%. Moreover, 75% of plants regenerated from PEG- and ABA-treated embryos formed roots while plants from non-treated embryos did not form roots.Abbreviations ABA (±)-Abscisic acid - BAP N ⁶-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA ₃ Gibberellic acid - Kin Kinetin - MS Murashige and Skoog medium - PEG 4000 Polyethylene glycol 4000 - PGR Plant growth regulators Communicated by H. van Onckelen     

Expression patterns of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">dpp</Emphasis> in the gastropod <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

We isolated the full-length cDNAs of engrailed and dpp-BMP2/4 orthologues from the pond snail Lymnaea stagnalis and examined their expression patterns during development by the whole mount in situ hybridization. At the gastrula and trochophore stages, engrailed is expressed in the peripheral ectoderm of the presumptive and invaginating shell gland, corroborating its role in the shell formation that is widely conserved among molluscs. At the same stages, dpp-BMP2/4 is expressed in the right-hand side ectoderm of the shell gland and in the invaginating stomodaeum. Unlike in the gastropod Patella vulgata, our results suggested that dpp-BMP2/4 has a role in the shell formation, rather than in the regional specification and that it could be involved in the specification pathway of the left–right asymmetry of the developing shell in L. stagnalis.     

Optimization of conditions for transient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated gene expression assays in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The β-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85–90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.     

Clustered root distribution in mature stands of <Emphasis Type="Italic">Fagus sylvatica</Emphasis> and <Emphasis Type="Italic">Picea abies</Emphasis>

Distribution of small roots (diameter between 2 mm and 5 mm) was studied in 19 pits with a total of 72 m² trench profile walls in pure stands of Fagus sylvatica and Picea abies. Root positions within the walls were marked and transformed into x-coordinates and y-coordinates. In a GIS-based evaluation, zones of potential influence around each root were calculated. The total potential influence produced isoline maps of relative root influence zones, thus indicating small root clustering. The questions studied were (1) whether there were marked clusters of small roots in the soil and (2) whether trees surrounding the pit (defined as tree density) correlate with the root abundance and distribution on the trench profile walls. Small roots of both species showed maximum abundance in the top 20 cm of the soil, where pronounced root clusters occurred next to areas with only low root accumulation. The area of root clusters did not differ significantly between the two stands. Weighted clumping, WC, calculated as a product of root class, and its area was used as an index of root clustering, which again did not differ between beech and spruce stands. However, evaluations on a single root level showed that beech achieved the same degree of clustering with lower number of roots. Regardless of soil properties related to root clusters, a significantly higher clustering acquired per root for beech than for spruce suggests beech to be more efficient in belowground acquisition of space. Because none of the parameters describing root clustering were correlated with tree density around the investigated soil profiles, clusters of small roots are inherently present within the tree stands.     

Factors affecting somatic embryogenesis in <Emphasis Type="Italic">Prunus incisa</Emphasis> cv. February Pink

Factors affecting somatic embryogenesis from root explants of Prunus incisa Thunb. cv. February Pink were investigated. Using a medium containing Murashige and Skoog salts and vitamins supplemented with 10 M 2,4-dichlorophenoxyacetic (2,4-D), we evaluated the effects of light, growth regulators, amino acids, carbohydrate source, and root induction medium. Explants cultured under light or dark conditions both resulted in the formation of embryos. Embryogenesis was inhibited by the addition of 6-benzyladenine, thidiazuron, or gibberellic acid to the medium. Amino acids were not effective in promoting embryogenesis, with high levels of amino acids actually inhibiting it. Sucrose and glucose effectively induced embryogenesis, while sorbitol and mannitol completely inhibited it. Sucrose and glucose also promoted secondary embryogenesis. Embryos that formed in medium containing 4% or 5% sucrose were abnormally shaped and did not fully develop, while those that formed in medium with sucrose concentrations of 2% or 3% were much more vigorous. Root explants that were induced on medium containing 1.0 M indole-3-butyric acid (IBA) produced more somatic embryos than explants induced on medium without IBA. Approximately 50% of the roots induced on medium containing 1.0 M IBA produced somatic embryos on medium containing 10 M 2,4-D and 3% sucrose.Abbreviations BA 6-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - TDZ Thidiazuron     

Changes in DNA methylation during somatic embryogenesis in <Emphasis Type="Italic">Cucurbita pepo</Emphasis> L.

Three pumpkin embryogenic lines were initiated on wounded zygotic embryos cultured on medium with or without 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryo development was controlled by the availability of various compounds in the medium: presence/absence of 2,4-D, nitrogen sources. The highest rate of DNA methylation was in the early embryo stages, predominantly on MSC medium with 2,4-D and on auxin-free medium supplemented with 1.0 mM NH₄Cl. DNA methylation was correlated with early embryo development in a manner that was not exclusively dependent on the presence/absence of exogenous auxin. DNA methylation decreased during embryo maturation on auxin-free MSC medium and on auxin-free MSC supplemented with 12.3 M 5-azacytidine (5-azaC). The embryogenic features of the pumpkin tissue were preserved, even after a 2-month treatment with 5-azaC.Abbreviations 5-azaC 5-Azacytidine - CRED-RA Coupled restriction enzyme digestion and random amplification - 2,4-D 2,4-Dichlorophenoxyacetic acid - DNMRT Duncans new multiple range test - IAA Indole-3-acetic acid - 5-mC 5-Methylcytosine     

Patterns of DNA Variation Among Three Centromere Satellite Families in <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> and <Emphasis Type="Italic">A</Emphasis>. <Emphasis Type="Italic">lyrata</Emphasis>

We describe patterns of DNA variation among the three centromeric satellite families in Arabidopsis halleri and lyrata. The newly studied subspecies (A. halleri ssp. halleri and A. lyrata ssp. lyrata and petraea), like the previously studied A. halleri ssp. gemmifera and A. lyrata ssp. kawasakiana, have three different centromeric satellite families, the older pAa family (also present in A. arenosa) and two families, pAge1 and pAge2, that probably evolved more recently. Sequence variability is high in all three satellite families, and the pAa sequences do not cluster by their species of origin. Diversity in the pAge2 family is complex, and different from variation among copies of the other two families, showing clear evidence for exchange events among family members, especially in A. halleri ssp. halleri. In A. lyrata ssp. lyrata there is some evidence for recent rapid spread of pAge2 variants, suggesting selection favoring these sequences. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Brian Morton]     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of poinsettia, <Emphasis Type="Italic">Euphorbia pulcherrima</Emphasis>, with virus-derived hairpin RNA constructs confers resistance to <Emphasis Type="Italic">Poinsettia mosaic virus</Emphasis>

Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2-3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.     

Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey     

Efficient <Emphasis Type="Italic">in vitro</Emphasis> bulblet regeneration from immature embryos of endangered <Emphasis Type="Italic">Sternbergia fischeriana</Emphasis>

Sternbergia fischeriana is an endangered geophyte and therefore in vitro micropropagation of this plant will have great importance for germplasm conservation and commercial production. Bulb scale and immature embryo explants of S. fischeriana were cultured on different nutrient media supplemented with various concentrations of plant growth regulators. Immature embryos produced higher number of bulblets than bulb scales. Large numbers of bulblets were regenerated (over 80 bulblets/explants) from immature embryos on Murashige and Skoog (MS) medium supplemented with 4 mg l^–1 6-benzylaminopurine (BA) and 0.25 mg l^–1 -naphthaleneacetic (NAA) or 2 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D) after 14 months of culture initiation. Regenerated bulblets were kept at 5 °C for 5 weeks and then transplanted to a potting mixture.