Characterization of the new human pleomorphic undifferentiated sarcoma <Emphasis Type="Italic">TP53</Emphasis>-<Emphasis Type="Italic">null</Emphasis> cell line <Emphasis Type="Italic">mfh</Emphasis>-<Emphasis Type="Italic">val2</Emphasis>

Pleomorphic undifferentiated sarcoma (PUS), also called malignant fibrous histiocytoma, is a soft tissue sarcoma which occurs predominantly in the extremities. Its origin is a poorly defined mesenchymal cell, which derives to histiocytic and fibroblastic cells. The patient, a 58 year-old man, presented a lesion located in the forearm composed by spindle cells and multinucleated giant cells, which expressed vimentin and adopted a histological pattern formed by irregular-swirling fascicles. Cells were cultured in vitro and a new cell line was established. We characterized this new cell line by histological analyses, cytogenetics (using G-bands and spectral karyotype technique) and cytometric analyses. Cells were grown in culture for more than 100 passages. They had elongated or polygonal morphology. The cells presented a saturation rate of 70,980 cells/cm², a plating efficiency of 21.5% and a mitotic index of 21 mitoses per field. The cell line was tumorigenic in nude mice. The ploidy study using flow cytometry revealed an aneuploid peak with a DNA index of 1.43. A side population was detected, demonstrating the presence of stem and progenitor cells. Cytogenetics showed a hypotriploid range with many clonal unbalanced rearrangements. Loss of p53 gene was evidenced by MLPA. We describe, for the first time, the characterization of a new human PUS TP53-null cell line called mfh-val2. Mfh-val2 presents a wide number of applications as a TP53-null cell line and a great interest in order to characterize genetic alterations influencing the oncogenesis or progression of PUS and to advance in the biological investigation of this tumor.     

Molecular mapping of a rust resistance gene <Emphasis Type="Italic">R</Emphasis><Subscript><Emphasis Type="Italic">14</Emphasis></Subscript> in cultivated sunflower line PH 3

Sunflower, the fifth largest oilseed crop in the world, plays an important role in human diets. Recently, sunflower production in North America has suffered serious yield losses from newly evolved races of sunflower rust (Puccinia helianthi Schwein.). The rust resistance gene, designated R ₁₄ , in a germplasm line PH 3 originated from a wild Helianthus annuus L. population resistant to 11 rust races. PH 3 has seedling with an extraordinary purple hypocotyl color. The objectives of this study were to map both the R ₁₄ rust resistance gene and the purple hypocotyl gene-designated PHC in PH 3, and to identify molecular markers for marker-assisted breeding for sunflower rust resistance. A set of 517 mapped SSR/InDel and four SNP markers was used to detect polymorphisms between the parents. Fourteen markers covering a genetic distance of 17.0 cM on linkage group (LG) 11 were linked to R ₁₄ . R ₁₄ was mapped to the middle of the LG, with a dominant SNP marker NSA_000064 as the closest marker at a distance of 0.7 cM, and another codominant marker ORS542 linked at 3.5 cM proximally. One dominant marker ZVG53 was linked on the distal side at 6.9 cM. The PHC gene was also linked to R ₁₄ with a distance of 6.2 cM. Chi-squared analysis of the segregation ratios of R ₁₄ , PHC, and ten linked markers indicated a deviation from an expected 1:2:1 or 3:1 ratio. The closely linked molecular or morphological markers could facilitate sunflower rust-resistant breeding and accelerate the development of rust-resistant hybrids.     

Fine mapping of the chromosome 5B region carrying closely linked rust resistance genes <Emphasis Type="Italic">Yr47</Emphasis> and <Emphasis Type="Italic">Lr52</Emphasis> in wheat

Key message

Fine mapping of Yr47 and Lr52 in chromosome arm 5BS of wheat identified close linkage of the marker sun180 to both genes and its robustness for marker-assisted selection was demonstrated.

Abstract

The widely effective and genetically linked rust resistance genes Yr47 and Lr52 have previously been mapped in the short arm of chromosome 5B in two F₃ populations (Aus28183/Aus27229 and Aus28187/Aus27229). The Aus28183/Aus27229 F₃ population was advanced to generate an F₆ recombinant inbred line (RIL) population to identify markers closely linked with Yr47 and Lr52. Diverse genomic resources including flow-sorted chromosome survey sequence contigs representing the orthologous region in Brachypodium distachyon, the physical map of chromosome arm 5BS, expressed sequence tags (ESTs) located in the 5BS6-0.81-1.00 deletion bin and resistance gene analog contigs of chromosome arm 5BS were used to develop markers to saturate the target region. Selective genotyping was also performed using the iSelect 90 K Infinium wheat SNP assay. A set of SSR, STS, gene-based and SNP markers were developed and genotyped on the Aus28183/Aus27229 RIL population. Yr47 and Lr52 are genetically distinct genes that mapped 0.4 cM apart in the RIL population. The SSR marker sun180 co-segregated with Lr52 and mapped 0.4 cM distal to Yr47. In a high resolution mapping population of 600 F₂ genotypes Yr47 and Lr52 mapped 0.2 cM apart and marker sun180 was placed 0.4 cM distal to Lr52. The amplification of a different sun180 amplicon (195 bp) than that linked with Yr47 and Lr52 (200 bp) in 204 diverse wheat genotypes demonstrated its robustness for marker-assisted selection of these genes.

     

Molecular evidence of symbiotic activity between <Emphasis Type="Italic">Symbiodinium</Emphasis> and <Emphasis Type="Italic">Tridacna maxima</Emphasis> larvae

Dinoflagellates in the genus Symbiodinium (zooxanthellae) provide the photosynthesis that sustains the majority of primary production in coral reefs. They occur symbiotically with several phyla, including mollusks such as giant clams (Tridacna spp.). This mutualistic association is obligatory for the giant clams, but the exact point in which this symbiosis is established and the main translocated photosynthate are unknown. In this study, we tracked the expression of specific genes for symbiosis and glycerol synthesis during a time course experiment. Giant clam larvae were raised until 75 h post-fertilization and then infected with cultured isolates of Symbiodinium clade A3. Expression of symbiosis-specific and housekeeping genes was monitored at four time points. The expression of H⁺-ATPase, a symbiosis-specific gene in Symbiodinium, was observed at 24 h after symbiont acquisition by the clam larvae. The expression of an enzyme responsible for glycerol synthesis was also observed. Together, these results show that the symbiotic relationship was already in place 24 h after Symbiodinium acquisition, during veliger larval stage. This is the first report using a molecular symbiosis-specific marker that supports symbiotic activity between Symbiodinium and a metazoan larva of an organism that acquires symbionts horizontally. From the expression of the glycerol-synthesizing gene, it was qualitatively determined that Symbiodinium cells may produce glycerol regardless of whether they are free-living or in symbiosis.     

<Emphasis Type="Italic">Braf</Emphasis>, <Emphasis Type="Italic">Kras</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis> epigenetic changes-associated chronic gastritis in Egyptian patients with and without gastric cancer

We aimed to study MLH1 and MGMT methylation status in Helicobacter pylori-associated chronic gastritis in Egyptian patients with and without gastric cancer. 39 patients were included in our study. They were divided into 2 groups; patients without (group I) and with gastric adenocarcinoma (group II). Patients were subjected to clinical examination, abdominal ultrasound and upper endoscopy for gastric biopsy. Biopsies were subjected to urease test, histological examination, and DNA purification. H. pylori, Braf, Kras, MLH1 and MGMT methylation were assessed by quantitative PCR. DNA sequencing was performed to assess Braf and Kras genes mutation. qPCR of H. pylori was significantly higher in patients with adenocarcinoma (group II) than those without adenocarcinoma (group I); with a p < 0.001 as well as in patients with age above 50 years with a p value = 0.008. By applying logistic regression analysis it was reported that the H. pylori qPCR is a significant predictor to the adenocarcinoma with OR = 1.025 (95 % CI: 1. 002–1.048), with sensitivity of 90 % and specificity of 100 %. Adenocarcinoma patients had a significantly higher mean age and levels of H. Pylori, Braf, K-ras, methylated MGMT and methylated MLH1 than those of gastritis patients. DNA sequence analysis of Braf (codon 12) and Kras (codon 600) had genes mutation in gastric adenocarcinoma versus chronic gastritis. Conclusion: H. pylori may cause epigenetic changes predisposing the patients to cancer stomach. Estimation of H. pylori by qPCR can be a good predictor to adenocarcinoma. Braf and Kras genes mutation were reveled in gastritis and adenocarcinoma patients.     

An intercalary translocation from <Emphasis Type="Italic">Agropyron</Emphasis><Emphasis Type="Italic">cristatum</Emphasis> 6P chromosome into common wheat confers enhanced kernel number per spike

Main conclusion

This study explored 6P chromosomal translocations in wheat, and determined the effects of 6P intercalary chromosome segments on kernel number per wheat spike. Exploiting and utilising gene(s) from wild relative species has become an essential strategy for wheat crop improvement. In the translocation line Pubing2978, the intercalary 6P chromosome segment from Agropyron cristatum (L.) Gaertn. (2n = 4x = 28, PPPP) carried valuable multi-kernel gene(s) and was selected from the offspring of the common wheat plant Fukuho and the irradiated wheat-A. cristatum 6P disomic substitution line 4844-8. Genomic in situ hybridisation (GISH), dual-colour fluorescence in situ hybridisation (FISH), and molecular markers were used to detect the small segmental 6P chromosome in the wheat background and its translocation breakpoint. Cytological studies demonstrated that Pubing2978 was a T1AS-6PL-1AS·1AL intercalary translocation with 42 chromosomes. The breakpoint was located near the centromeric region on the wheat chromosome 1AS and was flanked by the markers SSR12 and SSR283 based on an F₂ linkage map. The genotypic data, combined with the phenotypic information, implied that A. cristatum 6P chromosomal segment plays an important role in regulating the kernel number per spike (KPS). By comparison, the mean value of KPS in plants with translocations was approximately 10 higher than that in plants without translocations in three segregated populations. Moreover, the improvement in KPS was likely achieved by increasing both the spikelet number per spike (SNS) and the kernel number per spikelet. These excellent agronomic traits laid the foundation for further investigation of valuable genes and make the Pubing2978 line a promising germplasm for wheat breeding.

     

Development and characterization of <Emphasis Type="Italic">japonica</Emphasis> rice lines carrying the brown planthopper-resistance genes <Emphasis Type="Italic">BPH12</Emphasis> and <Emphasis Type="Italic">BPH6</Emphasis>

The brown planthopper (Nilaparvata lugens Stål; BPH) has become a severe constraint on rice production. Identification and pyramiding BPH-resistance genes is an economical and effective solution to increase the resistance level of rice varieties. All the BPH-resistance genes identified to date have been from indica rice or wild species. The BPH12 gene in the indica rice accession B14 is derived from the wild species Oryza latifolia. Using an F₂ population from a cross between the indica cultivar 93-11 and B14, we mapped the BPH12 gene to a 1.9-cM region on chromosome 4, flanked by the markers RM16459 and RM1305. In this population, BPH12 appeared to be partially dominant and explained 73.8% of the phenotypic variance in BPH resistance. A near-isogenic line (NIL) containing the BPH12 locus in the background of the susceptible japonica variety Nipponbare was developed and crossed with a NIL carrying BPH6 to generate a pyramid line (PYL) with both genes. BPH insects showed significant differences in non-preference in comparisons between the lines harboring resistance genes (NILs and PYL) and Nipponbare. BPH growth and development were inhibited and survival rates were lower on the NIL-BPH12 and NIL-BPH6 plants compared to the recurrent parent Nipponbare. PYL-BPH6 + BPH12 exhibited 46.4, 26.8 and 72.1% reductions in population growth rates (PGR) compared to NIL-BPH12, NIL-BPH6 and Nipponbare, respectively. Furthermore, insect survival rates were the lowest on the PYL-BPH6 + BPH12 plants. These results demonstrated that pyramiding different BPH-resistance genes resulted in stronger antixenotic and antibiotic effects on the BPH insects. This gene pyramiding strategy should be of great benefit for the breeding of BPH-resistant japonica rice varieties.     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

Characterization of morphology and resistance to <Emphasis Type="Italic">Blumeria graminis</Emphasis> of winter triticale monosomic addition lines with chromosome 2D of <Emphasis Type="Italic">Aegilops</Emphasis><Emphasis Type="Italic">tauschii</Emphasis>

Key message

Allocation of the chromosome 2D of Ae. tauschii in triticale background resulted in changes of its organization, what is related to varied expression of genes determining agronomically important traits.

Abstract

Monosomic alien addition lines (MAALs) are crucial for transfer of genes from wild relatives into cultivated varieties. This kind of genetic stocks is used for physical mapping of specific chromosomes and analyzing alien genes expression. The main aim of our study is to improve hexaploid triticale by transferring D-genome chromatin from Aegilops tauschii × Secale cereale (2n = 4x = 28, DDRR). In this paper, we demonstrate the molecular cytogenetics analysis and SSR markers screening combined with phenotype analysis and evaluation of powdery mildew infection of triticale monosomic addition lines carrying chromosome 2D of Ae. tauschii. We confirmed the inheritance of chromosome 2D from the BC₂F₄ to the BC₂F₆ generation of triticale hybrids. Moreover, we unveiled a high variable region on the short arm of chromosome 2D, where chromosome rearrangements were mapped. These events had direct influence on plant height of hybrids what might be connected with changes at Rht8 loci. We obtained 20 semi-dwarf plants of BC₂F₆ generation carrying 2D chromosome with the powdery mildew resistance, without changes in spike morphology, which can be used in the triticale breeding programs.

     

Comparison of molecular genetic utilities of TD,AFLP, and MSAP among the accessions of <Emphasis Type="Italic">japonica,indica</Emphasis>, and <Emphasis Type="Italic">Tongil</Emphasis> of <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

Rice is one of the most important food crops in the world. Genetic diversity is essential for cultivar improvement programs. We compared genetic diversity derived from insertion–deletion (in–del) or base substitutions by amplified fragment length polymorphism (AFLP), from transposon transposition mutations by transposon display (TD), and from cytosine methylation by methylation-sensitive amplified polymorphism (MSAP) in japonica, indica, and Tongil type varieties of Oryza sativa L. Polymorphic profiles from the three marker systems allowed us to clearly distinguish the three types of varieties. The indica type varieties showed the highest genetic diversity followed by the Tongil and japonica type varieties. Of the three marker systems, TD produced the highest marker indices, and AFLP and MSAP produced similar marker indices. Pair-wise comparisons of the three marker systems showed that the correlation between the two genetic markers systems (AFLP and TD, r = 0.959) was higher than the correlations between the genetic and epigenetic marker systems (AFLP and MSAP, r = 0.52; TD and MSAP, r = 0.505). Both genetic marker systems had similar levels of gene differentiation (G _ST ) and gene flow (N _m ), which differed in the epigenetic marker system. Although the G _ST of the epigenetic marker system was lower than the genetic marker systems, the N _m of the epigenetic marker system was higher than in the genetic marker systems, indicating that epigenetic variations have a greater influence than genetic variations among the O. sativa L. types.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) somatic embryos with fluorescent marker genes and optimization of transgenic plant recovery

Avocado globular somatic embryos were transformed with three binary vectors, pK7FNF2, pK7RNR2 and pK7S*NF2, harboring the marker genes gfp, DsRed and a gfp-gus fusion gene, respectively. GFP and DsRed fluorescence was detected in embryogenic lines growing in selection medium 2 months after Agrobacterium inoculation. The fluorescence signal was maintained thereafter in transgenic calli, as well as in mature somatic embryos. Red fluorescence in pK7RNR2 transgenic lines was higher and more easily observable than GFP fluorescence. Furthermore, calli transformed with pK7S*NF2, harboring gfp-gus, showed higher level of fluorescence than those transformed with pK7FNF2, containing two gfp. To improve plant recovery, maturated transgenic embryos that failed to germinate or showed an underdeveloped shoot were cultured for 4 weeks in a medium with 1 mg l^?1 TDZ and 1 mg l^?1 BA after partial removal of cotyledons. A 50% of embryos developed one or several shoots on the cut surface. These embryos were cultured for 4 additional weeks in a medium with 1 mg l^?1 BA for shoot elongation and then, shoots were grafted in vitro onto seedling rootstocks. Culture of micrografts in solid MS medium supplemented with 1 mg l^?1 BA allowed a 60–80% success rate. Young leaves from transgenic plants showed GFP or DsRed fluorescence located in the nucleus. The results obtained indicate that fluorescent marker genes, especially DsRed, could be useful for early selection of transgenic material and optimization of the transformation parameters in avocado. Furthermore, the protocol established allowed the successful recovery of transgenic plants, one of the main limiting steps in avocado transformation.     

Intertidal benthic communities associated with the macroalgae <Emphasis Type="Italic">Iridaea cordata</Emphasis> and <Emphasis Type="Italic">Adenocystis utricularis</Emphasis> in King George Island,Antarctica

Antarctic benthos has been a main target in Antarctic research, but very few quantitative studies have been carried out in the littoral zone, which may be seasonally covered by macroalgae. In this work, we studied (1) cover and biomass of the macroalgae Iridaea cordata and Adenocystis utricularis, and (2) composition of macrobenthic assemblage associated with these macroalgal species at three locations at King George Island: Mareograph Beach (1 M), Tank’s Bay (2R) and Ardley Bay (3R). Iridaea cordata was collected by completely detaching the algae from the substrate, while A. utricularis was scraped. Adenocystis utricularis covered more than 80 % of the substrate at all locations, while coverage of Iridaea cordata was below 53 % or absent (3R). Fresh biomass of I. cordata was 0.8–61.4 g/individual and 4.7–93.0 g/100 cm² for A. utricularis. The assemblage associated with both macroalgae differed significantly between sites. The studied fauna was composed mainly of amphipods, gastropods and bivalves. Species diversity was higher in the community associated with A. utricularis. A total of ~27 ind/g DW were found associated with I. cordata, while ~112 ind/g DW were found associated with A. utricularis. The most abundant groups associated with I. cordata were amphipods at 1 M (57 %) and gastropods at 2R (46 %). Both groups were responsible for the dissimilarity between localities (62.50 %). The most abundant groups associated with A. utricularis were the gastropods at all localities reaching up to 82 % at 1 M. This study provides a first baseline on the diversity and abundance of benthic assemblages associated with intertidal macroalgae in the southwest of King George Island.     

Histological and cytological studies of plant infection by <Emphasis Type="Italic">Erysiphe euonymi</Emphasis>-<Emphasis Type="Italic">japonici</Emphasis>

Powdery mildew caused by Erysiphe euonymi-japonici (Eej) is an increasingly serious fungal disease on Euonymus japonicus that is an important ornamental plant. However, little is currently known about infection and pathogenesis of Eej on E. japonicus. Here, we report plant infection by Eej at the histological and cytological levels. Eej caused severe disease symptoms with white and snow-like colonies on leaf surfaces of E. japonicus. Microscopic observations were conducted continuously to define infection process of Eej on E. japonicus. Eej conidia germinated to produce appressorial germ tubes on leaf surfaces and formed irregular haustoria in plant epidermal cells at 6 h post-inoculation (hpi) and 12 hpi, respectively. After uptaking nutrients from host cells by haustoria, Eej formed numerous hyphae and extensive colonization on leaf surfaces at 96 hpi and finally produced abundant conidiophores and new conidia on leaf surfaces at 168 hpi. In addition, there was consistently a single nucleus in different Eej infection structures and haustorial development could be divided into three major stages, including formation of penetration peg, formation of haustorial neck and initial haustorium, and maturation of haustorium. These results provide useful information for further determination of Eej pathogenesis and finally controlling the disease.     

Impact of elevated CO<Subscript>2</Subscript> in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> rooted stem cuttings inoculated with <Emphasis Type="Italic">Frankia</Emphasis>

Impact of different levels of elevated CO ₂ on the activity of Frankia (Nitrogen-fixing actinomycete) in Casuarina equisetifolia rooted stem cuttings has been studied to understand the relationship between C. equisetifolia, Frankia and CO₂. The stem cuttings of C. equietifolia were collected and treated with 2000 ppm of Indole Butyric Acid (IBA) for rooting. Thus vegetative propagated rooted stem cuttings of C. equisetifolia were inoculated with Frankia and placed in the Open top chambers (OTC) with elevated CO₂ facilities. These planting stocks were maintained in the OTC for 12 months under different levels of elevated CO₂ (ambient control, 600 ppm, 900 ppm). After 12 months, the nodule numbers, bio mass, growth, and photosynthesis of C. equisetifolia rooted stem cuttings inoculated with Frankia were improved under 600 ppm of CO₂. The rooted stem cuttings of C. equisetifolia inoculated with Frankia showed a higher number of nodules under 900 ppm of CO₂ and cuttings without Frankia inoculation exhibited poor growth. Tissue Nitrogen (N) content was also higher under 900 ppm of CO₂ than ambient control and 600 ppm levels. The photosynthetic rate was higher (17.8 μ mol CO₂ m^?2 s^?1) in 900 ppm of CO₂ than in 600 ppm (13.2 μ mol CO₂ m^?2 s^?1) and ambient control (8.3 μ mol CO₂ m^?2 s^?1). This study showed that Frankia can improve growth, N fixation and photosynthesis of C. equietifolia rooted stem cuttings under extreme elevated CO₂ level conditions (900 ppm).     

<Emphasis Type="Italic">Pseudomonas</Emphasis> spp. increases root biomass and tropane alkaloid yields in transgenic hairy roots of <Emphasis Type="Italic">Datura</Emphasis> spp.

Transgenic hairy roots of Datura spp., established using strain A4 of Agrobacterium rhizogenes, are genetically stable and produce high levels of tropane alkaloids. To increase biomass and tropane alkaloid content of this plant tissue, four Pseudomonas strains, Pseudomonas fluorescens P64, P66, C7R12, and Pseudomonas putida PP01 were assayed as biotic elicitors on transgenic hairy roots of Datura stramonium, Datura tatula, and Datura innoxia. Alkaloids were extracted from dried biomass, and hyoscyamine and scopolamine were quantified using liquid chromatography-tandem mass spectrometry analysis. D. stramonium and D. innoxia biomass production was stimulated by all Pseudomonas spp. strains after a 5-d treatment. All strains of P. fluorescens increased hyoscyamine yields compared to untreated cultures after both 5 and 10 d of treatment. Hyoscyamine yields were highest in D. tatula cultures exposed to a 5-d treatment with C7R12 (16.633 + 0.456 mg g^?1 dry weight, a 431% increase) although the highest yield increases compared to the control were observed in D. stramonium cultures exposed to strains P64 (511% increase) and C7R12 (583% increase) for 10 d. D. innoxia showed the highest scopolamine yields after elicitation with P. fluorescens strains P64 for 5 d (0.653 + 0.021 mg g^?1 dry weight, a 265% increase) and P66 for 5 and 10 d (5 d, 0.754 + 0.0.031 mg g^?1 dry weight, a 321% increase; 10 d 0.634 + 0.046 mg g^?1 dry weight, a 277% increase). These results show that the Pseudomonas strains studied here can positively and significantly affect biomass and the yields of hyoscyamine and scopolamine from transgenic roots of the three Datura species.