A Simple Method for In Vivo Expression Studies of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">citri</Emphasis>

A major problem in studying bacterial plant pathogens is obtaining the microorganism directly from the plant tissue to perform in vivo expression (protein or mRNA) analyses. Here we report an easy and fast protocol to isolate Xanthomonas axonopodis pv. citri directly from the host plant, in sufficient amounts to perform protein fingerprinting by 2-D gel electrophoresis as well as RNA expression assays. The protein profile obtained was very similar to that of X. axonopodis pv. citri grown in the presence of a leaf extract of Citrus sinensis; however, some differential proteins expressed in vivo were observed. Total RNA extraction revealed typical 16S and 23S bands in the agarose gel, and RT-PCR reactions using primers specific for genes of the bacterium confirmed the quality of the RNA preparation. Also, RT-PCR reactions using plant ribosomal primers were employed, and no amplification product was obtained, indicating that plant RNA is not present in the bacterium RNA sample.     

<Emphasis Type="Italic">In vitro</Emphasis> root induction in weedy <Emphasis Type="Italic">amaranthus</Emphasis> species to obtain mitotic chromosomes

Summary Mitotic chromosome analysis has proven to be an important tool in monitoring the potential for genetic exchange among related plant species. One major obstacle to using mitotic chromosome analysis in any species is obtaining large numbers of clear, well-spread metaphase chromosomes necessary to perform cytological techniques such as chromosome banding and fluorescent in situ hybridization. The ability to obtain good chromosome spreads is in part determined by the number and morphology of the roots, which contain the metaphase tissue. Many Amaranthus species produce very thin, delicate roots. The technique used in the process described herein provides for much more substantial roots, allowing for higher probability of obtaining well-spread metaphase chromosomes. Seeds were planted in a soilless mixture, and then cuttings and leaves were taken from the plants. The cuttings were sterilized and placed in Murashige and Skoog (MS) media, while leaf tissue was analyzed by flow cytometry, both pre-and post-propagation, to obtain DNA contents. No changes in DNA content were observed. The in vitro procedure produced significantly larger roots than were produced in soilless mix. Furthermore, all of the in vitro roots observed had 32 chromosomes of normal morphology. In vitro root propagation allowed large numbers of roots to be obtained from a single plant, thereby resulting in increased probability of obtaining cells with metaphase chromosomes that reflected the original plants' chromosome numbers and therefore may be used for molecular cytogenetic analysis.     

What does it take to be a plant pathogen: genomic insights from <Emphasis Type="Italic">Streptomyces</Emphasis> species

Plant pathogenicity is rare in the genus Streptomyces, with only a dozen or so species possessing this trait out of the more than 900 species described. Nevertheless, such species have had a significant impact on agricultural economies throughout the world due to their ability to cause important crop diseases such as potato common scab, which is characterized by lesions that form on the potato tuber surface. All pathogenic species that cause common scab produce a family of phytotoxins called the thaxtomins, which function as cellulose synthesis inhibitors. In addition, the nec1 and tomA genes are conserved in several pathogenic streptomycetes, the former of which is predicted to function in the suppression of plant defense responses. Streptomyces scabies is the oldest plant pathogen described and has a world-wide distribution, whereas species such as S. turgidiscabies and S. acidiscabies are believed to be newly emergent pathogens found in more limited geographical locations. The genome sequence of S. scabies 87-22 was recently completed, and comparative genomic analyses with other sequenced microbial pathogens have revealed the presence of additional genes that may play a role in plant pathogenicity, an idea that is supported by functional analysis of one such putative virulence locus. In addition, the availability of multiple genome sequences for both pathogenic and nonpathogenic streptomycetes has provided an opportunity for comparative genomic analyses to identify the Streptomyces pathogenome. Such genomic analyses will contribute to the fundamental understanding of the mechanisms and evolution of plant pathogenicity and plant-microbe biology within this genus.     

The influence of colonizing methylobacteria on morphogenesis and resistance of sugar beet and white cabbage plants to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

The influence of colonization of sugar beet (Beta vulgaris var. saccharifera (Alef) Krass) and white cabbage (Brassica oleracea var. capitata L.) plants by methylotrophic bacteria Methylovorus mays on the growth, rooting, and plant resistance to phytopathogen bacteria Erwinia carotovora was investigated. The colonization by methylobacteria led to their steady association with the plants which had increased growth speed, root formation and photosynthetic activity. The colonized plants had increased resistance to Erwinia carotovora phytopathogen and were better adapted to greenhouse conditions. The obtained results showed the perspectives for the practical implementation of methylobacteria in the ecologically clean microbiology substances used as the plant growth stimulators and for the plant protection from pathogens.     

A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: <Emphasis Type="Italic">Adonis aestivalis</Emphasis> as a case study

Carotenoids are indispensable pigments of the photosynthetic apparatus in plants, algae, and cyanobacteria and are produced, as well, by many bacteria and fungi. Elucidation of biochemical pathways leading to the carotenoids that function in the photosynthetic membranes of land plants has been greatly aided by the use of carotenoid-accumulating strains of Escherichia coli as heterologous hosts for functional assays, in vivo, of the otherwise difficult to study membrane-associated pathway enzymes. This same experimental approach is uniquely well-suited to the discovery and characterization of yet-to-be identified enzymes that lead to carotenoids of the photosynthetic membranes in algal cells, to the multitude of carotenoids found in nongreen plant tissues, and to the myriad flavor and aroma compounds that are derived from carotenoids in plant tissues. A portfolio of plasmids suitable for the production in E. coli of a variety of carotenoids is presented herein. The use of these carotenoid-producing E. coli for the identification of cDNAs encoding enzymes of carotenoid and isoprenoid biosynthesis, for characterization of the enzymes these cDNAs encode, and for the production of specific carotenoids for use as enzyme substrates and reference standards, is described using the flowering plant Adonis aestivalis to provide examples. cDNAs encoding nine different A. aestivalis enzymes of carotenoid and isoprenoid synthesis were identified and the enzymatic activity of their products verified. Those cDNAs newly described include ones that encode phytoene synthase, β-carotene hydroxylase, deoxyxylulose-5-phosphate synthase, isopentenyl diphosphate isomerase, and geranylgeranyl diphosphate synthase.     

Wilt and vascular root rot (<i>Pythium tracheiphilum</i>) of lettuce in Bahía Blanca,Argentina

A new disease of lettuce has been observed in the surroundings of Bahía Blanca, Argentina. The symptoms include dwarfing, general chlorosis, wilting, root rot, and leaf blight, sometimes followed by plant death. Pythium tracheiphilum Matta was found to be consistently associated with those symptoms. The morphological and morphometric characteristics of this oomycete are described. Isolates obtained from field-infected lettuce plants were inoculated to lettuce and caused similar symptoms as those found in natural infections. Seedling emergence was also severely affected following experimental inoculation. Koch’s Postulates were fulfilled by recovering the fungus from inoculated plants. The new disease shows a high destructive potential but currently has a limited prevalence and incidence.     

Molecular and phenotypic characterization of potential plant growth-promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> from rice and maize rhizospheres

In this study, Pseudomonas species were isolated from the rhizospheres of two plant hosts: rice (Oryza sativa cultivar Pathum Thani 1) and maize (Zea mays cultivar DK888). The genotypic diversity of isolates was determined on basis of amplified rDNA restriction analysis (ARDRA). This analysis showed that both plant varieties selected for two distinct populations of Pseudomonas. The actual biocontrol and plant promotion abilities of these strains was confirmed by bioassays on fungal (Verticillum sp., Rhizoctonia solani and Fusarium sp.) and bacterial (Ralstonia solanacearum and Bacillus subtilis) plant pathogens, as well as indole-3-acetic acid (IAA) production and carbon source utilization. There was a significant difference between isolates from rice and maize rhizosphere in terms of biological control against R.  solanacearum and B.  subtilis. Interestingly, none of the pseudomonads isolated from maize rhizosphere showed antagonistic activity against R.  solanacearum. This study indicated that the percentage of pseudomonad isolates obtained from rice rhizosphere which showed the ability to produce fluorescent pigments was almost threefold higher than pseudomonad isolates obtained from maize rhizosphere. Furthermore, the biocontrol assay results indicated that pseudomonad isolated from rice showed a higher ability to control bacterial and fungal root pathogens than pseudomonad isolates obtained from maize. This work clearly identified a number of isolates with potential for use as plant growth-promoting and biocontrol agents on rice and maize.     

DNA-Barcoding of Some Medicinal Plant Species in Saudi Arabia Using <i>rbcL</i> and <i>matK</i> Genes

In the Kingdom of Saudi Arabia (KSA), thousands of plants are considered to have therapeutic value. The ambiguous use of identification mainly morphological characteristics of many plants has resulted in the adulteration and displacement of plant products which undermine their therapeutic value and weak documentation of plant resources. The aims of this study were therefore to evaluate genetic variability and explore the phylogeographic architecture for Saudi medicinal plant samples using rbcL and matK genes as barcodes for genomic identification. The matK and rbcL sequences collected for these samples were used as key markers for examining the relationship between Saudi medicinal plant species based on genetic diversity. During our study we were successful in identifying and documenting 4 different species (Foeniculum vulgare, Nitraria retusa, Dodonaea viscosa, and Rumex nervosus) located in Saudi Arabia using DNA barcoding technique. A total number of 8 sequences were obtained with a total sequence length of 6176 bp, where it ranged from 617 bp to 878 bp with an average length of 772 bp. The total number of rbcL sequences length is 2801 bp, where it ranges from 617 bp to 807 bp with an average length of 700.2 bp. Out of the 4 plant samples used, only three samples were identified correctly on the species level with an identity percentage higher than 95% using rbcL gene. Additionally, 4 matK sequences have been retrieved belong to 4 species. The total number of matK sequences length is 3375 bp, where it ranges from 819 bp to 878 bp with an average length of 843.8 bp. Out of the 4 plant samples used, only two samples were identified correctly on the species level with an identity percentage higher than 98% using matK gene. Both rbcL and matK have been able to identify most of our collected plant samples by genus, and some by species. Using only one DNA-barcoding technique was not reliable for plant identification, where matK and rbcL must be used as a dual DNA-barcoding procedure.     

An efficient protocol for genetic transformation of <Emphasis Type="Italic">Platycodon grandiflorum</Emphasis> with <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>

The balloon flower (Platycodon grandiflorum) is a popular traditional medicinal plant used in Korea to treat conditions such as bronchitis, asthma, tuberculosis, diabetes, and inflammatory diseases. Recently, immunopharmacological research identified triterpenoid and saponin as important active compounds in P. grandiflorum. To study and extract these compounds and other metabolites from P. grandiflorum, a technique was developed for producing hairy root cultures, which are a reliable source of plant compounds. To achieve this, the activity of Agrobacterium rhizogenes was exploited, which can transfer DNA segments into plant genomes after infecting them. In this study, the A. rhizogenes strain R1000 was determined that had the highest infection frequency (87.5%) and induced the most hairy roots per plant, and the concentration of antibiotics (75 mg/l kanamycin) was elucidated for selection after transformation. Wild-type and transgenic hairy roots contained various phenolic compounds, although both of them had similar concentrations of phenolic compounds. In the future, the protocols described here should be useful for studying and extracting valuable metabolites such as phenolic compounds from P. grandiflorum hairy root cultures.     

Some propagation methods for cloning holm oak (<Emphasis Type="Italic">Quercus ilex</Emphasis> L.) plants

Holm oak (Quercus ilex L.), a typical evergreen tree of the Mediterranean area, is very important due to its ecological and economical values. Propagation of this species is extremely difficult and traditionally carried out only by seed germination. In this work, mature acorns were germinated in vitro and in peat substrate in aseptic and non-aseptic conditions. Explants from the seedlings obtained were propagated in vitro in WPM plus 4 μM BA. Plant regeneration was achieved from hypocotyls and root segments cultured in vitro on modified Gamborg medium plus 20 μM BA and 20 μM NAA. 13.8% of the hypocotyls and approximately 30% of the root segments developed both shoots and roots after 30 days of culture. Rooting of stem segments was obtained both in vitro and ex vitro by basal dipping in IBA solutions. Within ex vitro rooting, mother plant age had major influence on the percentage of rooting of the cuttings as the younger plants showed higher ability to root. In this way, Q. ilex plants could be propagated and cloned. The procedure described here would be a very useful tool for breeding programs since vegetative propagation of selected individuals can be achieved.     

Efficient transformation of<Emphasis Type="Italic"> Medicago truncatula</Emphasis> cv. Jemalong using the hypervirulent<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> strain AGL1

The efficiency of Agrobacterium tumefaciens transformation of the model legume Medicago truncatula cv. Jemalong (genotype 2HA) was evaluated for strains LBA 4404, C58pMP90, C58pGV2260 and AGL1. Binary vectors carrying promoter-gus/gfp reporter gene fusions and the nptII gene as selectable marker were used for plant in vitro transformation/regeneration. The highest transformation efficiency was obtained with the disarmed hypervirulent strain AGL1 (Ti plasmid TiBo542), for which the percentage of explants forming kanamycin (Km)-resistant calli was double that obtained with each of the other three strains. In addition, we were able to reduce the time necessary for plant regeneration using AGL1, with 24% of the explants generating Km-resistant transgenic plantlets within only 4–5 months of culture. Transgene expression in planta was analysed and found to be conserved in the T₁ descendents.Communicated by R.J. Rose     

Factors influencing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of monocotyledonous species

Summary Since the success of Agrobacterium-mediated transformation of rice in the early 1990s, significant advances in Agrobacterium-mediated transformation of monocotyledonous plant species have been achieved. Transgenic plants obtained via Agrobacterium-mediated transformation have been regenerated in more than a dozen monocotyledonous species, ranging from the most important cereal crops to ornamental plant species. Efficient transformation protocols for agronomically important cereal crops such as rice, wheat, maize, barley, and sorghum have been developed and transformation for some of these species has become routine. Many factors influencing Agrobacterium-mediated transformation of monocotyledonous plants have been investigated and elucidated. These factors include plant genotype, explant type, Agrobacterium strain, and binary vector. In addition, a wide variety of inoculation and co-culture conditions have been shown to be important for the transformation of monocots. For example, antinecrotic treatments using antioxidants and bactericides, osmotic treatments, desiccation of explants before or after Agrobacterium infection, and inoculation and co-culture medium compositions have influenced the ability to recover transgenic monocols. The plant selectable markers used and the promoters driving these marker genes have also been recognized as important factors influencing stable transformation frequency. Extension of transformation protocols to elite genotypes and to more readily available explants in agronomically important crop species will be the challenge of the future. Further evaluation of genes stimulating plant cell division or T-DNA integration, and genes increasing competency of plant cells to Agrobacterium, may increase transformation efficiency in various systems. Understanding mechanisms by which treatments such as desiccation and antioxidants impact T-DNA delivery and stable transformation will facilitate development of efficient transformation systems.     

Genetic variation of <Emphasis Type="Italic">Oplopanax elatus</Emphasis> (Nakai) Nakai (Araliaceae)

Using allozyme analysis, genetic and genotypic variation of rare relict species, a member of one of ancient angiosperm families, Araliaceae, the clonal plant Oplopanax elatus (Nakai) Nakai, was evaluated. Electrophoretic separation of the enzymes is described, and genetic interpretation of the enzymes variation patterns is presented. The values of genetic variation indices obtained were low (P = 25%; A = 1.45: H _o = 0.131; H _e = 0.113) and comparable with the data reported for the rare plant species and the representatives of the family Araliaceae. The main factors responsible for the polymorphism level observed might be the evolutionary history of the species and gene drift. The level of genotypic diversity (G/N = 0.76; D = 0.97) was substantially higher, compared to the values reported for species with vegetative reproduction (D = 0.62). These results suggest certain contribution of the propagation by seeds in the formation of the species of interest.     

Morphological and physiological characteristics of transgenic tobacco plants expressing expansin genes: <Emphasis Type="Italic">AtEXP10</Emphasis> from <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">PnEXPA1</Emphasis> from poplar

Expansins are non-enzymatic plant proteins breaking hydrogen bonds between cellulose microfibrils and hemicellulose polymer matrix. Each plant has many expansin genes, whose protein products participate in the regulation of plant growth and development mainly by regulating cell expansion. To analyze the effects of elevated expansin expression on the plant organ sizes, we cloned the AtEXPA10 gene from Arabidopsis thaliana and PnEXPA1 gene from Populus nigra. Transgenic tobacco plants expressing the target genes were obtained. The obtained transgenic tobacco plants were shown to have significantly larger leaves and longer stems compared to control plants. The flowers were quite insignificantly larger, but at the same time transgenic plants had more flowers. The microscopic studies showed that the organs of AtEXPA10-carrying plants were larger mainly due to stimulated cell proliferation, whereas the overexpression of the PnEXPA1 gene activated cell expansion.     

Improved genetic transformation protocol for cork oak (<Emphasis Type="Italic">Quercus suber</Emphasis> L.)

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of mature Quercus suber L. embryogenic masses is reported. In this work several variables were tested. Plant genotype, explant type and time elapsed between the last subculture and inoculation, i.e. the explant preculture period, were found to be very important. Interaction between inoculum density and cocultivation period influenced the transformation efficiency as well. A transformation efficiency (i.e. percentage of the inoculated explants that yielded independent transgenic embryogenic lines) of up to 43% was obtained, greatly improving the previously described method for plant transformation of adult-selected cork oak. It was also shown that this protocol could be applied to various genotypes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

(Chemotaxonomic) Implications of Postharvest/Storage‐Induced Changes in Plant Volatile Profiles – the Case of Artemisia absinthium L. Essential Oil

The plant volatile profile and the essential‐oil chemical composition change during the storage of plant material. The objective of this study was to develop a mathematical model able to predict, explain, and quantify these changes. Mathematical equations, derived under the assumption that the essential oil contained within plant material could be treated as an ideal solution (Raoult's law), were applied for tracking of postharvest changes in the volatile profile of Artemisia absinthium L. (the essential oils were analyzed by GC‐FID and GC/MS). Starting from a specific chemical composition of an essential‐oil sample obtained from plant material after a short drying period (typically 5–10 d), and by using the equations derived from this model, one could easily predict evaporation‐induced changes in the volatile profile of the plant material. Based on the composition of the essential‐oil sample obtained after a given storage time t, it is possible to identify those components that were involved in chemical reactions, both as reactants and possible products. The established model even allowed the recognition of pairs of transformation, i.e., ‘daughter’ products and their ‘parent’ compounds. The obtained results highlight that the essential‐oil composition is highly dependent on the storage period of any plant material and urges caution in different types of phytochemical studies, especially chemotaxonomic ones, or practical application.     

Quantification of<Emphasis Type="Italic">Bacillus</Emphasis> species in a wastewater treatment system by the molecular analyses

Bacillus species were observed and quantified by molecular approaches, using the 165 rDNA primers/probes, in a wastewater treatment plant designed for the purpose of stimulating the growth ofBacillus species. The plant has been operating as a test plant since 1997 in the city of Ina, Japan, with excellent treatment performance. Observations byin situ hybridization, usingBacillus-specific probes, indicated thatBacillus strains were inhabited in the plant and their numbers decreased during the treatment process. Similar results were obtained from a quantitative PCR analysis using aBacillus-specific primer set, and the amount of DNA originating from variousBacillus species was maximally 1.91% of the total DNA in the wastewater treatment tank. Clone library analysis using theBacillus-specific primers suggested that, while the population was noticeably increased, the phylogenetic diversity of the increasingBacillus species was very low.     

Larval Case Architecture and Implications of Host‐Plant Associations for North American Coleophora (Lepidoptera; Coleophoridae)1

The case‐bearing moths of North America are represented by a single genus, Coleophora, which contains approximately 144 described species. All are external seed or leaf miners that inhabit portable silk cases during most of the larval stage. Architectural and ecological characters from larval cases were used in cladistic analysis to investigate existing case groups for 32 North American species of Coleophora. Cladistic analysis confirmed monophyly of certain case groups, but not of others. Host‐plant preferences were also examined. The pattern of host plant use reflects more closely preference for certain plant tissues (seeds versus leaves) and growth forms (herbaceous versus woody) with exploitation of different plant taxa, rather than preference for certain plant taxa with exploitation of different plant tissues.     

Harvest-inducibility of the promoter of alfalfa<Emphasis Type="Italic"> S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-methionine:<Emphasis Type="Italic"> trans</Emphasis>-caffeoyl-CoA3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase gene

A major limitation on the expression of some foreign proteins in transgenic plants is the toxic effect of such proteins on the host plant resulting in inhibition of normal growth and development. A solution to this problem is to control the expression of genes for such proteins by means of inducible promoters, as is frequently done in microbial systems. A cDNA clone was obtained from subtractive hybridization of non-harvested and harvested alfalfa leaf tissue, named hi12. The hi12 cDNA was identified as part of the S-adenosyl-l-methionine: trans-caffeoyl-CoA3-O-methyltransferase gene of alfalfa, a gene encoding an essential key enzyme in lignin synthesis. The hi12 gene was strongly induced by harvesting and wounding but not by heat shock. The promoter of the hi12 gene, isolated by genomic walking, contained several stress response cis-elements. Transgenic plants of tobacco and Medicago truncatula containing the GUS gene driven by the promoter showed GUS expression following harvesting, demonstrating the activity of these regulatory regions in other plant species.     

Host‐plant use by two Orthomeria (Phasmida: Aschiphasmatini) species feeding on Macaranga myrmecophytes

Myrmecophytes depend on symbiotic ants (plant‐ants) to defend against herbivores. Although these defensive mechanisms are highly effective, some herbivorous insects can use myrmecophytes as their host‐plants. The feeding habits of these phytophages on myrmecophytes and the impacts of the plant‐ants on their feeding behavior have been poorly studied. We examined two phasmid species, Orthomeria alexis and O. cuprinus, which are known to feed on Macaranga (Euphorbiaceae) myrmecophytes in a Bornean primary forest. Our observations revealed that: (i) each phasmid species relied on two closely‐related myrmecophytic Macaranga species for its host‐plants in spite of their normal plant‐ant symbioses; and (ii) there was little overlap between their host‐plant preferences. More O. cuprinus adults and nymphs were found on new leaves, which were attended by more plant‐ants than mature leaves, while most adults and nymphs of O. alexis tended to avoid new leaves. In a feeding choice experiment under ant‐excluded conditions, O. alexis adults chose a non‐host Macaranga myrmecophyte that was more intensively defended by plant‐ants and was more palatable than their usual host‐plants almost as frequently as their usual host‐plant, suggesting that the host‐plant range of O. alexis was restricted by the presence of plant‐ants on non‐host‐plants. Phasmid behavior that appeared to minimize plant‐ant attacks is described.