Cadmium phytoextraction potential of poplar clones (Populus spp.)

Biomass production, leaf number and area, photosynthetic and dark respiration rates, leaf concentration of photosynthetic pigments, nitrate reductase activity, as well as cadmium concentrations in leaves, stem, and roots were measured in poplar clones PE 4/68, B-229, 665, and 45/51. Plants were grown hydroponically under controlled conditions and treated with two different cadmium (Cd) concentrations (10(-5) and 10(-7) M) in the same background solution (Hoagland's solution). The presence of Cd did not cause serious disturbance of growth and physiological parameters in the studied poplar clones. Cd concentrations in plant tissues reflected external concentrations. In treated plants, root contents increased from 38.57 to 511.51 ppm, leaf contents from 0.91 to 7.50, while stem contents ranged from 1.37 to 9.50 ppm.     

High efficiency poplar transformation

With the completion of the poplar tree genome database, Populus species have become one of the most useful model systems for the study of woody plant biology. Populus tremuloides (quaking aspen) is the most wide-spread tree species in North America, and its rapid growth generates the most abundant wood-based biomass out of any other plant species. To study such beneficial traits, there is a need for easier and more efficient transformation procedures that will allow the study of large numbers of tree genes. We have developed transformation procedures that are suitable for high-throughput format transformations using either Agrobacterium tumefaciens to produce transformed trees or Agrobacterium rhizogenes to generate hairy roots. Our method uses Agrobacterium inoculated aspen seedling hypocotyls followed by direct thidiazuron (TDZ)-mediated shoot regeneration on selective media. Transformation was verified through β-glucuronidase (GUS) reporter gene expression in all tree tissues, PCR amplification of appropriate vector products from isolated genomic DNA, and northern hybridization of incorporated and expressed transgenes. The hairy root protocol follows the same inoculation procedures and was tested using GUS reporter gene integration and antibiotic selection. The benefit of these procedures is that they are simple and efficient, requiring no maintenance of starting materials and allowing fully formed transgenic trees (or hairy roots) to be generated in only 3–4 months, rather than the 6–12 months required by more traditional methods. Likewise, the fact that the protocols are amenable to high-throughput formats makes them better suited for large-scale functional genomics studies in poplars.     

Hybrid rice (Oryza sativa L.): identification and parentage determination by RAPD fingerprinting

Summary DNA from three families of rice plants selected in Northern China (each comprising the male sterile, the restorer, the hybrid F1 and the maintainer lines) has been extracted and amplified by PCR with different random DNA primers (RAPD analysis). Then, DNA has been analysed by agarose gel electrophoresis and DNA bands scored as present or absent. The generated matrices are reproducible and amenable for identification of each single plant line. Thus, RAPD fingerprinting of the inbred parental lines and of the resulting hybrid is proposed as a convenient tool for the identification, protection and parentage determination of plant hybrids. Furthermore, by offering a molecular tool to verify the degree of dissimilarity between the parental lines, the RAPD analysis may also be used to search for new parental combinations.     

Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.)

A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary sequence. When separated electrophoretically in agarose gels, the amplification products give banding patterns that can be scored for genetic variation. The objective of this research was to apply these techniques to cultivated peanut (Arachis hypogaea L.) and related wild species to determine whether such an approach would be feasible for the construction of a genetic linkage map in peanut or for systematic studies of the genus. Two peanut cultivars, 25 unadapted germplasm lines of A. hypogaea, the wild allotetraploid progenitor of cultivated peanut (A. monticola), A. glabrata (a tetraploid species from section Rhizomatosae), and 29 diploid wild species of Arachis were evaluated for variability using primers of arbitrary sequence to amplify segments of genomic DNA. No variation in banding pattern was observed among the cultivars and germplasm lines of A. hypogaea, whereas the wild Arachis species were uniquely identified with most primers tested. Bands were scored (+/–) in the wild species and the PAUP computer program for phylogenetic analysis and the HyperRFLP program for genetic distance analysis were used to generate dendrograms showing genetic relationships among the diploid Arachis species evaluated. The two analyses produced nearly identical dendrograms of species relationships. In addition, approximately 100 F₂ progeny from each of two interspecific crosses were evaluated for segregation of banding patterns. Although normal segregation was observed among the F₂ progeny from both crosses, banding patterns were quite complex and undesirable for use in genetic mapping. The dominant behavior of the markers prevented the differentiation of heterozygotes from homozygotes with certainty, limiting the usefulness of arbitrary primer amplification products as markers in the construction of a genetic linkage map in peanut.     

Rapid identification of white-Engelmann spruce species by RAPD markers

Fragments of random amplified polymorphic DNA (RAPDs) were used as markers to distinguish Picea glauca (Moench) Voss (white spruce) and Picea engelmannii Parry (Engelmann spruce). These species and their putative hybrids are difficult to differentiate morphologically and are collectively known as interior spruce. Four oligodeoxynucleotide decamer primers showed species-specific amplification products between white spruce and Engelmann spruce. These fragments are highly conserved among seed lots and individual trees of each species from diverse geographic origins. The consistency and reproducibility of these species-specific amplification products were tested in more than two amplification reactions. Therefore, RAPD markers can provide genetic markers for easy and rapid identification of the specific genetic entry of these spruce species and their reported putative hybrids. According to the frequencies of the species-specific RAPD markers, it is possible to estimate the hybrid fraction, indicative of true introgression between the two species. These results are useful for quick identification of both species and their hybrid swarms at any stage in the sporophyte phase of the life cycle, for determining the occurrence and the magnitude of introgressive hybridization in an overlap zone between the two species, and for certification purposes in operational re-forestation and tree-improvement programs.     

Inhibition of cinnamyl-alcohol-dehydrogenase activity and lignin synthesis in poplar (Populus x euramericana Dode) tissues by two organic compounds

Two organic compounds, N-(O-hydroxyphenul)-and N-(O-aminophenyl)sulfinamoyltertiobutyl acetate (OHPAS and NH₂PAS, respectively) have been designed for inhibiting cinnamylalcohol dehydrogenase (EC 1.1.1.2), a zinc metalloenzyme involved specifically in lignification. This paper describes their effects in vitro on the activity of the enzyme isolated from poplar and in vivo on the lignification of poplar tissues. The enzyme inhibition was time- and dose-dependent and pseudoirreversible indicating that these compounds could act as suicide inhibitors. Neither OHPAS nor NH₂PAS exhibited affinity towards other plant zinc metalloenzymes or phenolic enzymes tested. Only NH₂PAS exerted an effect on cinnamoyl: CoA reductase, another specific enzyme of lignification. In addition, these inhibitors, at the concentration of 80 M, reduced the fluxes of lignin synthesis in poplar stems by 45%. These results show that OHPAS and NH₂PAS could become useful tools for reducing lignification in plants.Abbreviations CDH cinnamyl alcohol dehydrogenase - NH₂PAS N (O-aminophenyl) sulfinamoyltertiobutyl acetate - OHPAS N(O-hydroxyphenyl) sulfinamoyltertiobutyl acetate     

Species-specific RAPD fingerprints for the closely related Picea mariana and P. rubens

Species-specific molecular markers were designed to assist in the identification of closely related black spruce (Picea mariana [B.S.P.] Mill.) and red spruce (P. rubens Sarg.) in northeastern North America. Trees from six provenances of black spruce and three provenances of red spruce were sampled from outside the sympatric zone. They were first classified using a composite index of five qualitative morphological traits. The species-specific genetic markers were developed using random amplified polymorphic DNAs (RAPD) and a combination of bulk sample and individual tree analyses. Each species bulk sample was constructed from DNAs obtained from 12 trees that were from outside the sympatric zone and showed a morphological composite index specific of each species. A total of 161 primers were screened with the bulk samples. From these, 52 primers showing segregating fingerprints were further screened with the individual trees. Most of the markers observed were shared by the two species, and there was less diversity in P. rubens. A small number of markers were found to be monomorphic or nearly monomorphic and specific to either P. mariana or P. rubens. These markers remained species-specific when F₁ progenies derived from independent intraspecific crosses were screened, and they were subsequently found to co-segregate in hybrids derived from independent interspecific crosses here used as controls.     

Identification of somaclonal variants of sugarcane (Saccharum spp.) resistant to sugarcane mosaic virus via RAPD markers

Somaclonal variants resistant to sugarcane mosaic virus (SCMV) were obtained from susceptible sugarcane cv PR62258 through somatic embryogenesis by increasing the number of subcultures of the embryogenic callus tissue in MS medium with 3 mg/L 2,4-dichlorophenoxyacetic acid. Transfers were made at 30-day intervals for 1, 2 or 3 subcultures. Two somaclones, namely AT626 and BT627, were selected by their resistance to SCMV. These subclones have maintained the resistance trait over seven years of testing in the field. In this report we identified the somaclonal SCMV resistant variants from the maternal line and the nonresistant somaclones, using the RAPD technique.     

RAPD fingerprinting of blackcurrant (Ribes nigrum L.) cultivars

Ribes nigrum germplasm was screened for random amplified polymorphic DNA (RAPD) markers. Fiftyfour markers were identified which generated individual fingerprints for each of 21 cultivars. Genetic variation within R. nigrum germplasm, as detected by RAPDs, demonstrated that the genetic basis for improvement of blackcurrant is narrower than would be expected by the analysis of parentage.     

Effect of epiphytes on the extent of necrotic injuries of resistant and susceptible poplar clones infected with Dothichiza populea

Poplar cuttings of a resistant clone, Populus ‘Grandis’, and susceptible clones, Populus nigra ‘Italica’ and Populus ‘Robusta’, were infected with the pathogenic fungus Dothichiza populea alone, or with the pathogen and one of five strains of epiphytes antagonistic towards it (in vitro), isolated from poplar bark. The extent of injury was examined for 28 days after infection by determining the length of necrotic patches and their area as expressed in per cent of the total area of a cutting or the area of necrotic injuries caused by the pathogen alone.All the poplar cuttings of both the resistant and susceptible clones became diseased when infected with the pathogen alone. Surprisingly enough, however, the least affected clone was the susceptible P. ‘Robusta’, in which necrotic injuries covered 28% of the total area, as against 40% and 70% in the resistant P. ‘Grandis’ and the susceptible P. nigra ‘Italica’, respectively.When the cuttings were infected simultaneously with Dothichiza populea and its antagonistic epiphytes, the diseased area in the resistant clone diminished by as much as two-thirds, and in the susceptible P. nigra ‘Italica’, by one-third in comparison with the area affected by the pathogen alone. In turn, in the susceptible P. ‘Robusta’ the introduction of three out of five epiphytes stimulated the growth of the pathogenic fungus producing on average a double increase in the necrotic area. The differences in the response of the pathogen to the presence of epiphytes recorded in the susceptible clones indicate a marked influence of the plant on the nature of interactions between its epiphytic microflora and the pathogen.     

rDNA cloning and rapid hybrid identification inPopulus spp. (Salicaceae)

Ribosomal DNA genes fromP. deltoides have been cloned and specific sequences of the 25 S and 18 S rDNA region, labelled by digoxigenin, have been used to determine the rDNA structure ofPopulus tremula, P. fremontii, P. maximowiczii, P. yunnanensis, P. nigra, P. wislizenii, P. alba. The restriction maps of the coding region appeared to be similar among the examined species and with those ofP. deltoides andP. trichocarpa, reported in a previous paper. Inter- and intraspecific variation in rDNA repeat unit length have been revealed after EcoRI digestions. SstI and XbaI restriction sites have been found at different positions in the IGS of some species. The polymorphic fragments generated by SstI digestion allowed the identification of the hybrid origin of some genotypes. The number of rDNA genes in the genome ofP. deltoides has been estimated to be about 2 000 copies. Finally, the usefulness of these studies inPopulus spp. taxonomy and forestry genetics is discussed.Ribosomal RNA gene structure in somePopulus spp. (Salicaceae) and their hybrids 2.     

Constancy of RAPD primer amplification strength among distantly related taxa of flowering plants

A survey of 480 10-mer primers for RAPD markers revealed general consistency in primer amplification strength among the flowering plant generaDatisca, Helianthus andYucca. Six characteristics of primer base sequences were analyzed: total (G+C) content; the amounts of G, A, C, and T taken separately; and the (G+C) content in the last four bases of the 3′ end. Of these, total (G+C) content showed the most value in predicting primer amplification strength. Since the consistency of amplification strength is true only globally, there are still many primers showing high variation in amplification strength among genera, most probably due to DNA sequence differences, but perhaps also resulting from experimental artifact. Nevertheless, we suggest that this survey be used as a rough guide for prioritizing primer deployment in RAPD studies involving plants in the hope of improving efficiency during the search for adequate levels of polymorphism, with the understanding that taxon-specific differences in primer amplification strength are bound to occur.     

Phytomonas: analysis of polymorphism and genetic relatedness between isolates from plants and phytophagous insects from different geographic regions by RAPD fingerprints and synapomorphic markers

The random amplification of polymorphic DNA was used for easy, quick and sensitive assessment of genetic polymorphism within Phytomonas to discriminate isolates and determine genetic relationships within the genus. We examined 48 Phytomonas spp., 31 isolates from plants and 17 from insects, from different geographic regions. Topology of the dendrogram based on randomly amplified polymorphic DNA fingerprints segregated the Phytomonas spp. into 5 main clusters, despite the high genetic variability within this genus. Similar clustering could also be obtained by both visual and cross-hybridization analysis of randomly amplified synapomorphic DNA fragments. There was some concordance between the genetic relationship of isolates and their plant tissue tropism. Moreover, Phytomonas spp. from plants and insects were grouped according to geographic origin, thus revealing a complex structure of this taxon comprising several clusters of very closely related organisms.     

Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.     

The response of CO2 exchange rate to photosynthetic photon flux density for several Populus clones under laboratory conditions

No significant differences were found between four mathematical equations describing the response of CO₂ exchange rate to photosynthetic photon flux density in seven poplar clones under laboratory conditions. Choice of an optimal equation for poplar may be based on the contemplated aims. High significant differences (at p<0.001) were found among the clones.Research was supported by the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (I.W.O.N.L.), Brussels.     

Combination of ARDRA and RAPD genotyping techniques in identification of Acinetobacter spp. genomic species

A total of 10 non-repetitive multi-drug-resistant Acinetobacter strains were collected. With reference to A. calcoaceticus (ATCC23055), A. baumannii (ATCC19606), A. lwoffii (ATCC17986), and A. junii (NCTC5866),DNA fingerprint technique, amplified ribosomal DNA restriction analysis (ARDRA), and random amplified polymorphism DNA (RAPD) were carried out to identify the genomic species of Acinetobacter spp. The distances between them were calculated by the unweighted pair group method with arithmetic (UPGMA). Genotypes of Acinetobacter spp. were effectively classified and an A. junii together with nine A. baumannii isolates was genomically identified. The combination of ARDRA and RAPD DNA-fingerprint technique shows high complementarity, and could be a useful tool in Acinetobacter genomic species identification. __________ Translated from Microbiology, 2007, 34(2): 303–306 [译自:微生物学通报]     

Genetic variation in two sympatric European populations of Bosmina spp. (Cladocera) tested with RAPD markers

We used RAPDs (Random Amplified Polymorphic DNA) to test genetic divergence between two populations of Bosmina spp. in Lake Östersjön, Sweden. Previous taxonomic studies on European species within the genus Bosmina have been based on morphological characters alone. RAPD markers distinguished the two populations and supported the specific status of B. coregoni and B. longispina based on morphological characters. Furthermore, juveniles with a long antennule and a mucro were classified as B. coregoni. RAPDs also revealed genetic differences among the tested individuals, suggesting several clones within each species.     

Non-destructive RAPD genetic diagnostics of microspore-derivedBrassica embryos

We demonstrate the application of the RAPD genetic polymorphism assay (Williams et al., 1990, Welsh and McClelland 1990), to the determination of the genotype of immature, microspore-derived haploid embryos ofBrassica napus. Several hundred assays can be performed on the DNA obtained from a cotyledon fragment, and the remaining embryo can be regenerated. Thus, the assay can be used for ”prenatal” diagnostics of embryo-regenerated plants, and can facilitate selection of defined genotypes in plant breeding. A suitable population of embryos could also be used for the construction of RAPD genetic maps. The online version of the original article can be found at     

Non-destructive RAPD genetic diagnostics of microspore-derivedBrassica embryos

We demonstrate the application of the RAPD genetic polymorphism assay (Williams et al., 1990, Welsh and McClelland 1990), to the determination of the genotype of immature, microspore-derived haploid embryos ofBrassica napus. Several hundred assays can be performed on the DNA obtained from a cotyledon fragment, and the remaining embryo can be regenerated. Thus, the assay can be used for ”prenatal” diagnostics of embryo-regenerated plants, and can facilitate selection of defined genotypes in plant breeding. A suitable population of embryos could also be used for the construction of RAPD genetic maps.