Optimization of in vitro micropropagation and regeneration for Populus × interamericana and Populus × euramericana hybrids (P. deltoides, P. trichocarpa, and P. nigra)

A rapid and efficient micropropagation method has been established for six European poplar cultivars of economic interest - four Populus 2 interamericana and two Populus 2 euramericana. Using a three-step procedure, we were able to regenerate plantlets from callus and acclimate them within 4 months. In the first step, callogenesis was induced when explants were cultured for 25 days on culture medium supplemented with 10 µM !-naphthaleneacetic acid and 5 µM N⁶(2-isopentenyl)adenine. Bud regeneration followed by shoot elongation was then obtained from callus tissue by combining the cytokinin-like compound thidiazuron with the surfactant Pluronic F-68 at concentrations adjusted for each cultivar. The usefulness of this procedure in the area of genetic engineering is discussed.     

Genetic transformation of Populus deltoides and P.x euramericana clones using Agrobacterium tumefaciens

The susceptibility of different Populus euramericana (Neva, PE68-022 x P. nigra, 71-060 x P. nigra) and P. deltoides (PE68-022 x P. deltoides) clones to wild-type Agrobacterium tumefaciens strains (A281 and 82.139) was evaluated in an inoculation experiment, and differences in the frequency of tumor formation (0-48) were found. Co-cultivation experiments demonstrated high transformation ability of oncogenic binary A. tumefaciens strains as compared to disarmed strains. Using oncogenic binary strains, transgenic calluses were obtained from all tested clones. The presence of acetosyringone did not influence the transformation frequency of the disarmed strains. Co-inoculation experiments were performed using leaf discs and a bacterial suspension containing both wild-type and disarmed strains. No positive effects on transformation efficiency were noticed in these conditions either. The transformation of tumors and kanamycin resistant calluses was confirmed by DNA analysis.     

Dense genetic linkage maps of three Populus species (Populus deltoides, P. nigra and P. trichocarpa) based on AFLP and microsatellite markers

Populus deltoides, P. nigra, and P. trichocarpa are the most important species for poplar breeding programs worldwide. In addition, Populus has become a model for fundamental research on trees. Linkage maps were constructed for these three species by analyzing progeny of two controlled crosses sharing the same female parent, Populus deltoides cv. S9-2 x P. nigra cv. Ghoy and P. deltoides cv. S9-2 x P. trichocarpa cv. V24. The two-way pseudotestcross mapping strategy was used to construct the maps. Amplified fragment length polymorphism (AFLP) markers that segregated 1:1 were used to form the four parental maps. Microsatellites and sequence-tagged sites were used to align homoeologous groups between the maps and to merge linkage groups within the individual maps. Linkage analysis and alignment of the homoeologous groups resulted in 566 markers distributed over 19 groups for P. deltoides covering 86% of the genome, 339 markers distributed over 19 groups for P. trichocarpa covering 73%, and 369 markers distributed over 28 groups for P. nigra covering 61%. Several tests for randomness showed that the AFLP markers were randomly distributed over the genome.     

Structure, inheritance, and expression of hybrid poplar (Populus trichocarpa x Populus deltoides) phenylalanine ammonia-lyase genes.

A heterologous probe encoding phenylalanine ammonia-lyase (PAL) was used to identify PAL clones in cDNA libraries made with RNA from young leaf tissue of two Populus deltoides x P. trichocarpa F1 hybrid clones. Sequence analysis of a 2.4-kb cDNA confirmed its identity as a full-length PAl clone. The predicted amino acid sequence is conserved in comparison with that of PAL genes from several other plants. Southern blot analysis of popular genomic DNA from parental and hybrid individuals, restriction site polymorphism in PAL cDNA clones, and sequence heterogeneity in the 3' ends of several cDNA clones suggested that PAL is encoded by at least two genes that can be distinguished by HindIII restriction site polymorphisms. Clones containing each type of PAL gene were isolated from a poplar genomic library. Analysis of the segregation of PAL-specific HindIII restriction fragment-length polymorphisms demonstrated the existence of two independently segregating PAL loci, one of which was mapped to a linkage group of the poplar genetic map. Developmentally regulated PAL expression in poplar was analyzed using RNA blots. Highest expression was observed in young stems, apical buds, and young leaves. Expression was lower in older stems and undetectable in mature leaves. Cellular localization of PAL expression by in situ hybridization showed very high levels of expression in subepidermal cells of leaves early during leaf development. In stems and petioles, expression was associated with subepidermal cells and vascular tissues.     

Chloroplast DNA variation in Populus. II. Interspecific restriction fragment polymorphisms and genetic relationships among Populus deltoides,P. nigra,P. maximowiczii,and P. x canadensis

Restriction fragment analysis was conducted to determine interspecific chloroplast DNA (cpDNA) variation and genetic relationships among Populus deltoides, P. nigra, P. x canadensis (P. deltoides x P. nigra), and P. maximowiczii. Total cellular DNAs of these poplars were digested with 16 restriction endonucleases, and Southern blots of the restriction digests were probed with six different cloned cpDNA fragments from Petunia. P. deltoides, P. nigra, and P. maximowiczii each had a distinct chloroplast genome, separated by many restriction-site and restriction-fragment-length mutations, predominantly in the large single-copy region of the genome. P. x canadensis shared the same cpDNA restriction fragment patterns as P. deltoides var. deltoides. P. nigra was most diverged from P. deltoides, and P. deltoides showed close cpDNA relationships to P. maximowiczii. Nucleotide substitutions per site in cpDNA were 0.0036 between P. deltoides and P. maximowiczii, 0.0071 between P. nigra and P. maximowiczii, and 0.0077 between P. deltoides and P. nigra. We suggest that P. nigra should be classified in a new separate section, the Nigrae.     

Pollen competition among Populus deltoides Marsh., P. nigra L. and P. maximowiczii Henry in fertilizing P. deltoides ovules and siring its seed crop

Summary Interspecific pollen competition among Populus deltoides, P. nigra and P. maximowiczii in fertilizing P. deltoides ovules was studied by using a pollen mixture technique, allozymes and leaf morphology. The frequencies of F₁ seedlings of different paternities in pollen-mix crosses showed highly significant (P<0.01) departures from the 11 ratio expected if pollen selection was random. P. deltoides pollen was the most competitive. The mean percentages of F₁ seedlings of P. deltoides paternity in crosses with pollen mixes P. deltodes + P. nigra, P. deltoides + P. maximowiczii, and P. deltoides + P. nigra + P. maximowiczii were 95.0, 92.5, and 84.8, respectively. P. maximowiczii pollen was more competitive than P. nigra pollen, which was at a selective disadvantage. An average of 83.6% of F₁ progenies of the eight crosses with P. nigra + P. maximowiczii pollen showed P. maximowiczii paternity. Also, in four crosses with P. deltoides + P. nigra + P. maximowiczii pollen, the relative proportion of P. deltoides × P. maximowiczii seedlings (13.4%) was higher than that of P. deltoides × P. nigra seedlings (1.8%). Pollen proportions in the pollen mixes and pollen size did not significantly affect the competitive ability of the pollen. The relative pollen competitive ability indicated reproductive affinities among the species.     

Chloroplast DNA variation in Populus. I. Intraspecific restriction fragment diversity within Populus deltoides,P. nigra and P. maximowiczii

We examined intraspecific chloroplast (cp) DNA variation within Populus deltoides, P. nigra, and P. maximowiczii by restriction fragment analysis using 16 restriction endonucleases and six heterologous probes of cloned Petunia cpDNA fragments. All three Populus species showed intraspecific cpDNA variation, which was intra- and inter-varietal in P. deltoides, intervarietal in P. nigra, and origin-specific in P. maximowiczii. Two varieties of P. deltoides, var deltoides and var occidentalis, showed distinct cp genomes/DNA. Three distinct cp genomes/DNA, separated by a loss or gain of 1 EcoRV restriction site and/or 1 restriction fragment length polymorphism (RFLP), were observed among the individuals of P. deltoides var deltoides. Within P. nigra, cpDNA of var italica was distinct from that of vars nigra and plantierensis by one RFLP and by a loss or gain of one BamHI restriction site. Populus maximowiczii clones of Chinese origin were separated from those of Japanese origin by a gain or loss of one ClaI restriction site in their cpDNA. The estimate of nucleotide substitutions per site in cpDNA was 0.07% between two varieties of P. deltoides, 0.05% between var italica and var nigra or plantierensis of P. nigra, and 0.01% between Japanese and Chinese accessions of P. maximowiczii.     

Rapid Activation of Phenylpropanoid Metabolism in Elicitor-Treated Hybrid Poplar (Populus trichocarpa Torr. & Gray x Populus deltoides Marsh) Suspension-Cultured Cells

Elicitor induction of phenylpropanoid metabolism was investigated in suspension-cultured cells of the fast-growing poplar hybrid (Populus trichocarpa Torr. & Gray × Populus deltoides Marsh) H11-11. Treatment of cells with polygalacturonic acid lyase or two fungal elicitors resulted in rapid and transient increases in extractable l-phenylalanine ammonia lyase and 4-coumarate:coenzyme A ligase enzyme activities. The substrate specificity of the inducible 4-coumarate:coenzyme A ligase enzyme activity appeared to differ from substrate specificity of 4-coumarate:coenzyme A ligase enzyme activity in untreated control cells. Large and transient increases in the accumulation of l-phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase mRNAs preceded the increases in enzyme activities and were detectable by 30 minutes after the start of elicitor treatment. Chalcone synthase, cinnamyl alcohol dehydrogenase, and coniferin β-glucosidase enzyme activities were unaffected by the elicitors, but a large and transient increase in β-glucosidase activity capable of hydrolyzing 4-nitrophenyl-β-glucoside was observed. Subsequent to increases in l-phenylalanine ammonialyase and 4-coumarate:coenzyme A ligase enzyme activities, cell wall-bound thioglycolic acid-extractable compounds accumulated in elicitor-treated cultures, and these cells exhibited strong staining with phloroglucinol, suggesting the accumulation of wall-bound phenolic compounds.     

Genetic variation of stomatal traits and carbon isotope discrimination in two hybrid poplar families (Populus deltoides 'S9-2' x P. nigra 'Ghoy' and P. deltoides 'S9-2' x P. trichocarpa 'V24')

Background and Aims

Stomata play an important role in both the CO₂ assimilation and water relations of trees. Therefore, stomatal traits have been suggested as criteria for selection of clones or genotypes which are more productive and have larger water-use efficiency (WUE) than others. However, the relationships between plant growth, WUE and stomatal traits are still unclear depending on plant material (genus, species, families, genotypes) and, more precisely, on the strength of the relationships between the plants. In this study, the correlations between these three traits categories, i.e. plant growth, WUE and stomatal traits, were compared in two related poplar families.

Methods

Stomatal traits (stomatal density, length and ratio adaxial : abaxial stomatal densities) of a selection of F₁ genotypes and the parents of two hybrid poplar families Populus deltoides ‘S9-2’ × P. nigra ‘Ghoy’ (D × N family, 50 F₁) and P. deltoides ‘S9-2’ × P. trichocarpa ‘V24’ (D × T family, 50 F₁) were measured, together with stem height and circumference. Carbon isotope discrimination (Δ) was determined and used as an indicator of leaf-level intrinsic WUE.

Key Results

Leaves of hybrids and parents were amphistomatous, except for the P. trichocarpa parent. Both families displayed high values of heritability for stomatal traits and Δ. In the progeny, the relationship between stem circumference and Δ was weak for the D × N family, while abaxial and total stomatal density were positively associated with stem dimensions for the D × T family only.

Conclusions

Genetic variation in stomatal traits and Δ was large within as well as between the different poplar species and their hybrids, but there were no direct relationships between stomatal traits and plant growth or Δ. As already noticed in various poplar hybrids, the absence of, or the weak, relationship between Δ and plant growth allows the possibility of selecting poplar genotypes combining high productivity and high WUE. In this study, stomatal traits are of limited value as criteria for selection of genotypes with good growth and large WUE.Key words: Adaxial and abaxial stomatal density, stomatal length, heritability, water-use efficiency (WUE), F₁ hybrids, breeding, Populus deltoides, Populus nigra, Populus trichocarpa     

Betaines and free amino acids in salt stressed vitroplants and winter resting buds of Populus trichocarpa X deltoides

Organic solutes, in particular glycine betaine and proline, have been detected as osmoprotective compounds in many microorganisms and herbaceous species. However, for woody plants, very little information is available on mechanisms of adaptation to salt stress. In the present study, effects of NaCI treatment on betaine and free amino acid contents in a clone of Populus trichocarpa X deltoides micropropagated vitroplants were analyzed using HPLC and silicate plate chromatography. The application during 12 days of 50 to 200 m M NaCI to viroplants cultured on Murashige and Skoog medium (Murashige and Skoog 1962. Physiol. Plant. 15: 473–497) led to a progressive decrease of growth, leaf senescence, abscission, and apical necrosis. Populus trichocarpa X deltoides was characterized by the absence of glycine betainc and/or proline accumulation. However, trigonelline was found to increase in vitroplants subjected to 100 m M NaCI. Trigonelline was also present in dormant buds harvested in natural conditions and missing in active buds. In vitroplants, the content of some amino acids was strongly modified by salt stress. A progressive accumulation of alanine and γ-amino butyrate was particularly significant in roots, whereas the relative concentration of glutamine was strongly enhanced in leaves. Leaf content of glutamate and ornithine attained maximum in the presence of 100 and 150 m M NaCI concentrations, and then decreased. Arginine and serine pools were not significantly modified by salt treatment. The variations in vitroplants were of small amplitude compared to those observed in mature poplars where winter rest was associated with a very high arginine level and with a disappearance of nearly all other free amino acids. The results are discussed in relation to previous data obtained with herbaceous species and in relation to some metabolic pathways in poplars where trigonelline could be considered as a sensitive stress indicator.     

Genetic characterization and relationships of Populus alba,P. tremula,and P. x canescens,and their clones

Summary Isozyme analysis was conducted on individuals of Populus alba L., P. tremula L., and P. × canescens Smith to genetically characterize and differentiate species, hybrids, and individuals, and to determine genetic relationships among them. Thirty gene loci, with 71 alleles, coding for 15 enzymes were observed. Individuals could be identified on the basis of their multilocus genotypes. There were 21 unique multilocus genotypes among 23 P. alba clones. Five P. alba clones from Canada were genetically distinct from each other. Each of the 18 P. tremula and 15 P. × canescens clones had unique multilocus genotypes. Thirteen clones had a unique genotype at a single locus. Percentage of polymorphic loci, average number of alleles per locus, and mean observed heterozygosity were, respectively, 50.0, 1.86, and 0.085 in P. alba, 51.7, 1.66, and 0.096 in P. tremula, and 51.7, 1.86, and 0.157 in P. × canescens. Populus alba and P. tremula were genetically distinct from each other and could be distinguished by mutually exclusive alleles at Aco-3, P. tremula-specific gene Mdh-3, and allele frequency differences at 6 loci. Populus × canescens had allele contributions of P. alba and P. tremula. However, their allele frequencies were closer to those of P. alba than being truly intermediate. The mean genetic identity was 0.749 between P. alba and P. tremula, 0.987 between P. alba and P. × canescens, and 0.817 between P. tremula and P. × canescens. Canonical discriminant analysis of multilocus genotypes separated P. alba, P. tremula, and P. × canescens into three distinct groups and portrayed similar interspecific relationship as above. Our results suggested that the putative P. × canescens individuals consisted of a mixture of F₁ hybrids of P. alba and P. tremula and their backcrosses to P. alba.Presently with the University of Alberta, and BioGenetica Inc., P.O. Box 8261, Edmonton, Alberta, Canada T6H 4P1     

Productivity, leaf traits and carbon isotope discrimination in 29 Populus deltoides x P. nigra clones

Here we tested whether some leaf traits could be used as predictors for productivity in a range of Populus deltoides x P. nigra clones. These traits were assessed in 3-yr-old rooted cuttings from 29 clones growing in an open field trial, in a five randomized complete block design, under optimal irrigation. Variables were assigned to four groups describing productivity (above-ground biomass, total leaf area), leaf growth (total number of leaves increment rate), leaf structure (area of the largest leaf, specific leaf area, carbon and nitrogen contents), and carbon isotope discrimination in the leaves (Delta). High-yielding clones displayed larger total leaf area and individual leaf area, while no correlation could be detected between productivity and either leaf structure or Delta. By contrast, Delta was negatively correlated with number of leaves increment rate and leaf N content. Our study shows that there is a potential to improve water-use efficiency in poplar without necessarily reducing the overall productivity.     

Draft genome sequence of Rhizobium sp. strain PDO1-076, a bacterium isolated from Populus deltoides

Rhizobium sp. strain PDO1-076 is a plant-associated bacterium isolated from Populus deltoides, and its draft genome sequence is reported.     

Potassium,calcium and magnesium antagonism in clones of Populus trichocarpa

Two clones of Populus trichocarpa varying in growth rate were grown in nutrient solutions with a range of K supply. In the leaves an antagonism between the levels of K and Ca+Mg was found, this was most extreme in the older leaves. Analysis of the contents of these elements in leaf vacuoles and cytoplasm suggests that in the vacuoles the total contents of K+Ca+Mg are controlled to maintain ionic and osmotic balance. At low K supply, the K deficit in the cytoplasm is not balance by accumulation of Ca and Mg, and it is suggested that other organic osmotica may accumulate. The regulation of K+Mg+Ca supplied to the leaves seems to be regulated by the roots and is not simple competition of ions for uptake sites at the plasmalemma.     

A microarray-based genotyping and genetic mapping approach for highly heterozygous outcrossing species enables localization of a large fraction of the unassembled Populus trichocarpa genome sequence

Microarrays have demonstrated significant power for genome-wide analyses of gene expression, and recently have also revolutionized the genetic analysis of segregating populations by genotyping thousands of loci in a single assay. Although microarray-based genotyping approaches have been successfully applied in yeast and several inbred plant species, their power has not been proven in an outcrossing species with extensive genetic diversity. Here we have developed methods for high-throughput microarray-based genotyping in such species using a pseudo-backcross progeny of 154 individuals of Populus trichocarpa and P. deltoides analyzed with long-oligonucleotide in situ- synthesized microarray probes . Our analysis resulted in high-confidence genotypes for 719 single-feature polymorphism (SFP) and 1014 gene expression marker (GEM) candidates. Using these genotypes and an established microsatellite (SSR) framework map, we produced a high-density genetic map comprising over 600 SFPs, GEMs and SSRs. The abundance of gene-based markers allowed us to localize over 35 million base pairs of previously unplaced whole-genome shotgun (WGS) scaffold sequence to putative locations in the genome of P. trichocarpa . A high proportion of sampled scaffolds could be verified for their placement with independently mapped SSRs, demonstrating the previously un-utilized power that high-density genotyping can provide in the context of map-based WGS sequence reassembly. Our results provide a substantial contribution to the continued improvement of the Populus genome assembly, while demonstrating the feasibility of microarray-based genotyping in a highly heterozygous population. The strategies presented are applicable to genetic mapping efforts in all plant species with similarly high levels of genetic diversity.     

Comparative Distribution and Metabolism of Xylem-Borne Amino Compounds and Sucrose in Shoots of Populus deltoides

The transport and metabolism of xylem-borne amino compounds and sucrose were investigated in rapidly growing shoots of cottonwood (Populus deltoides Bartr. ex Marsh.). ¹⁴C-labeled glutamine, threonine, alanine, glutamic acid, aspartic acid, and sucrose were applied to the base of severed stems for transport in xylem. Distribution and metabolism of the compounds were followed with autoradiography, microautoradiography, and radioassay. Three utilization patterns were observed: (a) little alanine and sucrose was transported to the laminae of either mature leaves or developing leaves. These compounds were taken up from xylem free-space and utilized in adjacent tissue; (b) threonine also did not move into mature leaves but was translocated to developing leaves or utilized in the stem; (c) glutamic acid and aspartic acid were transported directly into the laminae of mature leaves via the xylem. Relatively less ¹⁴C was retained in stems compared to the other compounds.

Metabolism of the test compounds also differed considerably. ¹⁴C from amino acids moved primarily into organic acids and protein. The ¹⁴C from sucrose was widely distributed among the chemical fractions, with a high percentage found in structural carbohydrates. Clearly, cottonwood stems contain efficient uptake and transfer systems that differentiate among various compounds moving from root to shoot in xylem.

     

Multiple shoot regeneration from root organ cultures of Populus alba x P. grandidentata

Excised roots of various ages from Crandon and Hansen clones of Populus alba x P. grandidentata were tested for their regeneration capacity. Sixty-day-old excised roots that contained root tips were found to be most suitable. The highest number of shoots (an average of 111 shoots/root segment with Crandon and 98 with Hansen) was obtained by adding 22M and 14M zeatin to the medium, respectively. The two clones of hybrid poplar responded similarly to growth regulator treatments; however, the number of shoots produced was greater from the root organs derived from Crandon clones. Regenerated shoots were rooted in basal Woody Plant Medium without any growth regulators. Successful transplantation into soil and growth was achieved with all plants.     

银中杨树叶化学成分研究

采用硅胶柱色谱等分离方法,对银中杨Populus alba X P.berolinensis L.树叶的化学成分进行分离纯化,依据理化性质及波谱数据分析进行结构鉴定,从中分离鉴定了12个单体化合物,分别为:邻苯二酚(1),苯甲酸(2),水杨醇(3),东莨菪素(4),咖啡酸(5),水杨酸(6),2’-苯甲酰水杨苷(7),水杨苷(8),丁二酸(9),特里杨苷(10),3’-苯甲酰水杨苷(11)和6’-苯甲酰水杨苷(12),其中化合物3,4,7,9～12为首次从该植物中分离得到。