Short-distance gene flow in <Emphasis Type="Italic">Populus nigra</Emphasis> L. accounts for small-scale spatial genetic structures: implications for in situ conservation measures

The European black poplar (Populus nigra L.) is a major species of riparian softwood forests. Due to human influences, it is one of the most threatened tree species in Europe. For restoration purposes, remaining stands may act as source populations. We analysed a natural population of P. nigra for genetic diversity and spatial genetic structure using seven microsatellite markers. For the first time, paternity analysis of seedlings as well as juveniles from a restricted area of natural regeneration was used to quantify pollen- and seed-mediated gene flow, respectively. In both cases, cultivated P. x canadensis trees in vicinity could act as potential parents. Spatial genetic patterns of the adult tree population suggest small-scale isolation by distance due to short-distance gene flow, the major part of which (i.e. 70%) takes place within distances of less than 1 km. This helps to explain the reduced diversity in the juveniles. It has implications for the spatial management of natural regeneration areas within in situ conservation measures.     

Chromosome painting and comparative physical mapping of the sex chromosomes in <Emphasis Type="Italic">Populus tomentosa</Emphasis> and <Emphasis Type="Italic">Populus deltoides</Emphasis>

Dioecious species accounted for 6% of all plant species, including a number of crops and economically important species, such as poplar. However, sex determination and sex chromosome evolution have been studied only in few dioecious species. In poplar, the sex-determining locus was mapped to chromosome 19. Interestingly, this locus was mapped to either a peritelomeric or a centromeric region among different poplar species. We developed an oligonucleotide (oligo)-based chromosome painting probe based on the sequence of chromosome 19 from Populus trichocarpa. We performed chromosome painting in P. tomentosa and P. deltoides. Surprisingly, the distal end on the short arm of chromosome 19, which corresponds to the location of the sex-determining locus reported in several species, was not painted in both species. Thus, the DNA sequences associated with this region have not been anchored to the current chromosome 19 pseudomolecule, which was confirmed by painting of somatic metaphase chromosome 19 of P. trichocarpa. Interestingly, the unpainted distal ends of the two chromosome 19 did not pair at the pachytene stage in 22–24% of the meiotic cells in the two species, suggest that these regions from the sex chromosomes have structurally diverged from each other, resulting in the reduced pairing frequency. These results shed light on divergence of a pair of young sex chromosomes in poplar.     

Comparative genomic analysis of CIPK gene family in <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Populus</Emphasis>

Calcium serves as a second messenger in various signal transduction pathways in plants. CBL-interacting protein kinases (CIPKs), which have a variety of functions, are involved in calcium signal transduction. Previous, the studies on CIPK family members focused on Arabidopsis and rice. Here, we present a comparative genomic analysis of the CIPK gene family in Arabidopsis and poplar, a model tree species. Twenty-seven potential CIPKs were identified from poplar using genome-wide analysis. Like the CIPK gene family from Arabidopsis, CIPK genes from poplar were also divided into intron-free and intron-harboring groups. In the intron-harboring group, the intron distribution of CIPKs is rather conserved during the genome evolutionary process. Many homologous gene pairs were found in the CIPK gene family, indicating duplication events might contribute to the amplification of this gene family. The phylogenetic comparison of CIPKs in combination with intron distribution analysis revealed that CIPK genes from both Arabidopsis and poplar might have an ancient origin, which formed earlier than the separation of these two eudicot species. Our genomic and bioinformatic analysis will provide an important foundation for further functional dissection of the CBL-CIPK signaling network in poplars. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High-density genetic map of <Emphasis Type="Italic">Populus deltoides</Emphasis> constructed by using specific length amplified fragment sequencing

Populus deltoides is an important industrial tree species widely planted in many areas of the world, and de novo genome sequencing of this plant has been carried out by several research groups. A dense genetic map associating genome sequences is highly desirable for reconstructing the chromosome pseudomolecules using the obtained sequence scaffold assemblies. For this purpose, an intraspecific full-sib F₁ mapping pedigree was established by using the sequenced P. deltoides as the maternal parent. With this mapping pedigree, we constructed a high-density genetic map using 92 randomly selected progenies. Single nucleotide polymorphism (SNP) markers were discovered by using specific length amplified fragment sequencing (SLAF-seq). In total, 487,038 SLAFs were generated, of which 179,360 were polymorphic. A high-density genetic map was built using HighMap software, which included 11,680 Mendelian segregation markers distributing in 2851 marker bins. The established map consisted of 19 linkage groups (LGs) that equaled to the 19 haploid chromosomes in poplar genome, and spanned a total genetic length of 3494.66 cM, with an average distance of 1.23 cM per marker bin. The map presented here will be useful for anchoring the genome sequence assemblies along chromosomes, and for many other aspects of genetic studies on P. deltoides and the closely related species.     

Polyphenol oxidase overexpression in transgenic<Emphasis Type="Italic"> Populus</Emphasis> enhances resistance to herbivory by forest tent caterpillar (<Emphasis Type="Italic">Malacosoma disstria</Emphasis>)

In order to functionally analyze the predicted defensive role of leaf polyphenol oxidase (PPO; EC 1.10.3.1) in Populus, transgenic hybrid aspen (Populus tremula × P. alba) plants overexpressing a hybrid poplar (Populus trichocarpa × P. deltoides) PtdPPO1 gene were constructed. Regenerated transgenic plants showed high PPO enzyme activity, PtdPPO1 mRNA levels and PPO protein accumulation. In leaf disk bioassays, forest tent caterpillar (Malacosoma disstria) larvae feeding on PPO-overexpressing transgenics experienced significantly higher mortality and reduced average weight gain compared to larvae feeding on control leaves. However, this effect was observed only when older egg masses were used and the resulting larvae showed reduced growth and vigor. In choice tests, no effect of PPO overexpression was detected. Although PPO in poplar leaves is latent and requires activation with detergents or trypsin for full enzymatic activity, in caterpillar frass the enzyme was extracted in the fully activated form. This activation correlated with partial proteolytic cleavage, suggesting that PPO latency and activation during digestion could be an adaptive and defense-related feature of poplar PPO.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Resistance to herbivory of two populations of <Emphasis Type="Italic">Elodea</Emphasis><Emphasis Type="Italic">canadensis</Emphasis> Michaux and <Emphasis Type="Italic">Elodea nuttallii</Emphasis> Planchon St. John

In this article, we compared the resistance of two introduced populations of Elodea nuttallii and Elodea canadensis to two different herbivores. Samples were collected from the River Rhine and River Rhône in eastern France. The two populations of E. nuttallii differed in their introduction history, whereas E. canadensis was introduced at the same time in the two sites. The Daily Food Consumption (DFC) rates of the two macrophyte populations were evaluated in no-choice experiments using the scraper Lymnaea stagnalis and the shredder Gammarus roeseli. At the same time, we assessed four plant traits: dry matter content (DMC), total nitrogen content, carbon/nitrogen ratio and total phenolic content. The two populations of E. canadensis were consumed at low levels by both the herbivores. L. stagnalis showed a higher DFC on the Rhône population of E. nuttallii than on the Rhine population. No significant difference between the two populations was established with G. roeseli, but the level of DFC was high. This result demonstrates that the assessment of plant palatability should be carried out with several generalist herbivores belonging to various feeding groups (e.g. scrapers or shredders). Although the Rhône population of E. nuttallii had higher levels of phenols than the other populations, it was consistently consumed in greater quantities than E. canadensis. Neither the phenolic contents were not effective against these herbivores, nor the levels of phenolics too low to induce an efficient resistance. The higher DMC and the lower DFC of the two populations of E. canadensis suggest that this introduced plant has co-evolved with indigenous enemies in the introduced range.     

A fast and simple genetic survey reveals the spread of poplar hybrids at a natural Elbe river site

It is known that various poplar species and cultivated poplar hybrids have the potential to interbreed and produce fertile offspring. Conservation strategies for the genetic resources of the endangered Eurasian black poplar (Populus nigra L.) thus rely on a monitoring which enables the identification and verification of the pure species status. At the same time, the risk of hybrid dispersal and introgressive gene flow has to be estimated. In the present study a combination of two molecular markers, one from chloroplast DNA and the other from nuclear DNA, was applied to evaluate a large P. nigra population on the Elbe River. Hybrid clones of P. × canadensis are scattered within this population and also occur as plantations in the surrounding landscape. By means of the DNA markers the taxonomic status of 208 adult trees in the population and 140 young poplars along the riverbank was monitored. From the analysed young poplars, almost 20 percent were found to exhibit at least one of the two P. deltoides or P. × canadensis diagnostic alleles or genotypes, respectively. Possible vegetative spreads of F1 hybrids and precedent mating scenarios are discussed. Most interestingly we found clear evidence for a small number of backcross hybrids where P.  × canadensis acted as pollen donor. This case had long been debated and thought to be less probable, so far.     

Genetic diversity of the African poplar (<Emphasis Type="Italic">Populus ilicifolia</Emphasis>) populations in Kenya

We evaluated the genetic diversity of the African poplar (Populus ilicifolia) populations found in Kenya compared with reference samples of five poplar species from North America and one species introduced in Kenya from India (KEFRI-Kenya). Amplified fragment length polymorphism (AFLP) was used with the objective of providing important information for breeding and in situ/ex situ conservation of this species. Samples collected from three locations along the species’ natural range (Athi, Ewaso Nyiro, and Tana rivers) were compared with four samples of locally planted Populus deltoides stand introduced from India and ten reference samples from North America. Six AFLP primer combinations produced 521 clear bands for analysis. The percentage polymorphic loci were lowest in Tana (20.4 %) and highest in Athi (40.6 %). The average heterozygosity across the studied populations was between 0.07 and 0.3. AMOVA revealed more genetic variation partitioning within population (87 %; P?<?0.01) than among populations (13 %; P?<?0.01) suggesting significant genetic variation between populations. Further, UPGMA delineation showed two clusters of the Tana, Athi, and Ewaso Nyiro populations clustered together compared to the North America and India/KEFRI reference samples. Moreover, the study showed that the Athi population is more diverse than those of Tana and Ewaso Nyiro and may be important for conservation, domestication, and improvement studies. The genetic differentiation (F _ST ?=?0.134) among Kenyan P. ilicifolia populations suggests limited possibility of gene flow between these populations.     

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.     

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.     

Positioning of the major locus for <Emphasis Type="Italic">Puccinia psidii</Emphasis> rust resistance (<Emphasis Type="Italic">Ppr1</Emphasis>) on the <Emphasis Type="Italic">Eucalyptus</Emphasis> reference map and its validation across unrelated pedigrees

In this report the major locus for Puccinia psidii rust resistance, Ppr1, was positioned on the reference genetic map for Eucalyptus. Additionally, its position was validated by association genetics in a related and two unrelated pedigrees involving different Eucalyptus grandis resistant trees crossed to individuals of two other species, Eucalyptus tereticornis and Eucalyptus camaldulensis. These results are consistent with the hypothesis that Ppr1 controls a large proportion of the variation for rust resistance, strengthening its role as a major locus in Eucalyptus and providing its unequivocal genomic position on linkage group 3. A localized map with 19 microsatellite loci was built around Ppr1. Multiallelic profiles were observed at several mapped microsatellites suggesting recent tandem duplications in the genomic landscape surrounding Ppr1. Markers EMBRA125 and EMBRA1071 flank Ppr1 at 9.5% and 7% recombination, respectively, and were found to be in linkage equilibrium in a E. grandis breeding population, consistent with the expectations in outcrossed Eucalyptus. Their potential use for MAS will specifically be directed to identifying resistant offspring of P. psidii resistant parent trees that are heterozygous at Ppr1. In these circumstances, a significant amount of LD is expected to occur between specific alleles at flanking microsatellites and the resistance allele at Ppr1. Moreover, the positional information of Ppr1 paves the way for prospective undertakings in this genomic region with the upcoming availability of a draft genome for E. grandis.     

Leaf-rust resistance in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.). 2. Genetic analysis and mapping of resistance genes <Emphasis Type="Italic">Pr3</Emphasis>, <Emphasis Type="Italic">Pr4</Emphasis>, and <Emphasis Type="Italic">Pr5</Emphasis>

The conventional way to drive modifications in old forest tree seed orchards is to establish progeny trials involving each parent tree and then evaluate its contribution to the performance of the progeny by estimating its general and specific combining ability (GCA and SCA). In this work, we successfully applied an alternative parent selection tactic based on paternity testing of superior offspring derived from a hybrid seed orchard established with a single Eucalyptus grandis seed parents and six E. urophylla pollen parents. A battery of 14 microsatellite markers was used to carry out parentage tests of 256 progeny individuals including two independent samples of selected trees and one control unselected sample, all derived from 6-year-old forest stands in eastern Brazil. Paternity determination was carried out for all progeny individuals by a sequential paternity exclusion procedure. Exclusion was declared only when the obligatory paternal allele in the progeny tree was not present in the alleged parent tree for at least four independent markers to avoid false exclusions due to mutation or null alleles. After maternity checks to identify seed mixtures and selfed individuals, the paternity tests revealed that approximately 29% of the offspring was sired by pollen parents outside the orchard. No selfed progeny were found in the selected samples. Three pollen parents were found to have sired essentially all of the offspring in the samples of selected and non-selected progeny individuals. One of these three parents sired significantly more selected progeny than unselected ones (P0.0002 in a Fisher exact test). Based on these results, low-reproductive-successful parents were culled from the orchard, and management procedures were adopted to minimize external pollen contamination. A significant difference (P<0.01) in mean annual increment was observed between forest stands produced with seed from the orchard before and after selection of parents and revitalization of the orchard. An average realized gain of 24.3% in volume growth was obtained from the selection of parents as measured in forest stands at age 2–4 years. The marker-assisted tree-breeding tactic presented herein efficiently identified top parents in a seed orchard and resulted in an improved seed variety. It should be applicable for rapidly improving the output quality of seed orchards, especially when an emergency demand for improved seed is faced by the breeder.Communicated by D.B. Neale     

Hot water and dilute acid pretreatment of high and low specific gravity <Emphasis Type="Italic">Populus</Emphasis><Emphasis Type="Italic">deltoides</Emphasis> clones

Populus sp. are hardwood feedstocks that grow in forest management areas that are logged for softwoods; however, they are also being considered as an energy-destined feedstock. The objective of this work was to determine the effect of xylose yield from dilute acid and hot water pretreatments performed in unstirred batch stainless steel reactors at temperatures ranging from 140 to 200°C. Populus deltoides clones S13C20 and S7C15 used in this study originated from Eastern Texas and were cultivated for 14 years in Pine Tree, AR. P. deltoides clones S13C20 and S7C15 had specific gravities of 0.48 and 0.40, respectively. Bark and wood were examined separately. As expected, hot water pretreatments, in the tested temperature range, resulted in very little direct xylose recovery. However, the 140°C dilute acid pretreatment of the lower specific gravity clone, S7C15, wood yielded the highest average xylose recovery of 56%. This condition also yielded the highest concentration of furfural, 9 mg/g sample, which can be inhibitory to the fermentation step. The highest xylose recovery from bark samples, 31%, was obtained with clone S7C15, using the 160°C dilute acid pretreatment for 60 min.     

Molecular cloning and characterization of a profilin gene <Emphasis Type="Italic">BnPFN</Emphasis> from <Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">nigra</Emphasis> that expressing in a pollen-specific manner

Brassica nigra is a newly found invasive species in Zhejiang Province, China. It distributes alongside the roads, in vegetable fields and on riversides. When it blooms, some natives there will suffer from allergic rhinitis. We designed gene-specific primer pairs according to reported profilin genes and successfully isolated their homolog from flower bud cDNA of B. nigra. The gene, designated BnPFN, was submitted to GenBank under accession number EU004073. BnPFN was 405 bp in length encoding 134 amino acids. Expression analysis of BnPFN gene was carried out by means of RT-PCR. The results showed that BnPFN express only in anthers and pollens, and there was no detection in roots, leaves, stems, sepals, petals and pistils. We suggest that BnPFN is a pollen-specific gene and may be responsible for pollen anaphylactic reactions in those invading areas when B. nigra blooms.     

Alignment of a <Emphasis Type="Italic">Salix</Emphasis> linkage map to the <Emphasis Type="Italic">Populus</Emphasis> genomic sequence reveals macrosynteny between willow and poplar genomes

Poplars (genus Populus) and willows (genus Salix) are members of the Salicaceae, a family of catkin-bearing trees, shrubs and sub-shrubs. Poplar is considered the model system for biological studies in trees and considerable genetic and genomic resources have become available in recent years. The transfer of information to research studies in willow, for which fewer resources are currently available, would be highly beneficial. However, the extent of conservation between poplar and willow genomes has not yet been extensively studied. To address this, we have constructed a linkage map of willow based on a large mapping population derived from a cross between two Salix viminalis × (S. viminalis × S. schwerinii) hybrid sibs, and aligned this to the publicly available poplar genome sequence. A set of genome-wide, expressed poplar sequences was selected and used to design primer sets that efficiently amplified homeologous regions in willow. Direct sequencing of the willow products confirmed homology with the poplar target in the majority of instances and allowed identification of single nucleotide polymorphisms (SNPs) that were used to map these loci. In total, 202 amplified fragment length polymorphisms (AFLPs), 75 microsatellites and 79 SNPs were used to construct a willow consensus map that spanned 1,856.7 cM with an average interval between markers of 6.3 cM. Poplar sequences homologous to those of the mapped willow microsatellite loci were identified and used in addition to the SNP markers to putatively align all but two minor linkage groups to the poplar genome sequence. A high degree of macrosynteny was revealed.     

Responses to nonaeration and/or salinity stress in hydroponically cultured <Emphasis Type="Italic">Populus nigra</Emphasis> and <Emphasis Type="Italic">Populus alba</Emphasis> cuttings

Growth, photosynthesis, and Na⁺, K⁺, Ca²⁺, and Mg²⁺ distributions were examined in two-year-old hydroponically cultured Populus nigra and Populus alba cuttings exposed to salt stress (0, 50, or 100 mM NaCl) for four or six weeks and to nonaeration stress for one or three weeks, followed by a three-week aeration period in 2/5 Hoagland solution. Salt stress with 100 mM NaCl totally inhibited height increase in P. nigra cuttings. Combined salinity and nonaeration inhibited height increase to a greater degree than either stress alone in both species. Simple salt stress did not affect diameter increase in P. alba, whereas combined high salinity (100 mM NaCl) and nonaeration inhibited diameter increase. Growth and biomass accumulation were more sensitive to salt stress in P. nigra cuttings than in P. alba, although P. alba showed a more rapid decrease in photosynthesis in response to nonaeration stress. Ion distributions in the leaves and roots differed between species. P. alba was superior to P. nigra in terms of Na⁺ exclusion capacity, such that most of the absorbed Na⁺ was confined to the root system, with little reaching the leaves. The distributions of K⁺, Ca²⁺, and Mg²⁺ in the leaves and roots of each species under the two stressors were also analyzed. The lower Na⁺/K⁺ ratio in leaves indicated that P. alba was more tolerant to salt stress than P. nigra.     

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

The ectopic expression of apple <Emphasis Type="Italic">MYB1</Emphasis> and <Emphasis Type="Italic">bHLH3</Emphasis> differentially activates anthocyanin biosynthesis in tobacco

Two direct DNA transfer methods, biolistic transformation and a protoplast transformation approach using the INRA-clone 717 1B4 (Populus tremula?×?P. alba), are applied to poplars and compared. Both the in vitro culture and the transformation parameters were optimized to receive a maximum quantity of transformed cells to achieve a stable transformation. For the first time, the stable integration of gfp and dsred in the poplar genome and their expression as visual reporter genes in regenerated plantlets can be shown. For biolistic transformation, stem segments cut lengthwise and incubated for 10 days on a callus induction medium revealed the highest number of transient Gfp- and dsRed signals. After optimization of the in vitro culture parameter, Gfp and dsRed-expressing transgenic poplars were regenerated, proven by PCR and Southern blot analysis. For protoplast transformation, the focus was initially on the development of a highly efficient protoplast isolation and plant regeneration system. Using an enzyme solution consisting of 1.0% cellulase R10 and 0.24% macerozyme, 1?×?10⁷ protoplasts were obtained from 1 g fresh weight leaves. Following incubation of the protoplasts in 600 mOsm culture medium, a high number of microcalli were obtained, from which plantlets were regenerated. The parameters for isolation and regeneration were then complemented by an efficient protoplast transformation protocol with 40% PEG₁₅₀₀. The results of this study confirm that both the biolistic and the protoplast transformation methods can be considered suitable for transferring cisgenes directly into poplar.