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Floodplain forests as one of the most endangered ecosystems in Europe have recently been impacted by fungal pathogens Phytophtora sp. and Hymenoscyphus fraxineus (chalara). Grey poplar (Populus × canescens Aiton Sm.) can be used as one of well adapted tree species for the reforestation of withering stands of ash (Fraxinus sp.). A unique population of grey poplar (Populus × canescens Aiton Sm.) characterized by desirable phenotypic traits was used for this purpose. The gender distribution was asymmetric; out of 155 individuals, 113 were female. Out of 33 different genotypes determined in the population, 15 were used as a source of approved forest reproductive material. Vegetative reproduction methods (ex situ clonal reproduction by micropropagation and cuttings) were developed and used, to rapidly initiate the recovery of forest stands of grey poplar. In total, 940 explants were successfully micropropagated and adapted to natural conditions, to ensure a genetically diverse source of viable plants used for reforestation. Moreover, we used methodological procedures of micropropagation for setting up cryopreservation technique. With respect to long-term storage of valuable grey poplar genotypes, modulation of gene expression by cold hardening during cryopreservation revealed significant changes in a few candidate genes involved in plant cellular processes.

Insect infestation triggers multiple defense responses in plants, both locally at the infection site and systemically throughout the plant, including the production of feeding deterrents, toxins, defensive proteins, enzymes, and secondary metabolites. Our study aimed to compare the endogenous levels of antioxidative enzymes, photosynthetic pigments, phytohormones, total phenols, and flavonoids in bark-beetle-infested and uninfested trees. We evaluated the surviving trees in bark-beetle-infested stands, assessing both the condition and defense of uninfested and infested beech (Fagus sylvatica), pine (Pinus sylvestris), and fir (Abies alba) trees. Sampling was performed at six affected sites in the Czech Republic, targeting trees that were resilient to significant health deterioration caused by abiotic and biotic factors. The results showed different levels of most of the measured compounds in the three species. Among all the tested species, photosynthetic pigment levels showed the strongest association with infestation status, which was generally lower in the infested plants. For chlorophyll a, extremely significant reductions were observed from 123 ± 20.6 to 101 ± 17.9 μg/g dry weight (DW) in pine, from 231 ± 33.1 to 199 ± 22.2 μg/g DW in beech, and from 60 ± 5.66 to 51.3 ± 6.27 μg/g DW in fir. In contrast, enzymatic activities indicated only isolated instances of significant association, whereas antioxidative properties (total phenolic content, flavonoids, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity) were not significantly associated with infestation status. There was a statistically significant increase in glutathione reductase activity in infested fir and pine trees. However, this difference was not statistically significant in beech. In contrast, a significant increase in superoxide dismutase activity was detected in infected beech trees. Phytohormones have emerged as the most diverse group of analyzed compounds. Cytokinins were the most distinct, with many of them being significantly increased in infested pines, whereas both beech and fir showed only one significant association. Additionally, derivatives of jasmonic acid also showed a distinct pattern of change associated with bark beetle infestation, with the levels of three out of the four analyzed jasmonates being significantly decreased in infested pines, whereas no effects were observed in beeches and firs. Notably, many phytohormones were significantly elevated in the infested pine, whereas both beech and fir exhibited only one significant association. Overall, the data showed that pines responded differently to bark beetles than to beeches or firs. The greatest changes in phytohormones were observed in pine, whereas the most significant changes in photosynthetic pigments were observed in beech and fir trees. [ABSTRACT FROM AUTHOR]

The aim of the work is to determine the genetic diversity, genetic differentiation and genetic structure of subpopulations of the specially protected common yew in the area of South Moravian Podyjí (Czech Republic), Thayatal (Austria) and in the Svitavy region (Czech Republic) using DNA analyses (nSSR-nuclear Simple Sequence Repeats). The subjects of the analyses were yews of 1) natural occurrences in the territory of the Podyjí National Park (NP), 2) cultural origin in the Podyjí NP, 3) natural occurrences in the Thayatal NP (Austria), 4) natural occurrences outside the Podyjí NP, and 5) cultural origin in the Podyjí region outside the NP. For the comparison and better interpretation of results there also were analysed geographically more distant subpopulations, 6) natural occurrences in the Hřebečský ridge (NW Moravia), 7) cultural yew occurrences in the Svitavy region (NE Bohemia & NW Moravia), 8) natural occurrences in the Jílovské yews (N Bohemia), and 9) natural occurrences in the Moravian Karst (S Moravia). We observed higher level of genetic diversity (Ho = 0.54; He = 0.77), the FST with ENA correction ranged from 0.007 to 0.123. According to a STRUCTURE analysis, high genetic similarity was observed between samples from the Podyjí NP and Hřebečský ridge. The impact of the analysis results on the conservation management of tested populations was outlined. [ABSTRACT FROM AUTHOR]

Amurk cork tree (Phellodendron amurense Rupr.) is known for its anti-infl ammatory, antipyretic, cholagogue, and antibacterial properties, and it is also a potential source of industrial cork. Shoot organogenesis and subsequent plant multiplication of Amur cork tree were established from mature seeds of trees growing on research plots in the Czech Republic. Germination percentage was increased by mechanical disruption of the seed coat, the highest number of germinated plants was achieved within 8–10 weeks of culture on MS medium containing 2 mg.l-1 BAP and 0,2 mg.l-1 NAA. Within 4 weeks of transfer to the multiplication medium containing 2 mg.l-1 BAP and 0,2 mg.l-1 NAA, chlorosis and reduced growth were observed in all cultured microcuttings. Lowering the BAP concentration to 0,4 mg.l-1 led to a resumption of growth, the newly emerging shoots and leaves were vital and the multiplication rate was around 90%. Our developed plant regeneration method may be useful in future studies on ex vitro establishment of in vitro derived plants. [ABSTRACT FROM AUTHOR]

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Th e purpose of this study was to investigate the possibilities of using objective methods of DNA analysis to verify the declared origin of reproductive material of Scots pine in terms of the Czech Republic. Monitoring of reproductive material identity was carried out during four years, i.e., from seed collection to transplanted plants production. Reference samples from the 40 sets of reproductive material were obtained, analyses of microsatellite markers were performed, and the genetic compositions of sets were compared aft er statistical processing. Altogether, DNA analyses were performed on 2390 samples of plant material from 10 selected sources of forest reproductive material (units of forest reproductive material). Seven optimally polymorphic markers with sufficient informative value were selected for the subsequent evaluation of the genetic structure of the monitored sets of Scots pine reproductive material by Bayesian clustering. Using the performed Structure analysis, the obtained profiles of 10 monitored units of reproductive material (4 sample sets from one units) of different origin were distinguishable from each other. Th us, these methodological procedures could be used in the state control systems to certify the origin of forest reproductive material and increase consumer protection of forest owners and nursery production in the Czech Republic. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate the possibilities of using objective methods of DNA analysis to verify the declared origin of reproductive material of Norway spruce in terms of the Czech Republic. Monitoring of the identity of reproductive material was carried out during three years, i.e., from seed collection to transplanted plants production. Reference samples from the 32 sets of reproductive material were obtained, analyses of microsatellite markers were performed, and the genetic compositions of sets were compared after statistical processing. Altogether, DNA analyses were performed on 1920 samples of plant material from 8 selected sources of forest reproductive material (units of forest reproductive material). Seven optimally polymorphic markers with sufficient informative value were selected for the subsequent evaluation of the genetic structure of the monitored sets of Norway spruce reproductive material by Bayesian clustering. Using the performed Structure analysis, the obtained profiles of 8 monitored units of reproductive material (4 sample sets from one units) of different origin were distinguishable from each other. Thus, these methodological procedures could be used in the state control systems to certify the origin of forest reproductive material and increase consumer protection of forest owners and nursery production in the Czech Republic. Identity of forest tree reproductive material is essential in artificial forest regeneration. A certain proof of origin is important for tracing back forest reproductive material. The Czech Republic as a member state of the European Union has the obligation to create a functioning control system for determination of forest reproductive material. This commitment is also based on international legislation (Council Directive 1999/105/EC on the marketing of forest reproductive material on the market). Its aim is to ensure clear identification of reproductive material from the acquisition to delivery to the consumer. The Directive 1999/105/EC is transposed into national legislation by Act No. 149/2003 Coll., on the marketing of forest reproductive material of forestry importance and artificial hybrids, intended for forest regeneration and afforestation, and amending certain related acts (Act on Trade in reproductive material of forest trees), as amended, the provisions of which include appropriate adjustment control measures. The existing legal regulations on forest reproductive material in the Czech Republic provide only the inspection of the master certificates and delivery papers as a control measure. The aim of the work was to verify the level of genetic diversity, heterozygosity and other genetic characteristics of selected samples of Norway spruce reproductive material based on analyses of microsatellite (SSR) markers, to determine genetic similarity of monitored sources of forest reproductive material (units of forest reproductive material), and to verify suitability of methodology for monitoring identity of Norway spruce reproductive material. DNA analyses were performed on 1920 samples of plant material from 8 selected sources of reproductive material. Sampling of reference samples was performed from the sets and units of reproductive material listed in Table 1. Seed collections for units of reproductive material was done from 35–60 trees from 8 recognized stands. Sampling of material to analyses was carried out over 3 years. From each of 8 reproductive material units there were analysed 60 samples from raw cones seeds, 60 samples from seeds after extraction and cleaning, 60 samples from seedling production and 60 samples from transplanted plant. The genomic DNA was extracted by the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer´s instruction. Seven polymorphic markers (PAAC23, SpAG2, WS00111.K13, WS00716.F13, WS0022.B15, WS0073.H08, WS0023.B03) with the corresponding predictive value were selected from the original set of 26 tested microsatellite markers. PCR and fragment analysis procedures were optimized for selected markers. PCR products were separated by capillary electrophoresis using the Applied Biosystems 3500 genetic analyser. The statistical programs CERVUS (Kalinowski et al. 2007), GenAlEx 6.503 (Peakall, Smouse 2006, 2012), the Bayesian clustering method implemented in the software STRUCTURE 2.3.4 (Pritchard et al. 2000; Falush et al. 2003, 2007; Hubisz et al. 2009) were used to analyse the genetic data. PCR products of the selected markers generated simple patterns and provided variable expected sizes of alleles. The genetic diversity parameters with the primer sequences of the studied markers are reported in Table 2. The most polymorphic locus was WS0023.B03 and the least was WS0073.H08. Genetic diversity characteristics of the 32 Norway spruce sample sets are given in Table 3. Nei’s genetic distances among 32 Norway spruce sample sets ranged from 0.04 to 0.325, and they are presented in Fig. 1, constructed on the basis of principal coordinate analysis (PCoA). The results of the AMOVA showed that variation among individuals was 10% and among the observed sample sets it was 3%. Pairwise population FST values ranging from 0.003 to 0.038 indicated low genetic differentiation between observed sets of sample. The structuring of investigated Norway spruce sample sets was also confirmed by various proportions of genetic profiles according to the Bayesian clustering method results (Fig. 2). Using the performed Structure analysis, the obtained profiles of 8 monitored units of reproductive material (4 sample sets from one units) of different origin were distinguishable from each other. These methodological procedures could be used in state control systems of forest reproductive material origin, and in order to increase consumer protection of forest owners and nursery production in the Czech Republic. [ABSTRACT FROM AUTHOR]

Th e aim of the study was to determine the possibility of using SSR markers for assessing clonal identity of European larch (Larix decidua Mill.) trees in seed orchards and determine the suitability of markers for the analysis of genetic diversity in populations of European larch in the Czech Republic. Total genomic DNA was extracted by DNA Plant Mini Kit (QIAGEN) from young needles taken from 218 sampled trees of two seed orchards. Samples were screened using thirteen selected polymorphic nuclear microsatellite markers. Measuring of the size of amplification products was carried out using Applied Biosystems 3500 genetic analyser. Th e obtained data was analysed by statistical programs CERVUS and GenAlEx 6.503. 155 different alleles were detected at 13 loci of the 218 larch individuals from two seed orchards. By applying the 13 suitable markers to the 90 clones from model seed orchards we obtained multilocus genotypes (MLG). Th e obtained results illustrate the utility of the microsatellite loci for assessing spatial patterns of genetic diversity and for individual genotypes identification. 98.24% of the sampled trees could be assigned to the clones represented in the Bílovice seed orchard. Th e tested genetic loci were verified as highly polymorphic and could be further used for clonal identification and genetic diversity evaluation of European larch trees. [ABSTRACT FROM AUTHOR]

The Simple Sequence Repeats (SSR) method of DNA analyses was used to clonal identification of trees in a model lime (Tillia cordata Mill.) seed orchard. Total genomic DNA was extracted by DNA Plant Mini Kit (QIAGEN) from young leaves taken from 377 sampled trees of the seed orchard. Samples were screened using selected eight polymorphic nuclear microsatellite markers. Measuring of the size of amplification products was carried out using the genetic analyser Applied Biosystems 3500. The obtained data were analysed by statistical programs CERVUS, GenAlEx 6.503. There were detected 79 different alleles at 8 loci in the 377 lime individuals from seed orchard. By applying of the 8 suitable markers to the 38 clones from model seed orchard we obtained multilocus genotypes (MLG). The obtained results illustrate the utility of the microsatellite loci for assessing spatial patterns of genetic diversity and for individual genotypes identification. 93.6 % of the sampled trees could be assigned to the clones represented in the seed orchard. The identified genetic loci were verified as highly polymorphic and could be further used for clonal identification of lime trees. [ABSTRACT FROM AUTHOR]

The genetic structure of selected Norway spruce stands from the Jeseníky Mts., Orlické hory Mts., and Krušné hory Mts. (Czech Republic) was studied to verify the genetic quality. The genetic variability of tree populations ensures the stability and sustainability of forest ecosystems. DNA polymorphism at twelve nuclear microsatellites of fourteen Norway spruce stands was investigated. The level of genetic diversity within 14 investigated Czech Norway spruce stands was relatively high. Mean values for number of different alleles ranged from 12.25 (stand SM J51) to 15.92 (stand SM T1). The values of observed heterozygosity (Ho) ranged from 0.65 to 0.81, and expected heterozygosity (He) from 0.81 to 0.86. Pairwise population FST values ranging from 0.007 to 0.030 indicated low genetic differentiation between units, and values of Nei’s genetic distance among Norway spruce units ranged from 0.083 to 0.313. The structuring of investigated Norway spruce stands was also confirmed by different ratios of genetic profiles according to the Bayesian clustering method results. Closer genetic similarity was seen in the stands from the gene conservation unit in the Orlické hory than in other studied stands. The most genetically different stand was from the Krušné hory. Knowledge based on DNA analyses regarding the variability of genetic resources will contribute to the quality of the reproduction material and to creation of an optimal species composition in forests. [ABSTRACT FROM AUTHOR]

The main goal of the presented study was to evaluate genotypes of unique population of grey poplar (Populus × canescens Aiton Sm.), which has been selected for its valuable parameters and limited reproduction ability by classical methods of vegetative propagation for gene pool preservation and restoration at Dyjákovice village (South Moravia Region, Czech Republic). Nuclear microsatellite (SSR) analyses using 14 SSR markers together with phenotype characterization determined that 90 sampled trees of female bias belonged to one clone. According to these results, the collection of grey poplars has been extended for 155 individuals in totally including both female and male bias (120 and 35, respectively) to discriminate their genetic diversity. Our data revealed occurrence of 6 unique genotypes among all female samples while higher genetic variability pattern exhibited male samples among which 27 genotypes have been detected. These findings about species genetic identity as well as genetic variability indicate the importance of SSR method for selection of valuable grey poplar genotypes as a source of reproductive material for further reforestation. [ABSTRACT FROM AUTHOR]

The main goal of the presented study is to explore an effective micropropagation method for grey poplar (Populus ×canescens Aiton (Sm.)), which belongs to woody plant species characterized by rapid growth and early reproductive maturity. The influence of three culture media - Murashige and Skoog medium (MS), woody plant medium (WPM), and medium according to Lambardi et al. (2000) (LAM) differing in nutrition and phytohormone compositions have been followed in relation to shoot-tip segments growth. The best results have been achieved on MS medium enriched in glutamine (10 mg.l-1), glycine (2 mg.l-1), BAP (0.2 mg.l-1) and IBA (0.1 mg.l-1), even though the concentrations of photosynthetic pigments were comparable with apical segments cultivated on WPM medium. In addition, faster shoot regeneration and subsequent proliferation were observed in excised apical segments on selected MS medium in comparison to excised nodal segments, in which lateral buds formations was unequal and delayed due to excessive adventitious shoots formation at the base of segments. These results clearly indicate the importance of medium composition and a part of segments used for efficient in vitro micropropagation technique of grey poplar. [ABSTRACT FROM AUTHOR]

The Simple Sequence Repeats (SSR) method of DNA analyses was used to clonal identification of trees in a model wild cherry (Prunus avium L.) seed orchard. Total genomic DNA was extracted by DNA Plant Mini Kit (QIAGEN) from buds taken from 91 sampled trees of the seed orchard. Samples were screened using selected ten polymorphic nuclear microsatellite markers. Measuring of the size of amplification products was carried out using the genetic analyser Applied Biosystems 3500. The obtained data were analysed by statistical programs CERVUS, GenAlEx 6.501 and Micro-Checker. There were detected 65 different alleles at 10 loci in the 91 wild cherry individuals from seed orchard. By applying of the 10 suitable markers to the 18 clones from model seed orchard we obtained multilocus genotypes (MLG). The obtained results illustrate the utility of the microsatellite loci for assessing spatial patterns of genetic diversity and for individual genotypes identification. Declared clone affiliation was confirmed in 94% of sampled trees. The identified genetic loci were verified as highly polymorphic and could be further used for clonal identification of wild cherry trees. [ABSTRACT FROM AUTHOR]