Various effects of the expression of the xyloglucanase gene from Penicillium canescens in transgenic aspen under semi-natural conditions.

BACKGROUND: Recombinant carbohydrases genes are used to produce transgenic woody plants with improved phenotypic traits. However, cultivation of such plants in open field is challenging due to a number of problems. Therefore, additional research is needed to alleviate them. RESULTS: Results of successful cultivation of the transgenic aspens (Populus tremula) carrying the recombinant xyloglucanase gene (sp-Xeg) from Penicillium canescens in semi-natural conditions are reported in this paper for the first time. Change of carbohydrate composition of wood was observed in transgenic aspens carrying the sp-Xeg gene. The transformed transgenic line Xeg-2-1b demonstrated accelerated growth and increased content of cellulose in wood of trees growing in both greenhouse and outside in comparison with the control untransformed line Pt. The accelerated growth was observed also in the transgenic line Xeg-1-1c. Thicker cell-wall and longer xylem fiber were also observed in both these transgenic lines. Undescribed earlier considerable reduction in the wood decomposition rate of the transgenic aspen stems was also revealed for the transformed transgenic lines. The decomposition rate was approximately twice as lower for the transgenic line Xeg-2-3b in comparison with the control untransformed line Pt. CONCLUSION: A direct dependence of the phenotypic and biochemical traits on the expression of the recombinant gene sp-Xeg was demonstrated. The higher was the level of the sp-Xeg gene expression, the more pronounced were changes in the phenotypic and biochemical traits. All lines showed phenotypic changes in the leave traits. Our results showed that the plants carrying the recombinant sp-Xeg gene do not demonstrate a decrease in growth parameters in semi-natural conditions. In some transgenic lines, a change in the carbohydrate composition of the wood, an increase in the cell wall thickness, and a decrease in the rate of decomposition of wood were observed.

Fell Upas Sits, the Hydra-Tree of Death †, or the Phytotoxicity of Trees.

The use of natural products that can serve as natural herbicides and insecticides is a promising direction because of their greater safety for humans and environment. Secondary metabolites of plants that are toxic to plants and insects-allelochemicals-can be used as such products. Woody plants can produce allelochemicals, but they are studied much less than herbaceous species. Meanwhile, there is a problem of interaction of woody species with neighboring plants in the process of introduction or invasion, co-cultivation with agricultural crops (agroforestry) or in plantation forestry (multiclonal or multispecies plantations). This review describes woody plants with the greatest allelopathic potential, allelochemicals derived from them, and the prospects for their use as biopesticides. In addition, the achievement of and the prospects for the use of biotechnology methods in relation to the allelopathy of woody plants are presented and discussed.

Hormonal Status of Transgenic Birch with a Pine Glutamine Synthetase Gene during Rooting In Vitro and Budburst Outdoors.

Improving nitrogen use efficiency (NUE) is one of the main ways of increasing plant productivity through genetic engineering. The modification of nitrogen (N) metabolism can affect the hormonal content, but in transgenic plants, this aspect has not been sufficiently studied. Transgenic birch (Betula pubescens) plants with the pine glutamine synthetase gene GS1 were evaluated for hormone levels during rooting in vitro and budburst under outdoor conditions. In the shoots of the transgenic lines, the content of indoleacetic acid (IAA) was 1.5-3 times higher than in the wild type. The addition of phosphinothricin (PPT), a glutamine synthetase (GS) inhibitor, to the medium reduced the IAA content in transgenic plants, but it did not change in the control. In the roots of birch plants, PPT had the opposite effect. PPT decreased the content of free amino acids in the leaves of nontransgenic birch, but their content increased in GS-overexpressing plants. A three-year pot experiment with different N availability showed that the productivity of the transgenic birch line was significantly higher than in the control under N deficiency, but not excess, conditions. Nitrogen availability did not affect budburst in the spring of the fourth year; however, bud breaking in transgenic plants was delayed compared to the control. The IAA and abscisic acid (ABA) contents in the buds of birch plants at dormancy and budburst depended both on N availability and the transgenic status. These results enable a better understanding of the interaction between phytohormones and nutrients in woody plants.

Relationship between Phenolic Compounds and Antioxidant Activity in Berries and Leaves of Raspberry Genotypes and Their Genotyping by SSR Markers.

The red raspberry is one of the world's most popular berries. The main direction of its breeding has switched to nutritional quality, and the evaluation of raspberry germplasm for antioxidant content and activity is very important. As berries, raspberry leaves contain valuable bioactive compounds, but the optimal time for their collection is unknown. We evaluated 25 new breeding lines and standard raspberry cultivars for their polyphenolic content and antioxidant capacity. The antioxidant activity of berries correlated better with the content of total phenolics (0.88 and 0.92) and flavonoids (0.76 and 0.88) than with anthocyanins (0.37 and 0.66). Two breeding lines were significantly superior to the standard cultivars and can be used in further breeding. Leaves collected in three phenological phases of the raspberry contained more phenolics (5.4-fold) and flavonoids (4.1-fold) and showed higher antioxidant activities (2.4-fold in FRAP assay, 2.2-fold in ABTS) than berries. The optimal time for harvesting raspberry leaves is the fruit ripening stage, with exceptions for some cultivars. Genetic diversity analysis using microsatellite (SSR) markers from flavonoid biosynthesis genes divided the genotypes into five clusters, generally in agreement with their kinships. The relationship between genetic data based on metabolism-specific SSR markers and the chemical diversity of cultivars was first assessed. The biochemical and genetic results show a strong correlation (0.78). This study is useful for further the improvement of raspberry and other berry crops.

Expression of a Stilbene Synthase Gene from the Vitis labrusca x Vitis vinifera L. Hybrid Increases the Resistance of Transgenic Nicotiana tabacum L. Plants to Erwinia carotovora.

'Isabel' grape (Vitis labrusca x V. vinifera L. hybrid) is one of the main grape cultivars in Russia and some other countries for processing, due to its vigor, tolerance to the main fungal diseases, high yield and potential for sugar accumulation. The stilbene synthase gene VlvSTS was isolated from the hybrid grape cv. Isabel and cloned into a pSS plant transformation vector under the control of a constitutive 35S RNA double promoter of the cauliflower mosaic virus, CaMV 35SS. VlvSTS-gene containing transgenic tobacco lines were obtained and analyzed. For the first time plants expressing the VlvSTS gene were shown to have an enhanced resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora B15. Transgenic plants were tested for resistance to a number of fungal pathogens. The plants were resistant to the grey mould fungus Botrytis cinerea, but not to the fungi Fusarium oxysporum, F. sporotrichioides, or F. culmorum. According to the results of a high performance liquid chromatography-mass spectrometry analysis, the amount of trans-resveratrol in leaves of transgenic plants with the highest expression of the VlvSTS gene was in a range from 150 to 170 µg/g of raw biomass. Change in the color and a decreased anthocyanin content in the flower corollas of transgenic plants were observed in transgenic lines with the highest expression of VlvSTS. A decrease in total flavonoid content was found in the flower petals but not the leaves of these tobacco lines. High expression of the VlvSTS gene influenced pollen development and seed productivity in transgenic plants. The size of pollen grains increased, while their total number per anther decreased. A decrease in the number of fertile pollen grains resulted in a decreased average weight of a seed boll in transgenic plants.

Genetic Engineering and Genome Editing for Improving Nitrogen Use Efficiency in Plants.

Low nitrogen availability is one of the main limiting factors for plant growth and development, and high doses of N fertilizers are necessary to achieve high yields in agriculture. However, most N is not used by plants and pollutes the environment. This situation can be improved by enhancing the nitrogen use efficiency (NUE) in plants. NUE is a complex trait driven by multiple interactions between genetic and environmental factors, and its improvement requires a fundamental understanding of the key steps in plant N metabolism-uptake, assimilation, and remobilization. This review summarizes two decades of research into bioengineering modification of N metabolism to increase the biomass accumulation and yield in crops. The expression of structural and regulatory genes was most often altered using overexpression strategies, although RNAi and genome editing techniques were also used. Particular attention was paid to woody plants, which have great economic importance, play a crucial role in the ecosystems and have fundamental differences from herbaceous species. The review also considers the issue of unintended effects of transgenic plants with modified N metabolism, e.g., early flowering-a research topic which is currently receiving little attention. The future prospects of improving NUE in crops, essential for the development of sustainable agriculture, using various approaches and in the context of global climate change, are discussed.

Efficient Cryopreservation of Populus tremula by In Vitro-Grown Axillary Buds and Genetic Stability of Recovered Plants.

Axillary buds of in vitro microshoots were successfully frozen at -196 °C by the one-step freezing method using the protective vitrification solution 2 (PVS2). Microshoots were taken from 11 transgenic lines and three wild type lines. Influence of different explant pretreatments were analyzed from the point of their influence towards recovery after cryopreservation. It was found out that the use of axillary buds as explants after removal of the apical one increases recovery on average by 8%. The cultivation on growth medium of higher density insignificantly raises the regenerants survival rate. Pretreatment of the osmotic fluid (OF) shows the greatest influence on the survival rate. It leads to the increase in survival rate by 20%. The cryopreservation technology providing regenerants average survival rate of 83% was developed. It was based on the experimental results obtained with explant pretreatment. Incubation time in liquid nitrogen did not affect the explants survival rate after thawing. After six months cryostorage of samples their genetic variability was analyzed. Six variable simple sequence repeat (SSR) loci were used to analyze genotype variability after the freezing-thawing procedure. The microsatellite analysis showed the genetic status identity of plants after cryopreservation and of the original genotypes. The presence of the recombinant gene in the transgenic lines after cryostorage were confirmed so as the interclonal variation in the growth rate under greenhouse conditions. The developed technique is recommended for long-term storage of various breeding and genetically modified lines of aspen plants, as it provides a high percentage of explants survival with no changes in genotype.

Structure and Phylogeny of the Curly Birch Chloroplast Genome.

Curly birch [Betula pendula var. carelica (Merckl.) Hämet-Ahti] is a relatively rare variety of silver birch (B. pendula Roth) that occurs mainly in Northern Europe and northwest part of Russia (Karelia). It is famous for the beautiful decorative texture of wood. Abnormal xylogenesis underlying this trait is heritable, but its genetic mechanism has not yet been fully understood. The high number of potentially informative genetic markers can be identified through sequencing nuclear and organelle genomes. Here, the de novo assembly, complete nucleotide sequence, and annotation of the chloroplast genome (plastome) of curly birch are presented for the first time. The complete plastome length is 160,523 bp. It contains 82 genes encoding structural and enzymatic proteins, 37 transfer RNAs (tRNAs), and eight ribosomal RNAs (rRNAs). The chloroplast DNA (cpDNA) is AT-rich containing 31.5% of A and 32.5% of T nucleotides. The GC-rich regions represent inverted repeats IR1 and IR2 containing genes of rRNAs (5S, 4.5S, 23S, and 16S) and tRNAs (trnV, trnI, and trnA). A high content of GC was found in rRNA (55.2%) and tRNA (53.2%) genes, but only 37.0% in protein-coding genes. In total, 384 microsatellite or simple sequence repeat (SSR) loci were found, mostly with mononucleotide motifs (92% of all loci) and predominantly A or T motifs (94% of all mononucleotide motifs). Comparative analysis of cpDNA in different plant species revealed high structural and functional conservatism in organization of the angiosperm plastomes, while the level of differences depends on the phylogenetic relationship. The structural and functional organization of plastome in curly birch was similar to cpDNA in other species of woody plants. Finally, the identified cpDNA sequence variation will allow to develop useful genetic markers.

Transferability and Polymorphism of SSR Markers Located in Flavonoid Pathway Genes in Fragaria and Rubus Species.

Strawberry (Fragaria) and raspberry (Rubus) are very popular crops, and improving their nutritional quality and disease resistance are important tasks in their breeding programs that are becoming increasingly based on use of functional DNA markers. We identified 118 microsatellite (simple sequence repeat-SSR) loci in the nucleotide sequences of flavonoid biosynthesis and pathogenesis-related genes and developed 24 SSR markers representing some of these structural and regulatory genes. These markers were used to assess the genetic diversity of 48 Fragaria and Rubus specimens, including wild species and rare cultivars, which differ in berry color, ploidy, and origin. We have demonstrated that a high proportion of the developed markers are transferable within and between Fragaria and Rubus genera and are polymorphic. Transferability and polymorphism of the SSR markers depended on location of their polymerase chain reaction (PCR) primer annealing sites and microsatellite loci in genes, respectively. High polymorphism of the SSR markers in regulatory flavonoid biosynthesis genes suggests their allelic variability that can be potentially associated with differences in flavonoid accumulation and composition. This set of SSR markers may be a useful molecular tool in strawberry and raspberry breeding programs for improvement anthocyanin related traits.

Influence of Nitrogen Availability on Growth of Two Transgenic Birch Species Carrying the Pine GS1a Gene.

An alternative way to increase plant productivity through the use of nitrogen fertilizers is to improve the efficiency of nitrogen utilization via genetic engineering. The effects of overexpression of pine glutamine synthetase (GS) gene and nitrogen availability on growth and leaf pigment levels of two Betula species were studied. Untransformed and transgenic plants of downy birch (B. pubescens) and silver birch (B. pendula) were grown under open-air conditions at three nitrogen regimes (0, 1, or 10 mM) for one growing season. The transfer of the GS1a gene led to a significant increase in the height of only two transgenic lines of nine B. pubescens, but three of five B. pendula transgenic lines were higher than the controls. In general, nitrogen supply reduced the positive effect of the GS gene on the growth of transgenic birch plants. No differences in leaf pigment levels between control and transgenic plants were found. Nitrogen fertilization increased leaf chlorophyll content in untransformed plants but its effect on most of the transgenic lines was insignificant. The results suggest that birch plants carrying the GS gene use nitrogen more efficiently, especially when growing in nitrogen deficient soil. Transgenic lines were less responsive to nitrogen supply in comparison to wild-type plants.

Molecular Identification and Karyological Analysis of a Rampant Aspen Populus tremula L. (Salicaceae) Clone.

A rampant highly heterozygous aspen (Populus tremula L.) clone "Meshabash" has been revealed in course of population genetic diversity analysis in a native stand in the Republic of Tatarstan, Russia. Here we report the results of karyological analysis showing that this highly vigorous clone is diploid (2n = 38) while typically triploid aspen demonstrates increased growth rate and resistance to aspen trunk rot caused by fungus Phellinus tremulae. By means of DNA identification of a series of model trees using 14 SSR loci we outlined the area occupied by this clone (at least 1.94 ha) and demonstrated that its ramets constitute 40 out of 48 genotyped trunks on the plot with the maximal distance between ramets 254 m. Since aspen is able to regenerate after cutting or die-off of maternal tree by root suckers at a distance up to 20-35 m this assumed that current stand appeared as a result of such spreading from an ortet tree during at least 5 generations. Trunk rot damage in the wood of model trees indicated low influence of this pathogen on viability and performance of the studied clone that can be associated with its extreme heterozygosity level (0.926) exceeding all the studied trees in this research plot and in three other control samples.

Application of Microsatellite Loci for Molecular Identification of Elite Genotypes, Analysis of Clonality, and Genetic Diversity in Aspen Populus tremula L. (Salicaceae).

Testing systems for molecular identification of micropropagated elite aspen (Populus tremula L.) genotypes were developed on the base on microsatellite (SSR) loci. Out of 33 tested microsatellite loci, 14 were selected due to sustainable PCR amplification and substantial variability in elite clones of aspen aimed for establishment of fast-rotated forest plantations. All eight tested clones had different multilocus genotypes. Among 114 trees from three reference native stands located near the established plantations, 80 haplotypes were identified while some repeated genotypes were attributed to natural clones which appeared as a result of sprouting. The selected set of SSR markers showed reliable individual identification with low probability of appearance of identical aspen genotypes (a minimum of 4.8 · 10(-10) and 1 × 10(-4) for unrelated and related individuals, resp.). Case studies demonstrating practical applications of the test system are described including analysis of clonal structure and levels of genetic diversity in three natural aspen stands growing in the regions where plantations made of elite clones were established.