Christian Sig Jensen

Transgenic Populus tremula: a step-by-step protocol for its Agrobacterium-mediated transformation

In recent years, Populus species have acquired an important place in basic and applied research of woody plants. The practical role of Populus species in world forestry and their importance to research as a woody-plant model have led to increasing interest in tissue-culture and molecular techniques, as well as the development of transformation procedures for this genus. A simple technical procedure is described here step-by-step, for the first time, as a routine method for transforming Populus tremula using a disarmed Agrobacterium tumefaciens hypervirulent strain. The procedure begins with the inoculation of stem explants with bacterial suspension, followed by a short period of co-cultivation on a highly regenerative medium. Transformed shoots are selected on regeneration medium containing antibiotics and the presence of the inserted target genes is checked using a rapid and efficient PCR test. Selected shoots are transferred to a rooting medium, under the same selection pressure, and propagated via stem cuttings. Selected plants can be hardened and transferred to the green-house within 4 months of inoculation. The method has proven efficient for several gene constructs, selection on Kan or Hyg, and three different Agrobacterium strains.

Floral inhibition in red fescue (Festuca rubra L.) through expression of a heterologous flowering repressor from Lolium

Extension of the vegetative growth phase through delay of flowering is an important goal in todays breeding programs of both forage and turf grasses. In forage grasses, the stem and inflorescence production comprise a significant reduction in the digestibility, nutritional value and productivity of the crop, and in turf grasses the stems that start to emerge during the growth season suppress the formation of new shoots and affect the quality, density and persistence of the sward. We have tested the potential of the strong floral repressor LpTFL1 from perennial ryegrass (Lolium perenne L.) to manipulate the transition to flowering in red fescue (Festuca rubra L.), a cool-season turf grass. Expression of LpTFL1 from the constitutive maize ubiquitin promoter represses flowering in red fescue, and the flowering repression phenotype correlates well with the level of LpTFL expression. Transgenic lines showing low to intermediate expression of LpTFL1 flowered approximately two weeks later than the controls, and transgenic lines showing very high LpTFL1 expression levels still remained non-flowering after exposure to natural vernalization conditions (Danish winter) in two successive years. There were no other phenotypic effects associated with the LpTFL transgene expression during vegetative growth. However, there was a tendency towards an LpTFL1-mediated reduction in stem length among the flowering lines. Expression of a truncated LpTFL, caused by transgene rearrangements during the transformation, lead to increased flowering and stem production and a decrease in panicle size. This is to our knowledge the first report on full inhibition of floral development in a commercially important grass species.

Nucleotide diversity and linkage disequilibrium of nine genes with putative effects on flowering time in perennial ryegrass (Lolium perenne L.).

Optimization of flowering is an important breeding goal in forage and turf grasses, such as perennial ryegrass (Lolium perenne L.). Nine floral control genes including Lolium perenne CONSTANS (LpCO), SISTER OF FLOWERING LOCUS T (LpSFT), TERMINAL FLOWER1 (LpTFL1), VERNALIZATION1 (LpVRN1, identical to LpMADS1) and five additional MADS-box genes, were analyzed for nucleotide diversity and linkage disequilibrium (LD). For each gene, about 1 kb genomic fragments were isolated from 10 to 20 genotypes of perennial ryegrass of diverse origin. Four to twelve haplotypes per gene were observed. On average, one single nucleotide polymorphism (SNP) was present per 127 bp between two randomly sampled sequences for the nine genes (π = 0.00790). Two MADS-box genes, LpMADS1 and LpMADS10, involved in timing of flowering showed high nucleotide diversity and rapid LD decay, whereas MADS-box genes involved in floral organ identity were found to be highly conserved and showed extended LD. For LpMADS4, LpMADS5, LpCO, LpSFT and LpTFL1, LD extended over the entire region analyzed. The results are compared to previously published results on resistance genes within the same collection of genotypes and the prospects for association mapping of floral control in perennial ryegrass are discussed.

Seasonal changes in the metabolic fingerprint of 21 grass and legume cultivars studied by nuclear magnetic resonance-based metabolomics.

A nuclear magnetic resonance (NMR)-based approach was introduced for metabolic fingerprinting of 21 grass and legume cultivars in the present study. Applying principal component analysis (PCA) on the fingerprints obtained on water extracts, it was possible to elucidate the variation between cultivars and the magnitude of changes in the metabolic fingerprint between the spring growth and the second regrowth. Consequently, the potential of the method for tracking differences and changes related to cultivar and season was demonstrated. In addition, partial least-squares (PLS) regressions revealed correlations between the NMR fingerprints and the value of the grasses as animal feed evaluated as concentration of sugars, neutral detergent fibres (NDF) (R = 0.82), indigestible neutral detergent fibres (iNDF) (R = 0.90), and in vitro organic matter digestibility (IVOMD) (R = 0.75). The correlations between these parameters and the NMR fingerprint could mainly be ascribed to differences in spectral intensities from signals assigned to malic acid (2.40 and 4.70 ppm), choline (3.27 ppm), and glucose (5.24 ppm), and the biochemical rationale for this relation is discussed.

Genomic dissection and prediction of heading date in perennial ryegrass.

BackgroundGenomic selection (GS) has become a commonly used technology in animal breeding. In crops, it is expected to significantly improve the genetic gains per unit of time. So far, its implementation in plant breeding has been mainly investigated in species farmed as homogeneous varieties. Concerning crops farmed in family pools, only a few theoretical studies are currently available. Here, we test the opportunity to implement GS in breeding of perennial ryegrass, using real data from a forage breeding program. Heading date was chosen as a model trait, due to its high heritability and ease of assessment. Genome Wide Association analysis was performed to uncover the genetic architecture of the trait. Then, Genomic Prediction (GP) models were tested and prediction accuracy was compared to the one obtained in traditional Marker Assisted Selection (MAS) methods.ResultsSeveral markers were significantly associated with heading date, some locating within or proximal to genes with a well-established role in floral regulation. GP models gave very high accuracies, which were significantly better than those obtained through traditional MAS. Accuracies were higher when predictions were made from related families and from larger training populations, whereas predicting from unrelated families caused the variance of the estimated breeding values to be biased downwards.ConclusionsWe have demonstrated that there are good perspectives for GS implementation in perennial ryegrass breeding, and that problems resulting from low linkage disequilibrium (LD) can be reduced by the presence of structure and related families in the breeding population. While comprehensive Genome Wide Association analysis is difficult in species with extremely low LD, we did identify variants proximal to genes with a known role in flowering time (e.g. CONSTANS and Phytochrome C).

Arabidopsis thaliana endo-1,4-

Summary Transgenic aspen plants were transformed with a bacterial s-glucuronidase gene ( uidA ) under the control of the Arabidopsis thaliana elongation-specific endo-1,4-s-glucanase ( cel 1 ) promoter. Transformed plants exhibited a high level of s-glucuronidase (GUS) activity in young, fast-growing tissues. Unlike the cauliflower mosaique virus 35S promoter, the expression pattern of uidA in the shoots and leaves was clear-cut and highly controlled by cel 1 promoter. GUS-staining in the leaves mirrored the pattern of expanding cells in the organ. These results further substantiate the link between cel 1 expression and plant cell elongation. Moreover, our results suggest that the cel 1 promoter may be useful in the genetic engineering of forest trees and possibly other plants when gene expression is desired only in fast growing tissues.

-ß-glucanase (cell) Promoter Mediates uidA Expression in Elongating Tissues of Aspen (Populus tremula)

High accuracies for genomic prediction in a perennial ryegrass breeding program The additive genetic variance can be traced by genotyping assays Predictions work across different generations and in different traits Good prospects for the implementation of genomic selection in perennial ryegrass

Accuracy of Genomic Prediction in a Commercial Perennial Ryegrass Breeding Program

BackgroundReed canary grass (Phalaris arundinacea) is an economically important forage and bioenergy grass of the temperate regions of the world. Despite its economic importance, it is lacking in public genomic data. We explore comparative exomics of the grass cultivars in the context of response to salt exposure. The limited data set poses challenges to the computational pipeline.MethodsAs a prerequisite for the comparative study, we generate the Phalaris reference transcriptome sequence, one of the first steps in addressing the issue of paucity of processed genomic data in this species. In addition, the differential expression (DE) and active-but-stable genes for salt stress conditions were analyzed by a novel method that was experimentally verified on human RNA-seq data. For the comparative exomics, we focus on the DE and stable genic regions, with respect to salt stress, of the genome.Results and conclusionsIn our comparative study, we find that phylogeny of the DE and stable genic regions of the Phalaris cultivars are distinct. At the same time we find the phylogeny of the entire expressed reference transcriptome matches the phylogeny of only the stable genes. Thus the behavior of the different cultivars is distinguished by the salt stress response. This is also reflected in the genomic distinctions in the DE genic regions. These observations have important implications in the choice of cultivars, and their breeding, for bio-energy fuels. Further, we identified genes that are representative of DE under salt stress and could provide vital clues in our understanding of the stress handling mechanisms in general.

Comparative exomics of Phalaris cultivars under salt stress.

Adventitious root formation and root-system performance are important aspects in the clonal propagation of different plant species, both from cuttings and in vitro. Root formation from cuttings is affected by anatomical, physiological and environmental factors and by their interaction. In addition, it is largely dependent on selecting a suitable organ (e.g. stem, leaf or root cuttings). This decision is influenced mainly by the inherent ability of the cuttings to regenerate root primordia. Several techniques have been proposed to improve rooting ability in difficult-to-root species, including heavy pruning (Scott, 1987) and etiolating stock plants to obtain easy-to-root juvenile material (Maynard and Bassuk, 1987), using specific rooting media (Sabalka, 1986), fog (Harrison- Murray et al., 1988) or mist atmospheres (Rosenberg et al, 1992), and applying growth regulators directly to the target tissue.

Improved Rooting Ability and Root-System Performance in Transgenic Aspen Plants

BackgroundReed canary grass (Phalaris arundinacea) is an economically important forage and bioenergy grass of the temperate regions of the world. Despite its economic importance, it is lacking in public genomic data. We explore comparative exomics of the grass cultivars in the context of response to salt exposure. The limited data set poses challenges to the computational pipeline.MethodsAs a prerequisite for the comparative study, we generate the Phalaris reference transcriptome sequence, one of the first steps in addressing the issue of paucity of processed genomic data in this species. In addition, the differential expression (DE) and active-but-stable genes for salt stress conditions were analyzed by a novel method that was experimentally verified on human RNA-seq data. For the comparative exomics, we focus on the DE and stable genic regions, with respect to salt stress, of the genome.Results and conclusionsIn our comparative study, we find that phylogeny of the DE and stable genic regions of the Phalaris cultivars are distinct. At the same time we find the phylogeny of the entire expressed reference transcriptome matches the phylogeny of only the stable genes. Thus the behavior of the different cultivars is distinguished by the salt stress response. This is also reflected in the genomic distinctions in the DE genic regions. These observations have important implications in the choice of cultivars, and their breeding, for bio-energy fuels. Further, we identified genes that are representative of DE under salt stress and could provide vital clues in our understanding of the stress handling mechanisms in general.