Polyploidy - A tool in adapting trees to future climate changes? A review of polyploidy in trees

Polyploidy, or genome doubling, has occurred repeatedly through plant evolution. While polyploid plants are used extensively in agriculture and horticulture, they have so far found limited use in forestry. Here we review the potentials of polyploid trees under climate change, and investigate if there is support for increased use. We find that polyploid trees like other plants have consistent increases in cell sizes compared to diploids, and that leaf-area based rates of photosynthesis tend to increase with increasing levels of ploidy. While no particular trend could be discerned in terms of biomass between trees of different ploidy levels, physiology is affected by polyploidization and several studies point towards a high potential for polyploid trees to adapt to drought stress. The ploidy level of most tree species is unknown, and analysis of geographical patterns in frequencies of polyploid trees are inconclusive. Artificial polyploid trees are often created by colchicine and in a few cases these have been successfully applied in forestry, but the effects of induced polyploidization in many economically important tree species remains untested. Polyploids would also be increasingly useful in tree breeding programs, to create synthetic hybrids or sterile triploids that could control unwanted spreading of germplasm in nature. In conclusion, this review suggests that polyploid trees may be superior under climate change in some cases, but that the potential of polyploids is not yet fully known and should be evaluated on a case-to-case basis for different tree species

In silico characterization of putative gene homologues involved in somatic embryogenesis suggests that some conifer species may lack LEC2, one of the key regulators of initiation of the process

Background Somatic embryogenesis (SE) is the process in which somatic embryos develop from somatic tissue in vitro on medium in most cases supplemented with growth regulators. Knowledge of genes involved in regulation of initiation and of development of somatic embryos is crucial for application of SE as an efficient tool to enable genetic improvement across genotypes by clonal propagation. Results Current work presents in silico identification of putative homologues of central regulators of SE initiation and development in conifers focusing mainly on key transcription factors (TFs) e.g. BBM, LEC1, LEC1-LIKE, LEC2 and FUSCA3, based on sequence similarity using BLASTP. Protein sequences of well-characterised candidates genes from Arabidopsis thaliana were used to query the databases (Gymno PLAZA, Congenie, GenBank) including whole-genome sequence data from two representative species from the genus Picea (Picea abies) and Pinus (Pinus taeda), for finding putative conifer homologues, using BLASTP. Identification of corresponding conifer proteins was further confirmed by domain search (Conserved Domain Database), alignment (MUSCLE) with respective sequences of Arabidopsis thaliana proteins and phylogenetic analysis (Phylogeny.fr). Conclusions This in silico analysis suggests absence of LEC2 in Picea abies and Pinus taeda, the conifer species whose genomes have been sequenced. Based on available sequence data to date, LEC2 was also not detected in the other conifer species included in the study. LEC2 is one of the key TFs associated with initiation and regulation of the process of SE in angiosperms. Potential alternative mechanisms that might be functional in conifers to compensate the lack of LEC2 are discussed

Automation and Scale Up of Somatic Embryogenesis for Commercial Plant Production, With Emphasis on Conifers

For large scale production of clonal plants, somatic embryogenesis (SE) has many advantages over other clonal propagation methods such as the rooting of cuttings. In particular, the SE process is more suited to scale up and automation, thereby reducing labor costs and increasing the reliability of the production process. Furthermore, the plants resulting from SE closely resemble those from seeds, as somatic embryos, like zygotic (seed) embryos, develop with good connection between root and shoot, and without the plagiotropism often associated with propagation by cuttings. For practical purposes in breeding programs and for deployment of elite clones, it is valuable that a virtually unlimited number of SE plants can be generated from one original seed embryo; and SE cultures (clones) can be cryostored for at least 20 years, allowing long-term testing of clones. To date, there has however been limited use of SE for large-scale plant production mainly because without automation it is labor-intensive. Development of automation is particularly attractive in countries with high labor costs, where conifer forestry is often of great economic importance. Various approaches for automating SE processes are under investigation and the progress is reviewed here, with emphasis on conifers. These approaches include simplification of culture routines with preference for liquid rather than solid cultures, use of robotics and automation for the harvest of selected individual mature embryos, followed by automated handling of germination and subsequent planting. Different approaches to handle the processes of somatic embryogenesis in conifers are outlined below, followed by an update on efforts to automate the different steps, which are nearing an operational stage

Effect of Fluid Shear Stress on Endocytosis of Heparan Sulfate and Low-density Lipoproteins

Hemodynamic stress is a critical factor in the onset of atherosclerosis such that reduced rates of shear stress occurring at regions of high curvature are more prone to disease. The level of shear stress has direct influence on the thickness and integrity of the glycocalyx layer. Here we show that heparan sulfate, the main component of the glycocalyx layer, forms an intact layer only on cell surfaces subjected to shear, and not under static conditions. Furthermore, receptor-mediated endocytosis of heparan sulfate and low-density liporoteins is not detectable in cells exposed to shear stress. The internalized heparan sulfate and low-density lipoproteins are colocalized as shown by confocal imaging

Histological analysis reveals the formation of shoots rather than embryos in regenerating cultures of Eucalyptus globulus

Eucalyptus globulus is an important species in international forestry in regions with Mediterranean climates and comprises 65 % of the plantation hardwood in Australia. Propagation by somatic embryogenesis would offer many advantages and its development has received much attention. Structures regenerating on explants from hypocotyls of mature zygotic embryos of E. globulus cultured on medium with NAA, reported previously to be effective for embryogenic regeneration, were analyzed morphologically and histologically to clarify their pathway of development. Analysis of series of sections revealed organogenic, rather than embryogenic, pathways of regeneration in this system. We show that protocols for propagation of E. globulus must be carefully evaluated by microscopic examination of adequate numbers of serial sections

Stipagrostis pennata (Trin.) De Winter Artificial Seed Production and Seedlings Multiplication in Temporary Immersion Bioreactors

This study was conducted to develop the protocol for artificial seed production of Stipagrostis pennata (Trin.) De Winter via somatic embryo encapsulation as well as test a temporary bioreactor system for germination and seedling growth. Embryogenic calli were encapsulated using sodium alginate and calcium chloride and then sowed in the Murashige and Skoog (MS) germination medium in in vitro cultures. The experiments were conducted as a factorial based on a completely randomized design with three replications. The treatments include three concentrations of sodium alginate (1.5%, 2.5%, and 3.5%), two ion exchange times (20 and 30 min), and two artificial seed germination media (hormone-free MS and MS supplemented with zeatin riboside and L-proline). Germination percentage and number of days needed until the beginning of germination were studied. The highest percentage of artificial seed germination was obtained when 2.5% sodium alginate was used for 30 min (ion exchange time) and when the seeds were placed on the MS germination medium supplemented with zeatin riboside and L-proline. The results of the analysis of variance in the temporary immersion bioreactor system showed that the main effects observed on the seedling growth were associated with different growth hormones in culture media and the number of feeding cycles. Experimental results also indicated that the total protein analyses of zygotic seedlings and seedlings originating from the synthetic seeds showed no statistically significant differences between these samples

Accumulated effects of factors determining plant development from somatic embryos of Abies nordmanniana and Abies bornmuelleriana

Despite a much later inception of somatic embryogenesis (SE) propagation protocols for gymnosperms than for angiosperm species, SE is becoming increasingly important due to its applications for commercial forestry. For many conifers, there are however still major bottlenecks in the SE plant production process limiting the use of SE for forestry operations, Christmas tree production and research projects. In the present case study, the effects on plant growth from different cultural factors applied during the SE developmental process were studied in two conifer species of high value for Christmas tree production. Seven clones of Abies nordmanniana and two clones of Abies bornmuelleriana were included in the study. Accumulated effects from cultural treatments were recorded from the start of germination of mature embryos of different quality scores through development into plants in the third growing period. Experimental factors of the cultural treatments included were: germination temperature, germination time, light conditions, survival ex vitro and traits for plant growth and vitality. The results reveal that most of the studied experimental factors influenced plant growth during the first three years however their relative importance was different. Plant survival rate at end of the nursery stage was strongly impacted by germination temperature (p<0.001), initial embryo score (p=0.007), clone (p<0.001) and to a lesser extend week of germination (p=0.017). This case-study highlights and quantifies the strong interrelation between the developmental steps of somatic embryogenesis and show the importance of considering all cultural steps when optimizing SE plant production protocols

Comparison of standard exponential and linear techniques to amplify small cDNA samples for microarrays

BACKGROUND: The need to perform microarray experiments with small amounts of tissue has led to the development of several protocols for amplifying the target transcripts. The use of different amplification protocols could affect the comparability of microarray experiments. RESULTS: Here we compare expression data from Pinus taeda cDNA microarrays using transcripts amplified either exponentially by PCR or linearly by T7 transcription. The amplified transcripts vary significantly in estimated length, GC content and expression depending on amplification technique. Amplification by T7 RNA polymerase gives transcripts with a greater range of lengths, greater estimated mean length, and greater variation of expression levels, but lower average GC content, than those from PCR amplification. For genes with significantly higher expression after T7 transcription than after PCR, the transcripts were 27% longer and had about 2 percentage units lower GC content. The correlation of expression intensities between technical repeats was high for both methods (R(2 )= 0.98) whereas the correlation of expression intensities using the different methods was considerably lower (R(2 )= 0.52). Correlation of expression intensities between amplified and unamplified transcripts were intermediate (R(2 )= 0.68–0.77). CONCLUSION: Amplification with T7 transcription better reflects the variation of the unamplified transcriptome than PCR based methods owing to the better representation of long transcripts. If transcripts of particular interest are known to have high GC content and are of limited length, however, PCR-based methods may be preferable

Heterologous Array Analysis in Pinaceae: Hybridization of Pinus Taeda cDNA Arrays With cDNA From Needles and Embryogenic Cultures of P. Taeda, P. Sylvestris or Picea Abies

Hybridization of labelled cDNA from various cell types with high-density arrays of expressed sequence tags is a powerful technique for investigating gene expression. Few conifer cDNA libraries have been sequenced. Because of the high level of sequence conservation between Pinus and Picea we have investigated the use of arrays from one genus for studies of gene expression in the other. The partial cDNAs from 384 identifiable genes expressed in differentiating xylem of Pinus taeda were printed on nylon membranes in randomized replicates. These were hybridized with labelled cDNA from needles or embryogenic cultures of Pinus taeda, P. sylvestris and Picea abies, and with labelled cDNA from leaves of Nicotiana tabacum. The Spearman correlation of gene expression for pairs of conifer species was high for needles (r2 = 0.78 − 0.86), and somewhat lower for embryogenic cultures (r2 = 0.68 − 0.83). The correlation of gene expression for tobacco leaves and needles of each of the three conifer species was lower but sufficiently high (r2 = 0.52 − 0.63) to suggest that many partial gene sequences are conserved in angiosperms and gymnosperms. Heterologous probing was further used to identify tissue-specific gene expression over species boundaries. To evaluate the significance of differences in gene expression, conventional parametric tests were compared with permutation tests after four methods of normalization. Permutation tests after Z-normalization provide the highest degree of discrimination but may enhance the probability of type I errors. It is concluded that arrays of cDNA from loblolly pine are useful for studies of gene expression in other pines or spruces