Transient transformation of pollen and embryogenic tissues of white spruce (Picea glauca (Moench.) Voss) resulting from microprojectile bombardment

Detailed analyses of the physical parameters inherent in the microprojectile bombardment technology necessary to produce optimum transient -glucuronidase (GUS) expression were undertaken in pollen and embryogenic tissues of white spruce. Higher helium pressure used for microprojectile bombardment resulted in lower GUS expression in pollen, but in higher GUS expression in embryogenic tissues. Modification of the osmoticum of the culture medium had a limited effect on GUS transient expression in pollen but substantially increased the transient expression in embryogenic tissues. The viability of transformed pollen was not affected by the bombardment procedure. This is the first detailed analysis of microprojectile bombardment technology reporting the conditions needed for optimum transient transformation of pollen and embryogenic tissues of white spruce.     

High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large- scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene -glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co- cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.     

Pollination of Picea orientalis (Pinaceae): saccus morphology governs pollen

Sacci of conifer pollen do not function primarily to increase the efficiency of wind pollination as is widely thought. Rather, they are bladders and cause pollen to float upwards in a liquid drop into the ovules. This observation is seemingly unsupported in the case of oriental spruce (Picea orientalis (L.) Link), which has saccate pollen. Ovulate cones are pendant at the time of pollination, which requires that pollen sink into the ovules. Pollen of oriental spruce floats at first but within 1-2 min sinks into the ovule. As sinking does not occur in saccate pollen of other Pinaceae, a variety of techniques was used to determine anatomical differences leading to this uncharacteristic tendency. Light, scanning electron, and confocal microscopy of the pollen surface yielded no significant appearing difference between pollen of oriental spruce and white spruce. However, transmission electron microscopy of freeze-fixed/freeze-substituted hydrated pollen revealed that the ektexine of oriental spruce pollen sacci is porous compared to that of white spruce. Confocal microscopy allowed examination of pollen hydration dynamics. Water enters pollen at the distal pole between sacci, and resulting rapid expansion of the tube cell forces air out of the saccate space. White spruce pollen remains buoyant because of enclosed air pockets in the saccus ektexine. Evolutionary change in pollen wall anatomy with resultant loss of saccus function is correlated with a change in ovulate strobilus orientation at pollination in oriental spruce. A suite of characters interact in the conifer pollination mechanism, and concerted change in these characters may lead to speciation.     

Gas exchange and growth responses of ectomycorrhizal Picea mariana, Picea glauca, and Pinus banksiana seedlings to NaCl and Na2SO4

Abstract: Black spruce (Picea mariana), white spruce (Picea glauca), and jack pine (Pinus banksiana) seedlings were inoculated with Hebeloma crustuliniforme or Laccaria bicolor and subjected to NaCl and Na₂SO₄ treatments. The effects of ectomycorrhizas on salt uptake, growth, gas exchange, and needle necrosis varied depending on the tree and fungal species. In jack pine seedlings, ectomycorrhizal (ECM) fungi reduced shoot and root dry weights and in the ECM white spruce, there was a small increase in dry weights. Sodium chloride treatment reduced net photosynthesis and transpiration rates in the three studied tree species. However, NaCl-treated black spruce and jack pine colonized by H. crustuliniforme maintained relatively high photosynthetic and transpiration rates and needle necrosis of NaCl-treated black spruce seedlings was reduced by the ECM fungi. Higher concentrations of Na+ were found in shoots compared with roots of the three examined conifer species. ECM fungi reduced the concentrations of Na+ mainly in the shoots and this reduction was greater in plants treated with NaCl compared with Na₂SO₄. Shoots contained generally higher concentrations of Cl^- compared with roots. In the NaCl-treated black spruce and white spruce, both ECM species significantly reduced Cl^- concentrations. Our results point to overall greater phytotoxicity of NaCl compared with Na₂SO₄ and support our earlier findings which demonstrated beneficial effects of ECM fungi for woody plants exposed to NaCl stress.     

Transformation of white spruce (Picea glauca) somatic embryos by microprojectile bombardment

Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of 605 embryos (2.2%) remained GUS positive after two months in culture. Three of those thirteen (23%) embryo-derived tissues consistently showed GUS activity for eight months in culture. These putatively transfomed embryogenic tissues were subjected to Southern blot analysis and the results suggested integration of the gus::nptll gene expression cassette in the white spruce genome.Abbreviations ABA (±)abscisic acid - BA benzyladenine - bp base pair - 2,4-D 2,4-dichlorophenoxyacetic acid - kb kilobase - gus E. coli gene uid A for -glucuronidase - nptll neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronic acid     

Factors affecting transient gene expression in electroporated black spruce (Picea mariana) and jack pine (Pinus banksiana) protoplasts

Summary Methods were developed for transient gene expression in protoplasts of black spruce (Picea mariana) and jack pine (Pinus banksiana). Protoplasts were isolated from embryogenic suspension cultures of black spruce and from non-embryogenic suspensions of jack pine. Using electroporation, transient expression of the chloramphenicol acetyltransferase (CAT) gene was assayed and shown to be affected by the cell line used, by voltage, temperature, and by the plasmid concentration and conformation. Increasing the plasmid DNA concentration (0–150g ml^–1) resulted in higher levels of transient CAT expression. In jack pine, linearized plasmid gave 2.5 times higher levels of CAT enzyme activity than circular. Optimal voltage varied for each cell line of the two species within the range 200–350 V cm^–1 (960 F). A heat shock treatment of protoplasts for 5 min at 45 °C resulted in enhanced CAT gene expression for both species.NRCC No. 30491     

Do tree-level monocultures develop following Canadian boreal silviculture? Tree-level diversity tested using a new method

Concern about forestry practices creating tree-level monoculture plantations exists. Our study investigates tree diversity responses for six early seral boreal forest plantations in Ontario, Canada, representing three conifer species; black spruce (Picea mariana), white spruce (P. glauca), and jack pine (Pinus banksiana), 14 release treatments, and 94 experimental units. Dominance-diversity curves and Simpson’s indices of diversity and evenness indicate tree alpha diversity. We propose a new method for assessing diversity, using percentage of theoretical species maximum (%TSM) which is determined by comparing post-disturbance richness (S) with a theoretical species maximum (TSM). Our results support the hypothesis that alternative vegetation release treatments generally do not reduce tree species diversity levels (%TSM) relative to untreated plots. The only %TSM (P ≤ 0.05) comparison that produced less diversity than in control plots was repeated annual treatments of Vision herbicide at one of the black spruce study sites. Our results generally support the hypothesis that tree monocultures do not develop after vegetation release. Only one out of 94 experimental units developed into a tree layer monoculture (Simpson’s reciprocal diversity index = 1). Again this was one of the repeated annual treatments of Vision herbicide at one of the black spruce study sites—a treatment which is atypical of Canadian forest management.

Stable genetic transformation of white pine (Pinus strobus L.) after cocultivation of embryogenic tissues with Agrobacterium tumefaciens

A genetic transformation procedure for white pine has been developed after cocultivation of embryogenic tissues with Agrobacterium tumefaciens. This efficient transformation procedure led to an average of four independent transformed lines per gram of cocultivated embryogenic tissue and up to 50 transformed lines can be obtained in a routine experiment. Constructs bearing the uidA gene or the green fluorescent protein (GFP) gene were introduced and -glucuronidase (GUS) activity was followed over time. The expression of the uidA gene was lowest with a 35S-gus-intron construct and was 20-fold higher with a 35S-35S-AMVgus::nptII construct. The addition of scaffold attachment region (SAR) sequences surrounding the gus::nptII fusion did not significantly enhance the GUS activity. Transformed mature somatic embryos have been germinated and plantlets are presently being acclimatized.     

Insights into Conifer Giga-Genomes

Insights from sequenced genomes of major land plant lineages have advanced research in almost every aspect of plant biology. Until recently, however, assembled genome sequences of gymnosperms have been missing from this picture. Conifers of the pine family (Pinaceae) are a group of gymnosperms that dominate large parts of the world’s forests. Despite their ecological and economic importance, conifers seemed long out of reach for complete genome sequencing, due in part to their enormous genome size (20–30 Gb) and the highly repetitive nature of their genomes. Technological advances in genome sequencing and assembly enabled the recent publication of three conifer genomes: white spruce (Picea glauca), Norway spruce (Picea abies), and loblolly pine (Pinus taeda). These genome sequences revealed distinctive features compared with other plant genomes and may represent a window into the past of seed plant genomes. This Update highlights recent advances, remaining challenges, and opportunities in light of the publication of the first conifer and gymnosperm genomes.Conifers are the most widely distributed group of gymnosperms, with 600 to 630 species in 69 genera, including 220 to 250 species of the Pinaceae family (Wang and Ran, 2014). Coniferous forests cover an estimated 39% of the world’s forests (Armenise et al., 2012). Conifers dominate many natural and planted forests in the northern hemisphere and are also planted as exotics for commercial forestry in the southern hemisphere. The importance of conifers for global ecosystem services, their value for forestry-dependent economies, and their contrasting biology with angiosperms are major drivers behind efforts to understand the complex structure, functions, and evolution of their genomes. However, owing to their nonmodel system attributes (i.e. slow-growing and long-lived life history traits), extremely large genome size (Fig. 1), and repeat-rich genome sequence with repeats mostly in the form of transposable elements, no reports of a conifer genome assembly, or any gymnosperm genome for that matter (Soltis and Soltis, 2013), were published until recently. Following early releases of the white spruce (Picea glauca) and loblolly pine (Pinus taeda) genome sequences in public databases (e.g. National Center for Biotechnology Information and http://dendrome.ucdavis.edu/treegenes/), a series of articles described the first conifer genome assemblies for Norway spruce (Picea abies; Nystedt et al., 2013) and interior white spruce, a genetic admix of white spruce (Birol et al., 2013) and loblolly pine (Neale et al., 2014; Zimin et al., 2014). Norway spruce is a prominent forest tree in northern Europe. White spruce is a dominant tree species across the large Canadian forest landscape. Loblolly pine dominates commercial forestry in the southeastern United States. White spruce, Norway spruce, and loblolly pine represent some of the most economically important conifers worldwide, and they are the subjects of important tree improvement/breeding programs (Mullin et al., 2011). This Update highlights significant insights obtained from these genomes as well as some ongoing challenges and recent developments in conifer genomics.Open in a separate windowFigure 1.Size and assembly of conifer genomes compared with other plant genomes. Genome size is plotted against the number of scaffolds divided by the haploid chromosome number for a range of plant species. As such, an assembly that reconstructs a genome with perfect contiguity will have a value of 1, and values greater than 1 represent increasing genome fragmentation. Genome assemblies that utilized Sanger sequencing either in full or in part are represented as white circles. Assemblies constructed using only next generation sequencing technologies are represented as black circles. Both axes are plotted on a log₁₀ scale. With the exception of Populus tremula, Hordeum vulgare, and the three conifer genomes, all genomes were obtained from the Phytozome resource (version 10; http://phytozome.jgi.doe.gov/). The early release draft assembly of P. tremula was obtained from the PopGenIE.org FTP resource (ftp://popgenie.org/popgenie/UPSC_genomes/UPSC_Draft_Assemblies/Current/Genome/) and H. vulgare ‘Morex’ from the Munich Information Center for Protein Sequences barley genome database FTP resource (ftp://ftpmips.helmholtz-muenchen.de/plants/barley/public_data/sequences/). The conifer genomes are detailed by Birol et al. (2013), Nystedt et al. (2013), and Zimin et al. (2014).     

Transformation of white spruce and other conifer species byAgrobacterium tumefaciens

Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0–80% depending upon the strain ofAgrobacterium, and the conifer species. Thirty sixA. tumefaciens strains and oneA. rhizogenes strain were tested on 6 month old white spruce seedlings. NineA. tumefaciens strains induced gall formation on more than 50% of the inoculated trees and at greater than 10% of the inoculated sites. One strain, B2/74 gave rise to galls at 28% of the inoculated sites on white spruce and induced the highest overall frequency of gall formation on all the conifer species tested. Relative frequency of gall formation was consistent among species, although the overall frequency was much higher on Douglas-fir. Of the well characterized strains for which disarmed derivatives are available only A281 (carrying the supervirulent tumor inducing plasmid, pTiBo542) gave efficient transformation. Stable integration of T-DNA encoded genes has been confirmed by the expression of opine synthesis and hormone autonomous growth. The transfer and long-term stable expression of kanamycin resistance and firefly luciferase activity using binary vector systems was also achieved.     

From glacial refugia to modern populations: new assemblages of organelle genomes generated by differential cytoplasmic gene flow in transcontinental black spruce

Assessing species' range-wide cytoplasmic diversity provides valuable insights regarding their dispersal and adaptive potential in a changing environment. Transcontinental chloroplast (cpDNA) and mitochondrial DNA (mtDNA) population structures were compared to identify putative ancestral and new cytoplasmic genome assemblages in black spruce (Picea mariana), a North American boreal conifer. Mean within-population diversity and allelic richness for cpSSR markers were 0.80 and 4.21, respectively, and diminished westward. Population differentiation based on G(ST) was lower for cpDNA than for mtDNA (G(ST) =0.104 and 0.645, respectively) but appeared comparable when estimated using Jost differentiation index (D=0.459 and 0.537, respectively). Further analyses resulted in the delineation of at least three genetically distinct cpDNA lineages partially congruent with those inferred from mtDNA data, which roughly corresponded to western, central and eastern Canada. Additionally, the patterns of variation in Alaska for both cpDNA and mtDNA markers suggested that black spruce survived the last glacial maximum in this northern region. The range-wide comparison of the geographic extent of cytoplasmic DNA lineages revealed that extensive pollen gene flow between ancestral lineages occurred preferentially from west to east during the postglacial expansion of the species, while seed-mediated gene flow remained geographically restricted. This differential gene flow promoted intraspecific cytoplasmic capture that generated new assemblages of cpDNA and mtDNA genomes during the Holocene. Hence, black spruce postglacial colonization unexpectedly resulted in an increase in genetic diversity with possible adaptive consequences.     

β-Glucuronidase gene and green fluorescent protein gene expression in de-exined pollen of Nicotiana tabacum by microprojectile bombardment

 Gene constructs containing the β-glucuronidase (GUS) gene or green fluorescent protein (GFP) gene under the control of pollen-specific promoter Zm13-260 from maize were introduced by particle bombardment into de-exined pollen of Nicotiana tabacum. The de-exined pollen exhibited transient expression of the GUS or GFP gene as indicated by histochemical and fluorescent assay, respectively. The frequency of de-exined pollen transformation with the GUS or GFP gene was approximately 6 and 3 times higher, respectively, than that of pollen with intact walls, indicating that pollen deprived of the exine barrier responded better to foreign gene transfer than did the original. Cytological observation of GUS-expressing pollen grains showed that introduced gold particles were visible in the cytoplasm and vegetative nucleus as well as in the generative nucleus. GFP-expressing pollen tubes were observed in the style even after pollination. Received: 28 October 1997 / Revision accepted: 13 April 1998     

Variation in mitochondrial DNA reveals multiple distant glacial refugia in black spruce (Picea mariana), a transcontinental North American conifer

Range-wide genetic variation of black spruce (Picea mariana) was studied using polymerase chain reaction-random fragment length polymorphism markers of the mitochondrial genome. Four polymorphic mitochondrial DNA (mtDNA) loci were surveyed and two or three alleles were detected at each locus, resulting in 10 multilocus mtDNA types or mitotypes. A significant subdivision of population genetic diversity was detected (GST = 0.671; NST = 0.726), suggesting low levels of gene flow among populations. The distribution of mitotypes was not random (NST > GST; P < 0.05) and revealed four partially overlapping zones, presumably representative of different glacial populations. Comparison of the genetic structure derived from mtDNA markers and the colonization paths previously deduced from the fossil and pollen records allow us to infer at least three southern and one northeastern glacial populations for black spruce. The patterns revealed in this study suggest that black spruce shares its biogeographical history with other forest-associated North American species.     

The actin microfilament network within elongating pollen tubes of the gymnospermPicea abies (Norway spruce)

Summary Actin microfilaments form a dense network within pollen tubes of the gymnosperm Norway spruce (Picea abies). Microfilaments emanate from within the pollen grain and form long, branching arrays passing through the aperture and down the length of the pollen tube to the tip. Pollen tubes are densely packed with large amyloplasts, which are surrounded by branching microfilament bundles. The vegetative nucleus is suspended within the elongating pollen tube within a complex array of microfilaments oriented both parallel to and perpendicular with the growing axis. Microfilament bundles branch out along the nuclear surface, and some filaments terminate on or emanate from the surface. Microfilaments in the pollen tube tip form a 6 m thick, dense, uniform layer beneath the plasma membrane. This layer ensheathes an actin depleted core which contains cytoplasm and organelles, including small amyloplasts, and extends back 36 m from the tip. Behind the core region, the distinct actin layer is absent as microfilaments are present throughout the pollen tube. Organelle zonation is not always maintained in these conifer pollen tubes. Large amyloplasts will fill the pollen tube up to the growing tip, while the distinct layer of microfilaments and cytoplasm beneath the plasma membrane is maintained. The distinctive microfilament arrangement in the pollen tube tips of this conifer is similar to that seen in tip growth in fungi, ferns and mosses, but has not been reported previously in seed plants.     

Expression of inducible angiosperm promoters in a gymnosperm,Picea glauca (white spruce)

Electrical discharge particle acceleration was used to test the transient expression of numerous inducible angiosperm promoters in a gymnospermPicea glauca (white spruce). Promoter expression was assayed in three different tissues capable ofin vitro regeneration, zygotic embryos, seedlings and embryogenic callus. The promoters tested include the light-inducibleArabidopsis and soybean ribulose-1,5-bisphosphate small subunit promoters and a maize phosphoenolpyruvate carboxylase promoter; a soybean heat-shock-inducible promoter, a soybean auxin inducible promoter and a maize alcohol dehydrogenase promoter. Promoters were cloned into a promoter-less expression vector to form a promoter--glucuronidase-nopaline synthase 3 fusion. A similar construct was made using the cauliflower mosaic virus 35S (CaMV 35S) promoter as a control. All promoters were expressed in white spruce embryos, yet at levels lower than CaMV 35S. In addition, in the embryos the heat-shock and the alcohol dehydrogenase promoters showed inducible expression when given the proper induction stimulus. In seedlings, expression of all promoters was lower than in the embryos and expression was only inducible with the heat-shock promoter in the cotyledons. Of the tissues tested, the expression level of all promoters was lowest in embryogenic callus. Interestingly, the expression of the -glucuronidase gene in embryogenic callus was restricted to the proembryonal head cells regardless of the promoter used. These results clearly demonstrate the use of particle bombardment to test the transient expression of heterologous promoters in organized tissue and the expression of angiosperm promoters in a gymnosperm.     

Visual Recognition Software for Binary Classification and Its Application to Spruce Pollen Identification

Discriminating between black and white spruce (Picea mariana and Picea glauca) is a difficult palynological classification problem that, if solved, would provide valuable data for paleoclimate reconstructions. We developed an open-source visual recognition software (ARLO, Automated Recognition with Layered Optimization) capable of differentiating between these two species at an accuracy on par with human experts. The system applies pattern recognition and machine learning to the analysis of pollen images and discovers general-purpose image features, defined by simple features of lines and grids of pixels taken at different dimensions, size, spacing, and resolution. It adapts to a given problem by searching for the most effective combination of both feature representation and learning strategy. This results in a powerful and flexible framework for image classification. We worked with images acquired using an automated slide scanner. We first applied a hash-based “pollen spotting” model to segment pollen grains from the slide background. We next tested ARLO’s ability to reconstruct black to white spruce pollen ratios using artificially constructed slides of known ratios. We then developed a more scalable hash-based method of image analysis that was able to distinguish between the pollen of black and white spruce with an estimated accuracy of 83.61%, comparable to human expert performance. Our results demonstrate the capability of machine learning systems to automate challenging taxonomic classifications in pollen analysis, and our success with simple image representations suggests that our approach is generalizable to many other object recognition problems.     

Green fluorescent protein (GFP) as a marker during pollen development

The transient expression of three mutant forms of green fluorescent protein (GFP) genes, GFP4, GFP5ER, and GFP4S65C, under several constitutive and pollenspecific promoters throughout pollen development in Nicotianatabacum, thaliana and Antirrhinummajus is described. Immature pollen of tobacco, Arabidopsis and snapdragon, isolated at different developmental stages, were bombarded with plasmids containing the GFP and cultured in vitro for several days until maturity. The expression of GFP was monitored every day during in vitro maturation, germination and pollination, as well as after in situ pollination. The expression pattern of each construct was compared in parallel experiments to that of ßglucuronidase (GUS) constructs expressed by the same promoters. The results show that the expression level of all three GFP mutant forms was dependent on the strength of the promoter used. The strongest promoter was the DC3 promoter, and no notable differences in the intensity and brightness of all three versions of GFP were observed. GFPexpressing pollen from tobacco and snapdragon developed in vitro for several days until maturity and germinated in vitro as well as on the surface of stigmata, strongly suggesting that all three GFPs are not toxic for the development of functional pollen. Furthermore, stably transformed tobacco plants expressing GFP under the control of the strong pollenexpressed DC3 and LAT52 promoters were not impaired in reproductive function, confirming that GFP can be used as a nondestructive marker for plant reproductive biology and development.