Agrobacterium rhizogenes-mediated genetic transformation and regeneration of a conifer:Larix decidua

Summary Gene transfer and plant regeneration systems have been developed for European larch (Larix decidua Mill.) in our laboratory. Aseptically germinated young seedlings were hypocotyl wound-inoculated withAgrobacterium rhizogenes strains 11325 containing a wild-type Ri (root-inducing) plasmid. Swollen stems appeared at infected wounds followed by either abundant hairy roots or adventitious shoot buds that developed within 3 to 4 wk after inoculation. No symptoms were seen on wounded but uninoculated seedlings. We demonstrated agrobacteria attached to larch cells by examination of scanning electron micrographs. Subsequently, calli derived from symptomatic tissues exhibited phytohormone autotrophic growth. Adventitious buds were elongated and rooted in vitro before being transferred to the greenhouse where the transformed whole plants grew normally. Transformants tested positive for opine production and transformation was further confirmed by Southern blot analysis with larch genomic DNAs isolated from both proliferated calli and needle tissue of transgenic plants.     

Influence of climatic factors upon tree rings of Larix decidua and L. decidua × L. kaempferi from Pulawy,Poland

Summary Dendroclimatological techniques are used to assess the impact of climatic factors on tree-ring width of Larix decidua and L. decidua × L. kaempferi (= L. x eurolepis) growing in two experimental plots established in 1914 in south-west Poland. One plot included F₁ progeny grown from seeds of an artificial crossing between European and Japanese larch. The other plot included progeny from maternal trees of European larch. Total ring width, earlywood width and latewood widths were dated, standardized and related to monthly climatic data using response function and stepwise multiple regression analyses. Wide rings in larch are associated with high precipitation in May–July, cool conditions in July–September of the preceding year, and cool dry conditions in August. Ring widths in L. x eurolepis are more dependent upon precipitation than ring widths in L. decidua. Latewood widths in L. x eurolepis are more dependent on high temperatures in June and July than latewood in L. decidua as well as total width and earlywood measurements. Variations in latewood were relatively independent of variations in earlywood and total wood. The variability of ring widths in these larches was greater than the variability reported for larches in many alpine sites and for other conifer species in some regions of North America.     

Genetic relationships between European and Siberian larch,Larix spp. (Pinaceae), studied by allozymes. Is the Polish larch a hybrid between these two species?

Thirteen populations ofLarix decidua subsp.decidua and subsp.polonica, and three populations ofL. sibirica were investigated by starch-gel electrophoresis. In the populations assayed 61 alleles at 17 loci were revealed. The allozyme data support the earlier observations about close relationships between these two larch species. Nei's genetic distances betweenL. decidua andL. sibirica were relatively small (D = 0.057), however, almost five times larger, on average, than those between populations of the same species. Results obtained in this study disagree withBobrov's hypothesis about the hybrid origin of the Polish larch and suggest a direct origin from the European larch.     

Identification of larch species (Larix decidua, Larix kaempferi and Larix X eurolepis) and estimation of hybrid fraction in seed lots by RAPD fingerprints

Species-specific RAPD markers were used to identify the different larch species (Larix decidua and Larix kaempferi) and their interspecific hybrid (Larix X eurolepis). Although morphological differences between pure species and the hybrids exist, differentiation is not always possible, especially at an early stage (seed or plantlet). Eleven RAPD markers differentiated the two larch species, and 4 species-specific markers were sufficient to estimate the F₁ hybrid fraction in a seed lot. The species-specific markers were tested on individual trees of European and Japanese larches of diverse geographic origins and on several seed lots of different origins (F₁, F₂ hybrids and pure species). The 4 specific markers found for the European larch and the Japanese larch were monomorphic and present in all provenances and in all F₁ hybrid trees tested. Polymorphic SCAR fragments were obtained for 3 of the 11 fragments originally selected for the RAPD screening phase. For 2 of them, the sequence had some homology with the mitochondrial genome of other organisms and is thus mitochondrial. The two mitochondrial fragments and the OPF-13₁₀₀₀ fragment exhibited one polymorphic band, thereby maintaining its species-specific identity: OPF-13₁₀₀₀ is specific to the European larch. The 4 RAPD primers selected in this study offer a reliable, quick and cheap tool for the identification of different larch species (Larix decidua and Larix kaempferi) and their interspecific hybrid (Larix X eurolepis). Received: 28 February 1999 / Accepted: 29 April 1999     

Sensitivity of Siberian larch forests to climate change

The Northern Hemisphere's boreal forests, particularly the Siberian boreal forest, may have a strong effect on Earth's climate through changes in dominant vegetation and associated regional surface albedo. We show that warmer climate will likely convert Siberia's deciduous larch (Larix spp.) to evergreen conifer forests, and thus decrease regional surface albedo. The dynamic vegetation model, FAREAST, simulates Russian boreal forest composition and was used to explore the feedback between climate change and forest composition at continental, regional, and local scales. FAREAST was used to simulate the impact of changes in temperature and precipitation on total and genus‐level biomass at sites across Siberia and the Russian Far East (RFE), and for six high‐ and low‐diversity regions. Model runs with and without European Larch (Larix decidua) included in the available species pool were compared to assess the potential for this species, which is adapted to warmer climate conditions, to mitigate the effects of climate change, especially the shift to evergreen dominance. At the continental scale, when temperature is increased, larch‐dominated sites become vulnerable to early replacement by evergreen conifers. At the regional and local scales, the diverse Amur region of the RFE does not show a strong response to climate change, but the low‐diversity regions in central and southern Siberia have an abrupt vegetation shift from larch‐dominated forest to evergreen‐conifer forest in response to increased temperatures. The introduction of L. decidua prevents the collapse of larch in these low‐diversity areas and thus mitigates the response to warming. Using contemporary MODIS albedo measurements, we determined that a conversion from larch to evergreen stands in low‐diversity regions of southern Siberia would generate a local positive radiative forcing of 5.1±2.6 W m^?2. This radiative heating would reinforce the warming projected to occur in the area under climate change.     

Ectomycorrhizal fungal community of naturally regenerated European larch (Larix decidua) seedlings

An investigation was undertaken to assess the community structure of ectomycorrhizal (ECM) fungi on naturally regenerating European larch (Larix decidua Mill.) seedlings grown under forest conditions. The sites examined were in two managed monoculture larch forests, differentiated by soil chemistry and mature tree density. Morphological and molecular analyses revealed a total of 22 fungal taxa. From detected ECM fungal taxa, 13 were noted at Site I and 13 at Site II. Only four taxa were found in both sites (Russula ochroleuca, Thelephora terrestris, Lactarius tabidus and Paxillus involutus). The most abundant species at Site I (lower mineral concentration, high tree density) was Hydnotrya tulasnei (25.7?%), followed by Pseudotomentella tristis, Tomentella sublilacina and Russula puelaris. At Site II (higher mineral concentration, low tree density) the dominant fungal symbiont of larch seedlings was clearly Wilcoxina mikolae, which accounted for 74?% of mycorrhizal tips. The less abundant species comprised T. terrestris, L. tabidus, Xerocomus pruinatus and R. ochroleuca. The analysis of similarity (ANOSIM) and non-metric multidimensional scaling (NMDS) ordination clearly separated the ECM fungal assemblages in the two sites tested. Because our study sites were differentiated by many factors, it is not easy to distinguish one factor in particular to explain the differences observed between the ECM communities at Sites I and II. The results obtained significantly increase our knowledge about the diversity of the ECM fungi hosted by L. decidua. The large number of ECM fungi detected was the first observation showing these fungi as symbiotic partners of European larch.     

Accumulation of photosynthetic pigments in Larix decidua Mill. and Picea abies (L.) Karst. cotyledons treated with 5-aminolevulinic acid under different irradiation

European larch (Larix decidua Mill.) and Norway spruce [Picea abies (L.) Karst.] synthesize chlorophyll (Chl) in darkness. This paper compares Chl accumulation in 14-d-old dark-grown seedlings of L. decidua and P. abies after shortterm (24 h) feeding with 5-aminolevulinic acid (ALA). We used two ALA concentrations (1 and 10 mM) fed to cotyledons of both species in darkness and in continuous light. The dark-grown seedlings of L. decidua accumulated Chl only in trace amounts and the seedlings remained etiolated. In contrast, P. abies seedlings grown in darkness were green and had significantly higher Chl content. After ALA feeding, higher protochlorophyllide (Pchlide) content was observed in L. decidua than in P. abies cotyledons incubated in darkness. Although short-term ALA feeding stimulated the synthesis of Pchlide, Chl content did not change significantly in cotyledons incubated in darkness. The Chl accumulation in cotyledons fed with ALA was similar to the rate of Chl accumulation in the controls. Higher Chl accumulation was reported in control samples after illumination: 86.9% in L. decidua cotyledons and 46.4% in P. abies cotyledons. The Chl content decreased and bleaching occurred in cotyledons incubated with ALA in light due to photooxidation. Analyses of Chlbinding proteins (D1 and LHCIIb) by Western blotting proved differences between Chl biosynthesis in L. decidua and P. abies seedlings in the dark and in the light. No remarkable increase was found in protein accumulation (D1 and LHCIIb) after ALA application. Our results showed interspecific difference in Chl synthesis between two gymnosperms. Shortterm ALA feeding did not stimulate Chl synthesis, thus ALA synthesis was not the rate-limiting step in Chl synthesis in the dark.     

Transformation of Indica Rice (Oryza sativa L.) Mediated by Agrobacterium tumefaciens

A system has been developed for the transfer of genes by Agrobacteriumtumefaciens to indica rice (Oryza sativa L. cv. Taichung Native1), and optimal conditions for such Agrobacterium-mediated transformationhave been determined. Excised stems, leaves, and roots from3- to 4-day-old seedlings were infected with strains of Agrobacteriumthat carried plasmids with chimeric derivatives of genes forß-glucuronidase (GUS) and neomycin phosphotransferase(NPTII). After selection with the antibiotic G418, only rootssurvived and formed calli. The integration of chimeric genesin the rice genome was demonstrated by Southern analysis andassays of enzymatic activity. We obtained a transformation frequencyof 37.5% for transgenic callus. In addition, the absence ofhybridization with vir probes indicated that transformants werenot contaminated with Agrobacterium. The complete failure oftransformation in the absence of medium from potato suspensioncultures (PSC) indicates that PSC is essential for Ti-mediatedtransformation in indica rice. A high efficiency of transformationwas observed upon infection with the cointegrate vector pGV2260::NG1. (Received August 13, 1991; Accepted April 28, 1992)     

Tissue specific response of Agrobacterium tumefaciens attachment to Sorghum bicolor (L) Moench

Agrobacterium mediated genetic transformation of plants have advantages over other methods, especially for making single copy transgenic plants with reduced chances of gene silencing and instability. However, monocotyledonous plant species could not utilize the full potential of this system because of possible limitations in Agrobacterium interaction with monocot plant cells. Agrobacterium attachment as a factor in genetic transformation was studied in the leaf, shoot apex, and leaf derived callus of sorghum (Sorghum bicolor (L) Moench). Pre-induction of Agrobacterium with acetosyringone was found necessary for Agrobacterium attachment to sorghum tissues. All the explants responded positively, with preferential Agrobacterium attachment and colonization around the tissues having actively dividing cells. Callus proved to be the best explant for Agrobacterium attachment as observed in scanning electron microscopy and transient GUS expression. Loss of Agrobacterium attachment was observed with an increase in the degree of tissue differentiation.Key words: Genetic transformation, Acetosyringone, Scanning electron microscopy, Transient gene expression, GUS assays, qRT-PCR     

A novel insight into the regulation of light-independent chlorophyll biosynthesis in <Emphasis Type="Italic">Larix decidua</Emphasis> and <Emphasis Type="Italic">Picea abies</Emphasis> seedlings

Light-independent chlorophyll (Chl) biosynthesis is a prerequisite for the assembly of photosynthetic pigment–protein complexes in the dark. Dark-grown Larix decidua Mill. seedlings synthesize Chl only in the early developmental stages and their Chl level rapidly declines during the subsequent development. Our analysis of the key regulatory steps in Chl biosynthesis revealed that etiolation of initially green dark-grown larch cotyledons is connected with decreasing content of glutamyl-tRNA reductase and reduced 5-aminolevulinic acid synthesizing capacity. The level of the Chl precursor protochlorophyllide also declined in the developing larch cotyledons. Although the genes chlL, chlN and chlB encoding subunits of the light-independent protochlorophyllide oxidoreductase were constitutively expressed in the larch seedlings, the accumulation of the ChlB subunit was developmentally regulated and ChlB content decreased in the fully developed cotyledons. The efficiency of chlB RNA-editing was also reduced in the mature dark-grown larch seedlings. In contrast to larch, dark-grown seedlings of Picea abies (L.) Karst. accumulate Chl throughout their whole development and show a different control of ChlB expression. Analysis of the plastid ultrastructure, photosynthetic proteins by Western blotting and photosynthetic parameters by gas exchange and Chl fluorescence measurements provide additional experimental proofs for differences between dark and light Chl biosynthesis in spruce and larch seedlings.     

Paternal inheritance of chloroplast DNA in Larix

Restriction enzyme analysis was used to determine the inheritance of chloroplast DNA in conifers. The plant material studied included five individual trees of European larch (Larix decidua Mill.) and Japanese larch (Larix leptolepis Sieb. & Zucc.) and six hybrids from controlled crosses between these species. The chloroplast DNA fragment patterns generated by Bam-HI and Bcl-I were species-specific. Paternal inheritance of chloroplast DNA patterns was found in most Larix crosses. One hybrid showed maternal chloroplast DNA patterns. In addition, two other hybrids had mixed Bam-HI patterns suggesting recombination between maternal and paternal chloroplast DNA. The mechanisms favoring paternal inheritance in conifers are not known. Paternal inheritance of chloroplast DNA is suggested it to be a general phenomenon in conifers.     

Colour of larch heartwood and relationships to extractives and brown-rot decay resistance

Larch heartwood is appreciated for its good mechanical properties, its colour and its texture, and it is often used outdoors because of its natural durability (decay resistance). In this study the colour of larch heartwood was studied in relation to extractives and decay resistance, with the aim to estimate durability of larch heartwood from its colour. On a total of 293 trees colour in the CIE L*a*b* space (L* lightness, a* red/green axis, b* yellow/blue axis), extractives content (acetone and hot-water extractives, amount of phenolics) and the brown-rot decay resistance were determined. For calculating the relative decay resistance ( x), mass loss after inoculation for 16 weeks with two fungi [ Coniophora puteana (Schum.ex.Fr.) Karst., Poria placenta (Fr.) Cke, European standard EN 113] of larch heartwood samples was compared to Scots pine ( Pinus sylvestris L) sapwood reference samples (EN 350-1). Different species [Japanese larch ( Larix kaempferi Lamb.), Hybrid larch (Larix deciduax L. kaempferi) and European larch ( L. decidua Mill.)], provenances and age classes (38-year, >150-year) were included. Japanese larch heartwood turned out to be significantly more reddish (higher a*-values) compared to the European larch provenances. Reddishness of the hybrids was intermediate. The red hue (+a*) was strongly correlated with the amount of phenols ( r =0.84) and decay resistance ( r =0.63) and therefore suitable for prediction of both parameters. The results suggest that colour measurements of larch heartwood could be of benefit in tree breeding programs and for an optimised utilization of larch timber.     

Changes in the Arrangement of Microtubules and Microfibrils in Differentiating Conifer Tracheids During the Expansion of Cells

The arrangements of microtubules and the cellulose microfibrilsof radial walls in tracheids of Abies sachalinensis Mastersduring the expansion of cells were examined by immunofluorescenceand field-emission scanning electron microscopy. The radialdiameter of tracheids increased to three to four times thatof cambial initial cells. Microfibrils on the innermost surfaceof primary walls of conifer tracheids at early stages were notwell ordered and most of the microfibrils were oriented longitudinally.As each cell expanded, microfibrils in the process of depositionwere still not well ordered but their orientation changed fromlongitudinal to transverse. When cell expansion ceased, microfibrilswere well ordered and oriented transversely. Cortical microtubulesshowed a change in orientation similar to that of the microfibrils.These results indicate that the orientation of cortical microtubulesis correlated with that of microfibrils as they are being laiddown and with cell morphogenesis in conifer tracheids.Copyright1995, 1999 Academic Press Microfibril, microtubule, tracheid, cell expansion, Abies sachalinensis Masters, field-emission scanning electron microscopy, immunofluorescence microscopy     

Responses of young trees to wind and shading: effects on root architecture

Two wind tunnels were designed to detect influences of windon the development of the root systems of young trees: Piceasitchensis and Larix decidua were grown in the first and L.decidua only in the second. In the second experiment, the taproot of each L. decidua seedling was removed in order to mimicthe formation of a shallow root-plate. Responses of shoot growthto wind stress were small, although uneven irradiance levelsresulted in asymmetric growth of both shoots and roots; thedistribution of root biomass around the tree was related spatiallyto that of shoot growth In both experiments there was an increase in the number of largewindward and leeward roots in both species. In the first experiment,the sum of the crosssectional area (     

Application of AFLP, RAPD and ISSR markers to genetic mapping of European and Japanese larch

Genetic linkage maps have been increasingly developed for a wide variety of plants, using segregating populations such as F₂s or backcrosses between inbred lines. These pedigrees are rarely available in outbred species like forest trees which have long generation times. Thus genetic mapping studies have to use peculiar pedigrees and markers in appropriate configurations. We constructed single-tree genetic linkage maps of European larch (Larix decidua Mill.) and Japanese larch [Larix kaempferi (Lamb.) Carr.] using segregation data from 112 progeny individuals of an hybrid family. A total of 266 markers (114 AFLP, 149 RAPD and 3 ISSR loci) showing a testcross configuration, i.e.heterozygous in one parent and null in the other parent, were grouped at LOD 4.0, θ=0.3. The maternal parent map (L. decidua)consisted of 117 markers partitioned within 17 linkage groups (1152 cM) and the paternal parent map (L. kaempferi) had 125 markers assembled into 21 linkage groups (1206 cM). The map distance covered by markers was determined by adding a 34.7-cM independence distance at the end of each group and unlinked marker. It reached 2537 cM and 2997 cM respectively for European larch and Japanese larch, and represented respectively a 79.6% and 80.8% coverage of the overall genome. A few 3:1 segregating markers were used to identify homologous linkage groups between the European larch and the Japanese larch genetic maps. The PCR-based molecular markers allowed the construction of genetic maps, thus ensuring a good coverage of the larch genome for further QTL detection and mapping studies. Received: 15 March 1999 / Accepted: 29 March 1999     

Cytological and molecular relationships between Larix decidua,L. leptolepis and Larix x eurolepis: identification of species-specific Chromosoms and synchronization of mitotic cells

The effects of different concentrations of hydroxyurea (HU) and aphidicolin (APH) on the mitotic index (MI) were compared in cells of embryogenic cultures of Larix decidua, L. leptolepis, and L. decidua x L. leptolepis (Larix x eurolepis). The highest enhancement of the MI was obtained with HU at 1.25 mM and 0.6% colchicine. In general the MI decreased with an increase of HU or APH concentration (over 1.25 mM for HU and 5 M for APH). Detailed karyotype analyses were made on the somatic complement of L. decidua, L. leptolepis, and their hybrid. These karyotypes were asymmetrical and advanced, with the smaller chromosomes being more submedian than the larger ones. The topography of chromosome 7 of L. decidua and chromosome 9 of L. leptolepis was found to be the most significant cytotaxonomic characteristic in differentiating these two species. Cytological data indicate that Japanese larch (L. leptolepis) is phylogenetically closer to European larch (L. decidua) than the Siberian larch group (L. sibirica and L. sukaczewii). Chromosomes with unusually long kinetochores were found in both species and the hybrid. Hyperploid cells (2n = 25) were observed in the hybrid (Larix x eurolepis) material analyzed. A genomic L. decidua probe hybridized strongly to dots of DNA from L. leptolepis indicating that there is high sequence homology between these two species.     

Comparative investigations on the differentiation of the endodermis and the development of the Hartig net in mycorrhizae of Picea abies and Larix decidua

Summary The differentiation of the endodermis of mycorrhizal roots of Picea abies and Larix decidua was investigated by means of light and transmission electron microscopy and with fluorescence techniques. The initiation and differentiation of the Hartig net were recorded. Differences between the two tree species were found, as were differences between the two tree species and angiosperms. The Casparian band developed immediately after the origin of endodermal cells from the meristem in mycorrhizae of both tree species. In L. decidua only the primary endodermis was present in most mycorrhizal laterals. The secondary structure of the endodermis was restricted to main roots and proximal parts of larch mycorrhizae. In P. abies mycorrhizae, however, the secondary stage of the endodermis developed soon after the primary endodermis and was characterized by regular alternation of short, active passage cells and elongated, rapidly degenerating cells, the inner surface of which was covered by a thick suberin layer. Hartig net development started in P. abies short roots only after the differentiation of endodermis into the secondary stage, whereas in L. decidua, the Hartig net was already initiated at the primary endodermal stage. Differences were specific for tree species.     

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.     

Differences in nitrate and ammonium uptake between Scots pine and European larch

In forest soils, ammonium is usually the predominant form of inorganic nitrogen. However, the capacity of trees to utilize both NO₃ ^- and NH₃ ⁺ may provide greater flexibility in responding to changes of nitrogen supply from the environment. Such capacity has been studied in seedlings of Scots pine (Pinus sylvestris L.) and European larch (Larix decidua Mill.) grown in the presence or absence of either nitrate or ammonium. Nitrate-induced plants showed a higher nitrate uptake rate than non-induced plants; this difference was almost negligible after 24 h of exposure to NO₃ ^-. Ammonium uptake in both species was consistently higher than that of nitrate, regardless of prior nitrogen provision. In both nutrient conditions, larch showed a more efficient transport system in comparison with Scots pine, with higher ammonium and nitrate uptake rates in both induced and non-induced plants. This was consistent also with the activity of nitrate reductase, measured in vivo in roots and leaves. This revised version was published online in June 2006 with corrections to the Cover Date.