Plantlet differentiation in leaf and root cultures of birch (Betula Pendula roth.)

The newly-formed leaves on plantlets differentiated from shoot bud cultures of Betula pendula, when excised and grown on a fresh medium produced callus from the margins or regenerated leafy shoots, roots and plantlets. After 4 weeks, upon transfer to murashige and Skoog (MS) medium supplemented with 3-indoleacetic acid (IAA) + 6-(4-hydroxy-3-methyl-trans-2-enyl)aminopurine (zeatin) + 6-aminopurine (adenine), 15–20 plantlets were produced from each explant. Likewise, the roots also showed meristematic activity at several sites, and produced nodulated callus on MS + α-naphthaleneacetic acid (NAA) + 6-(3-methyl-2-butenyl-amino)purine (2-iP) + adenine, and ultimately differentiated plantlets. Anatomical studies showed that initiation of callus takes place by meristematic activity in epidermal cells of leaves, and cortical cells of roots. Cytological investigations revealed no change in chromosomal complement.     

Rapid adventitious shoot regeneration from leaf explants of European birch

The goal of this research was to develop a rapid and efficient system for regenerating shoots from leaf explants of European birch, Betula pendula Roth. Single-node stem explants were established in culture, and microshoots were subcultured every 4 weeks through 12 subcultures. Leaves from glasshouse plants or subcultured shoots were excised from stems, cut into approximately 35-mm² pieces, and placed on Woody Plant Medium (WPM) containing different combinations of naphthaleneacetic acid (NAA) (0, 3, 6 or 9 M) and benzyladenine (BA) (0, 7.5, 15 or 22.5 M) in a 4×4 factorial design. The percentage of leaf pieces forming shoots and the number of shoots regenerated per explant were recorded after 4 weeks. Only media containing BA without NAA stimulated shoot formation on leaf explants. Fifteen micromolar BA induced the most shoots to form on leaf explants compared to 30, 45 or 60 M of this cytokinin. In addition, shoot regeneration was enhanced up to four-fold between the first and eleventh subculture. Over 90% of the leaf explants regenerated shoots with an average of 18 buds formed per explant for the eleventh subculture. Almost twice as many explants formed shoots if their adaxial side was in contact with the medium rather than oriented away from it. The ability to regenerate shoots from leaves varied among plants, regardless of stock plant age. This reliable shoot regeneration system can be used for rapid shoot proliferation and potentially for genetic engineering of European birch.     

Spatial variation in sapwood area to leaf area ratio and specific leaf area within a crown of silver birch

Spatial variation in sapwood area to leaf area ratio (Huber value, HV) and specific leaf area (SLA) was examined in branches of closed-canopy trees of silver birch (Betula pendula Roth). HV increased basipetally within a crown and decreased with increasing branch order, but exhibited no significant radial trend along a primary branch. HV was primarily determined by branch position in a crown and branch diameter at the sampling point, being independent of the size of the tree and branch. Greater HV in the lower-crown branches is considered a means to mitigate differences in hydraulic transport capacity between the branches located in different canopy layers. Beside branch position and sampling location on a branch, SLA depended significantly on several other variables characterising tree and branch size. SLA increased basipetally within a crown and along a primary branch, but exhibited no significant trend with branch orders. Because height caused leaf area (A_L) to diminish more rapidly than leaf dry weight, A_L primarily determined the vertical variation in SLA.     

Cadmium and copper interactions on the accumulation and distribution of Cd and Cu in birch (Betula pendula Roth) seedlings

The effect of different external cadmium (Cd) and copper (Cu) regimes on the concentration of Cd and Cu in roots and shoots of birch (Betula pendula Roth.) seedlings was investigated. The seedlings were grown for 12 days in a weak nutrient solution (containing all essential nutrient elements including 0.025 µM Cu) at pH 4.2 with combinations of additional 0–2 µM CdCl₂ and 0–2 µM CuCl₂. Root and shoot concentrations of Cu were decreased by Cd in all treatments which included 0.1–2 µM of additional Cu in the treatment solution. When no extra Cu was added, only the shoot concentration of Cu was decreased by Cd whereas the root concentration was not affected. The shoot concentration of Cd was decreased by 0.5 and 2 µM of additional Cu in the treatment solution. The root concentration of Cd was decreased by Cu only when the concentration of additional Cu in the treatment solution was equal to or exceeded the concentration of Cd.     

Boron uptake by ectomycorrhizas of silver birch

Boron (B) is an essential micronutrient for plants but it is thought not to be essential for fungi. We studied whether the extraradical mycelia of Paxillus involutus in symbiosis with silver birch (Betula pendula) take up B and transport it to the host plant. We grew mycorrhizal plants in flat microcosms with a partitioning wall, below which there was only extraradical mycelium. A boric acid solution enriched in ¹⁰B was applied to these mycelia. Increased ¹⁰B/¹¹B isotope ratios were subsequently measured in birch leaves, stems, and roots plus mycorrhizas in the upper compartment. Boron was therefore taken up by the mycorrhizal mycelia and transported to the host plant in this species combination.     

Measuring and modelling leaf diffusive conductance in juvenile silver birch, <Emphasis Type="Italic">Betula pendula</Emphasis>

Leaf diffusive conductance for water (g_l) and twig xylem pressure (_xt) was measured in juvenile silver birch, Betula pendula, under field conditions in southern Sweden. Data from one site were used to parameterise two different multiplicative models for g_l (dependent data), and measurements from another site were used to validate these models (independent data). In addition, experiments were performed in controlled environments to validate the g_l response functions used in the models. The driving variables in the D-model were photosynthetic photon flux density, air temperature and water vapour pressure deficit of the air (D_a), while the DH-model also included the accumulated hours after sunrise each day with D_a above a certain threshold (H). Both models satisfactorily predicted the variation in g_l in dependent as well as in independent data, and the g_l response functions used were supported by the experiments in controlled environments. The DH-model was more successful in predicting g_l than the D-model by accounting for the observation that g_l was lower at higher H under similar weather conditions. There was a considerable variation in maximum g_l during the season, as well as between the two sites. On relatively warm and dry days _xt rapidly declined during the morning and then stabilized around a constant value until the late afternoon, with the stomatal regulation effectively preventing _xt from decreasing below this value. We suggest that these models could be used to simulate the g_l in juvenile birch if maximum g_l is locally estimated and if the response functions are not extrapolated beyond the climate range for this study.     

Epirrita autumnata induced VOC emission of silver birch differ from emission induced by leaf fungal pathogen

The production of volatile organic compounds (VOCs) through the activation of different signal-transduction pathways may be induced in various biotic and abiotic stress situations having importance e.g. in insect and disease resistance. We compared the emission of VOCs emitted from silver birch Betula pendula Roth (clones 4 and 80) twigs damaged either by larvae of Epirrita autumnata, or infected with pathogenic leaf spot causing fungus Marssonina betulae. We also analysed whether local herbivore damage can systemically induce the release of VOCs from the undamaged top of same sapling. The emissions of methylsalicylate (MeSA), (Z)-ocimene, (E)-β-ocimene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and linalool were induced from the twigs after 72 h feeding damage by E. autumnata larvae. However, 48 h feeding damage did not induce rapid systemic release of VOCs from undamaged top leaves of the same twigs. Pathogen-infected birch twigs had significantly greater emission of (Z)-ocimene and (E)-β-ocimene than intact control twigs. The emission of DMNT was not significantly induced and MeSA was not found at all after pathogen infection, both being significantly different from herbivore damaged twigs. According to our results leaf fungal pathogen induces VOC emission profile differs from that of arthropod herbivore-damaged leaves, suggesting that birch is able to transmit parasite-specific information via VOC emissions to conspecifics and natural enemies of herbivores. Handling editor: Yvan Rahbé     

Estimation of canopy density and irradiance in 20- to 40-year-old birch stands (Betula pubescens Ehrh. andBetula pendula Roth)

Three different experiments in birch (Betula pubescens Ehrh. andBetula pendula Roth) stands were conducted during summer 1991: an evaluation of the accuracy of the vertical tube method measuring crown free projection (CFP) in four birch stands; a study of relative irradiance (RI; photosynthetically active radiation, PAR), in birch stands differing in stem density and basal area; and an estimation of the CFP in different types of birch stands and the correlation between RI and CFP. The results indicate that the number of observation points well distributed over an area of 0.1 ha can vary between 50 and 200 to achieve sufficient accuracy. There were no significant differences in the CFP estimates made on the same plot by different observers including an untrained observer. CFP was related to basal area, stem number and diameter sum in a non-linear regression, CFP = A^*e^-BX. The correlations between RI and basal area, density and diameter sum, respectively, were 0.54, 0.57 and 0.67 for PAR. The coefficients of determination for RI (PAR) and CFP were high (0.86).     

Delayed induced responses of birch glandular trichomes and leaf surface lipophilic compounds to mechanical defoliation and simulated winter browsing

Changes in morphology and chemistry of leaf surface in response to herbivore damage may increase plant resistance to subsequent herbivore attack; however, there is lack of studies on induced responses of glandular trichomes and their exudates in woody plants and on effects of these changes on herbivores. We studied delayed induced responses in leaf surface traits of five clones of silver birch (Betula pendula Roth) subjected to various types of mechanical defoliation and simulated winter browsing. Glandular trichome density and concentrations of the majority of surface lipophilic compounds increased in trees defoliated during the previous summer. This induced response was systemic, since control branches in branch defoliated trees responded to the treatments similarly to defoliated branches, but differently from control trees. In contrast to defoliation treatments, simulated winter browsing reduced glandular trichome density on the following summer and had fewer effects on individual surface lipophilic compounds. Moreover, constitutive density of glandular trichomes was negatively correlated with induced total amount of lipophilic compounds per trichome, indicating a trade-off between constitutive and induced resistance in silver birch. Induced changes in leaf surface traits had no significant effect on leaf damage by chewers, miners and gall mites, but increased susceptibility of birch trees to aphids. However, leaf damage by chewers, miners and gall mites in defoliated (but not in control) trees was correlated with concentrations of some fatty acids and triterpenoids, although the direction of relationships varied among herbivore species. This indicates that induction of surface lipophilic compounds may influence birch resistance to herbivores. Our study thus demonstrated both specificity of elicitation of induced responses of birch leaf surface traits by different types of damage and specificity of the effects of these responses on different types of herbivores.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Characterization of a birch (Betula pendula Roth.) embryogenic gene,BP8

We have isolated the birch homologue (BP8) for the carrot embryogenic gene DC8 by heterologous hybridization. The birch BP8 gene encodes a putative protein of 53 kDa, showing 52% sequence identity with the DC8 gene at the amino acid level. The putative BP8 protein contains 20 repeats of 11 amino acids and thus belongs to the group of LEA proteins isolated from such plants as carrot, cotton and wheat. Northern hybridization of mRNA isolated from birch cells representing different stages of somatic embryogenesis and non-embryogenetic material with a PB8 probe gave no signals, suggesting a low expression level of the BP8 gene.     

Prevention of flower development in birch and other plants using a BpFULL1::BARNASE construct

The prevention of flower formation is important for avoiding the spread of transgenes from genetically modified plants into wild populations. Moreover, the resources not expended for the generation of flowers and fruits might be allocated to increased vegetative growth. We have been developing methods for preventing flower formation in silver birch (Betula pendula), a tree species of considerable economical importance in the boreal region. Here we study the suitability of the promoter of BpFRUITFULL-LIKE1 (BpFULL1, formerly BpMADS5) for tissue-specific ablation of inflorescences in Arabidopsis, tobacco and birch. With all these species, the development of inflorescences was successfully prevented. The results show that the BpFULL1::BARNASE construct has potential biotechnological applications in different plant species.This revised version was published online in March 2005 because the name of the second author (M. Hassinen) was missing.     

Somatic embryogenesis in cell cultures of birch (Betula pendula Roth.)

Somatic embryogenesis was induced in cell cultures of birch (Betula pendula Roth.) derived from juvenile tissue of seed embryos and from mature leaf tissue. Embryos were formed in liquid and on solidified medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylaminopurine (kinetin). Sometimes somatic embryos formed only after transfer to medium devoid of growth regulators. The embryos germinated on hormone-free medium and were potted in soil and grown in the greenhouse.Finnish Forest Research InstituteUniversity of Helsinki, School of Pharmacy;     

Impairment of gas exchange and structure in birch leaves (Betula pendula) caused by low ozone concentrations

Summary Injury caused by low O₃ concentrations (0, 0.05, 0.075, 0.1 l 1^-1) was analyzed in the epidermis and mesophyll of fully developed birch leaves by gas exchange experiments and low-temperature SEM: (I) after leaf formation in O₃-free and ozonated air, and (II) after transferring control plants into ozonated air. In control leaves, autumnal senescence also was studied in O₃-free air (III). As O₃ concentration increased, leaves of (I) stayed reduced in size, but showed increased specific weight and stomatal density. The declining photosynthetic capacity, quantum yield and carboxylation efficiency lowered the light saturation of CO₂ uptake and the water-use efficiency (WUE). Carbon gain was less limited by the reduced stomatal conductance than by the declining ability of CO₂ fixation in the mesophyll. The changes in gas exchange were related to the O₃ dose and were mediated by narrowed stomatal pores (overriding the increase in stomatal density) and by progressive collapse of mesophyll cells. The air space in the mesophyll increased, preceded by exudate formation on cell walls. Ozonated leaves, which had developed in O₃-free air (II), displayed a similar but more rapid decline than the leaves from (I). In senescent leaves (III), CO₂ uptake showed a similar decrease as in leaves with O₃ injury but no changes in mesophyll structure and WUE. The nitrogen concentration declined only in senescent leaves in parallel with the rate of CO₂ uptake. A thorough understanding of O₃ injury and natural senescence requires combined structural and functional analyses of leaves.     

Effects of light, magnesium and sucrose on leaf anatomy, photosynthesis, starch and total sugar accumulation, in kiwifruit culturedin vitro

Shootlets of kiwifruit plants (Actinidia deliciosa) were culturedin vitro. Combinations of light intensity, Mg and sucrose in the cultures showed that an increase of light intensity resulted in a corresponding increase of the relative size of the leaf mesophyll cells and in a decrease of the numbers of chloroplasts and contained starch grains. The addition of sucrose to the substrate media negatively affected the size of the mesophyll cells under normal Mg concentration (35 mg l⁻¹), and positively under high Mg concentration (105 mg l⁻¹ ). Sucrose further resulted in an increase in the numbers of chloroplasts and contained starch grains. The photosynthetic capacity of leaves greatly increased when Mg concentration was enhanced and sucrose was excluded from the nutrient substrate. Total sugar accumulation in all treatments was favoured by normal light intensity and addition of sucrose.     

Prevention of the flowering of a tree,silver birch

Genetic modification of trees presents great advantages but it is hampered by the possible spread of introduced genes to native populations. However, the spread would be prevented if the modified trees would be sterile. We have previously shown that the induction of sterility by the prevention of flowering is possible in tobacco and Arabidopsis by introducing a gene construct composed of the ribonuclease gene BARNASE ligated to the flower-specific promoter of the birch gene BpMADS1. In the present study, we test this gene construct in silver birch (Betula pendula Roth). When this gene construct was introduced into very early-flowering birch clones, 81 kanamycin resistant lines were obtained. In 38 lines, the vegetative development was disturbed, e.g., the leaves were small and the plants were short and bushy or the growth of plants was weak. More importantly, in 7 other lines no male inflorescences formed or they aborted early. If male inflorescences were formed, they did not contain any stamens. The initial growth of these lines was similar to the non-transgenic control lines. Later, however, the growth of the non-flowering lines differed from that of the controls in showing some dichotomic branching and a reduced number of branches. Preliminary results showed that the gene construct can prevent the development of female inflorescences as well. The results show clearly that BpMADS1::BARNASE can prevent the flowering in a tree but the prevention of flowering may cause some side effects. Studies with ordinary birch clones will show whether the side effects are a property of the early flowering clones or all birches.     

Differentiation and structural decline in the leaves and bark of birch (Betula pendula) under low ozone concentrations

Summary Leaf and bark structure of a birch clone (Betula pendula Roth) continuously exposed to charcoal-filtered air or charcoal-filtered air plus ozone (0.05, 0.075, 0.1 l 1^-1) was investigated throughout one growing season. Increasing ozone dose influenced leaf differentiation by reducing leaf area and increasing inner leaf air space, density of cells developing into stomata, scales and hairs. When approximately the same ozone dose had been reached, macroscopical and microscopical symptoms appeared irrespective of the ozone concentration used during treatment. Structural decline began in mesophyll cells around stomatal cavities (droplet-like exudates on the cell walls), continued with disintegration of the cytoplasma and ended in cell collapse. Epidermal cells showed shrinkage of the mucilaginous layer (related to water loss). Their collapse marked the final stage of leaf decline. When subsidiary cells collapsed, guard cells passively opened for a transitory period before collapsing and closing. With increasing ozone dose starch remained accumulated along the small leaf veins and in guard cells. IIK-positive grains were formed in the epidermal cells. This contrasted with the senescent leaves, where starch was entirely retranslocated. Injury symptoms in stem and petiole proceeded from the epidermis to the cambium. Reduced tissue area indicated reduced cambial activity. In plants grown in filtered air and transferred into ozone on 20 August, injury symptoms developed faster than in leaves formed in the presence of ozone. Results are discussed with regard to O₃-caused acclimation and injury mechanisms.     

Growth and nutrition of birch seedlings in relation to potassium supply rate

Summary Growth of hydroponically cultivated birch seedlings (Betula pendula Roth.) at sub- and supra-optimum potassium supply rates was investigated. Potassium was supplied either as a relative addition rate (r _k = 5, 10, 15 and 20% increase day^-1) or as fixed concentrations (0.2, 3, 6, 12 and 15 mM) in the culture solution. After an acclimation period the growth rate of the seedlings in the suboptimum treatments reached values close to the treatment variable, the relative rate of K-addition. Deficiency symptoms, in the form of chlorosis and necroses along the leaf margins, developed initially in all suboptimum treatments, but very few new symptoms appeared once the seedlings had reached the phase of steady-state nutrition and growth. At supra-optimum K-supply levels, i.e. at 0.2–15 mM K in the culture solution, no symptoms of deficiency or toxicity developed, and the relative growth rate of the seedlings remained maximum. The relative growth rate of the seedlings was linearly related to the plant K-status for K contents ranging from 0.2 to 1.0% of dry weight (DW). At higher internal K-concentrations, 1.0–3.0% DW, no further increase in relative growth rate was achieved. A shortage of K resulted in a decrease in the net assimilation rate. This effect was counterbalanced by the absence of shift in he leaf weight ratio as well as by the production of relatively thin leaves. The fraction of dry matter allocated to roots decreased in K-limited plants, as did the leaf contents of soluble carbohydrates and starch.     

Applicability of the co-inoculation technique using Agrobacterium tumefaciens shooty-tumour strain 82.139 in silver birch

In the co-inoculation technique, genetic transformation is performed using a mixture of Agrobacterium strains – shoot regeneration is induced by the wild-type strain 82.139, while the transferable genes are provided in a binary plasmid by another, non-oncogenic Agrobacterium strain. The aim of the present work was to study the applicability of co-inoculation under both in vitro and greenhouse conditions for in planta transformation in silver birch (Betula pendula Roth). In addition to the original method, several modifications of the technique including an genetically engineered 82.139 strain harbouring the binary pGUSINT were tested. The co-inoculations resulted in a gall formation frequency of 52–94% with greenhouse seedlings, and 4–63% with tissue-cultured plantlets, the shoot induction percentage varying by 0–13 in the greenhouse and 42–75 in vitro. PCR analysis verified that the majority of the regenerated material was non-transgenic, with a few individuals showing integration of the oncogenic T-DNA. According to the histochemical tests, however, some of the numerous differentiating buds and small shoots on gall tissues were transgenic, and contained the GUS reporter gene. The results show that it would have been necessary to apply selection pressure during differentiation in order to recover shoots transformed with the desired genes from the binary plasmid. The morphology and growth of all the regenerated plantlets was normal, suggesting that the oncogenic T-DNA was not expressed even though it was present. In conclusion, it was possible to obtain transgenic silver birch plantlets using the A. tumefaciens strain 82.139, but the co-inoculation method is not directly applicable as in planta transformation protocol.     

Carbon transfer associated with assimilation of organic nitrogen sources by silver birch (Betula pendula Roth.)

Summary Qualitative and quantitative aspects of heterotrophic carbon assimilation by mycorrhizal plants of birch (Betula pendula) were examined. Plants were grown aseptically from seed in the mycorrhizal condition with the fungus Hebeloma crustuliniforme and in the non-mycorrhizal condition, with protein as their sole exogenous nitrogen source. Yields and nitrogen contents were determined in some of the plants, while the roots of others were supplied with ¹⁴C-labelled protein and their shoots exposed for up to 72 h to different irradiance regimes. Only mycorrhizal plants utilised the organic nitrogen. Uptake of carbon associated with this utilisation and its translocation to the leaves was demonstrated directly by means of autoradiography. Amounts of activity transferred to shoots were greatest in low irradiance regimes. Calculation of net carbon gain from the heterotrophic source, based upon the assumption that breakdown products of protein are assimilated as amino-acids, indicates that over a 55-day growth period up to 9% of plant C may be derived from protein. The physiological and ecological significance of these findings are discussed.