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Shortening of telomeres has been connected with ageing and loss of cell replication or regeneration capacity. The aim of the present study was to examine potential variation in the length of telomeric repeats in silver birch (Betula pendula Roth) using clonal materials consisting of different-aged outdoor trees and tissue cultures of seven genotypes. The overall average length of telomeres was 13.6 kb (±0.3), the minimum length of repeats in the different genotypes varied from 5.9 kb (±0.5) to 9.6 kb (±0.6), and the maximum length varied from 15.3 kb (±1.1) to 22.8 kb (±0.4). When germinated seeds and leaf and cambium samples from 15- and 80-year-old trees were compared, no correlation of ageing and the length of telomeric repeats was found. Positional variation in the telomere length was, however, observed in the cambium of mature trees, the stem base having longer repeats than the upper parts of the tree. Tissue cultures were found to have shorter telomeres than outdoor trees: prolonged culture, callus culture and stressful conditions were all observed to shorten telomeric repeats and should thus be avoided in birch micropropagation. There were significant differences among the studied silver birch genotypes in their telomere length, and these differences were consistent over the sample types. This is the first report on variation of telomeric repeats in a long-living organism studied with clonal materials.

The shortening of telomeres, the specific structures at the end of the eukaryotic chromosomes, has been associated with ageing and loss of cell replication or regeneration capacity. The aim of this study was to determine the variation in telomeric repeats during the life-span of Scots pine (Pinus sylvestris L.) by studying different tissues and age groups ranging from immature embryos to cambium, buds, and needles of mature, 200-year-old trees. The telomeric repeats in Scots pine including interstitial and centromeric repeats ranged from 0.9 up to 25 kb, and true telomeres were evaluated to have a mean length of 19.3 kb (±SE 0.17). Telomeres were observed to shorten with increasing tissue differentiation, embryonal samples having the longest repeats with an average length of 21.1 (±0.34) to 21.7 kb (±0.42) and the needles having, on average, the shortest repeats of 18.1 kb (±0.24). After germination, ageing per se had no significant effect on the length of telomeric repeats in cambium, bud, or needle tissues. In the older trees (50–200 years of age), the telomeres in stem cambium showed shortening towards the tree top. This is the first observation on such a position-related variation in telomeric repeats. Finally, there was a remarkable genotypic variation in the length of telomeric repeats, and this was consistent over the tissue types. In the small number of tested donor trees, the genotypic differences were not related to regeneration ability in tissue culture.

Somatic embryogenesis (SE) is considered the most promising vegetative propagation method for Scots pine (Pinus sylvestris L.), and the aim of the present work was to study ways to improve the individual steps of the technique. According to the results, donor genotype has a significant effect on SE initiation in Scots pine. Relatively good initiation frequencies of 20–30% can be achieved, but there are also recalcitrant families. To obtain high-quality somatic embryos that also perform well as emblings in the greenhouse, the SE cultures should be proliferated and matured as a thin layer spread on filter paper, with a high (80–90 µM) abscisic acid concentration applied during maturation. Filter proliferation was found to result in a slightly smaller number of embryos, but of higher quality, than proliferation as tissue clumps. From maturation, the somatic embryos should be harvested for germination only during a limited period, and their germination performed in vitro on tissue culture media. Spec...

The aim of this study was to determine, whether interactions between a drug and hydrophobic polymer matrix are present, and if so, how they affect the drug release. In addition, the most important formulation parameters, for example porosity or structure of the tablet, which have the greatest impact on drug release, were defined. Six different drug compounds, that is allopurinol, acyclovir, metronidazole, paracetamol, salicylamide and theophylline, were used in different formulations with hydrophobic starch acetate (DS 2.7) as a matrix forming polymer. Results indicate that the formulation parameters describing directly or indirectly the structure of the matrix, such as porosity, compaction force and the particle size fraction of the filler-binder, have the strongest impact on drug release. The contribution of drug property based variables is not as high as formulation parameters, but they cannot be overlooked. The contribution of water solubility and dissolution rate of the compound are obvious, but there are other significant parameters, which describe the hydrophobic and hydrophilic regions of the molecule, that also affect the drug release. This can be seen especially with the salicylamide: compound which appears to have a strong and sufficiently high hydrophobic region that interacts with starch acetate and impairs the drug release.

Clonal trueness of micropropagated or cryopreserved material is essential, especially with long-living tree species. In this study, the growth rate and morphology of regenerated silver birch (Betula pendula Roth) plants growing in the nursery were evaluated after different treatments: short-term (14 months) and long-term (70 months) tissue culture periods, cryostorage of in vivo buds and cryopreservation of in vitro shoot apices using four different slow cooling cryopreservation protocols with PGD (10% PEG, 10% glucose, 10% DMSO) as cryoprotectant. Genetic fidelity of the regenerated plants compared to the original donor trees was evaluated using RAPD assays together with chromosome analysis. The regenerated plants showed no genetic or phenotypic changes, and can thus be considered as reliable material for any research, breeding or silvicultural activities.

The aim of the present study was to develop an effective cryopreservation method for hybrid aspen ( Populus tremula L.× Populus tremuloides Michx.), an economically important woody plant and widely used model system of forest scientists. Specific attention was paid to genetic fidelity of cryopreserved materials and transgene stability after retrieval. Three different kinds of cryopreservation protocols were applied, i.e. the slow cooling of PGD-treated in vitro apical segments, the vitrification of PVS2-treated in vitro buds and the slow cooling of dormant in vivo buds. The slow cooling of in vivo buds proved to be the most appropriate way to cryopreserve the samples, resulting in a generally excellent regrowth rate of 72–96%. According to random amplified polymorphic DNA (RAPD) assays the genetic fidelity of the cryostored materials was maintained during both slow cooling and vitrification. In addition, neomycinphosphotransferase II ( nptII ) gene was found to be stable in transgenic lines after cryopreservation. The results clearly indicate that it is possible to apply cryopreservation techniques to preservation of valuable clones and transgenic lines of hybrid aspen.

We describe the first ever expression of Vitreoscilla haemoglobin (VHb) in an economically important boreal woody plant hybrid aspen (Populus tremula x tremuloides). VHb has mainly been expressed in biotechnologically important unicellular organisms of both prokaryotic and eukaryotic origin. VHb expression, in this study, was analysed under different greenhouse cultivation conditions and under elevated UV-B illumination. Microscope analyses of leaves grown under optimized conditions revealed significant differences both in cell structure and size when the transgenic VHb lines were compared with the control lines. VHb lines displayed a higher relative volume of mitochondria and a significantly enhanced accumulation of starch in chloroplasts, all of which pointed towards changes in cellular energy production. Under elevated UV-B illumination, the differences between VHb lines became evident. Some specific VHb lines had elevated levels of total flavonoids, individual quercetin, kaempferol- and myricetin-derivatives relative to controls and other transgenic lines. This observation may reflect the availability of extra energy resources for secondary metabolite production and possibly an enhanced protection ability of these transgenic lines against UV-B illumination. Thus, all these findings point to changes in the energy metabolism of VHb lines. In the cultivation conditions tested this observation did not, however, result in a general improvement of elongation growth.

The aim of the study was to develop a preservation method for transgenic silver birch (Betula pendula Roth) lines based on cryopreservation. Specific attention was paid to transgene stability and functioning. Vegetative buds collected from one- or two-year-old silver birches representing four transgenic lines and two wild-type lines were used as explants. Generally, the average regeneration of either transgenic or wild-type, cryoperserved and non-cryopreserved control buds was excellent, and varied from 72 to 100 percent. The regeneration percentage of cryopreserved buds was, however, significantly lower than that of non-cryopreserved control buds when estimated two weeks after thawing, but the differences were no longer significant four weeks after thawing. Growth of the plants in the greenhouse was more dependent on the clone than on the cryopreservation treatment. The studied transgenic lines have three (line E/5) to nine (line R/3.2) copies of transferred neomycin phosphotransferase genes that were also found to be stable after cryopreservation. In general, the neomycin phosphotransferase transcript levels did not change due to cryopreservation. The results indicate that it is possible to apply the cryopreservation technique to preserve valuable transgenic lines of a forest tree, silver birch. The method presented here leads to high regeneration percentages combined with transgene stability and functioning.

Summary Silver birch shoot tips were cryopreserved after cold hardening of the mother shoots for 28 days at +5 °C under an 8/16 light/dark photoperiod on medium containing 100 μmol/L ABA. The aim of the present study was to investigate the effect of the elimination of ammonium ions on the recovery of cryopreserved silver birch shoot tips. Ammonium was substituted by different nitrogen sources; with KNO3 or L-glutamine already during cold hardening of the mother shoots, or with KNO3, L-glutamine, or a mixture of the two, during post-thaw cultivation of the shoot tips. According to the results, the substitution of ammonium nitrogen improved the recovery of the cryopreserved and regenerated silver birch shoots. The highest recovery and improved morphological appearance of the regenerated shoots were achieved when ammonium was substituted by KNO3 already during the cold hardening phase.

The aim of the present study was to apply cryopreservation for the embryogenic cultures of open-pollinated Abies cephalonica, as well as to test the genetic fidelity of the cryopreserved cell lines using random amplified polymorphic DNAs, i.e. RAPD markers. Dimethylsulfoxide (DMSO) and two mixtures of polyethylene glycol, glucose and DMSO (PGD I and PGD II), were used as cryoprotectants and the cryoprotectant treatments were performed with and without storage in liquid nitrogen. All the tested cryopreservation methods, i.e. DMSO, PGD I or PDG II used as cryoprotectant combined with slow cooling and fast thawing, proved to be applicable for A. cephalonica SE cultures. The best results, measured as survival percentage and mean growth ratio, were achieved using the PGD I treatment. The RAPD assays performed revealed considerable genetic variation in the DMSO treated but non-frozen samples, i.e. 16.8% of the produced RAPD profiles showed intraclonal variation while background variation was seen in 1.7% of the control amplifications. The present results show that cryoprotectants may cause a risk for genetic fidelity of plant material and thus, monitoring it is important. This is particularly true in the breeding of the long-living conifers, since the effects of occasional mutations or genetic rearrangements may not be readily observed in young plants but expressed substantially later on in mature trees.

Early spring buds of silver birch (Betula pendula Roth), collected with and without a female catkin in the middle of April, were cryopreserved using slow cooling followed by immersion in liquid nitrogen at -196°C for 8 days, 6 months, 1 year, 3 years, or 5 years. After fast thawing the buds were cultured in vitro according to the published protocol. The regrowth ability of the two types of buds was different. The regrowth of vegetative buds without a female catkin was good after all the cryopreservation times. There was a significant decrease in the regrowth ability of buds growing in the axil of a female catkin compared with the corresponding unfrozen controls after 1 year in cryostorage. In addition to the effect of the presence of a catkin on the regrowth ability, the late collecting time of the buds also probably decreased the regrowth and regrowth rates of both types of bud. The regrowth rates of buds without a catkin were 66, 67 and 24% after 1, 3, and 5 years of cryostorage, respectively, while those of buds with a catkin were 13.5, 32, and 2.6%, respectively.

Recovery of cryopreserved in vitro shoot tips of silver birch (Betula pendula Roth) was doubled by incorporating abscisic acid (ABA) in the culture medium during cold hardening of the mother shoots. The average recovery of shoot tips was over 40% after cold hardening for 28 days at +5 degreesC under an 8/16 light/dark photoperiod on medium containing 10(-4) M ABA. ABA was effective in combination with low temperature and short daylength only, although large genotypical differences were noted. ABA had two different effects: it enhanced cold hardening and increased callus formation during regeneration of cryopreserved shoot tips. Copyright 1998 Academic Press.

The aim of the study was to develop an effective cryopreservation method for Scots pine (Pinus sylvestris L.) embryogenic cultures. Altogether nine cell lines derived from three mother trees were cryopreserved after cold hardening using dimethylsulfoxide or two different mixtures of polyethyleneglycol 6000, glucose and dimethylsulfoxide as cryoprotectants. Seventy-eight percent of the cell lines remained viable after cryostorage, the best cryoprotectant treatment being 10% polyethyleneglycol 6000, 10% glucose, and 10% dimethylsulfoxide in water. This treatment resulted in significantly better regrowth of the embryogenic cultures than with the other cryoprotectants or with the controls. According to microscopical observations, the cells that retained their viability and regrowth ability after cryopreservation were the embryonal head cells, as well as some elliptic suspensor cells close to the embryonal head cell area. When proliferation growth of the frozen cultures had started, their morphological appearance was the same as the non-frozen cultures. In addition, the RAPD assays suggested that the cryostorage treatment used here preserved the genetic fidelity of the Scots pine embryogenic cultures.

Shoot tips of silver birch (Betulapendula Roth) derived from in vitro cultures originating from mature trees were used as explant material. Several pretreatments prior to cryopreservation were studied in order to enhance shoot tip recovery after storage in liquid nitrogen. Cold acclimation and the classical slow freezing procedure proved to be essential for successful survival, while both nonhardening and vitrification resulted in minimal survival. The optimum procedure was the following: Shoots were cold hardened (5 °C on a 8 h: 16 h (light:dark) cycle) for 3 weeks, and dissected shoot tips were then precultivated on woody plant medium with 5% dimethyl sulfoxide for 72 h. The material was transferred to cryotubes, and a mixture of 10% polyethylene glycol, 10% glucose, and 10% dimethyl sulfoxide was used as cryoprotectant. The cryotubes were slowly cooled to −38 °C before immersion in liquid nitrogen for 8 d. After fast thawing, axillary buds formed shoots without callus formation, but the growth of the new shoots was delayed from a few days to several weeks. Variability in regrowth was considerable as a result of genotype differences.

Cryopreservation was developed for the storage of in vivo buds of silver birch (Betulapendula Roth). The principles of the optimized method were the use of buds already acclimated in nature and slow freezing with a cooling velocity of 10 °C/h down to a terminal temperature of −38 °C. After 24 h at this temperature the buds were immersed in liquid nitrogen at − 196 °C for 8 days, 6 months, or 12 months. After fast thawing, 5 min in a water bath at 37 °C, the buds were surface sterilized and cultured according to the normal laboratory routine. The buds were examined after 2 and 4 weeks of cultivation. There were no significant differences in survival and growth between the unfrozen controls and buds stored in liquid nitrogen for different times. The growth percentage of buds without a female catkin was double that of buds with a catkin. These results indicate that cryopreservation would be an ideal method for the ex situ gene conservation of birch and retention of the juvenility of adult genotypes.

Cryopreservation—the storage of plant germplasm in liquid nitrogen—provides a modern tool for the conservation of forest genetic resources. It is especially applicable for species in which their micropropagation can be initiated from mature tree buds, e.g., silver birch (Betula pendula Roth), thus enabling the conservation of specific genotypes: endangered elite trees and trees expressing rare, valuable or interesting characteristics. The aim of the present study was to develop a vitrification protocol applicable for the cryostorage of silver birch that avoids the use of expensive sophisticated freezers. The average recovery of vitrified axillary silver birch buds was 71% using a protocol that started with four-week cold hardening of bud-bearing in vitro donor shoots on modified medium under short day conditions. After cold hardening, the excised axillary buds were precultivated on medium containing 0.7 M sucrose for 24 h under the same conditions as during the cold hardening period. Following preculture, the buds were treated with loading solution containing 2 M glycerol and 0.4 M sucrose for 20 min at room temperature. Finally, the buds were dehydrated with PVS2 cryoprotectant for 120 min followed by direct immersion in liquid nitrogen. According to the morphology and the RAPD profiles of regenerated plants in the greenhouse, the genetic fidelity of the vitrified birch material seems to have remained unchanged.

Growth, crown structure, flowering and seed production of silver birch ( Roth) seedlings, grafts and micropropagated plants was compared during four years in a polythene greenhouse experiment. The growth of the seedlings was clearly the most vigorous and that of the grafts the weakest, the micropropagated plants being intermediate. The seedlings had the highest and the grafts the lowest number of branches before cutting the tops of the plants, but the differences between the material types were no more significant after cutting the tops. The grafts had significantly shorter and thinner branches than the seedlings and the micropropagated plants, whereas the differences in branch length and branch thickness between the latter two groups were not significant. The grafts started flowering at the age of two years, one year earlier than the other two types of material. At the age of four years the micropropagated plants had abundant seed production, about 75% of that of the seedlings and about two times higher than that of the grafts. Thus, the micropropagated plants can be used instead of grafts when establishing polythene greenhouse seed orchards of birch.Betula pendula

Seed production of micropropagated plants, seedlings and grafts of silver birch (Betula pendula Roth) in a polythene greenhouse experiment was followed for five years. The grafts started flowering and seed production at the age of two years, one year earlier than other two types of material. At the age of three the seed production of both the micropropagated plants and seedlings was already more than two times higher than that of the grafts. Variation between the clones was high and plant type x clone interaction was significant. At the age of four, in 1993, seed production was high in all three types of material. Seed production of the micropropagated plants was two times higher than that of the grafts but about 75% of that of the seedlings. In 1994 seed production of all three plant types was very low, which shows large variation between different years. The early development of the plant material types suggests that micropropagated birch plants have higher seed production than grafts and could well be used instead of grafts in polythene greenhouse seed orchards.

This paper presents three interconnected developments made during the course of a recent collective research work, the development of a systematic graph-based search tool for physical contradictions, a ranking approach for defining the order of criticality of the design contradictions and the associated analysis of the different design strategies that can be used to solve those contradictions or to enhance performance indicators. The systematic graph-based search for physical contradictions is using the set of elementary variables necessary to describe the system as basic input. The initial set is extracted based on taxonomy of variables combining classification work from NIST and classification of variables derived from the Bond Graph theory. The contradiction search method is in a second step classifying the set of variables into three categories: the constraint variables imposed to the designers by the context and the environment, the design variables on which the designer as the possibility to act and the performance variables that are used to evaluate the performance of the designed system. In a third step, interactions between variables are searched using two possibilities: a causal ordering algorithm developed during the course of the research or via a collective work of experts. The result of this step is a directed graph starting from the constraints variables and ending with the performance variables. In the fourth step objectives have to be assigned to the performance variables (minimal value, maximal value or target value). Those objectives are propagated back into the graph by analyzing the impact of the variables interacting with the performance variables. A physical contradiction is detected each time it is discovered that a design variable is associated with two contradictory objectives. Following this approach, a contradiction is represented as a node in the directed graph. It is possible to systematically map the different design strategies that can be used and to rank the possible impact of those design strategies. The article presents a concrete application of the approach on the case study of an air bearing and demonstrates the novelty of the approach to generate new viewpoints and insight in the analysis of the early stages of the development process. The potential impact of such type of design support is potentially very important. A future step will consists of developing a computer aided tool implementing the method.

The curly-grained trait of Roth is inheritable, but it is persumably not question of only one Mendelian gene since, for instance, there are a number of different types of curly-birch. The progeny obtained from controlled crossing between two curly-birch individuals do not all posses the curly-grained trait.Betula pendulaBetula pendulacarelica