Hanna-Leena Pasonen

Effects of sugar beet chitinase IV on root-associated fungal community of transgenic silver birch in a field trial

Heterogenous chitinases have been introduced in many plant species with the aim to increase the resistance of plants to fungal diseases. We studied the effects of the heterologous expression of sugar beet chitinase IV on the intensity of ectomycorrhizal (ECM) colonization and the structure of fungal communities in the field trial of 15 transgenic and 8 wild-type silver birch (Betula pendula Roth) genotypes. Fungal sequences were separated in denaturing gradient gel electrophoresis and identified by sequencing the ITS1 region to reveal the operational taxonomic units. ECM colonization was less intense in 7 out of 15 transgenic lines than in the corresponding non-transgenic control plants, but the slight decrease in overall ECM colonization in transgenic lines could not be related to sugar beet chitinase IV expression or total endochitinase activity. One transgenic line showing fairly weak sugar beet chitinase IV expression without significantly increased total endochitinase activity differed significantly from the non-transgenic controls in the structure of fungal community. Five sequences belonging to three different fungal genera (Hebeloma, Inocybe, Laccaria) were indicative of wild-type genotypes, and one sequence (Lactarius) indicated one transgenic line. In cluster analysis, the non-transgenic control grouped together with the transgenic lines indicating that genotype was a more important factor determining the structure of fungal communities than the transgenic status of the plants. With the tested birch lines, no clear evidence for the effect of the heterologous expression of sugar beet chitinase IV on ECM colonization or the structure of fungal community was found.

Field performance of chitinase transgenic silver birch (Betula pendula Roth): growth and adaptive traits

Fifteen transgenic birch lines carrying a chitinase IV gene from sugar beet and non-transgenic control plants were grown in a field trial, and traits connected to growth, quality and adaptation were monitored. Significant variation among the transgenic lines was observed in the growth parameters as well as parameters linked to stress status and leaf phenology of the trees. It was hypothesized that the differences among the lines could be explained by the differences in the level of transgene expression measured as sugar beet chitinase IV transcript accumulation. The level of the transgene expression was not detected to have influence on growth or leaf phenology, but instead it had influence on the parameters related to stress status of a tree. The increased levels of red colour and decreased general condition of the transgenic plants compared to the control plants may indicate physiological stress among the transgenic plants. The proportion of trees infested by Phytobia, larvae of which cause an easthetic defect to birch wood, was lower among many transgenic lines than in controls but the variation in Phytobia occurrence was explained by the differences in plant size only. Three lines out of fifteen were frequently different from the control plants in growth and leaf phenology, and these differences are suggested to be due to the position effect of the transgene. The observed changes in individual transgenic lines were not correlated with the sugar beet chitinase IV expression, and were more likely to impair than improve the traits that are usually considered important in adaptation and birch breeding.

Transgenic Betula pendula expressing sugar beet chitinase IV forms normal ectomycorrhizae with Paxillus involutus in vitro

Abstract Mycorrhizal fungi contain chitin in their cell walls and may be influenced by transgenic chitinases. This study examined the ability of a transgenic tree, silver birch (Betula pendula Roth), constitutively expressing the sugar beet chitinase IV gene, to form ectomycorrhizae with Paxillus involutus (Batsch) Fr. Eight transgenic lines showing varying levels of sugar beet chitinase IV expression and the non-transgenic control plants were inoculated by P. involutus in vitro, and the morphology of the mycorrhizae, mycorrhization efficiency and shoot and root fresh weights were studied. All the transgenic birch lines were able to form normal ectomycorrhizae containing distinctive mantles and Hartig nets. The level of sugar beet chitinase IV expression had no significant influence on mycorrhizal colonization. The only significant differences between transgenic and control plants were detected in weight parameters. According to these results, the expression of sugar beet chitinase IV in silver birch is not deleterious to formation of ectomycorrhizae between birch and P. involutus.

Density and composition of an insect population in a field trial of chitinase transgenic and wild-type silver birch (Betula pendula) clones.

Abstract Fifteen silver birch (Betula pendula Roth) lines carrying a sugar beet chitinase IV gene and eight wild-type birch clones were grown in a field trial. The composition and density of the insect population and the leaf damage caused by insects were monitored and compared between transgenic and wild-type trees. The most abundant insect group in all trees was aphids, and the variation in total insect densities was mainly explained by the variation in aphid densities. Insect densities were generally higher in the transgenic than in the control trees, indicating that the expression of the sugar beet chitinase IV gene had an influence on the suitability of birch leaves to aphids. The level of leaf damage was higher among transgenic than among control trees. Chewing damage was the most common type of leaf damage in all trees. The number of different damage types was higher among the wild-type clones than among the transgenic lines or their controls. The results indicate that the chitinase transgenic trees are more susceptible to aphids and suffer higher levels of leaf damage than the control trees. In the composition of the damage types, the control trees were more similar to the transgenic than to other wild-type trees, indicating that the composition was mostly linked to the genotype of the tree and not to the expression of the transgene. This study provides important information on the ecological interactions of chitinase transgenic trees in the field trial. No clear harmful effects of transgenic chitinase on the biodiversity of insect population were detected.

Pollen–pollen interactions in Pinus sylvestris

Direct pollen–pollen interactions can influence pollen fertilization ability when pollen from different donors and origins germinate on the same flower. In this study, in vitro germination tests were used to study direct pollen–pollen interactions in Scots pine (Pinus sylvestris L.). Pollen from southern (S, latitude 61° N) and northern (N, latitude 68° N) origins was germinated separately and in pairs at two temperatures (16 and 20°C), and pollen germination percentages and pollen tube lengths of the samples were analysed. The observed pollen performances of the two-donor pollen mixtures were compared with the mean performances of the pure pollen samples germinated in isolation. The significant difference between the observed and expected pollen performances indicated pollen–pollen interactions. Among S×S mixtures, only negative pollen–pollen interactions (= interference) were detected, while only positive interactions were found among N×N mixtures. Both types of interaction were substantially more frequent at the lower temperature. In general, direct pollen–pollen interactions during in vitro germination were rather infrequent and are not likely to have substantial influence on fertilization abilities in vivo in seed orchards or in natural forest stands of Scots pine. However, direct pollen–pollen interactions can be more pronounced among certain genotypes, which should be considered in artificial crossing experiments using pollen mixtures.

Palatability of transgenic birch and aspen to roe deer and mountain hare

Abstract Genetic engineering of plant resistance characteristics against fungi may unintentionally influence traits that are important for plant–herbivore interactions. We studied the palatability of transgenic birch (Betula pendula), aspen (Populus tremula) and hybrid aspen (P. tremula x tremuloides) genetically modified with the aim to improve fungal disease resistance, to selective mammalian herbivores in cafeteria tests. Roe deer (Capreolus capreolus) were fed with transgenic birch carrying a sugar beet chitinase IV gene. In the experiment with roe deer, none of the six transgenic birch lines differed significantly from the wild-type control in the proportion of consumed plant biomass. Correlation analyses suggested that sugar content did not guide the feeding preferences of roe deer but revealed a positive correlation between starch content and proportion of mass consumed. However, the variation in starch content could not be related to the level of transgene expression. Mountain hares (Lepus timidus) were fed with plant material from chitinase transgenic birch and aspen and hybrid aspen that carried a pinosylvin synthase gene from Scots pine. One transgenic birch line was significantly less palatable to hares than the wild-type control. The results of this study suggest that plant genotype may be related to the palatability of plant material when transgenic and wild-type woody plant material is used as winter food for hares. The results of this case study did not reveal changes in the palatability of the studied transgenic lines that could be readily related to the functioning of the used transgenes.