This partner will provide seeds and plant material from full-sib families of Norway spruce produced after controlled pollination and seed production in different environments to the other partners at the beginning of the project. Earlier experiments have shown that the performance of these families as regards growth cessation and autumn frost hardiness to a large extent depends on the temperature conditions during the seed production. The other partners will investigate the mechanisms behind these differences in performance by isozyme and molecular genetic studies.

The first year we will do controlled pollination on Norway spruce grafts which are kept in eight phytotron rooms until the seeds are developed. Temperature and day length will be regulated in these rooms so that two rooms will have one of four climatic treatments, combining high and low temperatures and short and long days. Altogether five female and five male parents will be involved in these crosses. We will later test the seedlings from the seed lots produced from these full-sib families in artificial freezing tests. Seeds will also be distributed to the other project participants for isozyme and molecular genetic analyses.

In artificial freezing tests performed with two-year-old seedlings from families and provenances ten years ago we selected seedlings that survived temperatures of -6°C, -8°C and -12°C. These seedlings were planted in a field test in 1993 together with seedlings that had not been exposed to the low temperatures. In this experiment measurements will be made of bud phenology, freezing tests of twigs will be performed both in the spring and in the autumn and physiological measurements will be made. Vegetative tissue from the saplings will be distributed to other project participants. Altogether this will make it possible to estimate the genetic changes that has taken place in the population due to the early selection for frost hardiness.

A field trial was recently established with seedlings from seeds collected in spruce stands in Norway planted with provenances from Germany, Austria and Poland, and with seedlings from direct import of the same provenances. We will make measurements of the annual growth rhythm of this plant material to verify the early observations from freezing tests that a seemingly fast adaptation has taken place after one generation in the northern environment.

In a three-step approach we will test if methylation of DNA is a possible molecular mechanism explaining the environmental preconditioning phenomenon. The first step includes the use of northern and immuno-blot analyses of phytochromes O and P. Seedlings of one full-sib family will be studied under active growth and during growth cessation. If these experiments reveal different expression patterns in progenies from a cold and a warm crossing environment, we will continue by studying methylation patterns of the genes using Phy O and Phy P as probes. If no difference in expression pattern appears, the next step will be to screen an EST-library made from cDNAs of Norway spruce needles, using macro- or microarray techniques. If positive, we will pick up genes that are differentially expressed as a “memory” of the crossing environment. Candidate genes will be used to develop new probes, which will be used to study methylation patterns of the same genes. If this step fails, we would try to develop a probe to study the general methylation pattern of DNA from the full-sibs of the two contrasting crossing environments.

Materials					Methods
available seeds from cold and warm crossing environments					isoenzymes DNA markers G x E drought
produce seeds in controlled climatic conditions, cold, warm, long and short days					isoenzymes DNA markers phenology and hardiness
field trials with selected seedlings surviving freezing tests					isoenzymes DNA markers phenology and hardiness
field trials with progenies of southern provenances planted in Norway					isoenzymes DNA markers phenology and hardiness