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SEP
Propagation of European pines via somatic embryogenesis

A vegetative propagation method will be developed for two economically important pine species, Pinus syvestris and P. pinaster, using somatic embryos. The method will be based on knowledge of how somatic pine embryos proliferate and develop, and how the different steps involved are regulated. The best growth conditions for the in vitro steps, especially maturation, will be defined and this will enable vigorous plants under outdoor conditions to be developed. The method will be approved for seed families of high value to forestry. Embryogenic cultures will be cryopreserved and somatic embryo plants will be regenerated. The potential of the method will be assessed by end-users by comparing the early performance of regenerated somatic embryo plants to seedlings from the same families, and estimating whether the plant material concerned has multiplied in a satisfactory way or not.

Results
During the first two years, embryogenic cell lines from more than 2800 genotypes representing 78 families were established. The embryogenic cultures were initiated from immature zygotic embryos. More than 1300 cell lines were cryopreserved, and most have been thawed out and show a high potential for regrowth. New cultures have been established from thawed cell lines. Mature somatic embryos were obtained after maturation treatment. By the end of the project, 8200 embryo plants were regenerated, and more than 3100 were transferred to nurseries or fields.
Multivariate analysis was used to study important factors for proliferating embryogenic cultures. A standard protocol can be used for all tested genotypes. Genotype is the main factor affecting proliferation. Two major mechanisms of proliferation of early somatic embryos in pine were characterised: embryo formation through meristematic activity in the secondary suspensor, alternating with cleavage polyembryology. The multiplication rate is very high and proliferation is difficult to stop.
The ability to form mature somatic embryos varies significantly among genotypes. Water-related stress during the maturation step increases the number of mature somatic embryos. However, embryos developing under such stress conditions are usually smaller than those not exposed to stress.
Mature somatic embryos have been obtained from 60% of the established cell lines. However, their number and quality vary among cell lines. Therefore, presently a low number of genotypes can be used for large-scale plant production.
Expression of genes involved in nitrogen metabolism can be related to specific stages of development of somatic embryos and cell types. The spatial expression pattern of these genes is similar during somatic and zygotic embryogenesis. The expression profiles of genes coding for enzymes in the primary metabolism in somatic embryo plants matched to a great extent those ocurring in seed plants.
The current procedure of somatic embryogenesis gives rise to increased genetic instability as evidenced from the analysis of four nuclear microsatellite loci in somatic versus zygotic embryos of P. sylvestris. Investigations are under way to assess if/how instability in the studied microsatellite loci reflects alterations in fucntional genes.
Cuttings from somatic embryo plants grow in a similar way to cuttings from seedlings.

Classified in ARABLE CROPS, FORESTRY

Scientist responsible for the project

Ms SARA VON ARNOLD
Baeckloesavaegen 2 C Box 7027
750 07 Uppsala
Sweden - SE

Phone: +46 1867 3230
Fax: +46 1867 3279
E-mail: Sara.von.Arnold@sgen.slu.se

References

Project ID QLRT-1999-00679
Organisation Sveriges Lantbruksuniversitet
Area 5.3.1
Start date 01 February 2000
Duration (months) 48
Total cost 2346511 €
Total EC contribution   1660951 €
Status Completed

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