A method will be developed for vegetative propagation of two economically important pine species, Pinups Sylvester's and P. pilaster, by using somatic embryos. The method will be based on knowledge about how somatic embryos of pine proliferate and develop and how the different steps are regulated. The best growth conditions for the in vitro steps, especially for maturation, will be defined, allowing establishment of vigorous plants under outdoor conditions. The method will be approved for seed families of high value for forestry. Embryogenic cultures will be cryopreserved. Somatic embryo plants will be regenerated. The potential of the method will be assessed by the end users by comparing the early performance of regenarated somatic embryo plants to seedlings from the same families and estimating wether the plant material of interest is multiplied in a satisfactory way.
Part 1. Embryogenic cell lines from 433 genotypes of P. sylvestris and 1039 genotypes of P. pinaster selected by the forest industry were established and cryopreserved. More than 4400 somatic embryo plants of P. pinaster and more than 3700 of P. sylvestris were regenerated from the cell lines and about 2500 of the P. pinaster and about 680 of the P. sylvestris plants were transferred to nurseries or field.
Part 2. A. Increased understanding of how somatic embryos of pine proliferate and develop. Time-lapse tracking was carried out during proliferation and development of somatic embryos in order to learn how embryo formation and development proceeds. Two major mechanisms of multiplication of early somatic embryos in pine were characterized: embryo formation through meristematic activity in the secondary suspensor, alternating with cleavage polyembryony. The multiplication rate is very high and it is difficult to stop. In order to study which conditions influence growth of embryogenic cultures, 80 different conditions were tested for Pinus pinaster. It was found that culture medium composition, subculture frequency and genotype had significant effects on proliferation rate. Best results were obtained with a one-week subculture interval, removal of plant growth regulators and maltose as carbohydrate source. B. Production of mature somatic embryos. Different methods were used for optimizing yield and quality of mature somatic embryos. In P. sylvestris, the highest number of cotyledonary somatic embryos was obtained using a pre-maturation treatment for 1 to 4 weeks on solidified PGR-free medium in combination with a maturation treatment on medium containing both ABA and polyethylene glycol (PEG). Prematuration treatment of embryogenic cultures of P. pinaster had no stimulatory effect, neither had addition of PEG to the maturation medium.
It was found that the conditions given to the cultures during the proliferation stage did not influence embryo maturation. The ability to form mature somatic embryos varied significantly among genotypes. Mature somatic embryos were partially desiccated and germinated on solidified medium. C. Performance and growth of somatic embryo plants. Plants were acclimatized and grown in a greenhouse and then transferred to the nursery. A high frequency of P. sylvestris plants did not perform as expected, but showed early plagiotropism, indicating that parts of the method should be improved. In some batches of P. pinaster plants, some clones showed altered morphology but it was also shown for in vitro germinated seedlings. Further improvement of germination and plant transfer to greenhouse are needed in order to correct this difficulty. D. Nitrogen metabolism in proliferating and developing somatic embryo plants. The expression patterns of genes involved in N metabolism during proliferation, maturation and germination of somatic embryos and during growth of regenerated plants were described. Different gene expression patterns and distribution of transcripts were found for genes encoding a key enzyme in nitrogen metabolism, glutamine synthetase.
Specific changes were associated to developmentally regulated processes such as the assembly of photosynthetic machinery (GS1a gene) and vascular differentiation (GS1b gene). We also showed that changes in the relative abundance of transcripts for key genes involved in N-remobilisation and recycling can be used to test the performance of somatic embryo plants during germination and early stages of plant growth. E. Genetic stability in embryogenic cultures and regenerated somatic embryo plants. A system which applies microsatellite regions as reporter regions to monitor genetic stability in the genome of embryogenic culture cells of P. sylvestris was developed. The current procedure of somatic embryogenesis gives rise to increased genomic instability in the studied micosatellite loci. However, the genetic stability in somatic and zygotic embryos varied among families. Investigations are under way to address the question if/how instability in the studied loci reflects alterations in functional genes.
Funding SchemeCSC - Cost-sharing contracts
751 83 Uppsala
29071 Estepona (Malaga)