Multiplication by somatic embryogenesis is a part of forest tree breeding programmes. It allows fast production of large numbers of copies of a selected genotype. Nevertheless, with conifers, its application is limited to immature zygotic embryos and to young cotyledons. Only cotyledons taken from 7 day old seedlings will differentiate embryogenic calli. Older cotyledons or needles taken from the same plantlet are unable to differentiate embryogenic calli. This is due to differences in the expression of the genome of young and mature cotyledons, of young cotyledons and young needles and, of embryogenic and non embryogenic calli.
The project is concerned with the study of morphological and biochemical markers of somatic embryogenesis. As well as improving our understanding of the mechanisms involved, the results should allow us to extend the application of somatic embryogenesis to even older conifer needles.
The aims of the work are to provide a better understanding of the somatic embryogenesis in Picea abies to raise somatic embryos from mature tree tissues, and to optimize the culture conditions necessary for practical application of the method.
The main steps of the work can be summarized as follows.
Preparation of embryogenic and non-embryogenic plant cells and tissues.
Embryogenic (EC) and non-embryogenic (NEC) calli differentiated from the same genotype from old and young cotyledons and needles. Tissues will be taken from clones which have shown their cotyledons embryogenic potentialities at a very young stage. Therefore it will be possible to identify markers related to embryogenic competence independently of the genotype and the type and age of the tissues.
Research of protein markers.
Quantitative and qualitative analysis of the soluble and insoluble proteins. Attention will be focused on the membrane and storage proteins along the maturation of the tissue in relation to embryogenic competence.
Comparative study of the cytoskeleton components (microtubuli and microfilaments) and the cytoplasmic distribution of the organelles (chloroplasts and mitochondria) in EC and NE calli.
Localisation of the embryogenic cells and the protein synthesis sites.
Estimation of gene activity.
Quantitative and qualitative study of the mRNA translation products. It will be possible to determine patterns of gene expression related to embryogenesis processes and to identify the specific RNA transcripts products involved in somatic embryogenesis.
Microsequencing of these products in the same way as for the proteins markers. Comparison with known sequences may enable possible functions to be assigned to embryogenesis specific polypeptides.
Study of non-random rearrangements of mitochondrial genome during in vitro tissue cultures in relation to morphogenetic capacities.
To detect these modifications, the mtDNA genome will be cloned and characterized. A mtDNA library will be constructed in a cosmid cloning vehicle. Classes of repeated sequences will be defined. The RFLP studies will be achieved at the level of competent and non competent cotyledons and needles and in embryogenic and non embryogenic calli derived from them. mtDNA restriction patterns correlated with embryogenic capacities will be defined.
Utilisation of the markers to promote somatic embryogenesis from needles of elite trees
Characterisation of the biochemical markers in order to obtain a better understanding of their role in the metabolic pathways leading to embryogenesis from needles of old trees.
Funding SchemeCSC - Cost-sharing contracts
CV35 9EF Warwick
106 91 Stockholm