The Concerted Action brings together workers on molecular breeding of fruit trees, including transformation techniques development, pilot tests on transformation with available disease resistance genes, cloning of target genes from fruit trees and study of cell and tissue-specific gene expression.
A. Transformation Technique Development
Previous work performed independently at East Malling and Angers has shown that it is possible to obtain transgenic plants of apple by Agrobacterium-mediated transformation methods. Although numerous transgenic apple plants have presently been obtained, the inherent low efficiency of the method needs to be increased in order to make it a routine technique. Efforts are now underway in East Malling, Angers and in Leuven, to improve the method using an intron-containing B-glucuronidase (GUS) marker gene that can easily be scored.
The knowledge that will result from a systematic analysis of all factors contributing to transformation efficiency will be combined with the established experiences of the group members on organogenesis of different fruit tree cultivars, so that adapted transformation protocols can be devised for each cultivar.
For pear, quince, plum, apricot and kiwi fruit, Agrobacterium will be co-cultivated with leaf, cotyledon or immature embryo explants that can regenerate shoots, while cherry transformation will be based on embryogenic callus derived from roots.
In addition, an alternative transformation method will be developed that is based on electroporation of protoplasts, as previously used for kiwi fruit. Angers has already successfully developed protocols to regenerate mature plants from protoplasts of different apple, pear and cherry cultivars. This methods makes possible the regeneration of plants from single cells, thereby avoiding the formation of chimaeras.
Recent advances using the 'particle gun' (biolistics) whereby foreign DNA may be literally fired into cells, will also be tested. If stable expression can be achieved this method makes the process of transformation applicable to almost all fruit tree cultivars.
B. Pilot Tests on Transformation
The major pests and diseases from which fruit trees suffer are caused by insects (e.g. aphids, codling moth), fungi (e.g. Venturia spp., Podosphaera spp.), bacteria (e.g. Erwinia spp.) and viruses (e.g. plum pox virus). Except for bacteria and virus, control of these diseases is currently performed by spraying with pesticides and fungicides (up to 20 treatments per year for apple).
Genes are currently available that may be inhibitory to each category of disease-causing organisms (see references in section 3). These genes will be inserted in apple under the control of a strong constitutive promotor, and the resulting transgenic plants will be tested for improved resistance or tolerance.
If successful for apple, the transfer of resistance genes will be repeated on other fruit tree species.
Funding SchemeCON - Coordination of research actions
AA - 6700 Wageningen
DE3 1GB Derby