SOMATIC EMBRYOGENESIS IS A RAPIDLY DEVELOPING FIELD THAT SHOULD ALLOW PLANT EMBRYOLOGY TO CATCH UP WITH THE MORE ADVANCED ANIMAL EMBRYOLOGY. SOMATIC EMBRYOGENESIS ALSO IS A NECESSARY PATHWAY FROM IN VITRO-CULTURED PLANT CELLS TO A REGENERATED ADULT PLANT, AND ITS SUCCESSFUL COMPLETION THEREFORE CONSTITUTES A MAJOR LIMITING FACTOR BEFORE ANY GENETIC MODIFICATION CAN BE TRANSFERRED FROM THE TEST TUBE TO THE FIELD.
NOT ALL SPECIES ARE AMENABLE TO THIS SORT OF REGENERATION AND A BETTER UNDERSTANDING OF THE PROCESS COULD HELP MAKE RECALCITRANT SPECIES ABLE TO REGENERATIE.
A study has been made of regeneration through somatic embryogenesis in Daucus carota (carrot), a plant species that regenerates by the production of somatic embryos from suspension cultured cells. In order to characterize the critical steps in carrot somatic embryogenesis, mutants impaired in somaticembryogenesis, mutants impaired in somatic embryogenesis were obtained and analysed. The objective was to identify genes and their encoded products that were essential to the process of somatic embryogenesis.
The approaches that were used were:
the isolation and characterization of temperature sensitive mutants with impaired embryogenesis;
the complementation of these mutants and drug induced phenocopies of these mutants with purified, secreted glycoproteins;
the production of antisera and complementary deoxyribonucleic acid (cDNA) clones corresponding to these and other extracellular glycoproteins;
the molecular analysis of these clones and antibodies.
Temperature sensitive mutant cell lines with impaired somatic embryogenesis were isolated and tested for their ability to be rescued by the addition of extracellular glycoproteins at the nonpermissive temperature. One of these cell lines, ts11, was unable to properly glycosylate the extracellular proteins, and could be rescued by an extracellular 32 kDa protein. The protein responsible for the rescue of somatic embryogenesis in the presence of the glycosylation inhibitor tunicamycin was also purified; it was a 38 kDa cationic isoperoxidase. Several other proteins were identified that increased the number of embryos obtained and, in some cases, restored embryogenic potential in nonembryogenic suspension cultures. cDNA clones were isolated by expression screening of cDNA libraries with antibodies raised against other gel excised extracellular proteins. One of these was shown to be an absolute marker of embryogenic potential in suspension cultures and shown by in situ hybridization to be only expressed in the L1 layer of shoot apical meristems of carrot seedlings. It was concluded that several different extracellular glycoproteins of embyrogenic carrot suspension cultures have different promoting effects on somatic embryogenesis. Some of these proteins may have a function in the meristems in intact plants.
A GENETIC DISSECTION OF DEVELOPMENT WILL BE ATTEMPTED, USING PRESENT AND NEWLY ISOLATED CARROT CELL MUTANTS, UNABLE TO COMPLETE NORMAL EMBRYOGENESIS AT NON-PERMISSIVE TEMPERATURE. THE CHARACTERIZATION OF SUCH MUTANTS WILL BE MADE IN 3 STEPS : DEFINITION OF THE STAGE AFFECTED, DEFINITION OF THE DOMINANCE-RECESSIVENESS OF THE MUTATIONS, ANALYSIS OF THE ALTERED PROTEIN PATTERN IN BI-DIMENSIONAL GEL ELECTROPHORESIS.
ANTIBODIES WILL BE MADE AGAINST THE RELEVANT PROTEINS, AND CORRESPONDING GENES WILL BE CLONED.
THE ITALIAN GROUP WILL FOCUS ON SOMATIC CELL GENETICS AND DEVELOPMENTAL BIOLOGY, WHEREAS THE DUTCH GROUP WILL CONCENTRATE ON MOLECULAR BIOLOGY.