GENETIC TRANSFORMATION AND GENE EXPRESSION IN WHITE PINE (PINUS STROBUS)

IN RECENT YEARS, CONSIDERABLE ATTENTION HAS BEEN FOCUSED ON THE DEVELOPMENT OF TECHNIQUES FOR GENETIC TRANSFORMATION OF HIGHER PLANTS. MOST ADVANCES HAVE BEEN MADE USING EITHER PETUNIA OR NICOTIANA AS A MODEL EXPERIMENTAL SYSTEM, MAINLY BECAUSE THESE PLANTS RESPOND WELL FOR REGENERATION IN CELL CULTURE. THERE IS AN OBVIOUS NEED TO EXTEND THESE STUDIES TO OTHER SPECIES, FOREST TREES IN PARTICULAR, WHICH POSE UNIQUE PROBLEMS FOR REGENERATION IN CELL CULTURES. THE TWO MAIN OBJECTIVES OF THE PROPOSED STUDY ARE: TO DEVELOP PROTOCOLS FOR TRANSFORMATION OF WHITE PINE (PINUS STROBUS) EMBRYONIC TISSUE; AND TO ANALYZE THE REGULATION OF FOREIGN GENE EXPRESSION BY LIGHT IN PINUS STROBUS. THE PROPOSED STUDIES WILL INVOLVE BOTH THE DEVELOPMENT OF PROTOCOLS FOR TRANSFORMATION AS WELL AS GATHERING OF INFORMATION ON REGULATION OF GENE EXPRESSION IN THE TRANSFORMED PLANTS. SELECTED GENOTYPES OF EASTERN WHITE PINE WILL BE USED FOR GENETIC TRANSFORMATION BY AGROBACTERIUM TUMEFACIENS STRAINS CONTAINING SPECIFIC MARKER GENES WHICH WILL ALLOW AN EASY AND RELIABLE SYSTEM FOR SELECTION OF THE TRANSFORMANTS, AND THE STUDY OF CONSTITUTIVE AS WELL AS LIGHT-INDUCED REGULATION OF GENE EXPRESSION. A TUMEFACIENS STRAINS TO BE USED IN THIS STUDY CONTAIN CODING SEQUENCES OF TWO BACTERIAL GENES, NEOMYCINE PHOSPHOTRANSFERASE (NPTII) AND CHLORAMPHENICOL ACETYL TRANSFERASE (CAT), COINTEGRATED WITH PROMOTERS OF EITHER AGROBACTERIUM OR PLANT ORIGIN. THE TRANSFORMED SHOOTS WILL BE SELECTED BY THEIR ABILITY TO GROW ON KANAMYCIN (NPT GENE RESPONSE) AND ANALYZED FOR ENZYME ACTIVITIES OF NPT AND CAT. THE COVALENT INTEGRATION OF THESE SEQUENCES IN THE DNA OF PLANT CELLS WILL BE CONFIRMED BY SOUTHERN HYBRIDIZATION OF GENOMIC DNA WITH SPECIFIC PROBES FOR EACH GENE.