Improvemed methods for cloning and mapping plant genes by DNA-tagging

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In Agrobacterium-mediated trans formation of a plant, eventual insertion of the T-DNA into a functional gene caused a mutation giving a recessive phenotype and being detectable in the progeny of the primary tranformant, and then the T-DNA-tagged gene can be isolated by using the inserted T-DNA as a probe. Since a large number of transformants are necessary to be screened for detecting T-DNA insertional mutations, an efficient transformation system must be employed for plant material.In this study, tomato and Arabidopsis thaliana were chosen as materials, and suitable transformation systems were established for the both species. Using a tomato plant that carries a part of the genome of Lycopersicon peruvianum as a material, it was confirmed that acetosyringon, when added to the medium for inoculation of Agrobacterium, showed a promoting effect on transformation. In Arabidopsis, root segments of ecotype Wassilewskija were found to be and excellent material for transformation.A mutant characteristic of abnormal flowers on the top of the stems were found among transformants of Arabidopsis. Results from genetic analysis and Southern hybridization suggested that the mutation was caused by a T-DNA insertion into an allele that had been described as "TERMINAL FLOWER" gene. A chromosomal DNA fragment franking the inserted T-DNA was cloned, and using the fragment as a probe, DNA clones having the homologous sequence were selected from a genomic DNA library of the wild type plants.Besides of these studies, a binary vector for screening promoters of plant genes was constructed, and obtained a result supporting its usefulness in a transformation experiment.Using FISH (fluorescent in situ hybridization), in A.thaliana, the 180 bp family of repetitive sequences and a 10 kb single-copy sequence were successfully localized on specific chromosomes.