T-DNA tagging In Brassica carinata with a promoterless gus : NPTII gene fusion vector

Abstract

An efficient system for or 'Agrobacterium'-mediated transformation of Brassica carinata was used together with a promoterless gus::nptII gene fusion to isolate putative promoter sequences. Cotyledonary petioles were transformed using the promoterless gene fusion construct. Only transformation events in which the promoterless gene fusion had integrated downstream from plant regulatory sequences were expected to produce viable tissue under kanamycin selection. Forty-two transgenic plants were recovered. Transformation efficiency was approximately 0.6%. Regenerated plants were screened for GUS expression in different tissues and organs by histological and fluorometric assays. Tissue-specific GUS expression was detected (stigmas, seed coat, leaf edges and vascular tissue) in some plants, while strong constitutive GUS expression was detected in others (based on GUS histological assays). Using subgenomic libraries, putative promoter fragments were isolated from the plants which exhibited GUS expression in stigmas, leaf edges and constitutively. A putative promoter fragment from a plant which exhibited GUS expression only in the stigma was fused with the gus gene and reintroduced by Agrobacterium -mediated transformation into B. napus, B. carinata, Arabidopsis' and tobacco . GUS expression was observed in the stigma of B. napus but not in ' B. carinata'. In Arabidopsis and tobacco GUS expression. was not tissue specific (weakly constitutive or restricted to two or more tissues). The 3' DNA sequence (15 kb) flanking the gus::nptII insert in the plant with GUS expression in the stigma was also isolated using a subgenomic library. A gene for a cytochrome P450 like protein was discovered on the minus DNA strand of the 3' sequence with a start codon approximately 6.5 kb from the T-DNA left border.