RNA-RNA in situ hybridization using digoxigenin-labeled probes: the use of high-molecular-weight polyvinyl alcohol in the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction.

A nonradioactive RNA-RNA in situ hybridization method using digoxigenin-labeled probes is described. The visualization of hybrids is done using the indoxyl-nitro blue tetrazolium alkaline phosphatase reaction. The addition of polyvinyl alcohols of high molecular weight (40-100 kDa) to the reaction medium enhances the alkaline phosphatase reaction and prevents diffusion of reaction intermediates, resulting in a 20-fold increase in sensitivity without increasing the background. Due to the more localized precipitation of the formazan, the site of hybridization can be determined more precisely.

Two T-DNA's co-transformed intoBrassica napus by a doubleAgrobacterium tumefaciens infection are mainly integrated at the same locus.

Hypocotyl explants of threeBrassica napus varieties were infected with two nopaline typeAgrobacterium strains each carrying a distinct disarmed T-DNA containing different selectable markers. Selection was done for only one of the markers, after which the regenerated plants were screened for the presence of the second marker. High co-transformation frequencies of both T-DNA's were obtained (39%-85% of the transformants). Where the two T-DNA's were integrated linked, they were usually present in an inverted orientation relative to each other; in all of the cases observed the two right borders were adjacent. Tandem orientations occurred less frequently. The T-DNA's were mainly integrated as intact copies and deletions did not often occur. The co-transformation system described favors a genetically linked integration of the two T-DNA's (78%), although in a single transformed plant both linked and unlinked copies of both T-DNA's may be present.