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In this paper we describe the isolation and characterization of a genomic clone (Bp4) from Brassica napus which contains three members of a pollen-specific multigene family. This family is composed of 10 to 15 closely related genes which are expressed in early stages of microspore development. The complete nucleotide sequence of the clone Bp4 and of three homologous cDNA clones is reported. One of the genes (Bp4B) contained in the genomic clone is believed to be non-functional because of sequence rearrangements in its 5' region and intron splicing sites. The remaining genes (Bp4A and Bp4C), as well as the cDNA clones, appear to code for small proteins of unique structure. Three different types of proteins can be predicted as a result of the deletion of carboxy or amino terminal portions of a conserved core protein. These proteins all share a common alternation of hydrophobic and hydrophilic domains. A fragment of the genomic clone containing the gene Bp4A, as well as the non-functional gene Bp4B, was introduced into tobacco plants via Agrobacterium-mediated transformation. The functional gene Bp4A is expressed in transgenic tobacco plants and shows spatial and temporal regulation consistent with the expression patterns seen in Brassica napus.

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Antisense RNA was used to specifically inhibit the expression of a GUS gene introduced in a transgenic plant. A tobacco transformant containing a single intact copy of the GUS gene and showing relatively high constitutive levels of GUS activity (GUS +) was re-transformed with an Agrobacterium Ti-derived binary vector containing an antisense version of this reporter gene. The sense and antisense GUS genes were each under the regulation of the CaMV 35S promoter. Re-transformed plants contained 1-5 copies of the antisense construct and all showed a greater than 90% reduction in GUS activity relative to the original GUS + plant. This reduction in GUS activity correlated closely with the levels of GUS enzyme and steady state GUS mRNA observed in these plants. The relatively low levels of sense and antisense GUS transcripts found in the re-transformed plants may indicate a rapid degradation of the RNA:RNA duplex in the cell.

The genomic clone for BN115, a low-temperature-responsive gene, was isolated from winter Brassica napus and its sequence was determined. A 1.2-kb fragment of the 5' regulatory region (from bp -1 107 to +loo) was fused to the &glucuronidase (GUS) reporter gene and EN1 15-promoted GUS expression was observed in green tissues of transgenic E. napus plants only after incubation at 2°C. No expression was observed after incubation at 22"C, either in the presence or the absence of ABA. Microprojedile bombardment of winter B. napus leaves with a EN115 promoter/ GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the lowtemperature regulation of B N I 15 expression involves a possible enhancer region between bp -1107 and -802 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated cauliflower mosaic virus 35s promoter suggest that the minimal size required for any maintenance of low-temperature GUS expression is a -300-bp fragment. Within this fragment are two 8-bp elements with the sequence TCGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5-bp core sequence in the low-temperature-and dehydration-responsive elements identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes.

Oxalic acid is an important pathogenic factor for the fungus Sclerotinia sclerotiorum (Lib.) de Bary. An oxalate degrading enzyme, oxalate oxidase (OxO), in transgenic soybean [Glycine max (L.) Merr.] has reduced pathogen growth in indoor seedling studies. The objective of this study was to characterize the response of this OxO transgenic soybean line to white mold under field conditions and also to characterize the agronomic performance of this transgenic line under noninfected conditions. The transgenic line 80(30)‐1contains the wheat germin gene gf‐2.8 that codes for OxO. This line was compared with 80(30)‐9, a sib line which does not contain the gene, as well as resistant and susceptible cultivars. Field tests were conducted at three sites infested with white mold in Ontario and Quebec across 3 yr. The Sainte‐Foy site provided the highest infection potential and the transgenic line had a disease severity index (DSI) of 7 compared with 80(30)‐9 with a DSI of 46, across 3 yr. Across 2 yr, resistant commercial cultivars had an average DSI of 2 and susceptible cultivars a DSI of 46. Stem inoculations were performed in the field at Elora and the transgenic line was significantly less infected compared with 80(30)‐9. In noninfested trials, no significant differences were found between the transgenic, the negative sib, and the parental lines for seed yield, maturity, seed weight, or seed protein and oil content. The transgene provided white mold resistance equivalent to the best commercial cultivars in a white mold susceptible background.

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