Leslie Cass

Isolation and characterization of a polygalacturonase gene highly expressed in Brassica napus pollen

A cDNA clone, Sta 44-4, corresponding to a mRNA highly expressed in Brassica napus cv. Westar stamens, was isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 44-4 is synthesized in pollen beginning at the late uninucleate stage and reaches a maximum in trinucleate microspores. Sta 44-4 displayed significant sequence similarity to known pollen polygalacturonase genes. The B. napus pollen polygalacturonase gene was shown to be part of a small gene family and to display some polymorphism among different cultivars.

Reduced herbivory of the European corn borer (Ostrinia nubilalis) on corn transformed with germin, a wheat oxalate oxidase gene

Abstract The strategy of targeting expression of a constitutively regulated gene to generate H 2 O 2 in the extracellular matrix, to reduce herbivory of the European corn borer (ECB) [ Ostrinia nubilalis (Hubner)] was tested by transforming corn with germin, a wheat oxalate oxidase (OXO) gene, regulated by the rice actin promoter elements (pAct-OXO). With two independent transformation events, enzyme activity was stable over seven generations of backcrossing into three maize inbred lines. Enzyme activity remained associated with the cell wall debris fraction of water extracted tissues. Leaf tissue of the germin transgenics had elevated levels of H 2 O 2 . In vitro leaf feeding bioassays demonstrated that ECB larvae feeding was significantly reduced and larval growth and development were delayed on all ECB infested germin transgenic lines. This reduced ECB feeding was confirmed under field conditions. Most significantly, stalk tunneling damage, measured at plant harvest, was substantially reduced in all germin transgenic lines. The reduction of tunneling by 50% in the transgenic lines is indicative of lower levels of ECB survival which should be significant in ECB epidemiology. Possible mechanisms of resistance include modifications in plant cell wall chemistry, activation of pathogen resistance genes and effects of H 2 O 2 and germin on insect physiology are discussed.

Developmental and environmental regulation of anthocyanin pigmentation in wheat tissues transformed with anthocyanin regulatory genes

SummaryCell autonomous anthocyanin pigmentation, produced by the anthocyanin regulatory genes B and C1 controlled by the constitutive CaMV35s promoter (pBC1-7), was used to optimize biolistic gene delivery into embryogenic wheat (Triticum aestivum L. cv ‘Chris’) scutellum cultures. Intensely pigmented callus cells were observed 24 h postbombardment but these cells did not continue to divide and were developmentally terminal. A population of nonexpressing cells generated transgenic sectors which showed light-dependent anthocyanin pigmentation. Anthocyanin pigmentation was suppressed in regenerating shoot cultures but reverted to light-dependent production in the pericarp of developing seeds. Similarly, following microtargeted gene delivery into apical meristems, anthocyanin production was developmentally suppressed in leaf base meristems but prominent anthocyanin sectors developed in mature tissues beyond this region and persisted throughout leaf growth. In three developmental situations, callus proliferation, plant regeneration, and leaf growth, perpetuation of cells with anthocyanin regulator genes under the control of constitutive promoters was dependent on a higher level of regulation to suppress pigmentation at developmentally sensitive stages of meristematic activity. These findings provide additional evidence that the anthocyanin regulatory genes may be responsive to a variety of developmental and environmental stimuli.

Oxalate oxidase: a novel reporter gene for monocot and dicot transformations

A wheat germin gene, with oxalate oxidase (OxO) activity, can be used as a sensitive reporter gene in both monocot and dicot transformations. Detection of H2O2 generated from OxO oxidation of oxalate provides simple, rapid detection of gene expression. Inexpensive substrates are required for both assays. OxO activity, could be detected histochemically in minutes, without chlorophyll clearing procedures. This assay was used to optimize transformation procedures and to track stable transgene expression in breeding populations over many generations. A simple spectrophotometric quantitative enzyme activity assay was used to select lines with various levels of transgene expression and to monitor transgene silencing phenomena. The quantitative OxO assay can also be used as an internal DNA delivery standard with a second reporter gene used in gene expression studies. The simplicity of the assay is ideal for screening large populations to identify primary transgenics, for monitoring transgene segregation in large populations in field studies and for assessing stability of transgene expression over numerous generations.

Molecular analysis of two Brassica napus genes expressed in the stigma.

A partial cDNA clone, Pis 63, corresponding to a mRNA highly expressed in Brassica napus pistils, was isolated by differential screening. PCR was used to complete the Pis 63 sequence (Pis 63-1) and to obtain the sequence of another related cDNA (Pis 63-2). Northern blot and in situ analyses demonstrated that these transcripts are expressed in the stigma throughout flower development. Pis 63-1 and Pis 63-2 display similarity to a cotton fibre cDNA clone.