Isolation and evaluation of three novel native promoters in Brassica napus

To provide effective and specific native promoters for canola (Brassica napus L.) genetic modification, three promoters were isolated by genome walking from this species. These three promoters were fused to the uidA reporter gene (GUS) and were independently used to generate populations of transgenic canola plants. Plants transformed with BnPGPro-GUS (B. napus putative germin promoter) exhibited GUS activity in all the tissues tested at a level comparable to those transformed with CaMV35 S promoter. This indicates that BnPGPro may serve as a native constitutive promoter for canola. The other two promoters, BnPro3-GUS and BnPro5-GUS (B. napus, promoter 3 and 5), exhibited GUS activity in various tissues. None of these two promoters expressed in embryo, however. These novel Brassica native promoters can be used to modify canola genes for various purposes.

Microprojectile-mediated genetic transformation and regeneration of Chinese elm

A protocol for the genetic transformation and regeneration of Chinese elm ( Ulmus parvifolia Ja cq.) is reported. Cell suspension cultures were initiated from hypocotyls and were maintained on Murashige and Skoog (MS) basal semi-solid medium supplemented with varying concentrations of thiadiazuron (TDZ) and 1-naphthaleneacetic acid (NAA). Two week- old cell suspension cultures were bombarded with gold particles coated with plasmid harboring the Basta®resistance and â -glucuronidase genes. Elm cultures produced green calli within a month on selection media containing 20 mg L-1phosphinothrycin. These c alli were transferred to the same medium without the herbicide and were regenerated into shoots within 2 mo. The expression of uidA reporter gene in transformed elms was detected by histochemical analysis for â-glucuronidase activity. The presence of the Basta®resistance insert was demonstrated by polymerase chain reaction analysis. Key words:

An efficient Biolistic® transformation protocol for Picea abies embryogenic tissue and regeneration of transgenic plants

An efficient Biolistic® transformation technology was developed to stably transform Picea abies (L.) Karst. Several embryogenic tissue lines were tested for proliferation on standard embryogenesis media. Transient transformation studies with these lines were performed to optimize the parameters for genetic transformation. Selection conditions for transgenic tissue based on the nptII resistance gene in combination with the antibiotic geneticin were defined such that only transgenic P. abies lines were able to develop. Nontransgenic tissue was completely inhibited under these conditions. Stable integration of a uidA reporter gene and a nptII resistance gene into the genome of P. abies was achieved and more than 200 mature embryos were regenerated for every transformation event. Histochemical and fluorometric analysis indicated strong expression of the uidA gene in transgenic material. ELISA studies to detect and quantify the nptII gene product as well as polymerase chain reaction and Southern blotting confirmed the presence and integration of uidA and nptII genes into the P. abies genome. Transgenic P. abies plants from nine independent transformation events were recovered and are currently growing in a greenhouse for genetically modified organisms, awaiting field release.

A transient assay for evaluating promoters in wheat endosperm tissue

A transient assay was developed for the evaluation of promoter sequences in wheat endosperm tissue. A deletion series from an omega-secalin gene promoter, located on chromosome 1RS.1DL of specific wheat lines, were translationally fused to a uidA reporter gene. These promoters were evaluated for expression in wheat endosperm tissue after integration of the DNA into the cell using microprojectile bombardment. The results were compared with those obtained using other transient assay systems.Key words: particle bombardment, transient assay, omega-secalin gene, wheat endosperm.

Discrete spatial and temporal cis-acting elements regulate transcription of the Arabidopsis floral homeotic gene APETALA3

The APETALA3 floral homeotic gene is required for petal and stamen development in Arabidopsis. APETALA3 transcripts are first detected in a meristematic region that will give rise to the petal and stamen primordia, and expression is maintained in this region during subsequent development of these organs. To dissect how the APETALA3 gene is expressed in this spatially and temporally restricted domain, various APETALA3 promoter fragments were fused to the uidA reporter gene encoding beta-glucuronidase and assayed for the resulting patterns of expression in transgenic Arabidopsis plants. Based on these promoter analyses, we defined cis-acting elements required for distinct phases of APETALA3 expression, as well as for petal-specific and stamen-specific expression. By crossing the petal-specific construct into different mutant backgrounds, we have shown that several floral genes, including APETALA3, PISTILLATA, UNUSUAL FLORAL ORGANS, and APETALA1, encode trans-acting factors required for second-whorl-specific APETALA3 expression. We have also shown that the products of the APETALA1, APETALA3, PISTILLATA and AGAMOUS genes bind to several conserved sequence motifs within the APETALA3 promoter. We present a model whereby spatially and temporally restricted APETALA3 transcription is controlled via interactions between proteins binding to different domains of the APETALA3 promoter.

Transformation of an Australian Variety of Carica papaya Using Microprojectile Bombardment

We have developed a method for the stable transformation and regeneration of a dioecious Australian cultivar of Carica papaya (papaw or papaya) by microprojectile bombardment. This method was developed after investigation of both zygotic and somatic embryos as target tissue and optimisation of a number of parameters using transient expression of the uidA reporter gene. The tissue culture regime prior to bombardment was critical in optimisation of transformation. Factors such as age of embryos and various treatments prior to bombardment, increased transient expression by up to 22-fold. Highest uidA transient expression results were obtained when somatic embryos 3 weeks since last subculture on solid medium were given a 3 day treatment in liquid medium, and a 2 h osmotic treatment pre- and post-bombardment. Stably transformed plants were obtained 6 months after bombardment using this system. Transformation efficiency was high with two experiments yielding 45% and 37.5% of bombarded plates regenerating plantlets on media containing kanamycin. The presence of both the uidA and aphA genes, (selectable marker) which code for the enzymes β-glucuronidase (GUS), and neomycin phosphotransferase II (NPTII) respectively, was confirmed in regenerated plantlets by Southern hybridisation.