Executive Summary

Our project successfully implemented Agrobacterium tumefaciens as a vehicle to deliver transgenes into maize. The optimized protocol utilizes immature maize embryos derived from the Hi-II genotype and the FDA-approved neomycin phosphotransferaseII (nptII) selectable marker gene to identify transgenic lines. Transformation frequencies average approximately 1%, which are comparable to other public sector efforts. The results of this work were presented at scientific meetings and are being prepared for scientific publication. Progress was also made in adapting Agrobacterium transformation to the H99 inbred line, which is more relevant to modern commercial germplasm than the Hi-II hybrid genotype. Efforts to optimize Agrobacterium infection parameters and media components that maintain embryogenic calli produced some transgenic events in H99 and suggest clear paths to improve future protocols. Initial attempts to develop an in planta transformation system by pre-infection of maize ears with Agrobacterium prior to pollination were not successful.

The above efforts have provided significant training opportunities to undergraduate students, graduate students, and visiting research scientists in the area of maize transformation. Transgenic maize lines were produced that are being used in studies of heat stress response, vegetative phase change, nitrogen metabolism, the influence of programmed cell death on transformation efficiency, and improved reporter genes for non-destructive monitoring of transgene activity in living tissues. These transgenic lines will continue to be valuable tools for both basic and applied research.

Project Highlights

Objective 1a: Assemble alternative strains of Agrobacterium tumefaciens.

We evaluated different Agrobacterium strains for their infection efficiency and then optimized the Type II embryogenic callus response of immature embryos infected with the best strains. Three Agrobacterium strains (C58C1/pMP90, NTL4/pTiKPS2 and Cry5/pTIEHA105) were identified that efficiently infected immature embryos from Hi-II, yet still permitted Type II embryogenic callus proliferation.

We confirmed the findings of Hansen (Molecular Plant-Microbe Interactions 13: 649) that A. tumefaciens can induce a necrotic response that negatively impacts regeneration potential. Moreover, the apoptotic-like response triggered by A. tumefaciens was alleviated in transgenic maize immature embryos expressing known negative regulators of programmed cell death, which in turn negated the impact on embryogenesis. This information suggests that strategies targeting reduction in the A. tumefaciens induced necrosis response in maize immature embryos may enhance transformation frequencies in the crop. We have generated transgenic maize lines expressing the negative regulator of programmed cell death from chicken, bcl-xl. We are now poised to directly test the hypothesis put forth by Hansen.

Information has been gleaned from the private sector that suggests immature maize embryos subjected to a brief heat shock display a reduced necrotic response following inoculation with A. tumefaciens. To test this a series of experiments were conducted investigating heat shock treatment (45°C for 5 min.) of maize immature embryos just prior to the inoculation step with either C58C1, LBA4404, or NTL4/pTiKpSF2 harboring a T-DNA with the GUS gene driven by the constitutive maize Ubiquitin1 promoter. Agrobacterium infection was monitored by transient GUS expression and transformation efficiencies were assessed for six transformation experiments by nptII ELISA and Southern blot assays. Though the heat shock treatment did not significantly increase transient expression, it did appear to improve stable transformation efficiencies. Additional experiments are in progress to confirm the beneficial effects of heat shock treatment.

Objective 1b: Expand the range of maize genotypes for Agrobacterium-mediated transformation.

A significant amount of effort has been devoted to establishing Agrobacterium-mediated transformation with a Type I regeneration system, which is more readily established in a range of different maize inbred lines. The biggest obstacle has been the variation in embryogenic culture response and severity of necrosis induced by Agrobacterium infection, which we believe is due to the slower establishment and growth of embryogenic callus in the Type I compared to the Type II system. Tests of the heat shock treatment with immature embryos from the H99 and Tom Thumb inbreds (the best at producing Type I calli) indicated similar transformation efficiencies with or without heat shock. Studies are continuing that focus primarily on modifications to media auxin concentrations and a more aggressive antibiotic regime to counter select A. tumefaciens.

We developed a protocol for Agrobacterium-mediated transformation of the maize inbred genotype H99, coupled with nptII as the selectable marker gene. Transgene integration was confirmed using molecular, biochemical, and genetic approaches, including faithful inheritance of the transgenes. To our knowledge this is the first demonstration of a public sector laboratory implementing non-"super-binary" plasmids to successfully deliver transgenes directly to a maize inbred genotype using nptII as a selectable marker. It is noteworthy that the H99 Type I Agrobacterium-mediated protocol has only been successful with one A. tumefaciens strain, Cry5/pTiEHA105.

Objective 2: Evaluate selectable marker genes for use in Agrobacterium-mediated maize transformation

We initiated a project aimed at investigating the utility of the cyanamide hydratase (cah) gene as a selectable marker. Though the results do not indicate the cah gene will be useful as a selectable marker, the transgenic lines that were generated will be subsequently evaluated for their potential in using cyanamide as an alternative fertilizer and herbicidal compound for the maize crop.

We have also synthesized a series of visual marker genes where codon usage has been optimized for expression in maize. These include green fluorescent protein (gfp), blue fluorescent protein (bfp), cyan fluorescent protein (cfp) and yellow fluorescent protein (yfp). The activity of these reporter genes can be monitored in living tissues, making them particularly useful for studies of regulatory sequence activities and protein localization. A set of expression cassettes has been assembled with the various fluorescent protein genes under the control of an array of different promoter elements. These constructs will be made available to the maize community upon request.

Objective 3: Investigate approaches for in planta maize transformation.

A maize transformation system that bypassed the requirement for a tissue culture step would represent a "quantum leap" in transformation efficiency. Attempts were made to adapt to maize the in planta transformation system developed for Arabidopsis, where immature carpels are infected with Agrobacterium that transforms early embryos. The silks (stigmas) of maize ears were incubated with Agrobacterium solutions the evening prior to pollination the next morning. It was established that Agrobacterium capable of infection remained on the silks for a period of days provided the temperature remained below 30oC (hence the evening treatments) and that adequate seed sets could be obtained after Agrobacterium treatments. Over 2000 seeds from Agrobacterium-treated ears were germinated and assayed for transgene expression based on complementation of the glossy15 mutant seedling phenotype; however, none were observed to express the transgene. Though these initial results were discouraging, modifications to the infection procedure coupled with determinations of the ability of Agrobacterium to infect pollen tubes and early embryos (using a GUS reporter gene construct) may make in plant maize transformation a future possibility.

Publications and Abstracts

• Moose, S.P (2001) Maize transformation in an academic research laboratory. Talk presented at the Plant and Animal Genome IX Conference, San Diego, CA.
• Moose, S.P. (2002) Gain-of-function analyses of Glossy15 and its role in regulating phase change in the leaf epidermis. Talk presented at 44th Annual Maize Genetics Conference, Orlando, FL.
• Sato, S., Moose, S., Clemente, T. (2002) Agrobacterium-mediated transformation of maize. Poster abstract presented at 44th Annual Maize Genetics Conference, Orlando, FL.
• Lauter, N., Kampani, A., Carlson, S., Goebel, M. and Moose, S.P. (2004) microRNA172 downregulates glossy15 to promote vegetative phase change in maize. Proc. Natl. Acad. Sci., in press
• Sato, S., Utomo, D., Moose, S.P., and Clemente, T. Agrobacterium-mediated transformation of maize using non super-binary T-DNA plasmids. In preparation for submission to Plant Cell Reports.

Maize Transgenic Lines

The support provided by this IMBA project has resulted in the production of maize transgenic lines that will be valuable for future maize research. A brief listing of thee transgenes and the number of lines generated is provided below:

• glossy15 regulator of leaf identity traits, 7 lines
• heat shock protein22 (hsp22), 15 lines
• alternative oxidase1 (aox1), 12 lines
• alternative oxidase3 (aox3), 17 lines
• rice actin1 promoter-gfp, approximately 25 lines
• 35S CaMV promoter-gfp, approximately 20 lines
• Ubiquitn1 promoter-cyanamide hydratase, 15 lines
• Ubiquitin1 promoter-bcl-xl apoptosis inhibitor, 7 lines
• 35S CaMV promoter-Rubisco small subunit RNAi construct, approximately 5 lines
• A number of other transgenic lines where different promoters are driving the expression of the GUS reporter gene in both the Hi II and H99 genotypes.

Leveraged Research Support from IMBA Funding

The monies provided by the IMBA program permitted our groups to establish in-house Agrobacterium-mediated maize transformation capabilities using Type II callus regeneration regime. This in turn allowed us to assist other maize laboratories, outside our home institutions, in studies requiring genetic engineering. To this end we have provided maize transformation services in support of two funded maize programs, "Bioengineering stress resistant maize" (IMBA project: K. Newton, Univ. of Missouri and T. Elthon, U. of Nebraska, PIs) and "From Proplastid to chloroplast: Understanding plastid differentiation in maize through microarray and proteome analysis" (NSF project: K. van Wijk, Cornell University, PI).

Training

Dr. Clemente participated as an instructor at the National Science Foundation's Maize Transformation Workshop held at Madison, Wisconsin in March, 2003. This workshop gave approximately 20 participants hands-on training in maize transformation protocols.

Through the IMBA-supported project the Moose lab has trained two undergraduates, two graduate students, and two postdoctoral research associates in maize transformation protocols, from dissection of immature embryos to harvesting of transgenic seed. This training serves to build future capacity for maize transformation technology in the public sector as well as provide employees to agricultural biotechnology companies. As one example, Ms. Hena Guo recently completed her M.S. degree in the Moose lab and now has a position with Pioneer Hi-Bred International to help characterize transgenic maize lines with altered seed traits.

The Clemente lab at Nebraska has two post doctorate associates and two research technologists fully trained in all aspects of maize transformation, including stock plant maintenance, A. tumefaciens handling, embryo isolation and culture, and characterization of transgenic plants.