Friable, embryogenic maize (Zea mays L.), inbred line A188, callus was established and maintained for more than one year without apparent loss of friability or embryogenic potential. Embryoid development was abundant in these cultures and plants were easily regenerated. Frequencies of friable-callus initiation and somatic-embryoid formation increased linearly with addition to N6 medium (C.C. Chu et al. 1975, Sci. Sin. [Peking] 18, 659-668) of up to 25 mM L-proline. Proline additions up to 9 mM to MS medium (inorganic elements of T. Murashige and F. Skoog 1962, Physiol. Plant. 15, 473-497, plus 0.5 mg 1(-1) thiamine hydrochloride and 150 mg 1(-1) L-asparagine monohydrate) did not stimulate embryoid formation. A major part of the difference between MS and N6 media could be attributed to their respective inorganic nitrogen components. L-Glutamine was not a satisfactory substitute for L-proline. Of 111 regenerated plants grown to maturity from three independent friable, embryogenic cell lines ranging in age from three to seven months, only four plants were abnormal based on morphology and pollen sterility. Seed was produced by 77% of the regenerated plants.

We obtained transgenic maize plants by using high-velocity microprojectiles to transfer genes into embryongenic cells. Two selectable genes were used to confer resistance to either chlorsulfuron or phosphinothricin, and genes encoding either E. coli beta-glucuronidase or firefly luciferase were used as markers to provide convenient assays for transformation. When regenerated without selection, only two of the eight transformed embryogenic calli obtained produced transgenic maize plants. With selection, transgenic plants were obtained from three of the other eight calli. One of the two initial lines produced 15 fertile transgenic plants. The progeny of these plants contained and expressed the foreign genes. Luciferase expression could be visualized, in the presence of added luciferin, by overlaying leaf sections with color film.

After the initial transformation and tissue culture process is complete, selectable marker genes, which are used in virtually all transformation approaches, are not required for the expression of the gene of interest in the transgenic plants. There are several advantages to removing the selectable marker gene after it is no longer needed, such as enabling the reuse of selectable markers and simplifying transgene arrays. We have tested the Cre/ lox system from bacteriophage P1 for its ability to precisely excise stably integrated marker genes from chromosomes in transgenic maize plants. Two strategies, crossing and autoexcision, have been tested and demonstrated. In the crossing strategy, plants expressing the Cre recombinase are crossed with plants bearing a transgene construct in which the selectable marker gene is flanked by directly repeated lox sites. Unlike previous reports in which incomplete somatic and germline excision were common, in our experiments complete somatic and germline marker gene excision occurred in the F(1) plants from most crosses with multiple independent Cre and lox lines. In the autoexcision strategy, the cre gene, under the control of a heat shock-inducible promoter, is excised along with the nptII marker gene. Our results show that a transient heat shock treatment of primary transgenic callus is sufficient for inducing cre and excising the cre and nptII genes. Genetic segregation and molecular analysis confirmed that marker gene removal is precise, complete and stable. The autoexcision strategy provides a way of removing the selectable marker gene from callus or other tissues such as embryos and kernels.

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We have developed a high-throughput Agrobacterium-mediated transformation model system using both nptII and the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens strain CP4 (cp4) based selections in MicroTom, a miniature rapid-cycling cherry tomato variety. With the NPTII selection system, transformation frequency calculated as independent transgenic events per inoculated explant ranged from 24 to 80% with an average of 56%, in industrial production scale transformation experiments. For CP4, with glyphosate selection, the average transformation frequency was 57%. Stable transformation frequency was positively correlated with transient expression (R=0.85), and variable with the genes of interest. DNA integration and germline transformation were confirmed by biological assay, Southern Blot analysis, and R(1) phenotype segregation. Transgene expression was observed in leaf, root, stem, flower, and fruit tissues of the transgenic plants. Ninety-five percent of transgenic events coexpressed two introduced genes based on beta-glucuronidase (GUS) and neonmycin phosphotransferase II (NPTII) expression. Seventy-five percent of transgenic events contained one to two copies of the introduced uidA (GUS) gene based on Southern analysis. Transgenic plants from the cotyledon explants to the transgenic plants transferred to soil were produced within about 2-3 months depending on the genes of interest. The utility of this MicroTom model transformation system for functional genomic studies, such as identification of genes related to important agricultural traits and gene function, is discussed.

Since the first demonstration of GFP from the jellyfish Aequorea victoria as a vital reporter for gene expression in both bacteria and Caenorkabditis elegans (Chalfie et al., 1994), GFP has attracted widespread interest and is considered to have severa1 advantages over other visual marker genes. First, the fluorescence emission of GFP does not require a cofactor or a substrate; fluorescence results from an interna1 p-hydroxybenzylidene-imidazo-lidinone chromophore generated by cyclization and oxidation of a SerTyr-Gly sequence at amino acid residues 65 to 67 (Cody et al., 1993). Detection of GFP in living cells thus only requires excitation by light at 395 or 470 nm. In contrast, the assay of GUS expression is cytotoxic, firefly luciferase (Ow et al., 1986;Millar et al., 1995) requires an exogenous substrate (luciferin) for detection, and plant anthocyanins (Klein et al., 1989;Lloyd et al., 1992) are generally useful only in mature, differentiated cells.The second advantage of GFP is that it is relatively small (26.9 kD) and can tolerate both N-and C-terminal protein fusions, lending itself to studies of protein localization and intracellular protein trafficking (Wang and Hazelrigg, 1994;Davis et al., 1995;Kaether and Gerdes, 1995). Another advantage of GFP is that GFP mutants with shifted wave-

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Over the last several decades, increased agricultural production has been driven by improved agronomic practices and a dramatic increase in the use of nitrogen-containing fertilizers to maximize the yield potential of crops. To reduce input costs and to minimize the potential environmental impacts of nitrogen fertilizer that has been used to optimize yield, an increased understanding of the molecular responses to nitrogen under field conditions is critical for our ability to further improve agricultural sustainability. Using maize (Zea mays) as a model, we have characterized the transcriptional response of plants grown under limiting and sufficient nitrogen conditions and during the recovery of nitrogen-starved plants. We show that a large percentage (approximately 7%) of the maize transcriptome is nitrogen responsive, similar to previous observations in other plant species. Furthermore, we have used statistical approaches to identify a small set of genes whose expression profiles can quantitatively assess the response of plants to varying nitrogen conditions. Using a composite gene expression scoring system, this single set of biomarker genes can accurately assess nitrogen responses independently of genotype, developmental stage, tissue type, or environment, including in plants grown under controlled environments or in the field. Importantly, the biomarker composite expression response is much more rapid and quantitative than phenotypic observations. Consequently, we have successfully used these biomarkers to monitor nitrogen status in real-time assays of field-grown maize plants under typical production conditions. Our results suggest that biomarkers have the potential to be used as agronomic tools to monitor and optimize nitrogen fertilizer usage to help achieve maximal crop yields.