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We have studied the activity of a wheat histone H4 promoter in transgenic maize plants produced by direct DNA uptake into embryogenic cell suspension protoplasts. Expression patterns revealed by fluorimetric as well as histochemical GUS reporter enzyme assays and Northern hybridisation indicated the cell division-dependent expression of the GUS gene driven by the histone H4 promoter in proliferating cells and meristems. Changes in gene expression associated with the progression through the cell cycle in maize cell-suspension cultures were also investigated after partial synchronisation of cell division by aphidicolin or hydroxyurea. The transgene expression regulated by the wheat histone H4 promoter coincided with elevated levels of mRNAs from other S phase-associated genes such as maize histone H4, histone H2B and proliferating cell nuclear antigen (PCNA). The reporter gene expression was low during mitosis when accumulation of the transcripts of a B-type mitotic cyclin CycB1;zm;1 could be detected. The presented data indicate that the used 720-bp-long promoter region can provide replication-dependent expression for the GUS reporter gene in transgenic maize. In these maize plants, external hormone stimuli generated by a synthetic auxin 2,4-dichlorophenoxy acetic acid (2,4-D) or abscisic acid (ABA) have modified the histone H4 promoter-driven GUS gene transcription. These findings support the usefulness of the H4::GUS transgenic plants for further studies on cell cycle activation/inactivation by mitogenic or stress related stimuli in maize.

Direct uptake of reporter gene constructs with the bacterial beta-glucuronidase (GUS) gene fused to various promoters was achieved to embryogenic cell suspension culture-derived protoplasts of GK Ságvári winter wheat (Triticum aestivum L.) with polyethylene glycol (PEG) treatment. Based on GUS specific activity values, it was found that Mg2+ with PEG at 20% final concentration can significantly increase the transient expression in wheat protoplasts in comparison to the Ca(2+)-containing medium. The optimum incubation time in transformation mixture was 5-10 min at 25 degrees C. Transient GUS expression as detected by spectrofluorimetry was positively correlated with the time elapsed after DNA uptake (with maximum activity at 48 h), and the incubation time in GUS reaction mixture. It was also found that the protoplast culture medium plays an important role in the efficiency, and the treated wheat protoplasts cultured in KM medium showed a higher GUS activity than those kept in GM medium. Among the five plasmid constructs 6-16-fold higher promoter activity has been achieved with pKM794 driven by CaMV 35S promoter plus two enhancer elements than with the other constructs tested.

A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the beta-glucuronidase (GUS) gene, under the control of the doubled enhancer element (the -208 to -46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to -389 bp from ATG) promoter of wheat, alpha-amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10-20 resistant calli, or GUS-expressing colonies after treatment of 10(6) protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80-90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.

The recent achievements in production of transgenic cereal plants are based on the variety of transformation protocols including DNA uptake into protoplasts (Shimamoto et al. 1989; Datta et al. 1990; Golovkin et al. 1993), electroporation of enzyme-treated tissues (D’Halluin et al. 1992), biolistic-mediated gene delivery into cultured cells or organized tissues (Fromm et al. 1990; Gordon-Kamm et al. 1990; Somers et al. 1992; Vasil et al. 1992), or Agrobacterium infection (Chan et al. 1993). Considering the published frequency data, the efficiency of various methods cannot be judged convincingly, since the majority of publications treat only a few transformants. The extensive use of transgenic plants for both gene expression studies and crop improvement requires the generation and analysis of a large number of independent transformants. For this purpose, we have developed a synthetic maize genotype (HE/89) with unique plant regeneration potential in the protoplast-derived tissues (Morocz et al. 1990). Protoplasts from rapidly cycling cells exposed to osmotic and wound stresses may represent a highly competent cell type for efficient uptake and integration of foreign DNA molecules (for review see Feher and Dudits 1994). Incubation of 106 protoplasts from HE/89 suspension culture with plasmid DNA and PEG solution reproducibly results in 30–60 transgenic clones with plant regeneration capability (Omirulleh et al. 1993). Consequently, in a routine transformation experiment, several hundreds of transformed maize plants can be produced that reach the flowering state within 4 months.

The recent methodology of introducing foreign genes into the genome of monocot cereal plants, including maize, is based on a variety of experimental approaches. Several laboratories have demonstrated the potentials in the use of microprojectile bombardment to transform embryogenic maize tissue cultures (Fromm et al. 1990; Gordon-Kamm et al. 1990; Walters et al. 1992). Furthermore, the production of transgenic maize plants expressing agronomically significant traits was also achieved by this method (Koziel et al. 1993). Regeneration of fertile transgenic maize plants was recently reported by D’Halluin et al. (1992) too, who electroporated plasmid molecules into wounded tissues of immature zygotic embryos or of embryogenic callus cultures. Both of these methods are proposed to be widely applicable for transformation of different breeding stocks. However, the regeneration capability was shown to be genotype-dependent by several tissue culture studies (Tomes and Smith 1985; Lupotto 1986; Petersen et al. 1992).