For this research, long-term endosperm cell cultures (callus) and cell suspension cultures were established in the inbred line A69y, in the wild-type and o2 mutant version, as described in Balconi et al. (Plant J. 3: 325-334, 1993). These endosperm cell cultures are not completely de-differentiated, and maintain in part the specific synthesis of starch and proteins characteristic of the genotypes.The analysis of zein and starch content indicates that the cell suspension cultures have a higher starch and a lower zein content in comparison with callus tissue. A more detailed analysis showed that the protein/starch ratio varies depending on the age of the cells, and along the four week time span of subculture.

Endosperm callus cultures, as described above, were subcultured to homogeneity, and utilized for the transformation experiments when the tissues were in the optimal stage of proliferation. This part of the work turned out to be essential for the success of transformation experiments, in order to guarantee constant operative conditions. Introduction of the plasmid vectors into endosperm-derived cells was performed via microbombardment using the Particle Gun device PDS He/1000 BioRad, with an operative pressure of 1100 PSI, and the target cells at about 7 cm from the stopping screen, in a partial vacuum atmosphere of 26 in Hg. The cells were bombarded with a series of plasmids all carrying the coding region of the GUS gene driven by: 35S CaMV, Ubiquitin-1 (maize), and Actin-1 (rice) promoter sequences in a similar plasmid backbone (pUC19). After bombardment, the cells were incubated 48h at 27 C in the dark, and subsequently histochemically stained for visualization and counting of the blue foci. Evaluations were performed calculating the number of blue foci per gram of fresh weight of tissue bombarded. Great attention was paid to reproducing the exact experimental conditions in each bombardment as far as size and amount of the cells used. A first set of experiments was indicative of good expression efficiency of the fusion p35S/GUS, with and without intron as enhancer. In addition, these results showed a dramatic positive effect of a short pre-treatment of the cells in the presence of osmotic pressure obtained by including 0.5M mannitol in the culture medium. The effect of the pretreatment enhanced up to 10 fold the number of blue foci recorded per bombardment (from 40 up to 900 on the average). The cells were also maintained in the same osmotic conditions 16h after transformation, and subsequently returned back to the original medium. Data gathered on the growth rate of the cells after bombardment including pre- and post-treatment in high osmotic medium, indicated that the whole procedure did not interfere with subsequent cell proliferation processes. These results are in good agreement with the general idea that a high osmotic pressure treatment of cells to be bombarded causes plasmolysis; this treatment which can reduce damage to cell membranes resulting in a better cell survival (Ye et al., Plant Mol. Biol. 15:809-819 Vain; and McMullen, Plant Cell Rep. 12:84-88,1993).

The procedure described was subsequently used for further studies aimed at defining the rate of expression of the GUS gene in endosperm cells, when driven by different promoter sequences. To this purpose, the cells were bombarded with plasmids carrying the GUS coding region under the promoter sequences of Ubi1 and Act1 genes, respectively, and the results obtained are compared with the data obtained in the case of p35SCaMV and p35SCaMVINT. For the osmotic treatment we chose a pre-treatment of 4h followed by a post-treatment of 16h in 0.5M mannitol added to the culture medium. In each experiment, the data obtained from osmotically pretreated cells were compared with those obtained from non-treated cells. For both genotypes (wt and o2 versions), and for all plasmids used, the osmotic treatment enhanced particle bombardment-mediated expression of the GUS gene in the maize endosperm cells. By comparing the rate of expression obtained with the various promoters, we detected the highest values for pAct1 and pUbi1 promoters; these values were approximately twice the values obtained with p35SCaMV. In general, in the best conditions, we obtained with the fusion pUbi1/GUS an average value of 1200 blue foci/g fresh weight tissue. This value was also compared with fluorimetric determination of the GUS activity measured after enzymatic assays; the results obtained were in good agreement with the previous results. Interestingly, with respect to somatic cell systems, no enhancement in the GUS activity was detected when using p35SCaMV with the Adh1 intron respect to the p35SCaMV without intron. The data were compared to non treated cells during one year culture of the target tissues, and at different timing of growth during subculture. The results obtained allowed us to set up the technique for detailed investigations of promoter/GUS fusions in the maize endosperm system, by means of particle bombardment, in the study of regulatory mechanisms of genes involved in seed protein synthesis.

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