Search

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.

Fertile transgenic maize plants were regenerated after direct transfer of a chimeric gene into maize protoplasts. Plasmid DNA containing mutant dihydrofolate reductase (DHFR) mouse gene, that confers methotrexate (MTX) resistance, under the control of the CaMV 35S promoter was introduced into maize embryogenic protoplasts by polyethylene glycol (PEG) treatment. Transformation was also carried out with a modified plasmid in which the selective marker gene casette was cloned into the BstBI site of the Ds 1 maize transposable element. Resistant callus tissues grown in the presence of 10−6 or 10−7 M MTX were selected and shoot or plant regeneration was achieved under hormone-free culture conditions. The presence of the introduced DHFR gene in DNA isolated from the selected colonies and the primary regenerants (T0) was shown by Southern hybridization and PCR analysis. PCR primers for the 35S promoter and for two regions of the coding sequence of the DHFR gene were used for amplification of the foreign sequence present in maize genomic DNA. The PCR products were hybridized with a mouse DHFR gene specific probe. Synthesis of the mouse DHFR in MTX resistant maize tissues was detected by staining for enzyme activity after native PAGE. The in vitro regenerated plants could be grown up to maturity in the greenhouse. Cross pollination has resulted in seeds and the F1 progenies were also analyzed. In addition to the segregation of MTX-resistant and-sensitive offsprings, molecular evidences based on Southern data and PCR analysis have indicated that the introduced gene was transferred in the first sexual generation. This report provides a new example for potentials in the use of embryogenic cereal protoplasts for production of fertile transgenic crop plants.

Regenerants from a 30-month-old haploid and a 10-month-old diploid tissue culture were cross-pollinated to generate a synthetic genotype (HE/89) with improved competence for maintenance of totipotency in various cultured expiants. The HE/89 zygotic embryos developed friable, embryogenic cultures in the commonly used MS-and N6-based media without the addition of L-proline. By optimalization and changing the culture conditions, we were able to regulate the maintenance of the earlier, more synchronous (Type II) and the later, asynchronous (Type I) in vitro embryogenesis, as well as the shift between different ontogenic stages. Within 70 days after the inoculation of immature embryos a relatively homogeneous, early-embryogenic suspension culture usable for protoplast isolation was established from the initially surface-grown cultures. Using modified solutions for protoplast isolation and culture, viable protoplasts were reproducibly obtained from which plants were regenerated via defined ontogenic steps. Despite the long in vitro history of the parental genotypes, 60-70% of the more than 500 plants derived from the HE/89 protoplasts set seeds following self or sib-pollination.

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).