Cowpea (Vigna unguiculata Walp) embryos mechanically isolated from mature seeds and incubated in the presence of plasmid DNA harboring chimeric gus genes were shown to germinate into seedlings expressing β-glucuronidase activity in a variety of tissues, including the apical meristem. Embryo electroporation in the presence of DNA and protectants such as spermine and Lipofectin(TM) increased both the proportion of embryo-derived seedlings expressing the chimeric gene and the level of gene expression. Microscopic observations of thin sections showed that the blue crystals representing the end product of transgene activity on X-glu were exclusively located inside the treated cells. Histological localization of the blue dye crystals varied with the promoter used to drive the transgene.

Electroporation-mediated gene transfer into intact plant tissues was demonstrated in pea, cowpea, lentil, and soybean plants. Transient expression of a chimeric gus reporter gene was used to monitor the uptake and expression of the introduced DNA in electroporated nodal axillary buds in vivo. The branches that grew out of the nodal meristems were chimeric and expressed the introduced gene up to 20 d after electroporation. Transgenic R1 pea, lentil, and cowpea plants were recovered from seeds originating on these chimeric branches as shown by Southern blot hybridization and GUS expression. Transgenic R2 soybean and lentil plants were also obtained. Segregation ratios in these populations showed a strong bias against transgene presence or expression.

Transient expression and stable integration and expression of transgenes were observed in the tissues and offspring of certain leguminous plants after electroporation of DNA into intact nodal meristems in planta. The method described in this article thus allows the study of transgene expression in tissues differentiating from meristematic cells present in the treated buds. In addition, transgenic plants can be recovered in the offspring of electroporated individuals. Therefore, this technique allows the production of transgenic leguminous plants without the need for in vitro tissue culture, often a major hurdle with this family.

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