An alternative method for transforming sweet orange [Citrus sinensis (L.) Osbeck] has been developed. Plasmid DNA encoding the non-destructive selectable marker enhanced green fluorescent protein gene was introduced using polyethylene glycol into protoplasts of`Itaborai' sweet orange isolated from an embryogenic nucellar-derived suspension culture. Following protoplast culture in liquid medium and transfer to solid medium, transformed calluses were identified via expression of the green fluorescent protein, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. Transgenic plantlets were recovered from germinating somatic embryos and by in vitro rooting of shoots. To expedite transgenic plant recovery, regenerated shoots were also micrografted onto sour orange seedling rootstocks. Presence of the transgene in calluses and regenerated sweet orange plants was verified by gene amplification and Southern analyses. Potential advantages of this transformation system over the commonly used Agrobacterium methods for citrus are discussed.

Comparative genomic and cDNA sequence analysis of ara h 2, a major peanut allergen, and a related conglutin ara h 6 were performed in Arachis hypogaea L. and its putative progenitors, Arachis duranensis and Arachis ipaensis. The complete identity between sequences encoding Ara h 2 isoforms demonstrated that these are homeologous genes and represent orthologs from diploid ancestors. Three copies of ara h 6 were identified in A. hypogaea, one of them located in the A-genome and the other two in the B-genome. Expression analysis showed higher levels of ara h 2 transcripts compared with ara h 6. Dual-labeled genomic in situ hybridization permitted identification of two subgenomes, each of which contained one pair of ara h 2-ara h 6 signals localized by fluorescence in situ hybridization. Characterization of genomic clones showed close genetic linkage between Ara h 2.02 and one copy of ara h 6 in the B-genome. The physical linkage may have arisen by tandem duplication and divergence of an ancestral gene. A gene duplication event specific to the B-genome progenitor has resulted in ara h 6 paralogs. These data provide further evidence for progenitor relationships and genomic organization of the conglutin gene family in the genus Arachis and could contribute to the development of a hypoallergenic peanut.

Pearl millet [ Pennisetum glaucum (L.) R. Br.] is a drought-tolerant cereal crop used for grain and forage. Novel traits from outside of the gene pool could be introduced provided a reliable gene-transfer method were available. We have obtained herbicide-resistant transgenic pearl millet plants by microprojectile bombardment of embryogenic tissues with the bar gene. Embryogenic tissues derived from immature embryos, inflorescences and apical meristems from diploid and tetraploid pearl millet genotypes were used as target tissues. Transformed cells were selected in the dark on Murashige and Skoog medium supplemented with 2 mg/l 2,4-D and 15 mg/l phosphinothricin (PPT). After 3-10 weeks in the dark, herbicide-resistant somatic embryos were induced to germinate on MS medium containing 0.1 mg/l thidiazuron and 0.1 mg/l 6-benzylaminopurine. Plants were transferred to the greenhouse after they were rooted in the presence of PPT and had passed a chlorophenol red assay (the medium turned from red to yellow). Transgenic plants were recovered from bombardments using intact pAHC25 plasmid DNA, a gel-purified bar fragment, or a mixture of pAHC25 plasmid or bar fragment and a plasmid containing the enhanced green fluorescent protein ( gfp) gene (p524EGFP.1). Analyses by the polymerase chain reaction, Southern blot hybridization, GFP expression, resistance to herbicide application, and segregation of the bar and gfp genes confirmed the presence and stable integration of the foreign DNA. Transformed plants were recovered from all three explants, although transformation conditions were optimized using only the tetraploid inflorescence. Time from culture initiation to rooted transgenic plant using the tetraploid inflorescence ranged from 3-4 months. Seven independent DNA/gold precipitations were used to bombard 52 plates, 29 of which produced an average of 5.5 herbicide-resistant plants per plate. The number of herbicide-resistant plants recovered per successful bombardment ranged from one to 28 and the frequency of co-transformation with gfp ranged from 5% to 85%.

A protocol was developed for biolistic transformation of hybrid bermudagrass cv. TifEagle using the bar gene. TifEagle is an ultradwarf used exclusively on golf greens. Herbicide resistance should serve as a useful management tool, especially if methyl-bromide is unavailable for fumigation prior to plant establishment. Hybrid bermudagrass is completely sterile, which should limit the chance of gene escape via out-crossing. Sliced nodes were used to initiate embryogenic tissue cultures on MS medium supplemented with 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01 mg/l 6-benzylaminopurine (BA). Embryogenic tissue was bombarded with the bar gene, and herbicide-resistant tissue was selected in the dark on medium supplemented with 0.75 mg/l 2,4-D, 0.01 mg/l BA and 5-15 mg/l phosphinothricin (PPT). Resistant somatic embryos were induced to germinate in the light on MS medium supplemented with 0.13 mg/l 2,4-D and 0.5 mg/l BA. Plants were transferred to the greenhouse after rooting in the presence of 10-15 mg/l PPT and testing positive in a chlorophenol red assay. A total of 89 herbicide-resistant plants were recovered from at least nine independent events from six separate bombardments, although the number of independent transformation events was not confirmed for the entire group. Flow cytometry indicated that most of the plants (82/89) were hexaploid and that the remaining seven plants were triploid. The hexaploid plants were a darker green than the triploids or TifEagle control. Other variation, present only in the hexaploids, included an increased leaf width and length. Southern blot hybridization confirmed genomic integration of the bar gene in triploid and a subset of hexaploid herbicide-resistant plants. AFLP analysis did not indicate changes in DNA profiles using [33P] and a sample of 32 hexaploid plants recovered from a single bombardment. DNA profiles were very similar to that of the TifEagle control with a semi-automated fluorescence-based AFLP.