Progeny recovered from backcrossed transgenic maize tissue culture regenerants (R0) were analyzed to determine the segregation, expression, and stability of the introduced genes. Transgenic A188 x B73 R0 plants (regenerated from embryogenic suspension culture cells transformed by microprojectile bombardment; see [9]) were pollinated with nontransformed B73 pollen. Inheritance of a selectable marker gene, bar, and a nonselectable marker gene, uidA, was analyzed in progeny (R1) representing four independent transformation events. Activity of the bar gene product, phosphinothricin acetyltransferase (PAT), was assessed in plants comprising the four R1 populations. The number of R1 plants containing PAT activity per total number of R1 plants recovered for each population was 2/7, 19/34, 3/14 and 73/73. Molecular analysis confirmed the segregation of bar in three R1 populations and the lack of segregation in one R1 population. Cosegregation analysis indicated genetic linkage of bar and uidA in all four R1 populations. Analysis of numerous R2 plants derived from crossing transformed R1 plants with nontransformed inbreds revealed 1:1 segregation of PAT activity in three of four lines, including the line that failed to segregate in the R1 generation. Integrated copies of bar in one line appeared to be unstable or poorly transmitted.

Introduced transgenes, uidA, sgfp (S65T) and/or bar, were localized using fluorescence in situ hybridization (FISH) on metaphase chromosomes of transgenic barley produced by microparticle bombardment of immature embryos. Of the 19 independent transgenic lines (eight diploid and 11 tetraploid), nine had uidA and ten had s gfp (S65T). All lines tested had three or more copies of the transgenes and 18 out of 19 lines had visibly different integration sites. At a gross level, it appeared that no preferential integration sites of foreign DNA among chromosomes were present in the lines tested; however, a distal preference for transgene integration was observed within the chromosome. In diploid T0 plants that gave a 3:1 segregation ratio of transgene expression in the T1, only single integration sites were detected on one of the homologous chromosomes. Homozygous diploid plants had doublet signals on a pair of homologous chromosomes. All tetraploid T0 plants that gave a 3:1 segregation ratio in the T1 generation had only a single integration site on one of the homologous chromosomes. In contrast, the single tetraploid T0 plant with a 35:1 segregation ratio in the T1 generation had doublet signals on a pair of homologous chromosomes. In the one tetraploid T0 line, which had a homozygote-like segregation ratio (45:0), there were doublet signals at two loci on separate chromosomes. We conclude that the application of FISH for analysis of transgenic plants is useful for the gross localization of transgene(s) and for early screening of homozygous plants.

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic oat ( Avena sativa L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by a rice actin promoter. In vitro shoot meristematic cultures (SMCs) induced from shoot apices of germinating mature seeds of a commercial oat cultivar, Garry, were used as a transformation target. Proliferating SMCs were bombarded with a mixture of plasmids containing the sgfp(S65T) gene and one of three selectable marker genes, phosphinothricin acetyltransferase (bar), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase (nptII). Cultures were selected with bialaphos, hygromycin B and geneticin (G418), respectively, to identify transgenic tissues. From 289 individual explants bombarded with the sgfp(S65T) gene and one of the three selectable marker genes, 23 independent transgenic events were obtained, giving a 8.0% transformation frequency. All 23 transgenic events were regenerable, and 64% produced fertile plants. Strong GFP expression driven by the rice actin promoter was observed in a variety of tissues of the T(0) plants and their progeny in 13 out of 23 independent transgenic lines. Stable GFP expression was observed in T(2) progeny from five independent GFP-expressing lines tested, and homozygous plants from two lines were obtained. Transgene silencing was observed in T(0) plants and their progeny of some transgenic lines.

Rapid environmental change can lead to extinction of populations or evolutionary rescue via genetic adaptation. In the past several years, smallholder and commercial cultivation of sorghum (Sorghum bicolor), a global cereal and forage crop, has been threatened by a global outbreak of an aggressive new biotype of sugarcane aphid (SCA; Melanaphis sacchari). Here we characterized genomic signatures of adaptation in a Haitian sorghum breeding population, which had been recently founded from admixed global germplasm, extensively intercrossed, and subjected to intense selection under SCA infestation. We conducted evolutionary population genomics analyses of 296 post-selection Haitian lines compared to 767 global accessions at 159,683 single nucleotide polymorphisms. Despite intense selection, the Haitian population retains high nucleotide diversity through much of the genome due to diverse founders and an intercrossing strategy. A genome-wide fixation (FST) scan and geographic analyses suggests that adaptation to SCA in the Haiti is conferred by a globally-rare East African allele of RMES1, which has also spread to breeding programs in Africa, Asia, and the Americas. De novo genome sequencing data for SCA resistant and susceptible lines revealed putative causative variants at RMES1. Convenient low-cost markers were developed from the RMES1 selective sweep and successfully predicted resistance in independent U.S. x African breeding lines and eight U.S. commercial and public breeding programs, demonstrating the global relevance of the findings. Together, the findings highlight the potential of evolutionary genomics to develop adaptive trait breeding technology and the value of global germplasm exchange to facilitate evolutionary rescue.

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