Segregation of transgenes in maize.

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.

Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants.

A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos. Integration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R0) were regenerated, and of 53 progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of herbicide to leaves of bar-transformed R0 and R1 plants resulted in no necrosis, confirming functional activity of PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one encoding PAT and one containing the nonselected gene encoding [beta]-glucuronidase. R0 plants regenerated from co-transformed callus expressed both genes. These results describe and confirm the development of a system for introduction of DNA into maize.

Bialaphos selection of stable transformants from maize cell culture.

Stable transformed Black Mexican Sweet (BMS) maize callus was recovered from suspension culture cells bombarded with plasmid DNA that conferred resistance to the herbicide bialaphos. Suspension culture cells were bombarded with a mixture of two plasmids. One plasmid contained a selectable marker gene, bar, which encoded phosphinothricin acetyl transferase (PAT), and the other plasmid encoded a screenable marker for ß-glucuronidase (GUS). Bombarded cells were selected on medium containing the herbicide bialaphos, which is cleaved in plant cells to yield phosphinothricin (PPT), an inhibitor of glutamine synthetase. The bialaphos-resistant callus contained the bar gene and expressed PAT as assayed by PPT inactivation. Transformants that expressed high levels of PAT grew more rapidly on increasing concentrations of bialaphos than transformants expressing low levels of PAT. Fifty percent of the bialaphos-resistant transformants tested (8 of 16) expressed the nonselected gene encoding GUS.

Two soybean seed lipoxygenase nulls accumulate reduced levels of lipoxygenase transcripts.

Soybean (Glycine max L. [Merrill]) seed lipoxygenase cDNA clones were recovered from two cDNA libraries: a size-selected library in pBR322 and an expression library in pUC8. The pUC8 library was made with total poly(A)(+) embryo RNA and was screened with antiserum to lipoxygenase-1, one of 3 seed lipoxygenase isozymes. Three lipoxygenase antigen-producing clones were identified: two with identical cDNA inserts of 977 nucleotides representing an open-reading frame and a third truncated clone bearing a 3' end common to the longer clones. A long clone, pAL-134, was chosen for further study and was used to screen the size-selected cDNA library from which sixteen clones were identified. They fall into two homology classes represented by pLX-10 (ca. 1360 bp) and pLX-65 (2047 bp).The lipoxygenase expression clone pAL-134 hybridized much more strongly to pLX-65 than to pLX-10. pAL-134 and pLX-65 share 89% nucleotide homology and 75% deduced amino acid homology along their common sequence. Their deduced amino acid sequences each show 80% homology to sequences determined for isolated peptides of the lipoxygenase-1 isozyme.pAL-134 hybridizes poorly with a 3.8 kb RNA from LOX-1 null (lx1) embryos while pLX-65 hybridizes more strongly, but still to a lesser extent than its hybridization to standard embryo RNA or to RNA from embryos lacking lipoxygenase-2 (lx2) or lipoxygenase-3 (lx3) protein.The lx3 null lacks almost all embryo 3.8 kb RNA homologous to pLX-10. This hybridization pattern suggests that pLX-10 encodes LOX-3. Thus, the lx1 and lx3 genotypes accumulate little, if any, mRNA for the lipoxygenase-1 and lipoxygenase-3 isozymes, respectively.