Site-specific recombination for genetic engineering in plants.

Site-specific recombination has been developed into a genetic engineering tool for higher eukaryotes. The manipulation of newly introduced DNA is now possible in the course of genetic transformation procedures, thus making the process more predictable and reliable. Also, a wide variety of chromosomal rearrangements using site-specific recombination have been documented both in metazoan and plant species. Applying such methods to plants opens new avenues for large-scale chromosome engineering in the future.

Characterization of maize (Zea mays L.) Wee1 and its activity in developing endosperm.

We report the characterization of a maize Wee1 homologue and its expression in developing endosperm. Using a 0.8-kb cDNA from an expressed sequence tag project, we isolated a 1.6-kb cDNA (ZmWee1), which encodes a protein of 403 aa with a calculated molecular size of 45.6 kDa. The deduced amino acid sequence shows 50% identity to the protein kinase domain of human Wee1. Overexpression of ZmWee1 in Schizosaccharomyces pombe inhibited cell division and caused the cells to enlarge significantly. Recombinant ZmWee1 obtained from Escherichia coli is able to inhibit the activity of p13(suc1)-adsorbed cyclin-dependent kinase from maize. ZmWee1 is encoded by a single gene at a locus on the long arm of chromosome 4. RNA gel blots showed the ZmWee1 transcript is about 2.4 kb in length and that its abundance reaches a maximum 15 days after pollination in endosperm tissue. High levels of expression of ZmWee1 at this stage of endosperm development imply that ZmWee1 plays a role in endoreduplication. Our results show that control of cyclin-dependent kinase activity by Wee1 is conserved among eukaryotes, from fungi to animals and plants.

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.

Lamellar-to-hexagonalII phase transitions in the plasma membrane of isolated protoplasts after freeze-induced dehydration.

In protoplasts isolated from nonacclimated rye leaves (Secale cereale L. cultivar Puma), cooling to -- 10 degrees C at a rate of 1 degrees C/min results in extensive freeze-induced dehydration (osmotic contraction), and injury is manifested as the loss of osmotic responsiveness during warming. Under these conditions, several changes were observed in the freeze-fracture morphology of the plasma membrane. These included (i) lateral phase separations in the plasma membrane, (ii) aparticulate lamellae lying next to the plasma membrane, and (iii) regions of the plasma membrane and associated lamellae in various stages of lamellar-to-hexagonalII transition. These morphological changes also were observed after equilibration in 5.37 osmolal sorbitol at 0 degrees C, which produced a similar extent of dehydration as did freezing to -- 10 degrees C. In contrast, only small areas of lateral phase separation in the plasma membrane, with no observable aparticulate lamellae or hexagonalII configurations, were observed in protoplasts supercooled to -- 10 degrees C. Therefore, freeze-induced lamellar-to-hexagonalII phase transitions in the plasma membrane are a consequence of dehydration rather than subzero temperature per se. When suspensions of protoplasts isolated from cold-acclimated leaves were frozen to -- 10 degrees C, no injury was incurred, and hexagonalII phase transitions were not observed. No hexagonalII phase was observed even at -- 35 degrees C, though acclimated protoplasts are injured at this temperature.

Destabilization of the plasma membrane of isolated plant protoplasts during a freeze-thaw cycle: the influence of cold acclimation.

In conclusion, isolated protoplasts are an excellent arena in which destabilization of the plasma membrane can be directly observed during a freeze-thaw cycle by cryomicroscopy. Destabilization is manifested in various ways--intracellular ice formation, loss of osmotic responsiveness, or expansion-induced lysis. The incidence of any particular form of injury will depend on the freeze-thaw protocol and hardiness of the tissue from which the protoplasts were isolated. In all cases, however, cold acclimation directly increases the stability of the plasma membrane to the multiple stresses that arise during a freeze-thaw cycle. Such observations provide for functional differences in the plasma membrane that may now be used to consider the significance of any compositional changes in the membrane that might be determined.