Site-specific heritable mutations in maize genes were engineered by introducing chimeric RNA͞DNA oligonucleotides. Two independent targets within the endogenous maize acetohydroxyacid synthase gene sequence were modified in a site-specific fashion, thereby conferring resistance to either imidazolinone or sulfonylurea herbicides. Similarly, an engineered green f luorescence protein transgene was site-specifically modified in vivo. Expression of the introduced inactive green f luorescence protein was restored, and plants containing the modified transgene were regenerated. Progeny analysis indicated Mendelian transmission of the converted transgene. The efficiency of gene conversion mediated by chimeric oligonucleotides in maize was estimated as 10 ؊4 , which is 1-3 orders of magnitude higher than frequencies reported for gene targeting by homologous recombination in plants. The heritable changes in maize genes engineered by this approach create opportunities for basic studies of plant gene function and agricultural trait manipulation and also provide a system for studying mismatch repair mechanisms in maize.

Maize plants resistant to imidazolinone herbicides were engineered through targeted modification of endogenous genes using chimeric RNA/DNA oligonucleotides. A precise single-point mutation was introduced into genes encoding acetohydroxyacid synthase (AHAS), at a position known to confer imidazolinone resistance. Phenotypically normal plants from the converted events (C0) were regenerated from resistant calli and grown to maturity. Herbicide leaf painting confirmed the resistance phenotype in C0 plants and demonstrated the anticipated segregation pattern in C1 progeny. DNA cloning and sequencing of the targeted region in resistant calli and derived C0 and C1 plants confirmed the expected mutation. These results demonstrate that oligonucleotide-mediated gene manipulation can be applied to crop improvement. This approach does not involve genomic integration of transgenes. Since the new trait is obtained through modifying a gene within its normal chromosomal context, position effects, transgene silencing, or other concerns that arise as part of developing transgenic events are avoided.

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter-beta-glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.

Subjecting 5-day-old plumules of corn (Zea mays L.) to elevated temperatures for brief periods of time causes the pattern of protein synthesis to shift from the production of a broad spectrum of proteins to the new and (or) enhanced synthesis of a small number of heat-shock polypeptides (HSPs). Most notable is the depressed synthesis of a major polypeptide (relative mass (Mr) = 93 000 and isoelectric point = 8.0) normally made at 27 degrees C and the enhanced and (or) new synthesis of polypeptides with MrS of 108 000, 89 000, 84 000, 76 000, 73 000, and 18 000, following 1 h of heat shock. These six HSPs is observed within 120 min following heat shock. Recovery from heat shock is rapid; after 6 to 8 h at 27 degrees C following heat shock, the polypeptide pattern is indistinguishable from the control. Extracts from individual heat-shocked shoots produced polypeptide synthetic patterns identical to those from extracts from 20 shoots, regardless of whether single shoots were intact or excised during labelling. Single 5-day-old primary roots exhibited polypeptide synthetic patterns and responded to heat shock in a manner similar to shoots. This is the first demonstration of the induction of heat-shock polypeptides in a whole, intact higher plant.

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