Glyphosate (N‐phosphonomethyl‐glycine) is the active ingredient the nonselective herbicide Roundup. The sensitivity of crop plants to glyphosate has limited its in‐season use as a postemergence herbicide. The extension of the use of Roundup herbicide to allow in‐season application in major crops such as soybeans [Glycine max (L.) Merr.] would provide new weed control options for farmers. A glyphosate‐tolerant soybean line, 40‐3‐2, was obtained through expression of the bacterial 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSP synthase, EPSPS) enzyme from Agrobacterium sp. strain CP4. Line 40‐3‐2 is highly tolerant to glyphosate, showing no visual injury after application of up to 1.68 kg acid equivalent (a.e.) ha−1 of glyphosate under field conditions. Molecular characterization studies determined that the single genetic insert in line 40‐3‐2 contains only a portion of the cauliflower mosaic virus 35S promoter (P‐E35S), the Petunia hybrida EPSPS chloroplast transit peptide (CTP), the CP4 EPSPS gene, and a portion of the 3' nontranslated region of the nopaline synthase gene (NOS 3') terminator. Inheritance studies have shown that the transgene behaves as a single dominant gene and is stable over several generations.

Starch, a major storage metabolite in plants, positively affects the agricultural yield of a number of crops. Its biosynthetic reactions use adenosine diphosphate glucose (ADPGlc) as a substrate; ADPGlc pyrophosphorylase, the enzyme involved in ADPGlc formation, is regulated by allosteric effectors. Evidence that this plastidial enzyme catalyzes a rate-limiting reaction in starch biosynthesis was derived by expression in plants of a gene that encodes a regulatory variant of this enzyme. Allosteric regulation was demonstrated to be the major physiological mechanism that controls starch biosynthesis. Thus, plant and bacterial systems for starch and glycogen biosynthesis are similar and distinct from yeast and mammalian systems, wherein glycogen synthase has been demonstrated to be the rate-limiting regulatory step.

The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells.

More than two billion people are micronutrient deficient. Polished grains of popular rice varieties have concentration of approximately 2 μg g−1 iron (Fe) and 16 μg g−1 zinc (Zn). The HarvestPlus breeding programs for biofortified rice target 13 μg g−1 Fe and 28 μg g−1 Zn to reach approximately 30% of the estimated average requirement (EAR). Reports on engineering Fe content in rice have shown an increase up to 18 μg g−1 in glasshouse settings; in contrast, under field conditions, 4 μg g−1 was the highest reported concentration. Here, we report on selected transgenic events, field evaluated in two countries, showing 15 μg g−1 Fe and 45.7 μg g−1 Zn in polished grain. Rigorous selection was applied to 1,689 IR64 transgenic events for insert cleanliness and, trait and agronomic performances. Event NASFer-274 containing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH-1) genes showed a single locus insertion without a yield penalty or altered grain quality. Endosperm Fe and Zn enrichment was visualized by X-ray fluorescence imaging. The Caco-2 cell assay indicated that Fe is bioavailable. No harmful heavy metals were detected in the grain. The trait remained stable in different genotype backgrounds.

A small region of the Ti plasmid (the tmr locus), thought to be involved in phytohormone metabolism in Agrobacterium tumefaciens-transformed plant tissue, was cloned and expressed in Escherichia coli. By enzyme assay, the tmr locus was shown to encode isopentenyltransferase, an enzyme that catalyzes the first step in cytokinin biosynthesis.Crown gall disease, characterized by the development of neoplastic growth on the infected plant, affects many dicotyledonous plants and is caused by the soil bacterium Agrobacterium tumefaciens (1). The induction and maintenance of the tumor is dependent upon the integration into the plant genome and expression of a DNA fragment (T-DNA) from the large virulence plasmid (Ti) harbored by the bacterium (2-7). In tissue culture the Agrobacterium-induced tumors have been shown to be autotrophic for phytohormones (8), specifically for auxins and cytokinins. That the transforming T-DNA was involved in the alteration in the endogenous hormone synthesis was suggested by the identification of A. tumefaciens isolates that induced tumors with abnormal morphologies, characteristic of hormonal imbalance, such as the overproduction of shoots or roots (9-13). The loci responsible for such altered morphologies have been mapped to the T-DNA and have been shown to correlate with alterations in the phytohormone levels in the tumors (14, 15). Specifically, mutants in the tms ("shooty") and tml ("large") loci result in over production of cytokinins, while those in the tmr ("rooty") locus result in a very large decrease in endogenous cytokinin levels.The enzyme central to all cytokinin biosynthesis is A2-isopentenyl pyrophosphate:5'-AMP A2-isopentenyltransferase (EC 2.5.1.-) commonly referred to as isopentenyl transferase (16). The product of this reaction is isopentenyladenosine monophosphate (iPeAMP) (Fig. 1), which by dephosphorylation is converted to the cytokinin isopentenyladenosine (iPeA) and which is the precursor to all other cytokinins (17). The tms and tml defects result in a large increase in isopentenyltransferase activity in the tumors, while the tmr defects result in a decrease in activity to a level lower than that found even in normal tissue (14). While the tms, tml, and tmr results are consistent with the regulatory role of these loci on the expression or activity of endogenous cytokinin biosynthesis, the results are also consistent with the tmr locus directly encoding a biosynthetic enzyme.Transcriptional and translation maps of the T-DNA regions have been determined recently for both the octopine and nopaline Ti plasmids, and the tmr gene transcript has been identified in tumors induced by both plasmid types (11,(18)(19)(20)(21)(22). The tmr region from the nopaline plasmid pTi T37 was cloned and the nucleotide sequence was determined (23). An open reading frame encoding a protein of 27 kilodaltons (kDa) was identified in this sequence, as has also been found for the same region of the octopine Ti plasmid (24). A protein of this size has been shown to be expressed from ...