Genetically engineered crops: from idea to product.

Genetically engineered crops were first commercialized in 1994 and since then have been rapidly adopted, enabling growers to more effectively manage pests and increase crop productivity while ensuring food, feed, and environmental safety. The development of these crops is complex and based on rigorous science that must be well coordinated to create a plant with desired beneficial phenotypes. This article describes the general process by which a genetically engineered crop is developed from an initial concept to a commercialized product.

Posttranscriptional gene silencing in nuclei.

In plants, small interfering RNAs (siRNAs) with sequence homology to transcribed regions of genes can guide the sequence-specific degradation of corresponding mRNAs, leading to posttranscriptional gene silencing (PTGS). The current consensus is that siRNA-mediated PTGS occurs primarily in the cytoplasm where target mRNAs are localized and translated into proteins. However, expression of an inverted-repeat double-stranded RNA corresponding to the soybean FAD2-1A desaturase intron is sufficient to silence FAD2-1, implicating nuclear precursor mRNA (pre-mRNA) rather than cytosolic mRNA as the target of PTGS. Silencing FAD2-1 using intronic or 3'-UTR sequences does not affect transcription rates of the target genes but results in the strong reduction of target transcript levels in the nucleus. Moreover, siRNAs corresponding to pre-mRNA-specific sequences accumulate in the nucleus. In Arabidopsis, we find that two enzymes involved in PTGS, Dicer-like 4 and RNA-dependent RNA polymerase 6, are localized in the nucleus. Collectively, these results demonstrate that siRNA-directed RNA degradation can take place in the nucleus, suggesting the need for a more complex view of the subcellular compartmentation of PTGS in plants.

RNAi trigger fragment truncation attenuates soybean FAD2-1 transcript suppression and yields intermediate oil phenotypes.

Suppression of the microsomal ω6 oleate desaturase during the seed development of soybean (Glycine max) with the 420-bp soybean FAD2-1A intron as RNAi trigger shifts the conventional fatty acid composition of soybean oil from 20% oleic and 60% polyunsaturates to one containing greater than 80% oleic acid and less than 10% polyunsaturates. To determine whether RNAi could be attenuated by reducing the trigger fragment length, transgenic plants were generated to express successively shorter 5' or 3' deletion derivatives of the FAD2-1A intron. We observed a gradual reduction in transcript suppression with shorter trigger fragments. Fatty acid composition was less affected with shorter triggers, and triggers less than 60 bp had no phenotypic effect. No trigger sequences conferring significantly higher or lower suppression efficiencies were found, and the primary determinant of suppression effect was sequence length. The observed relationship of transcript suppression with the induced fatty acid phenotype indicates that RNAi is a saturation process and not a step change between suppressed and nonsuppressed states and intermediate suppression states can be achieved.

An intron sense suppression construct targeting soybean FAD2-1 requires a double-stranded RNA-producing inverted repeat T-DNA insert.

We demonstrate that the transformation of soybean (Glycine max) with sense suppression constructs using intron sequences from the fatty acid oleyl Delta12 desaturase gene FAD2-1A leads to efficient and specific reduction of FAD2-1 transcripts in developing seeds, increased oleic acid, and decreased polyunsaturated fatty acids. The related FAD2-2 transcripts are only marginally affected. Despite screening a large number of independent transformants, no single-copy efficacious transformants could be found. Invariably, all the least complex transgenic loci have two T-DNA copies in an inverted repeat configuration, centered at the right borders. We show that this T-DNA configuration produces an inverted repeat transcript and that small interfering RNAs accumulate against the target sequence.

VARIATIONS IN THE BIOSYNTHESIS OF SEED-STORAGE LIPIDS.

In many plants lipids represent up to 80% of dry weight of storage tissues. In seeds, lipids accumulate as triacylglycerols (TAGs), which are formed by an extension of the membrane-lipid biosynthetic pathway common to all plant tissues. In contrast to the conserved fatty acid (FA) composition of membrane lipids, the observed divergence in seed oil acyl chains among different species is very high. The acyl groups of seed TAGs can vary in their chain length (from 8 to 24) as well as in their degree of unsaturation. In addition to methylene-interrupted double bonds, many seeds contain TAGs that have unusual functional groups in their FAs, such as hydroxyl, oxirane, or acetylene groups. All of the major steps in the biosynthetic pathway to TAG are now known and sequence information for genes encoding most of the enzymes involved is available. Here we present the current knowledge of the metabolic mechanisms involved in the divergence from the membrane-lipid biosynthetic pathway during storage lipid formation.