Validation of a rapid and sensitive HPLC/MS method for measuring sucrose, fructose and glucose in plant tissues.

Sucrose and its constituent components, glucose and fructose, are major determinants of sweetness in fruits and cooking quality in plant storage organs, such as potato tubers. Accurate monitoring of these sugars provides a predictive assessment of plant product quality. High Performance Liquid Chromatography (HPLC) coupled to a variety of detectors has been the method of choice for selective detection and quantitation of sugars in plant tissues. However, sugar extraction can be time-consuming to obtain a clean and concentrated sample suitable for accurate quantitation. Herein, we took advantage of a modern HPLC system coupled to a highly sensitive high-resolution high-accuracy mass spectrometer to develop a rapid method for measuring glucose, fructose, and sucrose in plant tissues. The method was validated in mature potato tubers and strawberry fruits and it proved to be highly accurate and robust with minimal sample care requirements.

Vacuolar invertase gene silencing in potato (Solanum tuberosum L.) improves processing quality by decreasing the frequency of sugar-end defects.

Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.

Employment of cytokinin vectors for marker-free and backbone-free transformation.

Marker-free methods of plant transformation sacrifice the advantages of a selectable marker during regeneration or add work after regeneration to remove the marker. On the positive side, there is no stably integrated marker gene in the plant genome to present regulatory hurdles or potential biosafety hazards once the plant is released to the environment. A marker-free method that is simple and adaptable to multiple crop species-even asexually propagated species-is presented herein. This method employs an engineered vector that utilizes the isopentenyltransferase (ipt) to drive the regeneration of intragenic cells containing the gene(s) of interest. The ipt gene also acts as a marker to screen against events where the vector backbone is stably integrated.

Overexpression of the wild potato eIF4E-1 variant Eva1 elicits Potato virus Y resistance in plants silenced for native eIF4E-1.

Potato virus Y (PVY) is the most important viral pathogen of cultivated potato (Solanum tuberosum) from a commercial perspective, causing severe losses in both tuber quality and yield worldwide. Specific accessions of wild potato species exhibit resistance against PVY but efforts to transfer the trait to cultivated material have not yielded widely adopted varieties. Because amino acid substitutions at specific domains of host factor eIF4E-1 often confer resistance to various crops, we sequenced the associated genes expressed in wild potato plants. A novel eIF4E-1 variant, designated here as Eva1, was identified in S. chacoense, S. demissum, and S. etuberosum. The protein contains amino acid substitutions at ten different positions when compared to its cultivated potato (S. tuberosum) homolog. In the yeast two-hybrid system, Eva1 failed to bind VPg, a viral protein required for infectivity. Overexpression of the associated cDNA conferred PVY resistance to transgenic potato plants silenced for the native eIF4E-1 gene. Because the gene sources of Eva1 are sexually compatible with potato, the molecular strategies described can be employed to develop 'intragenic' potato cultivars.

Low-acrylamide French fries and potato chips.

SUMMARY: Acrylamide is produced in starchy foods that are baked, roasted or fried at high temperatures. Concerns about the potential health issues associated with the dietary intake of this reactive compound led us to reduce the accumulation of asparagine, one of its main precursors, in the tubers of potato (Solanum tuberosum). This metabolic change was accomplished by silencing two asparagine synthetase genes through 'all-native DNA' transformation. Glasshouse-grown tubers of the transformed intragenic plants contained up to 20-fold reduced levels of free asparagine. This metabolic change coincided with a small increase in the formation of glutamine and did not affect tuber shape or yield. Heat-processed products derived from the low-asparagine tubers were also indistinguishable from their untransformed counterparts in terms of sensory characteristics. However, both French fries and potato chips accumulated as little as 5% of the acrylamide present in wild-type controls. Given the important role of processed potato products in the modern Western diet, a replacement of current varieties with intragenic potatoes could reduce the average daily intake of acrylamide by almost one-third.

Engineered native pathways for high kaempferol and caffeoylquinate production in potato.

Flavonols and caffeoylquinates represent important groups of phenolic antioxidants with health-promoting activities. The genetic potential of potato (Solanum tuberosum) to produce high levels of these dietary compounds has not been realized in currently available commodity varieties. In this article, it is demonstrated that tuber-specific expression of the native and slightly modified MYB transcription factor gene StMtf1(M) activates the phenylpropanoid biosynthetic pathway. Compared with untransformed controls, transgenic tubers contained fourfold increased levels of caffeoylquinates, including chlorogenic acid (CGA) (1.80 mg/g dry weight), whilst also accumulating various flavonols and anthocyanins. Subsequent impairment of anthocyanin biosynthesis through silencing of the flavonoid-3',5'-hydroxylase (F3'5'h) gene resulted in the accumulation of kaempferol-rut (KAR) to levels that were approximately 100-fold higher than in controls (0.12 mg/g dry weight). The biochemical changes were associated with increased expression of both the CGA biosynthetic hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (Hqt) gene and the upstream chorismate mutase (Cm) and prephenate dehydratase (Pdh) genes. Field trials indicated that transgenic lines produced similar tuber yields to the original potato variety Bintje. Processed products of these lines retained most of their phenylpropanoids and were indistinguishable from untransformed controls in texture and taste.

Improving potato storage and processing characteristics through all-native DNA transformation.

The dominant potato (Solanum tuberosum) variety for French fry production in the United States is the 131-year-old Russet Burbank. Market penetration of the higher yielding and more uniform Ranger Russet variety is limited to about one-fifth of that of the Russet Burbank because of two storage deficits: black spot bruise sensitivity and high levels of cold-induced sweetening. Here, these trait weaknesses are turned into strengths by simultaneously lowering the expression of Ranger Russet's tuber-expressed polyphenol oxidase (Ppo), starch-associated R1, and phosphorylase-L (PhL) genes. This genetic modification was accomplished without inserting any foreign DNA into the plant genome. French fries from the intragenic potatoes also contained reduced amounts of the antinutritional compound acrylamide while, unexpectedly, displaying enhanced sensory characteristics.

Crop improvement through modification of the plant's own genome.

Plant genetic engineering has, until now, relied on the incorporation of foreign DNA into plant genomes. Public concern about the extent to which transgenic crops differ from their traditionally bred counterparts has resulted in molecular strategies and gene choices that limit, but not eliminate, the introduction of foreign DNA. Here, we demonstrate that a plant-derived (P-) DNA fragment can be used to replace the universally employed Agrobacterium transfer (T-) DNA. Marker-free P-DNAs are transferred to plant cell nuclei together with conventional T-DNAs carrying a selectable marker gene. By subsequently linking a positive selection for temporary marker gene expression to a negative selection against marker gene integration, 29% of derived regeneration events contain P-DNA insertions but lack any copies of the T-DNA. Further refinements are accomplished by employing Omega-mutated virD2 and isopentenyl transferase cytokinin genes to impair T-DNA integration and select against backbone integration, respectively. The presented methods are used to produce hundreds of marker-free and backbone-free potato (Solanum tuberosum) plants displaying reduced expression of a tuber-specific polyphenol oxidase gene in potato. The modified plants represent the first example of genetically engineered plants that only contain native DNA.