Examples of the Intragenic Modification in Potato

Despite the importance of potato as the most frequently consumed vegetable, issues such as inbreeding depression, a high degree of heterozygosity, and poor fertility have hampered efforts to improve the yield and quality of this crop. Each year, millions of potato plants are evaluated in the United States for the basic input and storage traits required by the industry. The few clones selected through this rigorous process are subsequently processed and assayed for sensory traits associated with taste, texture, and color. There are currently only about ten potato varieties that display most of the traits required by the French fry, potato chip, and retail industries. Together, they occupy ^70% of the total potato acreage in the United States. Interestingly, the predominantly grown variety is also the oldest: this century-old "Russet Burbank" displays unsurpassable storage and processing characteristics, but it suffers from multiple bacterial, viral, and fungal diseases, while also displaying high levels of sensitivity against environmental stresses including salt, drought, and frost. Farmers generally prefer to grow higher-yielding and more stress-tolerant and uniform varieties. However, each variety has its own issues, most of which translate directly into specific risks for growers, processors, and retailers.

Given the urgent need for potato improvement, it may not be surprising that intragenic methods were first applied to the farmers' favorite variety "Ranger Russet". This variety combines superior yield with disease resistance, adaptability, tuber uniformity, and high levels of starch. However, Ranger Russet is particularly sensitive to tuber discolorations linked to impact-induced bruise, and it also accumulates high levels of glucose and fructose during cold storage. These reducing sugars react with amino acids during high-temperature processing of potato to produce Maillard reaction products that darken the color of French fries from golden-yellow to brown. The main weaknesses of Ranger Russet were turned into strengths by transforming it with a specific potato-derived transfer DNA (Rommens et al., 2006). This plant-derived transfer (P-)DNA carried a silencing construct designed to simultaneously silence the tuber-expressed polyphenol oxidase (Ppo), phosphorylase-L (PhL), and starch-associated R1 genes (Rommens et al. 2006). Tubers of the resulting intragenic plants were down-regulated in Ppo gene expression and, consequently, displayed resistance to black spot bruise. Additionally, the silencing of genes involved in starch phosphorylation and degradation lowered the formation of reducing sugars during cold storage. This reduced cold-sweetening not only extended tuber storability but also limited French fry discoloration.

Interestingly, the slight modification of potato tuber starch was found to enhance crispness and French fry taste as well (Rommens et al. 2006).

The second intragenic modification of potato addressed one of the most important issues of the processing industry. This issue relates to the accumulation of large amounts of asparagine in tubers. Upon heat processing, the amide amino acid reacts with glucose and other reducing sugars to produce neurotoxic acrylamide (Fig. 4.1). Dietary intake levels of acrylamide have been rising in the Western world since the early 1900s, in part because of the increased consumption of French fries and potato chips, and are currently estimated at 40 mg person1 day1. Indeed, acrylamide has become a signature ingredient of the modern Western diet and may represent a minor factor in the emergence of certain "modern" diseases.

A preferred route to lowering the accumulation of acrylamide would shift to crops that are naturally poor in acrylamide precursors. However, there are currently no varieties available that also display all the additional input, processing, and quality traits demanded by the processing industry. Therefore, methods were developed to reduce the acrylamide potential of existing varieties through intra-genic modification (Rommens et al. 2008a). These methods were based on the finding that simultaneous tuber-specific silencing of the asparagine synthetase-1 and -2 genes lowered asparagine levels by up to tenfold. The dramatic decrease of tuber asparagine levels was associated with slightly elevated levels of glutamine but did not affect the production of other amino acids, and also did not alter total protein yield. Both French fries and potato chips from the intragenic potatoes accumulated much less acrylamide than was present in controls. This modification did not alter the color, texture, and taste of the final product. Furthermore, preliminary greenhouse data indicated that the intragenic lines displayed the same agronomic features as their untransformed counterparts. If confirmed by follow-up studies, all-native fry and chip products with very low levels of acrylamide may be offered as a new market choice within the next five years. Given the important role of processed potato products in the modern Western diet, a replacement of current varieties with intragenic potatoes would reduce the average daily intake of acrylamide by almost one-third (Rommens et al. 2008a).

A third application of intragenic potato modification is directed towards enhancing the crop's antioxidant potential. Until recently, this quality trait was not considered in potato breeding programs. New initiatives to produce colored high-antioxidant potatoes still need to overcome many issues associated with the genetic complexity that underlies antioxidant product formation. For instance, several powerful flavo-nols such as kaempferol and quercetin are mainly produced in the anthers, where they support the production of viable pollen (Guyon et al. 2000). It is difficult to divert the underlying biosynthetic pathway to tubers by simply relying on random recombination processes. However, recent experiments have shown that this pathway can be activated in tubers through overexpression of the transcription factor gene StMtf1M (Rommens et al. 2008b). The subsequent down-regulation of flavo-noid-3',5'-hydroxylase gene expression limited the formation of anthocyanins and, instead, resulted in a 100-fold increased accumulation of kaempferol, to 0.27 mg g-1 dry weight (DW; Fig. 4.2). This genetic modification did not alter tuber yields and also had no effect on the sensory characteristics of processed food.

Ramus Dorsalis Plexus
  1. 4.1 Toxicity of heat-induced acrylamide formation in starchy foods (a) Nitrogen is acquired from the environment via nitrate reduction or ammonia uptake, and assimilated through the action of enzymes such as glutamine synthetase (GS), ferredoxin-dependent glutamate synthase (GOGAT), aspartate aminotransferase (ASPAT), asparagine synthetase (AS), asparaginase (ASPASE), and glutamate dehydrogenase (GDH). GLN Glutamine, aKG a-ketoglutarate, OXA oxaloacetate, ASP aspartate. (b) Asparagine plays a role in the long-distance transport of nitrogen, and in the storage of this compound in sink organs such as tubers and seeds. (c) At temperatures exceeding 120°C, the a-NH2 group of asparagine reacts with the carbonyl group of reducing sugars to produce a Schiff base which, especially under high moisture conditions, forms the Amadori compound N-(D-glucos-1-yl)-L-asparagine. This unstable compound then forms acryl-amide through decarboxylative deamination. (d) Upon intake of processed starchy foods, acrylamide is readily absorbed and distributed among tissues. Some acrylamide is detoxified (detox.) whereas another part is converted to glycidamide. Both acrylamide and glycidamide bind DNA and proteins to form adducts. This adduct formation is linked to various diseases
  2. 4.1 Toxicity of heat-induced acrylamide formation in starchy foods (a) Nitrogen is acquired from the environment via nitrate reduction or ammonia uptake, and assimilated through the action of enzymes such as glutamine synthetase (GS), ferredoxin-dependent glutamate synthase (GOGAT), aspartate aminotransferase (ASPAT), asparagine synthetase (AS), asparaginase (ASPASE), and glutamate dehydrogenase (GDH). GLN Glutamine, aKG a-ketoglutarate, OXA oxaloacetate, ASP aspartate. (b) Asparagine plays a role in the long-distance transport of nitrogen, and in the storage of this compound in sink organs such as tubers and seeds. (c) At temperatures exceeding 120°C, the a-NH2 group of asparagine reacts with the carbonyl group of reducing sugars to produce a Schiff base which, especially under high moisture conditions, forms the Amadori compound N-(D-glucos-1-yl)-L-asparagine. This unstable compound then forms acryl-amide through decarboxylative deamination. (d) Upon intake of processed starchy foods, acrylamide is readily absorbed and distributed among tissues. Some acrylamide is detoxified (detox.) whereas another part is converted to glycidamide. Both acrylamide and glycidamide bind DNA and proteins to form adducts. This adduct formation is linked to various diseases
  1. 4.2 Potato phenotypes. An untransformed Bintje potato tuber (left) is compared with two tubers from plants expressing StMtf1M (middle pair) and a tuber that expresses StMtf1M but is silenced for the F3'5'h gene (right). This last tuber contains kaempferol levels that are 12-fold higher than those of the primary transformant and almost 100-fold higher than the untransformed control
  2. 4.2 Potato phenotypes. An untransformed Bintje potato tuber (left) is compared with two tubers from plants expressing StMtf1M (middle pair) and a tuber that expresses StMtf1M but is silenced for the F3'5'h gene (right). This last tuber contains kaempferol levels that are 12-fold higher than those of the primary transformant and almost 100-fold higher than the untransformed control

Given the large amounts of potatoes that are consumed on a daily basis, estimated at 171 g person-1, replacement of currently available commodity potatoes by varieties overexpressing StMtf1M would, on average, double the average daily intake of kaempferol (Rommens et al. 2008b).

Another important target for intragenic modification relates to the lingering presence of plant-produced toxins or allergens in crops including potato. It is estimated that 0.2% of Americans are allergic to potatoes. Furthermore, consumption of potato products can occasionally result in the intake of glycoalkaloids at levels that are acutely toxic. Regulatory agencies oppose the intentional employment of genes that are known to produce allergens, toxins, or anti-nutritional compounds (Kaeppler 2000) but can do little to prevent the unchecked transfer of such genes through conventional breeding (Bradford et al. 2005). Many plant-derived toxins are effective against plant pathogens and insects, and breeders may have unknowingly selected for the presence of such genes by seeking to enhance disease tolerance levels. However, given the advances in integrated strategies to control diseases and pests, it may be possible now to start lowering toxin levels in the edible parts of food crops. Although some allergen-encoding Patatin genes could be inactivated through mutagenesis, it would be difficult to eliminate all these functional genes from potato. In contrast, a carefully designed silencing approach was recently shown to substantially reduce the formation of all Patatin storage proteins in potato tubers (Kim et al. 2008).

The above examples demonstrate the significance of intragenic crop modification for quality improvements. However, it is also important to support breeders in their efforts to enhance potato's tolerance to various biotic and abiotic stresses. One of the most important diseases that threatens the potato industry is late blight. Breeding programs have not been able to markedly increase the level of resistance of current potato varieties, and chemical control is under pressure as late blight becomes increasingly aggressive and there is societal resistance against the use of environmentally unfriendly fungicides. Consequently, groups of scientists at Wageningen University, the United States Department of Agriculture, Simplot, and elsewhere have embraced intragenic approaches to transfer multiple resistance (R-)genes from wild potatoes to important varieties with proven adaptation, such as Desiree and Atlantic. Two R-genes of particular interest are the Rpi-blb1 and Rpi-blb2 genes from Solanum bulbocastanum (van der Vossen et al. 2003, 2005; see also Chap. 20). Unlike other known R-genes, this gene combination appears to provide durable resistance to potato.

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