Transgenic Potato Tubers Research Articles

Increased accumulation of anthocyanins in transgenic potato tubers by overexpressing the 3GT gene

In order to explore a biotechnological method for improving potato tuber color and creating plants with increased anthocyanin contents, a potato UDP-glucose: flavonoid-3-O-glucosyltransferase (3GT) gene was inserted behind the GBSSI promoter of pBin19, and this construct was introduced into Solanumtuberosum L. cultivar Desiree plants by Agrobacterium-mediated transformation. Six independent transgenic lines overexpressing the 3GT gene were identified by PCR and Southern blot analysis from 18 kanamycin-resistant plants. Due to the expression of 3GT gene, the tuber color and the anthocyanin content were enhanced noticeably in the transgenic plants compared to the wild-type control plants. This result suggests that the 3GT gene can potentially be used to improve potato color and enhance levels of antioxidants in the diet.

Altering trehalose-6-phosphate content in transgenic potato tubers affects tuber growth and alters responsiveness to hormones during sprouting.

Trehalose-6-phosphate (T6P) is a signaling metabolite that regulates carbon metabolism, developmental processes, and growth in plants. In Arabidopsis (Arabidopsis thaliana), T6P signaling is, at least in part, mediated through inhibition of the SNF1-related protein kinase SnRK1. To investigate the role of T6P signaling in a heterotrophic, starch-accumulating storage organ, transgenic potato (Solanum tuberosum) plants with altered T6P levels specifically in their tubers were generated. Transgenic lines with elevated T6P levels (B33-TPS, expressing Escherichia coli osmoregulatory trehalose synthesis A [OtsA], which encodes a T6P synthase) displayed reduced starch content, decreased ATP contents, and increased respiration rate diagnostic for high metabolic activity. On the other hand, lines with significantly reduced T6P (B33-TPP, expressing E. coli OtsB, which encodes a T6P phosphatase) showed accumulation of soluble carbohydrates, hexose phosphates, and ATP, no change in starch when calculated on a fresh weight basis, and a strongly reduced tuber yield. [¹⁴C]glucose feeding to transgenic tubers indicated that carbon partitioning between starch and soluble carbohydrates was not altered. Transcriptional profiling of B33-TPP tubers revealed that target genes of SnRK1 were strongly up-regulated and that T6P inhibited potato tuber SnRK1 activity in vitro. Among the SnRK1 target genes in B33-TPP tubers, those involved in the promotion of cell proliferation and growth were down-regulated, while an inhibitor of cell cycle progression was up-regulated. T6P-accumulating tubers were strongly delayed in sprouting, while those with reduced T6P sprouted earlier than the wild type. Early sprouting of B33-TPP tubers correlated with a reduced abscisic acid content. Collectively, our data indicate that T6P plays an important role for potato tuber growth.

A Transgenic Study on Affecting Potato Tuber Yield by Expressing the Rice Sucrose Transporter Genes OsSUT5Z and OsSUT2MF

In many plants, sucrose transporters are essential for both sucrose exports from sources and imports into sinks, indicating a function in assimilate partitioning. To investigate whether sucrose transporters can improve the yield of starch plant, potato plants (Solanum tuberosum L. cv. Désirée) were transformed with cDNAs of the rice sucrose transporter genes OsSUT5Z and OsSUT2M under the control of a tuber-specific, class-I patatin promoter. Compared to the controls, the average fructose content of OsSUT5Z transgenic tubers significantly increased. However, the content of the sugars and starch in the OsSUT2M transgenic potato tubers showed no obvious difference. Correspondingly, the average tuber yield, average number of tubers per plant and average weight of single tuber showed no significant difference in OsSUT2M transgenic tubers with controls. In the OsSUT5Z transgenic lines, the average tuber yield per plant was 1.9-fold higher than the controls, and the average number of tubers per plant increased by more than 10 tubers on average, whereas the average weight of a single tuber did not increase significantly. These results suggested that the average number of tubers per plant showed more contribution than the average weight of a single tuber to the tuber yield per plant.

Resistance levels and fitness of glufosinate-resistant transgenic sweet potato in field experiments

Transgenic herbicide-resistant sweet potato plants [ Ipomoea batatas (L.) Lam.] produced through a biolistic transformation were used in this study. The objectives of this research were to (a) quantify resistance levels of transgenic sweet potato expressing the bar gene to glutamine synthetase (GS)-inhibitors, glufosinate and methionine sulfoximine, protoporphyrinogen oxidase (PPO)-inhibiting herbicide, oxyfluorfen and photosynthesis-inhibiting herbicide, paraquat; (b) compare the relative chlorophyll content, quantum yield, growth, and tuberous root yield between transgenic and wild-type sweet potato; (c) compare the response of transgenic and wild-type sweet potato plants to chilling and high temperature, and (d) compare amino acids, minerals and sugar contents of transgenic and wild-type sweet potato tubers. The transgenic sweet potato lines were 20–82-fold more resistant to glufosinate than the wild-type. The representative transgenic line 7171 also showed resistance to methionine sulfoximine, but was not resistant to oxyfluorfen and paraquat, which have different target sites. The stem length, number of leaves, petiole length, shoot fresh weight, and storage roots of transgenic lines were lower than those of the wild-type in field experiments. Reduced growth and root yield were not due to reduced quantum yield and relative chlorophyll contents. Glufosinate increased the ammonium accumulation in leaves of wild-type plants, but had minimal or no effect on leaves of transgenic line 7171. There were differences in mineral contents, free sugars, total amino acids, and free amino acids in the petiole and storage roots between wild-type and line 7171. There was no difference in drought tolerance between wild-type and line 7171, but the transgenic line was more tolerant to chilling temperature than wild-type plants. Follow-up research is required to elucidate the mechanisms underlying the growth and yield differences, different nutritional values, and differential response to low temperature between wild-type and transgenic sweet potato.

Biochemical analysis of enhanced tolerance in transgenic potato plants overexpressing d-galacturonic acid reductase gene in response to various abiotic stresses

Upregulation of the antioxidant enzyme system in plants provides protection against various abiotic stresses. Transgenic potato plants overexpressing the strawberry d-galacturonicacidreductase (GalUR) gene with enhanced accumulation of ascorbate (AsA) were used to study the antioxidant system involving the ascorbate–glutathione cycle in order to understand the tolerance mechanism in plants in response to various abiotic stresses under in vitro conditions. Transgenic potato tubers subjected to various abiotic stresses induced by methyl viologen, sodium chloride and zinc chloride showed enhanced activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.1.1.1.6) and enzymes of the ascorbate–glutathione cycle such as ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.8.1.7), as well as increased levels of ascorbate, glutathione (GSH) and proline when compared to untransformed tubers. The increased enzyme activities correlated with the mRNA transcript levels in the stressed transgenic tubers. Significant differences in redox status of AsA and GSH were also observed in stressed transgenic potato tubers that showed increased tolerance to abiotic stresses compared to untransformed tubers. This study suggests that the increased accumulation of AsA could upregulate the antioxidant system which imparts improved tolerance against various abiotic stresses in transgenic tubers compared to untransformed tubers.

Oral transgenic plant-based vaccine for hepatitis B

In addition to improving vaccine formulations in order to elicit robust and long-lasting immune responses, there is also an increasing need for improving the manner in which these vaccines are delivered. As most current vaccines are administered by injection by a health care giver, there is the ever-present danger of needlestick injuries. Therefore, needle-free vaccinations are a viable option toward limiting needle-associated injuries and additionally increasing compliance with vaccination schedules, as both children and adults have an aversion to injections. Noninvasive methods of vaccination will also facilitate speed of vaccine delivery and likely also reduce cost, both important factors for health care in developing countries. One alternative to current injectable immunizations is orally delivered vaccines. A specific approach that we and others are evaluating is the use of transgenic plant tissue that expresses vaccine antigens for oral immunization. Herein, we review the development of an oral HBV vaccine expressed in transgenic potato tubers and the immune responses generated in human subjects given this novel vaccine.

Enhancing Sucrose Synthase Activity in Transgenic Potato (Solanum tuberosum L.) Tubers Results in Increased Levels of Starch, ADPglucose and UDPglucose and Total Yield

Sucrose synthase (SuSy) is a highly regulated cytosolic enzyme that catalyzes the conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate glucose and fructose. To determine the impact of SuSy activity in starch metabolism and yield in potato (Solanum tuberosum L.) tubers we measured sugar levels and enzyme activities in tubers of SuSy-overexpressing potato plants grown in greenhouse and open field conditions. We also transcriptionally characterized tubers of SuSy-overexpressing and -antisensed potato plants. SuSy-overexpressing tubers exhibited a substantial increase in starch, UDPglucose and ADPglucose content when compared with controls. Tuber dry weight, starch content per plant and total yield of SuSy-overexpressing tubers increased significantly over those of control plants. In contrast, activities of enzymes directly involved in starch metabolism in SuSy-overexpressing tubers were normal when compared with controls. Transcriptomic analyses using POCI arrays and the MapMan software revealed that changes in SuSy activity affect the expression of genes involved in multiple biological processes, but not that of genes directly involved in starch metabolism. These analyses also revealed a reverse correlation between the expressions of acid invertase and SuSy-encoding genes, indicating that the balance between SuSy- and acid invertase-mediated sucrolytic pathways is a major determinant of starch accumulation in potato tubers. Results presented in this work show that SuSy strongly determines the intracellular levels of UDPglucose, ADPglucose and starch, and total yield in potato tubers. We also show that enhancement of SuSy activity represents a useful strategy for increasing starch accumulation and yield in potato tubers.

The sweet potato sporamin promoter confers high-level phytase expression and improves organic phosphorus acquisition and tuber yield of transgenic potato

The sweet potato sporamin promoter was used to control the expression in transgenic potato of the E. coli appA gene, which encodes a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The sporamin promoter was highly active in leaves, stems and different size tubers of transgenic potato, with levels of phytase expression ranging from 3.8 to 7.4% of total soluble proteins. Phytase expression levels in transgenic potato tubers were stable over several cycles of propagation. Field tests showed that tuber size, number and yield increased in transgenic potato. Improved phosphorus (P) acquisition when phytate was provided as a sole P source and enhanced microtuber formation in cultured transgenic potato seedlings when phytate was provided as an additional P source were observed, which may account for the increase in leaf chloroplast accumulation (important for photosynthesis) and tuber yield of field-grown transgenic potato supplemented with organic fertilizers. Animal feeding tests indicated that the potato-produced phytase supplement was as effective as a commercially available microbial phytase in increasing the availability of phytate-P to weanling pigs. This study demonstrates that the sporamin promoter can effectively direct high-level recombinant protein expression in potato tubers. Moreover, overexpression of phytase in transgenic potato not only offers an ideal feed additive for improving phytate-P digestibility in monogastric animals but also improves tuber yield, enhances P acquisition from organic fertilizers, and has a potential for phytoremediation.

Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers

Transgenic plants have facilitated our understanding of the functional roles of genes and the metabolic processes affected in plants. Recently, the Or gene was isolated from an orange cauliflower mutant and it was shown that the Or gene could serve as a novel genetic tool to enrich carotenoid content in transgenic potato tubers. An in-depth characterization of these Or transgenic lines is presented here. It was found that the Or transgene may facilitate the identification of potential rate-limiting step(s) of the carotenoid biosynthetic pathway. The Or transgenic tubers accumulated not only increased levels of carotenoids that normally are present in the controls, but also three additional metabolite intermediates of phytoene, phytofluene, and zeta-carotene, indicating that the desaturation steps became limiting following the expression of the Or transgene. Moreover, we observed that long-term cold storage greatly enhanced carotenoid content in the Or transgenic tubers to a level of 10-fold over controls. Expression of the Or transgene in the transgenic plants caused no dramatic changes in the transcript levels of the endogenous carotenoid biosynthetic genes, which is in agreement with the Or gene not directly controlling carotenoid biosynthesis. Microscope analysis revealed that the Or transgene conferred the formation of chromoplasts containing carotenoid sequestering structures in a heterologous system. Such structures were not observed in tubers of potato cultivars that accumulate high levels of carotenoids. Collectively, these results provide direct evidence demonstrating that the Or gene indeed controls chromoplast differentiation and that regulation of chromoplast formation can have a profound effect on carotenoid accumulation in plants.

Decreased expression of plastidial adenylate kinase in potato tubers results in an enhanced rate of respiration and a stimulation of starch synthesis that is attributable to post-translational redox-activation of ADP-glucose pyrophosphorylase

Adenine nucleotides are of general importance for many aspects of cell function, but their role in the regulation of biosynthetic processes is still unclear. It was previously reported that decreased expression of plastidial adenylate kinase, catalysing the interconversion of ATP and AMP to ADP, leads to increased adenylate pools and starch content in transgenic potato tubers. However, the underlying mechanisms were not elucidated. Here, it is shown that decreased expression of plastidial adenylate kinase in growing tubers leads to increased rates of respiratory oxygen consumption and increased carbon fluxes into starch. Increased rates of starch synthesis were accompanied by post-translational redox-activation of ADP-glucose pyrophosphorylase (AGPase), catalysing the key regulatory step of starch synthesis in the plastid, while there were no substantial changes in metabolic intermediates or sugar levels. A similar increase in post-translational redox-activation of AGPase was found after supplying adenine to wild-type potato tuber discs to increase adenine nucleotide levels. Results provide first evidence for a link between redox-activation of AGPase and adenine nucleotide levels in plants.

Evaluation of Neomycin Phosphotransferase II and Cry3A as Targets for the Serological Detection of Transgenic Potato Tubers

Neomycin phosphotransferase II (NptII) and Cry3A were evaluated as potential targets in ELISA for the detection of transgenic potato tubers. The highest concen- trations of NptII (0.23 - 2.95 μg /g) were measured in cultivar NL 10-ATL tubers, while very low (<0.007 μ g/ g) or undetectable levels of NptII were found in tuber tissue of the other cultivars. Polymerase chain reaction (PCR) analysis revealed that the nptII transgene could not be detected in cultivars NL 10-SUP or in NL-30-RBK-082. It was concluded that a serological method targeting NptII would not provide an effective approach for detecting transgenic potato tubers. Cry3A could be detected in all the cultivars, ranging between 0.02 μ g/ g in NL 20-SHE and NL 10-ATL to 1.98 μg /g in NL 10-SUP. Environmental conditions appeared to influence Cry3A protein accumulation in tubers ofcultivarNL30-RBK-350butnosucheffectoncultivarsNL 10-ATL or NL 20-SHE could be detected. It was concluded that ELISA methods for Cry3A detection could be used for confirming the presence of cry3A in individual tubers. With improved sensitivity for Cry3A, serological methods may be useful in screening larger lots for the presence of transgenic potato tubers. Resumen Se evaluaron neomicina fosfotransferasa II (NptII) y Cry 3A como objetivos potenciales en ELISA para la deteccion de tuberculos de papa transgenica. Las concentraciones mas altas de NptII (0.23-2.95 µg/g) se midieron en tuberculos del cultivar NL-10-ATL, mientras

RNA interference‐mediated repression of sucrose‐phosphatase in transgenic potato tubers (Solanum tuberosum) strongly affects the hexose‐to‐sucrose ratio upon cold storage with only minor effects on total soluble carbohydrate accumulation

Storage of potato tubers at low temperatures leads to the accumulation of glucose and fructose in a process called 'cold sweetening'. The aim of this work was to investigate the role of sucrose-phosphatase (SPP) in potato tuber carbohydrate metabolism at low temperature (4 degrees C). To this end, RNA interference (RNAi) was used to reduce SPP expression in transgenic potato tubers. Analysis of SPP specific small interfering RNAs (siRNAs), SPP protein accumulation and enzyme activity indicated that SPP silencing in transgenic tubers was stable during the cold treatment. Analysis of soluble carbohydrates showed that in transgenic tubers, cold-induced hexogenesis was inhibited while, despite strongly reduced SPP activity, sucrose levels exceeded wild-type (WT) values four- to fivefold after 34 d of cold treatment. This led to a drastic change in the hexose-to-sucrose ratio from 1.9 in WT tubers to 0.15 to 0.11 in transgenic tubers, while the total amount of soluble sugars was largely unchanged in both genotypes. Sucrose-6(F)-phosphate (Suc6P), the substrate of SPP, accumulated in transgenic tubers in the cold which most likely enables the residual enzyme to operate with maximal catalytic activity in vivo and thus, in the long term, counterbalances reduced SPP activity in the transformants. Northern analysis revealed that cold-induced expression of vacuolar invertase (VI) was blocked in SPP-silenced tubers explaining a reduced sucrose-to-hexose conversion. Suc6P levels were found to negatively correlate with VI expression. A possible role of Suc6P in regulating VI expression is discussed.

Decreased amount of reducing sugars in transgenic potato tubers and its influence on yield characteristics

This work focuses on the comparison of field characteristics and amounts of reducing sugars in cold-stored tubers of transgenic plants derived from two potato cultivars. The bacterial gene coding for phosphofructokinase under the tuber-specific promoter was used to support the glycolysis in stored tubers. While the tubers from untransformed control plants steadily accumulated reducing sugars during cold storage, the tubers from transformed plants regardless the genotype were characterized by subsequent decrease in the sugar content. After long period of cold storage the greatest reduction in the reducing sugar content was by more than 60 % compared to control. Before the storage, however, the content of reducing sugars was in 80 % of transgenic lines higher than in control ones. The plants evaluated in field trials for their appearance showed any changes in growth characteristics in about 25 % of the transgenic lines. Despite the introduced modification of sugar metabolism the yield of transgenic plants with normal appearance did not differ significantly from the yield of control plants.

Resistance to &lt;I&gt;Tecia solanivora&lt;/I&gt; (Lepidoptera: Gelechiidae) in Three Transgenic Andean Varieties of Potato Expressing &lt;I&gt;Bacillus thuringiensis&lt;/I&gt; Cry1Ac Protein

Transgenic potato, Solanum tuberosum L., plants containing a synthetic cry1Ac gene coding for the Bacillus thuringiensis (Bt) crystalline insecticidal protein were produced and evaluated for resistance to Tecia solanivora Povolny (Lepidoptera: Gelechiidae), the larvae of which attack potato tubers. In total, 43 transgenic lines of commercial Andean potato varieties Diacol Capiro, Pardo Pastusa, and Pandeazúcar were obtained. These transgenic lines were found to have one to four copies of cry1Ac per genome and expression levels of Cry1Ac protein varying from 0.02 to 17 microg/g fresh tuber tissue. Bioassays of T. solanivora larvae on these transgenic potato tubers showed 83.7-100% mortality, whereas the mortality levels on nontransgenic lines were 0-2.67%. Our data indicate the capability of Bt transgenic technology to control the T. solanivora while reducing the use of chemical insecticides. Further studies under controlled field conditions will be helpful in exploring the potential of CrylAc potatoes in the insect pest management strategies.

Assessing the potential for unintended effects in genetically modified potatoes perturbed in metabolic and developmental processes. Targeted analysis of key nutrients and anti-nutrients

Targeted compositional analysis was carried out on transgenic potato tubers of either cultivar (cv.) Record or cv. Desirée to assess the potential for unintended effects caused by the genetic modification process. The range of transgenic lines analysed included those modified in primary carbohydrate metabolism, polyamine biosynthesis and glycoprotein processing. Controls included wildtype tubers, tubers produced from plants regenerated through tissue culture (including a callus phase) and tubers derived from transformation with the 'empty vector' i.e. no specific target gene included (with the exception of the kanamycin resistance gene as a selectable marker). Metabolite analysis included soluble carbohydrates, glycoalkaloids, vitamin C, total nitrogen and fatty acids. Trypsin inhibitor activity was also assayed. These cover the major compounds recommended by the OECD in their Consensus Document on Compositional Considerations for New Varieties of Potatoes: Key Food and Feed Nutrients, Anti-Nutrients and Toxicants (2002). Data was statistically analysed using analysis of variance (ANOVA) for individual compounds and, where applicable, principal component analysis (PCA). In general, targeted compositional analysis revealed no consistent differences between GM lines and respective controls. No construct specifically induced unintended effects. Statistically significant differences between wildtype controls and specific GM lines did occur but appeared to be random and not associated with any specific construct. Indeed such significant differences were also found between wildtypes and both tissue culture derived tubers and tubers derived from transformation with the empty vector. This raises the possibility that somaclonal variation (known to occur significantly in potato, depending on genotype) may be responsible for an unknown proportion of any differences observed between specific GM lines and the wildtype. The most obvious differences seen in GC-MS profiles were between the two potato varieties used in the study.

Transgenic potato expressing Aβ reduce Aβ burden in Alzheimer’s disease mouse model

Beta amyloid (Aβ) is believed one of the major pathogens of Alzheimer’s disease (AD), and the reduction of Aβ is considered a primary therapeutic target. Immunization with Aβ can reduce Aβ burden and pathological features in transgenic AD model mice. Transgenic potato plants were made using genes encoding 5 tandem repeats of Aβ1–42 peptides with an ER retention signal. Amyloid precursor protein transgenic mice (Tg2576) fed with transgenic potato tubers with adjuvant showed a primary immune response and a partial reduction of Aβ burden in the brain. Thus, Aβ tandem repeats can be expressed in transgenic potato plants to form immunologically functional Aβ, and these potatoes has a potential to be used for the prevention and treatment of AD.

Transgenic potato tubers with overexpression of 14-3-3 protein in growing rat diets. 2. Redox indices in blood and brain

Transgenic potato tubers with overexpression of 14-3-3 protein in growing rat diets. 2. Redox indices in blood and brain

Transgenic potato tubers with overexpression of 14-3-3 protein in growing rat diets. 1. Selected hormone activities and liver function status

Transgenic potato tubers with overexpression of 14-3-3 protein in growing rat diets. 1. Selected hormone activities and liver function status

Transgenic potato tubers as a source of phenolic compounds. Localization of anthocyanins in the peridermis

The localization of phenolic compounds in tubers of transgenic potatoes overexpressing enzymes of the flavonoid synthesis pathway, i.e. chalcone synthase (CHS), chalcone isomerase (CHI) and dihydroflavonol dehydrogenase (DFR), was examined by fluorescence microscopy. The study revealed the presence of flavonoids (anthocyanins) in cell vacuoles, mainly in the peridermis. A relatively higher concentration of anthocyanin compounds was observed in the tissue of transgenic potato tubers, revealing the expression of chalcone synthase (CHS). The increased content of flavonoids in transgenic potato tubers may affect their agrotechnical traits and may increase the nutritive value of the tubers.

Caecal fermentation in rats fed diets containing transgenic potato tubers

Caecal fermentation in rats fed diets containing transgenic potato tubers