Transgenic Potato Research Articles

Increased ribonuclease activity in Solanum tuberosum L. transformed with heterologous genes of apoplastic ribonucleases as a putative approach for production of virus resistant plants.

Viral pathogens seriously decrease the yield of potato (Solanum tuberosum L.) –an important agricultural crop. Therefore, there is a demand for potato cultivars resistant to multiple viruses. Ribonucleases (RNases) are supposed to be engaged to antiviral response in plants. Heterologous RNase gene expression provides a tool for production of cultivars with multiple resistance to viruses and viroids. Transgenic potato cultivars Luhivs’ka and Lasynak with heterologous genes bov and ZRNase II of apoplastic RNases from Bos taurus and Zinnia elegans respectively were obtained via Agrobacterium-mediated transformation. The presence of bov and ZRNase II transgenes was confirmed by PCR analysis. RNase activity was examined by modified Oleshko method. Plants with heterologous ribonuclease genes had higher level of RNase activity compared to nontransgenic ones. Transgenic plants inoculated with Potato virus Y, PVY (genus Potyvirus, family Potyviridae) demonstrated delayed and less severe symptoms of viral infection. DAS-ELISA confirmed the presence of viral antigens both in transformed and control plants. Visual manifestations of viral infection in transgenic potatoes were milder than in control plants and their development was delayed, but complete elimination of the virus did not occur.

Food for thought – how tuberization and drought tolerance are linked in potato

Food for thought – how tuberization and drought tolerance are linked in potato

Expression of ice recrystallization inhibition protein in transgenic potato lines associated with reduced electrolyte leakage and efficient recovery post freezing injury

Potato (Solanum tuberosum L.) is considered to be frost-susceptible as short spells of frost can reduce the tuber yield and quality. Ice recrystallization inhibition (IRI) protein helps prevent growth of ice crystals in the cell apoplast during frost and help prevent damage associated with freezing stress. In this study, we investigated the in planta potential of Lolium perenne derived IRI3 transgene in improving the tolerance of transgenic potato lines for freezing stress. The codon optimized IRI3 transgene was introduced into potato cultivar Diamant through Agrobacterium mediated transformation. Three transgenic potato lines were successfully generated which were confirmed for transgene insertion and genomic integration by polymerase chain reaction and Southern blot. It was evident that the IRI3 transcript decreased in initial 24 h of cold stress treatment while the IRI3 mRNA expression up regulated in subsequent hours of cold treatment with maximum increase to 20 folds at 96 h post stress. A similar trend was also revealed in ion-leakage assay which showed that during cold stress, the transgenic potato lines depicted reduced ion leakage of 14–22% as compared to non-transgenic control plants. Further, the generated transgenic potato lines were tolerant to the frost spell in quarantine field conditions as compared to the non-transgenic potato lines. Additionally, the transgenic lines exhibited efficient recovery post frost injury in field conditions. The biochemical profiles of chlorophyll, proline and higher levels of antioxidant enzyme (superoxide dismutase, Catalase) activity and malondialdehyde levels showed that despite the phenotypic impact of low temperature, the transgenic potato lines quickly adjusted to maintain their cellular homeostasis post freezing stress by increasing the antioxidant defenses. This study suggests that up regulation of IRI3 transcript and regulatory network of cold stress response in transgenic potato lines improve frost tolerance and help stabilize yield in cultivated potato.

Future-Proofing Potato for Drought and Heat Tolerance by Overexpression of Hexokinase and SP6A.

Crop yield is largely affected by global climate change. Especially periods of heat and drought limit crop productivity worldwide. According to current models of future climate scenarios, heatwaves and periods of drought are likely to increase. Potato, as an important food crop of temperate latitudes, is very sensitive to heat and drought which impact tuber yield and quality. To improve abiotic stress resilience of potato plants, we aimed at co-expressing hexokinase 1 from Arabidopsis thaliana (AtHXK1) in guard cells and SELF-PRUNING 6A (SP6A) using the leaf/stem-specific StLS1 promoter in order to increase water use efficiency as well as tuberization under drought and heat stress. Guard cell-specific expression of AtHXK1 decreased stomatal conductance and improved water use efficiency of transgenic potato plants as has been shown for other crop plants. Additionally, co-expression with the FT-homolog SP6A stimulated tuberization and improved assimilate allocation to developing tubers under control as well as under single and combined drought and heat stress conditions. Thus, co-expression of both proteins provides a novel strategy to improve abiotic stress tolerance of potato plants.

Expression of the Tomato pot-1 Gene Confers Potato Virus Y (PVY) Resistance in Susceptible Potato Varieties

The translation initiation factor 4E (eIF4E) has been implicated in naturally-occurring resistance to the Potato Virus Y (PVY) determined by the pot-1 locus in tomato. The susceptible potato varieties Russet Norkotah, Silverton Russet, and Classic Russet, along with the Michigan State University breeding line MSE149-5Y, were transformed with the pot-1 gene from Solanum hirsutum to achieve PVY resistance. Transgenic potato events and their non-transgenic controls were screened for PVY resistance against three PVY strains (PVYO, PVYN:O, PVYNTN) by enzyme-linked immunosorbent assay (ELISA) in greenhouse and in field trials. The event A95.87.05 (Classic Russet) and MSE149.87.22 (MSE149-5Y) had resistance against all three PVY isolates tested in both foliage and tuber under either greenhouse or field conditions. In contrast, SR.87.62 (Silverton Russet) and RN1.87.09 (Russet Norkotah), displayed moderate susceptibility to PVY in foliage during the growing season and increased level of infection in tubers. Transformation of PVY-susceptible potato varieties with the tomato pot-1 gene can be a useful tool for improving PVY resistance.

Genetic engineering of potato (Solanum tuberosum L.) for enhanced α-tocopherols and abiotic stress tolerance.

Vitamin E (α-tocopherol) is a lipid-soluble essential vitamin recognized for improvement in degenerative health conditions, abating cancer risk, and coronary heart diseases in humans. While in plants, it acts as a free radical scavenger that protects cells against oxidative and photooxidative damages. The daily consumption of potato makes it a key target for biofortification with vitamins for eliminating vitamin deficiency in large populations. Vitamin E biosynthetic pathway genes have been overexpressed in plants via genetic engineering to enhance the α-tocopherol content. Major genes involved in the vitamin E biosynthesis in plants viz. the homogentisate-phytyltransferase (At-HPT) and γ-tocopherol-methyltransferase (At-γ-TMT), isolated from Arabidopsis were constitutively overexpressed in potato (Solanum tuberosum L.). The molecular analyses of independent transgenic lines revealed a stable integration of both the genes in the plant genome. The transgenic potato exhibited significantly improved vitamin E contents up to 173-258% in comparison to the untransformed control plants. Transgenic tissues also exhibited increased cellular antioxidant enzymes, proline, osmolyte, and glutathione content that are directly correlated with the ability of the plant to withstand abiotic stresses imposed by salt (NaCl) and heavy metal (CdCl2 ). Therefore, the current strategy of increasing the vitamin E content in potato with enhanced tolerance to abiotic stresses might greatly aid efforts to engineer crops for human health benefits and greater yield under adverse environmental conditions.

A novel aspartic protease inhibitor inhibits the enzymatic browning of potatoes

• StASPI expressed highly in browning-less potato than in browning-susceptible potato. • Both exogenous Pepstatin A and overexpressed -StASPI inhibited potato browning. • Overexpressed StASPI reduced protease activity and contents of free amino acids. • Overexpressed StASPI decreased PPO activity. • Overexpressed StASPI enhanced SOD and CAT activity, and reduced ROS contents. Enzymatic browning greatly affects the quality of fresh-cut potatoes ( Solanum tuberosum L.). The transcript level of a novel aspartic protease inhibitor gene, StASPI , was found significantly higher in browning-less potatoes than in browning-susceptible potatoes, and exogenous aspartic protease inhibitor Pepstatin A inhibited the browning of potato pulp. Therefore, we speculated that StASPI plays important roles in browning-resistance of potatoes. The effects of overexpressed- StASPI on browning, total free amino acids (FAAs), antioxidant enzyme activity and ROS accumulation in potatoes were investigated in this study. Results showed that overexpression of StASPI effectively reduced enzymatic browning after cutting, significantly decreased protease activity, and reduced the accumulation of total FAAs in potatoes. Browning degrees of transgenic potato mash were increased by supplementing exogenous FAAs, but the degrees were still significantly lower than that of wild-type (WT) mash. Furthermore, overexpression of StASPI decreased PPO activity, enhanced the activities of antioxidant enzymes SOD and CAT, and reduced H 2 O 2 and O 2 ˙ ― contents. These results indicated that overexpression of StASPI inhibited the enzymatic browning of potatoes, decreasing FAAs accumulation, reducing PPO activity, and enhancing activity of antioxidant enzymes. This study provides a new perspective on the strategies for inhibiting enzymatic browning of potatoes.

Tissue-specific and stress-inducible promoters establish their suitability for containment of foreign gene(s) expression in transgenic potatoes.

The present study was conducted to determine efficiency of green tissue-specific (pRCA) and stress-inducible promoters (pRD29A) to express E. coli beta-glucuronidase (gusA) genein transgenic potatoes compared with constitutive promoter (35S CaMV). The promoter fragments were isolated from their original source and cloned upstream to gusA in pCAMBIA-1301 binary vector to develop plant expression constructs, i.e., pRCA-pCAMBIA and pRD29A-pCAMBIA. Agrobacterium strain GV2260 harboring recombinant plasmids were used to infect leaf discs and internodal explant of Lady Olympia cultivar. GUS histochemical analysis was performed at different stages to determine GUS activity in transgenic plants. To determine activity of stress-inducible promoter (pRD29A), transgenic plants were exposed to heat, drought and combination of both heat and drought stress. The real time (RT-qPCR) and GUS florimetric assays revealed that pRD29A promoter gets more activated under drought, heat and combination of both stresses. GUS expression levels were more than 10 folds high with pRD29A promoter compared to control. Likewise, the reduced transcripts levels of gusA gene under control of pRCA promoter were found in tuber/roots of transgenic plants compared to 35S promoter. GUS florimetric assays also showed decreased or no GUS expression in tubers. In conclusion, the results encourage the appropriate use of promoters to drive the expression of foreign gene(s) for the development of potato lines tolerant to biotic and abiotic stress while minimizing the risks of transgenic technology in potatoes.

StRac1 plays an important role in potato resistance against Phytophthora infestans via regulating H2O2 production

ROP GTPases (Rho-related GTPases from plant), a unique subgroup of the Rho family in plants, is a group of key regulators of different signaling pathways controlling plant growth and development, cell polarity and differentiation, and plant response against biotic and abiotic stresses. The present study determined the potential regulatory mechanism of potato ROP GTPase (StRac1) against Phytophthora infestans (P. infestans) infection. Protein secondary structure analysis indicated that StRAC1 is a Rho GTPase. The expression level of StRac1 was variable in different tissues of potato, with the highest expression in young leaves of both Shepody and Hutou potato varieties. After challenging with P. infestans, the expression level of StRac1was higher in resistance varieties Zihuabai and Longshu 7 than in susceptible varieties Shepody and Desiree. StRAC1 fusion with GFP subcellularly localized at the plasma membrane (PM) in tobacco epidermal cells. The potato with transient or stable over-expression of CA-StRac1 (constitutively active form of StRac1)exhibited a dramatic enhancement of its resistance against P. infestans infections. The increased resistance level in transgenic potato was accompanied with elevated H2O2 levels. Importantly, silencing StRac1 via virus-induced gene silencing (VIGS) in potato resulted in higher susceptibility to P. infestans infection than in control plants. In summary, our data reveal that StRac1 regulates potato resistance against P. infestans via positively modulating the accumulation of H2O2.

Overexpression of StCYS1 gene enhances tolerance to salt stress in the transgenic potato (Solanum tuberosum L.) plant

Salt stress seriously restricts the growth and yield of potatoes. Plant cystatins are vital players in biotic stress and development, however, their roles in salt stress resistance remain elusive. Here, we report that StCYS1 positively regulates salt tolerance in potato plants. An in vitro biochemical test demonstrated that StCYS1 is a bona fide cystatin. Overexpression of StCYS1 in both Escherichia coli and potato plants significantly increased their resistance to high salinity. Further analysis revealed that the transgenic plants accumulated more proline and chlorophyll under salt stress conditions. Moreover, the transgenic plants displayed higher H2O2 scavenging capability and cell membrane integrity compared with wild-type potato. These results demonstrate that StCYS1 is closely correlated with salt stress and its overaccumulation can substantially enhance salt stress resistance.

Evaluation of two promoters for generating transgenic potato plants as salicylic acid biosensors

Plants are severely affected by many biotic stresses, which cause a reduction in crop quality and quantity. One of the strategies to manage biotic stresses is the generation of transgenic plant lines that can be used as biosensors. These biosensor plants can trigger an early warning upon any pathogen infection. Two promoters with β-glucuronidase reporter gene fusions were constructed. The first contained the flagellin sensing 2 gene promoter, whereas the second contained synthetic promoter containing four repeats of cis-acting elements from the pathogen-related protein 1 gene and two transcription enhancers from the 35S promoter. Transformed leaves were treated with a phytohormone salicylic acid to mimic the occurrence of biotic stress. Validation of reporter gene expression induced from both constructs in transformed potato leaves displayed an increase upon salicylic acid treatment. The results reflect that both constructs could serve in the production of potato biotic stress biosensors.

Manipulating genome of diploid potato inbred line Solanumchacoense M6 using selectable marker gene

The development of transgenic potatoes is imperative to investigate various gene functions as well as to develop robust potato varieties resistant to different biotic and abiotic stresses. Directing a potato breeding program from cultivated tetraploids to self-compatible diploid lines will be highly helpful for potato breeders. However, diploid potatoes are considered recalcitrant to regeneration. We aimed to develop a protocol for the transformation of diploid Solanum chacoense M6 potatoes using leaf, internodes, and microtubers as the explants. Agrobacterium tumefaciens strain GV2260 harboring pBIN19 expression vector containing gusA gene (interrupted by an intron to induce expression from eukaryotic cells) was used for this purpose. Different inoculation times (10, 20, 30 min) were applied in aforementioned explants for gene transfer. After cocultivation, explants were transferred to the media with various hormone concentrations [6-benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA), trans-zeatin, kinetin, and 2, 4-Dichlorophenoxyacetic acid (2,4-D)]. The calli generated were then transferred to shoot generating medium supplemented with thidiazuron (TDZ) and gibberellic acid (GA3). According to histochemical GUS analysis, we found a 20 min inoculation time to be optimal for gene transfer and the medium containing 2 mg L-1 BAP and 2 mg L-1 NAA was the most suitable medium for callus induction from 20 min inoculated explants (41% callus formation for internodes and 65% for leaf explants). Abundant transcripts levels of gusA gene was found in primary transformants when subjected to RT-qPCR. GUS fluorometric assay further confirmed the primary transformants at protein level. The present study can serve as a gateway to transfer gene(s) of interest in diploid potatoes.

Overexpression of phosphatidylserine synthase IbPSS1 affords cellular Na+ homeostasis and salt tolerance by activating plasma membrane Na+/H+ antiport activity in sweet potato roots

Phosphatidylserine synthase (PSS)-mediated phosphatidylserine (PS) synthesis is crucial for plant development. However, little is known about the contribution of PSS to Na+ homeostasis regulation and salt tolerance in plants. Here, we cloned the IbPSS1 gene, which encodes an ortholog of Arabidopsis AtPSS1, from sweet potato (Ipomoea batatas (L.) Lam.). The transient expression of IbPSS1 in Nicotiana benthamiana leaves increased PS abundance. We then established an efficient Agrobacterium rhizogenes-mediated in vivo root transgenic system for sweet potato. Overexpression of IbPSS1 through this system markedly decreased cellular Na+ accumulation in salinized transgenic roots (TRs) compared with adventitious roots. The overexpression of IbPSS1 enhanced salt-induced Na+/H+ antiport activity and increased plasma membrane (PM) Ca2+-permeable channel sensitivity to NaCl and H2O2 in the TRs. We confirmed the important role of IbPSS1 in improving salt tolerance in transgenic sweet potato lines obtained from an Agrobacterium tumefaciens-mediated transformation system. Similarly, compared with the wild-type (WT) plants, the transgenic lines presented decreased Na+ accumulation, enhanced Na+ exclusion, and increased PM Ca2+-permeable channel sensitivity to NaCl and H2O2 in the roots. Exogenous application of lysophosphatidylserine triggered similar shifts in Na+ accumulation and Na+ and Ca2+ fluxes in the salinized roots of WT. Overall, this study provides an efficient and reliable transgenic method for functional genomic studies of sweet potato. Our results revealed that IbPSS1 contributes to the salt tolerance of sweet potato by enabling Na+ homeostasis and Na+ exclusion in the roots, and the latter process is possibly controlled by PS reinforcing Ca2+ signaling in the roots.

Rapid analysis of GBSS1 and Vinv genes expressed in potato tubers using microtubers produced in liquid culture medium.

This study established a rapid method for the gene expression analysis in potato tubers. The use of microtubers would be useful for primary evaluation of tuber-expressed genes. In the development of transgenic potato or of potato with other genome modifications (e.g., genome editing or RNA-directed DNA methylation (RdDM) and so on) to improve tuber traits, analysis of the target gene is often difficult because of the long cultivation cycle (3-4months), large areas required, numerous materials for plant cultivation, and considerable efforts needed to obtain transgenic tubers. We demonstrate here rapid and convenient analysis of gene expression in potato microtubers. Enough microtubers for expression analysis can be induced over about 4weeks in a simple liquid medium in an Erlenmeyer flask. High-quality RNA and protein can be easily prepared from microtubers and used for northern blot, qRT-PCR, and western blot analyses without further purification. We investigated the expression of two tuber-expressed genes (GBSS1 and Vinv) in microtubers derived from the wild-type and from lines derived from RdDM-mediated transcriptional gene silencing. As expected, the expression of both genes was similar between microtubers and normal tubers. Furthermore, we demonstrated that microtubers can be used in western blot and confocal immunofluorescent microscopy analyses. These results suggest that expression analysis using microtubers is a convenient tool for the analysis of tuber-expressed genes such as GBSS1 and Vinv in potato.

Potato E3 ubiquitin ligase PUB27 negatively regulates drought tolerance by mediating stomatal movement

The ubiquitin-proteasome system (UPS) is one of the main ways of eukaryotic protein degradation and post-translational modification. It has proven as an essential process for plants to respond to abiotic stresses. Plant U-box (PUB) protein acts as a ubiquitin ligase, which recognizes and ubiquitinates the target proteins. Many PUBs have been involved in water stress in Arabidopsis and rice, but similar comprehensive studies in potato remained limited. In this study, the overexpressed and interfered transgenic potato plants of StPUB27 were obtained and their performances were evaluated under osmotic stress. The result showed that overexpression of StPUB27 accelerated the dehydration of detached leaves companied with greater stomatal conductance, while the down-regulated StPUB27 expression by RNA interference (RNAi) showed a smaller stomatal conductance and a lower rate of water loss in detached leaves, thus showing higher tolerance to osmotic stress. In addition, no significant changes in the proline content were observed between StPUB27 overexpressed and RNAi potato plants. The result demonstrated that potato E3 ubiquitin ligase PUB27 may negatively regulate drought tolerance by mediating stomatal conductance.

Activation of the peroxidase gene in potato plants expressing the gene of antimicrobial peptide MsrA1

Antimicrobial peptide MsrA1 is a synthetic hybrid molecule based on cecropin A from giant silk moth larvae and on melittin from melliferous bee venom. Transgenic potato plants of the Belarusian variety Odyssey with the constitutive expressiоn of msrA1 gene are shown to exhibit increased resistance to fungal pathogens Phytophthora infestans and Alternaria solani. Peroxidase genes expression studies using cDNA-PCR and DNA sequencing revealed the activation of the POX peroxidase gene in transgenic plants in the absence of pathogens. This may be indirect evidence of the increased formation of reactive oxygen species, which may explain special resistance to fungal pathogens. The data obtained also confirm a possible role of intracellular antimicrobial peptide in making the plants more resistant to oxidative stress by the way of activation of the host plant defense system.

A sucrose non-fermenting-1-related protein kinase-1 gene, IbSnRK1, confers salt, drought and cold tolerance in sweet potato

Sucrose non-fermenting-1-related protein kinase-1 (SnRK1) regulates carbon and nitrogen metabolism in plants. However, its roles and their underlying mechanisms in tolerance to abiotic stresses are little known. The present study indicated that the IbSnRK1 gene was strongly induced by NaCl, polyethylene glycol (PEG) 6000, hydrogen peroxide (H2O2), cold (4 °C), and abscisic acid (ABA). Its overexpression significantly increased salt, drought, and cold tolerance in transgenic sweet potato plants. ABA, proline, and K+ contents were significantly increased, whereas malondialdehyde (MDA), Na+ and H2O2 contents and O2− production rate were significantly decreased in the transgenic plants under salt, drought, and cold stresses. Overexpression of the gene up-regulated genes involved in ABA biosynthesis, stress response, and stomatal closure; increased enzyme activities in the reactive oxygen species (ROS) scavenging system; and controlled stomatal closure under salt, drought, and cold stresses. These results show that the IbSnRK1 gene confers salt, drought, and cold tolerance in sweet potato by activating the ROS scavenging system and controlling stomatal closure via the ABA signaling pathway.

Identification of disease resistance to soft rot in transgenic potato plants that overexpress the soybeancalmodulin-4gene (GmCaM-4)

Identification of disease resistance to soft rot in transgenic potato plants that overexpress the soybean<i>calmodulin-4</i>gene (<i>GmCaM-4</i>)

Compositional Analysis of the Transgenic Potato with High-level Phytase Expression

Phosphorus in the phytate form is undigestible by non-ruminant animals, causing environmental problems. The transgenic potato line 2-1 was generated with high phytase expression, and it could be a potential feed to improve phytate-P digestibility in non-ruminant animals. Compositional characteristics of genetically modified crops are one of key steps in safety assessment. To evaluate the feed safety of the transgenic potato line 2-1, the comparative analysis of compositions between the transgenic line 2-1 and the conventional line CK potato tubers for two consequent years was conducted. Only proline and zinc contents were shown to be significantly lower in both years, but within the database range. Trypsin inhibitors, lectins, and glycoalkaloids, including α-chaconines, and α-solanines, were also analyzed, and the contents had no significant differences between the two potato lines. The results suggested that the composition of the transgenic potato line 2-1 is substantially equivalent to that of the conventional line CK.

Comparison of defense responses of transgenic potato lines expressing three different Rpi genes to specific Phytophthora infestans races based on transcriptome profiling.

Potato late blight, one of the most devastating diseases in potato, is caused by the oomycete Phytophthora infestans. Over 20 resistance genes have been cloned including R1, R3a, and R3b. The distinctions between defense response mechanisms mediated by different resistance genes are still unclear. Here we performed transcriptome profiling in three transgenic lines, R1, R3a, and R3b, and wild-type Desiree under inoculation with two P. infestans isolates, 89148 (race 0) and CN152 (super race), using RNA-seq. Compared with wild type, specific differentially expressed genes (DEGs) were identified in the three transgenic lines. The highest number of DEGs occurred in transgenic R3b, with 779 DEGs in response to isolate 89148 and 864 DEGs in response to infection by CN152, followed by transgenic R1 lines with 408 DEGs for isolate 89148 and 267 DEGs for CN152. Based on gene ontology, the most common GO terms (15 for 89148 and 20 for CN152) were enriched in transgenic R3a and R3b lines. This indicates that the defense pathways mediated by R3a and R3b are more similar than those mediated by R1. Further separate GO analysis of up- or down-regulated DEGs showed that the down-regulated DEGs mainly functioned in mediating the resistance of potato to P. infestans 89148 by response to stress biological process and to CN152 by oxidation reduction biological process. KEGG pathways of DNA replication, plant-pathogen interaction and pentose and glucuronate interconversions are unique for transgenic R1, R3a, and R3b lines in incompatible interactions. Quantitative real-time PCR experimental validation confirmed the induced expression of DEGs in the late blight resistance signaling pathway. Our results will lay a solid foundation for further understanding the mechanisms of plant-pathogen interactions, and provide a theoretical reference for durable resistance in potato.