Transgenic Potato Research Articles

Destruction of transgenic potato plants (Solanum tuberosum L.) remnants under the influence of soil microbiome

Scientific community has been considering various hypotheses regarding the possible impact of GM plants on non-target soil microorganisms for decades, distinguishing direct and indirect effects. Direct effects are usually easier to detect, as transgenic proteins, characterized by a range of elimination against certain types of insects, pathogenic fungi and bacteria, can affect non-target beneficial symbionts and soil microbial groups involved in the transformation of organic substances. Indirect effects are difficult to assess, as many different factors can affect the composition of root exudates and the metabolic activity of plants. Changes in metabolic pathways can affect the composition of soil exudates and gene expression in plant tissues. At the same time, these changes can affect the decomposition of organic matter and the course of microbiological processes in the soil. Based on experimental studies, an assessment of the rate of destruction of plant residues and DNA degradation in transgenic potato plants in soddy-podzolic soil of potato crop rotation was carried out. The results of the studies have established a significant role of the microbiome of soddy-podzolic soil of potato crop rotation in the destruction of residues of transgenic potato plants and DNA degradation. Destruction of residues of transgenic plants under the influence of microflora of soddy-podzolic soil of potato crop rotation was 30 days with a loss of raw mass 87.14±1.11%. DNA degradation over this period is 88% (186 ng/mg) from the initial amount of total DNA, and degradation of nptII and 35S genes — about 90% from their initial amount in PCR products. It was found that about 90% of nptII and 35S genes in DNA residues of transgenic potato plants under the influence of microbiome soddy-podzolic soil occurs on day 30.

New potential roles of StDREB1 and VvWRKY2 transcription factors in potato dormancy and sprouting patterns

In a previous study, transgenic potato plants overexpressing the StDREB1 or the VvWRKY2 transcription factor (TF) proved to have higher productivity and ameliorated tuber quality in comparison to wildtype (WT; the BF15 variety) plants. Interestingly, when stored at 4 °C, we noticed that the tubers of transgenic potato plants exhibited a delay in sprouting, hence a longer dormancy period. Therefore, we decided to study the dormancy and sprouting of these tubers (the two transgenic and the WT lines) through a physiological and biochemical characterization. WT and genetically modified (GM) tubers were stored at 4 °C for different periods (0, 30, 90, 180, and 240 days) followed by placing them in a germination chamber and the sprouting parameters were then monitored. According to our findings, the overexpression of these two TFs led to modifications in the sprouting kinetic of tubers through an extension of the dormancy period and changes in the sprouting process. Indeed, WT tubers emitted apical and lateral sprouts while those from GM plants showed mainly apical sprouts. In addition, higher reactive oxygen species (ROS) rates, indicators of tuber aging, were recorded in WT tubers compared to GM ones. The higher antioxidant enzyme activities in GM tubers seem to be responsible for aging modification in comparison to WT. The above results suggest the first report on new roles of the StDREB1 and VvWRKY2 TF which seemed to be involved in the regulation of potato tuber aging via a reduction of the main biochemical factors concentration and the ROS content leading to a longer dormancy period and a modified sprouting pattern.

Cas13d-mediated multiplex RNA targeting confers a broad-spectrum resistance against RNA viruses in potato

CRISPR-Cas systems endow the bacterial and archaeal species with adaptive immune mechanisms to fend off invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13d has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However a vast number of different viruses can potentially infect the same host plant resulting in mixed infection, thus necessitating the generation of crops with broad-spectrum resistance to multiple viruses. Here we report the repurposing of CRISPR/Cas13d coupled with an endogenous tRNA-processing system (polycistronic tRNA-gRNA, PTG) to target the multiple potato RNA viruses. Expression of Cas13d and four different gRNAs were observed in transgenic potato lines expressing the Cas13d/PTG construct. We show that the Cas13d/PTG transgenic plants exhibit resistance to either PVY, PVS, PVX or PLRV alone or two/three viruses simultaneously by reducing viral accumulation in plant cells. In sum, our findings provide an efficient strategy for engineering crops that can simultaneously resist infection by multiple RNA viruses.

Potential of endochitinase gene to control Fusarium wilt and early blight disease in transgenic potato lines

Potential of endochitinase gene to control Fusarium wilt and early blight disease in transgenic potato lines

CRISPR/Cas9-mediated Inhibition of Vacuolar Invertase (VInv) Gene in Potato

Potato is the third most important food crop worldwide after the wheat and rice. It has potential to overcome the issues of world’s food security. Potato crop faces some challenges during storage as Cold Induce Sweetening (CIS). During cold storage, the accumulation of reducing sugars such as fructose and glucose takes place in potato tubers. These reducing sugars later react with free amino acid and produce dark-brown pigmentation on potato products during frying. That leads towards reduction of potato quality and consumption. The increased quantity of sucrose is further hydrolyzed by Vacuolar Invertase (VInv) gene into reducing sugars. VInv gene acts as a key player in inducing CIS in potato. The objective of study was cloning of CRISPR/Cas9 construct and successful inhibition of VInv gene function in potato. Agrobacterium-mediated transformation of VInv gene in potato tissues was performed. The knockout of VInv gene in transformed plants was analyzed by using specific primers through polymerase chain reaction (PCR). Real time polymerase chain reaction (RT-PCR) was carried out to analyze the expression of transgene in transgenic potato plants. The knockout of VInv gene using CRISPR/Cas9 strategy could maintain the quality of potato tubers during cold storage.

A Review of Potato Salt Tolerance.

Potato is the world's fourth largest food crop. Due to limited arable land and an ever-increasing demand for food from a growing population, it is critical to increase crop yields on existing acreage. Soil salinization is an increasing problem that dramatically impacts crop yields and restricts the growing area of potato. One possible solution to this problem is the development of salt-tolerant transgenic potato cultivars. In this work, we review the current potato planting distribution and the ways in which it overlaps with salinized land, in addition to covering the development and utilization of potato salt-tolerant cultivars. We also provide an overview of the current progress toward identifying potato salt tolerance genes and how they may be deployed to overcome the current challenges facing potato growers.

Overexpression of 9-cis-Epoxycarotenoid Dioxygenase Gene, IbNCED1, Negatively Regulates Plant Height in Transgenic Sweet Potato.

Plant height is one of the key agronomic traits for improving the yield of sweet potato. Phytohormones, especially gibberellins (GAs), are crucial to regulate plant height. The enzyme 9-cis-epoxycarotenoid dioxygenase (NCED) is the key enzyme for abscisic acid (ABA) biosynthesis signalling in higher plants. However, its role in regulating plant height has not been reported to date. Here, we cloned a new NCED gene, IbNCED1, from the sweet potato cultivar Jishu26. This gene encoded the 587-amino acid polypeptide containing an NCED superfamily domain. The expression level of IbNCED1 was highest in the stem and the old tissues in the in vitro-grown and field-grown Jishu26, respectively. The expression of IbNCED1 was induced by ABA and GA3. Overexpression of IbNCED1 promoted the accumulation of ABA and inhibited the content of active GA3 and plant height and affected the expression levels of genes involved in the GA metabolic pathway. Exogenous application of GA3 could rescue the dwarf phenotype. In conclusion, we suggest that IbNCED1 regulates plant height and development by controlling the ABA and GA signalling pathways in transgenic sweet potato.

StMAPK1 functions as a thermos-tolerant gene in regulating heat stress tolerance in potato (Solanum tuberosum).

Mitogen-activated protein kinases (MAPKs) have been reported to respond to various stimuli including heat stress. This research aimed to investigate whether StMAPK1 is implicated in the transduction of the heat stress signal to adapt heat stress as a thermos-tolerant gene. Potato plants were cultivated under mild (30°C) and acute (35°C) heat stress conditions to analyze mRNA expression of StMAPKs and physiological indicators. StMAPK1 was up-regulated and down-regulated by transfection. Subcellular localization of StMAPK1 protein was observed by fluorescence microscope. The transgenic potato plants were assayed for physiological indexes, photosynthesis, cellular membrane integrity, and heat stress response gene expression. Heat stress altered the expression prolife of StMAPKs. StMAPK1 overexpression changed the physiological characteristics and phenotypes of potato plants under heat stresses. StMAPK1 mediates photosynthesis and maintains membrane integrity of potato plants in response to heat stress. Stress response genes (StP5CS, StCAT, StSOD, and StPOD) in potato plants were altered by StMAPK1 dysregulation. mRNA expression of heat stress genes (StHSP90, StHSP70, StHSP20, and StHSFA3) was affected by StMAPK1. StMAPK1 overexpression increases the heat-tolerant capacity of potato plants at the morphological, physiological, molecular, and genetic levels.

Cloning and gene function of dicarboxylate-tricarboxylate carrier protein in Gastrodia elata

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.

Molecular mechanism by which StSN2 overexpression inhibits the enzymatic browning of potato

Enzymatic browning of fresh-cut potatoes (Solanum tuberosum L.) is an urgent problem to be solved in the potato industry. The transcript level of snakin-2 (StSN2) increased with prolonged fresh-cut potato storage time, and StSN2 overexpression reduced the enzymatic browning of fresh-cut potato compared to wild type (WT). Further studies suggested that overexpression of StSN2 effectively enhanced polyphenol oxidase (PPO), peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) activity, decreased phenolic and soluble quinone contents. The measurement of total free amino acid results showed that the contents of proline, isoleucine, and gamma-aminobutyric acid were increased, and the contents of cystine, methionine, and tyrosine were decreased in StSN2-overexpressing transgenic lines. Quantitative phosphoproteome analysis showed that 1515 proteins were phosphorylated in transgenic fresh-cut potato. Among them, 123 kinases were found to change phosphorylation levels, and a kinase-substrate network of serine/arginine proteins and mitogen-activated protein kinases was predicted. Our results indicated that overexpression of StSN2 inhibited the enzymatic browning of fresh-cut potatoes by enhancing antioxidant enzyme activity and decreasing phenolic and soluble quinone contents while also changing vital kinase and other protein phosphorylation levels. This study demonstrated a new function of StSN2 and provided target genes for enzymatic browning resistance breeding of potatoes.

Early Blight Resistance of Transgenic Potato Plants Expressing the ProSmAMP1 Gene for Antimicrobial Peptides under the Control of a Light-Inducible Cab Promoter

Early Blight Resistance of Transgenic Potato Plants Expressing the ProSmAMP1 Gene for Antimicrobial Peptides under the Control of a Light-Inducible Cab Promoter

Expression of Chitinase and shRNA Gene Exhibits Resistance to Fungi and Virus.

With the increasing global population, saving crops from diseases caused by different kinds of bacteria, fungi, viruses, and nematodes is essential. Potato is affected by various diseases, destroying many crops in the field and storage. In this study, we developed potato lines resistant to fungi and viruses, Potato Virus X (PVX) and Potato Virus Y (PVY), by inoculating chitinase for fungi and shRNA designed against the mRNA of the coat protein of PVX and PVY, respectively. The construct was developed using the pCAMBIA2301 vector and transformed into AGB-R (red skin) potato cultivar using Agrobacterium tumefaciens. The crude protein extract of the transgenic potato plant inhibited the growth of Fusarium oxysporum from ~13 to 63%. The detached leaf assay of the transgenic line (SP-21) showed decreased necrotic spots compared to the non-transgenic control when challenged with Fusarium oxysporum. The transgenic line, SP-21, showed maximum knockdown when challenged with PVX and PVY, i.e., 89 and 86%, while transgenic line SP-148 showed 68 and 70% knockdown in the PVX- and PVY-challenged conditions, respectively. It is concluded from this study that the developed transgenic potato cultivar AGB-R showed resistance against fungi and viruses (PVX and PVY).

A novel effector RipBT contributes to Ralstonia solanacearum virulence on potato.

Ralstonia solanacearum is one of the most destructive plant-pathogenic bacteria, infecting more than 200 plant species, including potato (Solanum tuberosum) and many other solanaceous crops. R. solanacearum has numerous pathogenicity factors, and type III effectors secreted through type III secretion system (T3SS) are key factors to counteract host immunity. Here, we show that RipBT is a novel T3SS-secreted effector by using a cyaA reporter system. Transient expression of RipBT in Nicotiania benthamiana induced strong cell death in a plasma membrane-localization dependent manner. Notably, mutation of RipBT in R. solanacearum showed attenuated virulence on potato, while RipBT transgenic potato plants exhibited enhanced susceptibility to R. solanacearum. Interestingly, transcriptomic analyses suggest that RipBT may interfere with plant reactive oxygen species (ROS) metabolism during the R. solanacearum infection of potato roots. In addition, the expression of RipBT remarkably suppressed the flg22-induced pathogen-associated molecular pattern-triggered immunity responses, such as the ROS burst. Taken together, RipBT acts as a T3SS effector, promoting R. solanacearum infection on potato and presumably disturbing ROS homeostasis.

Early Blight Resistance of Transgenic Potato Plants Expressingthe ProSmAMP1 Gene for Antimicrobial Peptides under the Control of a Light-Inducible Cab Promoter

The genome of Stellaria media contains a gene family for hevein-like antimicrobial peptides, some of which are known to encode two peptides released from the translation product as a result of post-translational proteolysis. These peptides have been shown to inhibit the growth of bacteria and fungi, including potato pathogens Alternaria solani and Alternaria alternata. One of these genes, ProSmAMP1, was introduced into the potato genome under the control of the light-inducible promoter of Cab gene from common wheat. The resulting transgenic lines expressed ProSmAMP1 mRNA during several vegetative passages, and their resistance to early blight was assessed by several indicators of detached leaf infection, with plants having the highest expression of the transgene also showing the highest resistance.

Heat, drought, and combined stress effect ontransgenic potato plants overexpressing theStERF94 transcription factor.

Despite their economic importance worldwide, potato plants are sensitive to various abiotic constraints, such as drought and high temperatures, which cause significant losses in yields and tuber quality. Moreover, because of the climate change phenomenon, plants are frequently subjected to combined stresses, mainly high temperatures and drought. In this context, breeding for tolerant varieties should consider not only plant response to drought or high temperature but also to combined stresses. In the current study, we studied transgenic potato plants overexpressing an ethylene response transcription factor (TF; StERF94) involved in abiotic stress response signaling pathways. Our previous results showed that these transgenic plants display tolerance to salt stress more than wildtype (WT). In this work, we aimed to investigate the effects of drought, heat, and combined stresses on transgenic potato plants overexpressing StERF94 TF under in vitro culture conditions. The obtained results revealed that StERF94 overexpression improved the tolerance of the transgenic plants to drought, heat, and combined stresses through better control of the leaf water and chlorophyll contents, activation of antioxidant enzymes, and an accumulation of proline, especially in the leaves. Indeed, the expression level of antioxidant enzyme-encoding genes (CuZnSOD, FeSOD, CAT1, and CAT2) was significantly induced by the different stress conditions in the transgenic potato plants compared with the WT plants. This study further confirms that StERF94 TF may be implicated in regulating the expression of target genes encoding antioxidant enzymes.

Sweet potato NAC transcription factor NAC43 negatively regulates plant growth by causing leaf curling and reducing photosynthetic efficiency.

Leaves comprise one of the most important organs for plant growth and development. Although there have been some reports on leaf development and the establishment of leaf polarity, their regulatory mechanisms are not very clear. In this study, we isolated a NAC (NAM, ATAF, and CUC) transcription factor (TF), i.e., IbNAC43, from Ipomoea trifida, which is a wild ancestor of sweet potato. This TF was highly expressed in the leaves and encoded a nuclear localization protein. The overexpression of IbNAC43 caused leaf curling and inhibited the growth and development of transgenic sweet potato plants. The chlorophyll content and photosynthetic rate in transgenic sweet potato plants were significantly lower than those in wild-type (WT) plants. Scanning electron microscopy (SEM) and paraffin sections showed that the ratio of cells in the upper and lower epidermis of the transgenic plant leaves was unbalanced; moreover, the abaxial epidermal cells were irregular and uneven in transgenic plants. In addition, the xylem of transgenic plants was more developed than that of WT plants, while their lignin and cellulose contents were significantly higher than those of WT. Quantitative real-time PCR (qRT-PCR) analysis showed that the overexpression of IbNAC43 upregulated the genes involved in leaf polarity development and lignin biosynthesis in transgenic plants. Moreover, it was found that IbNAC43 could directly activate the expression of the leaf adaxial polarity-related genes IbREV and IbAS1 by binding to their promoters. These results indicate that IbNAC43 might play a critical role in plant growth by affecting the establishment of leaf adaxial polarity. This study provides new insights regarding leaf development.

Amelioration of cold-induced sweetening in potato by RNAi mediated silencing of StUGPase encoding UDP-glucose pyrophosphorylase.

Cold-induced sweetening (CIS) is an unwanted physiological phenomenon in which reducing sugars (RS) get accumulated in potato (Solanum tuberosum) upon cold storage. High RS content makes potato commercially unsuitable for processing due to the unacceptable brown color in processed products like chips, fries, etc., and the production of a potential carcinogen, acrylamide. UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose towards the synthesis of sucrose and is also involved in the regulation of CIS in potato. The objective of the present work was RNAi-mediated downregulation of the StUGPase expression level in potato for the development of CIS tolerant potato. Hairpin RNA (hpRNA) gene construct was developed by placing UGPase cDNA fragment in sense and antisense orientation intervened by GBSS intron. Internodal stem explants (cv. Kufri Chipsona-4) were transformed with hpRNA gene construct, and 22 transgenic lines were obtained by PCR screening of putative transformants. Four transgenic lines showed the highest level of RS content reduction following 30 days of cold storage, with reductions in sucrose and RS (glucose & fructose) levels of up to 46% and 57.5%, respectively. Cold stored transgenic potato of these four lines produced acceptable chip colour upon processing. The selected transgenic lines carried two to five copies of the transgene. Northern hybridization revealed an accumulation of siRNA with a concomitant decrease in the StUGPase transcript level in these selected transgenic lines. The present work demonstrates the efficacy of StUGPase silencing in controlling CIS in potato, and the strategy can be employed for the development of CIS tolerant potato varieties.

Metabolic engineering of betacyanin in vegetables for anti-inflammatory therapy.

Betalains, which consist of the subgroups betaxanthins and betacyanins, are hydrophilic pigments that have classically been used for food colorants. Owing to their strong antioxidant property, their usefulness for application for therapeutic use is also expected. In addition, as betalains are mainly naturally available from plants of the order Caryophyllales, including beet (Beta vulgaris), metabolic engineering for betalain production in crops such as vegetables, fruits and cereals may provide new food resources useful for healthcare. Here we conducted metabolic engineering of betacyanins in tomato fruits and potato tubers. The transgenic tomato fruits and potato tubers with coexpression of betacyanin biosynthesis genes, CYP76AD1 from B. vulgaris, DOD (DOPA 4,5-dioxygenase) and 5GT (cyclo-DOPA 5-O-glucosyltransferase) from Mirabilis jalapa, under control of suitable specific promoters, possessed dark red tissues with enriched accumulation of betacyanins (betanin and isobetanin). The anti-inflammatory activity of transgenic tomato fruit extract was superior to that of wild-type fruit extract on macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS), as a result of decreased LPS-stimulated transcript levels of proinflammatory genes. These findings were in accord with the observation that administration of the transgenic tomato fruits ameliorated dextran sulfate sodium (DSS)-induced colitis as well as body weight loss and disease activity index in mice, via suppression of DSS-stimulated transcript levels of pro-inflammatory genes, including Tnf (encoding TNF-alpha), Il6, and Ptgs2 (encoding cyclooxygenae 2). Intriguingly, given the fact that the transgenic potato tuber extract failed to enrich the anti-inflammatory activity of macrophage cells, it is likely that metabolic engineering of betacyanins will be a powerful way of increasing the anti-inflammatory property of ordinary foods such as tomato.

The transcription factor IbNAC29 positively regulates the carotenoid accumulation in sweet potato.

Carotenoid is a tetraterpene pigment beneficial for human health. Although the carotenoid biosynthesis pathway has been extensively studied in plants, relatively little is known about their regulation in sweet potato. Previously, we conducted the transcriptome database of differentially expressed genes between the sweet potato (Ipomoea batatas) cultivar 'Weiduoli' and its high-carotenoid mutant 'HVB-3'. In this study, we selected one of these candidate genes, IbNAC29, for subsequent analyses. IbNAC29 belongs to the plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factor family. Relative IbNAC29 mRNA level in the HVB-3 storage roots was ~1.71-fold higher than Weiduoli. Additional experiments showed that the contents of α-carotene, lutein, β-carotene, zeaxanthin, and capsanthin are obviously increased in the storage roots of transgenic sweet potato plants overexpressing IbNAC29. Moreover, the levels of carotenoid biosynthesis genes in transgenic plants were also up-regulated. Nevertheless, yeast one-hybrid assays indicated that IbNAC29 could not directly bind to the promoters of these carotenoid biosynthesis genes. Furthermore, the level of IbSGR1 was down-regulated, whose homologous genes in tomato can negatively regulate carotene accumulation. Yeast three-hybrid analysis revealed that the IbNAC29-IbMYB1R1-IbAITR5 could form a regulatory module. Yeast one-hybrid, electrophoretic mobility shift assay, quantitative PCR analysis of chromatin immunoprecipitation and dual-luciferase reporter assay showed that IbAITR5 directly binds to and inhibits the promoter activity of IbSGR1, up-regulating carotenoid biosynthesis gene IbPSY. Taken together, IbNAC29 is a potential candidate gene for the genetic improvement of nutritive value in sweet potato.

Fast track to obtain heritable transgenic sweet potato inspired by its evolutionary history as a naturally transgenic plant.

Fast track to obtain heritable transgenic sweet potato inspired by its evolutionary history as a naturally transgenic plant.